517525

ÇUKUROVA UNIVERSITY
INSTITUTE OF NATURAL AND APPLIED SCIENCES
PhD THESIS
Cennet Pelin BOYACI GÜNDÜZ
MOLECULAR CHARACTERIZATION OF THE

PREDOMINANT LACTIC ACID BACTERIA AND YEASTS IN
THE SOURDOUGH AND CHICKPEA FERMENTATIONS
AND INVESTIGATION OF SOME LACTIC ACID BACTERIA
FOR POTENTIAL STARTER CULTURE USAGE
DEPARTMENT OF FOOD ENGINEERING
ADANA-2018
MOLECULAR CHARACTERIZATION OF THE PREDOMINANT

LACTIC ACID BACTERIA AND YEASTS IN THE SOURDOUGH AND
CHICKPEA FERMENTATIONS AND INVESTIGATION OF SOME
LACTIC ACID BACTERIA FOR POTENTIAL STARTER CULTURE
USAGE
PhD THESIS
We certify that the thesis titled above was reviewed and approved for the
award of degree of the Doctor of Philosophy by the board of jury on
19/07/2018.
………………………….. ……………………........ …………………………………

Prof. Dr. Hüseyin ERTEN Prof. Dr. Filiz ÖZÇELİK Prof. Dr. Turgut CABAROĞLU
Supervisor Member Member
…………………………......... …………………………….
Assoc. Prof. Dr. Luca Settanni Prof. Dr. Haşim KELEBEK
Member Member
This PhD Thesis is written at the Food Engineering Department of Institute of

Natural and Applied Sciences of Çukurova University.
Registration No:
Prof. Dr. Mustafa GÖK

Director
Institute of Natural and Applied Sciences
A part of this study was financially supported by Çukurova University Academic

Research Projects Unit.
Project No: FBA-2017-9035
Not: The usage of the presented specific declarations, tables, figures, and photographs
either in this thesis or in any other reference without citation is subjected to “The law
of Arts and Intellectual Products” number of 5846 of Turkish Republic.
ABSTRACT
PHD THESIS
MOLECULAR CHARACTERIZATION OF THE PREDOMINANT

LACTIC ACID BACTERIA AND YEASTS IN THE SOURDOUGH AND
CHICKPEA FERMENTATIONS AND INVESTIGATION OF SOME
LACTIC ACID BACTERIA FOR POTENTIAL STARTER CULTURE
USAGE
Supervisor : Prof. Dr. Hüseyin ERTEN

Year: 2018, Pages: 317
Jury : Prof. Dr. Hüseyin ERTEN
: Assoc. Prof. Dr. Luca SETTANNI
: Prof. Dr. Filiz ÖZÇELİK
: Prof. Dr. Turgut CABAROĞLU
: Prof. Dr. Haşim KELEBEK
In the present study, a total of 20 sourdough, chickpea liquid starter and dough samples
were collected from different bakeries at two different times. Sourdough and chickpea
fermentations were also conducted under laboratory conditions. Microbiological and chemical
properties of collected samples were investigated and lactic acid bacteria and yeasts were
isolated and identified by molecular methods. In sourdough fermentations, analysis by 16S
rRNA gene sequencing grouped the strains into 18 lactic acid bacteria species and the most
frequent isolates were Lactobacillus sanfranciscensis (32.7%), Lactobacillus plantarum (18.6%)
and Lactobacillus paralimentarious (15.9%). In chickpea fermentations, 12 lactic acid bacteria
species were identified and the most prevalent species were Weissella confusa (44.6%),
Enterococcus faecium (25.6%) and Weissella cibaria (11.6%). PCR-RFLP analysis identified
Saccharomyces cerevisiae in the sourdough (72.5%) and chickpea fermentations (40.7%) as the
most frequent yeast species. Other isolated yeast species were Kazachstania bulderi, Pichia
membranifaciens, Kazachstania servazzii, Kazachstania unispora and Hanseniaspora
valbyensis for sourdoughs and Candida parapsilosis, Meyerozyma guilliermondii and
Cryptococcus albidosimilis for chickpea fermentations. Pichia fermentans was isolated from
both of the fermentations. According to the technological potential, Lactobacillus plantarum
XL23 and Lactobacillus sanfranciscensis RL976 strains were used as mono- and dual-culture in
the production of experimental sourdoughs and Weissella confusa RL1139 strain was used as
mono-culture in the production of experimental chickpea liquid starters.
Key Words: Sourdough, chickpea liquid starter, LAB, yeasts, PCR
I
ÖZ
DOKTORA TEZİ
NOHUT MAYASI VE EKŞİ HAMUR FERMANTASYONLARINDAKİ

LAKTİK ASİT BAKTERİLERİNİN VE MAYALARIN MOLEKÜLER
YÖNTEMLERLE TANIMLANMASI VE BAZI LAKTİK ASİT
BAKTERİLERİNİN STARTER KÜLTÜR OLARAK KULLANILMA
POTANSİYELLERİNİN ARAŞTIRILMASI
ÇUKUROVA ÜNİVERSİTESİ
FEN BİLİMLERİ ENSTİTÜSÜ
GIDA MÜHENDİSLİĞİ BÖLÜMÜ
Danışman : Prof. Dr. Hüseyin ERTEN

Yıl: 2018, Sayfa: 317
Jüri : Prof. Dr. Hüseyin ERTEN
: Doç. Dr. Luca SETTANNI
: Prof. Dr. Filiz ÖZÇELİK
: Prof. Dr. Turgut CABAROĞLU
: Prof. Dr. Haşim KELEBEK
Bu çalışmada, farklı yerlerden iki farklı zamanda 20 adet ekşi hamur, nohut süzüntüsü
mayası ve nohut mayası hamuru örneği toplanmıştır. Ayrıca laboratuvar koşullarında da ekşi
hamur ve nohut mayası üretimi gerçekleştirilmiştir. Toplanan örneklerin mikrobiyolojik ve
kimyasal özellikleri araştırılmıştır. Laktik asit bakterileri ve mayalar izole edilerek moleküler
yöntemlerle tanımlanmışlardır. Ekşi hamurlarda 16S rRNA gen sekans analizleri izolatları 18
türe ayırmıştır. Lactobacillus sanfranciscensis (%32.7), Lactobacillus plantarum (%18.6) ve
Lactobacillus paralimentarious (%15.9) en sık izole edilen türlerdir. Nohut mayası
fermentasyonlarında, Weissella confusa (%44.6), Enterococcus faecium (%25.6) ve Weissella
cibaria (%11.6) en sık izole edilenler olmakla birlikte toplamda 12 farklı tür tanımlanmıştır.
PCR-RFLP sonuçlarına göre, Saccharomyces cerevisiae ekşi hamur (%72.5) ve nohut mayası
fermantasyonlarında (%40.7) en çok bulunan maya türüdür. Diğer izole edilen maya türleri ise
ekşi hamurlarda Kazachstania bulderi, Pichia membranifaciens, Kazachstania servazzii,
Kazachstania unispora ve Hanseniaspora valbyensis, nohut mayalarında Candida parapsilosis,
Meyerozyma guilliermondii ve Cryptococcus albidosimilis olarak belirlenmiştir. Pichia
fermentans her iki fermantasyondan da izole edilmiştir. Teknolojik potansiyellerine göre,
Lactobacillus plantarum XL23 ve Lactobacillus sanfranciscensis RL976 saf ve karışık kültür
olarak ekşi hamur fermantasyonlarında kullanılırken, nohut mayası fermantasyonlarında
Weissella confusa RL1139 mono kültür olarak kullanılmıştır.
Anahtar kelimeler: Ekşi hamur, nohut süzüntüsü mayası, LAB, maya, PCR
II
EXTENDED SUMMARY
In the present study, LAB and yeasts that populate the sourdough and
chickpea fermentations were investigated by molecular methods on the samples
collected from different locations at two different times. Also chemical and
microbiological properties of the collected samples were examined. Some LAB
strains were further analysed for their potential to be used as starter culture and
selected strains were used in the experimental sourdough and chickpea dough
productions.
Totally 20 samples including sourdough (8), chickpea liquid starter (6) and
dough (6) samples were collected from different bakeries at two different times.
Also sourdough and chickpea fermentations were conducted under laboratory
conditions. Microbiological and chemical properties of the collected samples were
investigated and a total of 834 lactic acid bacteria and 473 yeast colonies were
isolated from samples for molecular identification.
The pH and total acidity levels of the collected sourdough samples were in
the range of 3.71-3.96 and 6.78-23.93 mL 0.1 N NaOH /10 g dough, respectively.
According to the HPLC analysis, maltose+sucrose, glucose, fructose, ethanol,
lactic acid and acetic acid contents were in the range of <LOQ-6.24, 0.81-2.30,
0.78-6.96, 4.39-14.94, 5.15-14.12 and 0.58-2.40 g/kg, respectively. Fermentation
quotient of the sourdoughs were in the range of 2.48-5.90. The cell counts of
presumptive lactic acid bacteria varied from 4.78 to 11.96 log CFU/g and the
highest cell density on mMRS agar were counted as 11.67 log CFU/g in the rye
sourdough sample. Presumptive total and non-Saccharomyces yeast counts varied
from 6.75 to 10.02 log CFU/g on YPD and 2.70 to 8.22 log CFU/g on L-lysine agar
media. The highest cell density on YPD agar was counted in the rye sourdough
sample. Under laboratory conditions, sourdough was produced at 28°C by
propagating over a period of 7 days using the daily back-slopping (refreshment)
procedure. At the final refreshment, pH and TTA was determined as 3.60 and
III
17.56 mL 0.1 N NaOH/10 g dough, respectively. Fermentation quotient of the
laboratory produced sourdough was determined as 10.84. Presumptive lactic acid
bacteria cell counts on mMRS were 12 log CFU/g at the end of the fermentation.
A total of 439 LAB and 235 yeast isolates were collected from sourdough
samples including laboratory scale production. A total of 84 strains representing
178 isolates were confirmed to be members of the lactic acid bacteria. Analysis by
16S rRNA gene sequencing grouped the strains into 18 LAB species, which
belonged to six genera: Lactobacillus, Pediococcus, Enterococcus, Leuconostoc,
Weissella and Lactococcus. Lactobacillus sanfranciscensis (32.7%) was the
dominant species and followed by Lactobacillus plantarum (18.6%) and
Lactobacillus paralimentarious (15.9%). Also Lactobacillus paracasei (7.1%),
Leuconostoc mesenteroides (4.4%), Weissella confusa (3.5%), Lactobacillus
curvatus (3.5%) and Lactobacillus brevis (2.7%) were found as minor species. On
the other hand, Lactobacillus pentosus, Leuconostoc citreum, Lactobacillus
paraplantarum, Lactobacillus acidophilus, Enterococcus faecium, Pediococcus
inopinatus, Lactobacillus parabrevis, Lactococcus lactis subsp. cremoris,
Weissella cibaria and Pediococcus pentosaceus were only isolated from 1 or 2
samples.
A total of 153 isolates belonging to 7 yeast species were identified by 26S
rRNA gene sequencing. Saccharomyces cerevisiae (72.5%) was the dominant yeast
species. Other isolated yeast species were Kazachstania bulderi (7.2%), Pichia
fermentans (5.9%), Pichia membranifaciens (5.2%), Kazachstania servazzii
(4.6%), Kazachstania unispora (2.6%) and Hanseniaspora valbyensis (2%).
The pH and total acidity levels of the collected chickpea liquid starter
samples were in the range of 4.82-5.67 and 1.65-3.20 mL 0.1 N NaOH/10 g
sample, respectively. The pH and total acidity levels of the collected chickpea
dough samples were in the range of 5.12-5.53 and 3.03-5.40 mL 0.1 N NaOH/10 g
sample, respectively. According to the HPLC analysis, the content of
maltose+sucrose, glucose, fructose, ethanol, lactic acid and acetic acid in the
IV
chickpea liquid starter samples were in the range of 1.25-4.50, 2.59-6.94, 2.18-
6.44, 2.49-2.59, <LOQ-0.93 and 0.86-1.23 g/kg, respectively. The content of
maltose+sucrose, glucose, fructose, ethanol, lactic acid and acetic acid in the
chickpea dough samples were in the range of 20.38-29.38, 5.54-9.80, 4.35-8.44,
2.45-2.81, <LOQ-0.94 and <LOQ-<LOQ g/kg, respectively. Cell counts of
presumptive lactic acid bacteria in collected chickpea liquid starters were found to
be in the range of 1.60-7.18 log CFU/g on mMRS medium. The mean cell counts
of presumptive lactic acid bacteria in collected chickpea dough samples were
determined to be in the range of 4.30-6.89 on mMRS medium. Presumptive yeast
cells in chickpea liquid and dough samples were in the range of 0-5.85 and <1.00-
6.83 log CFU/g on two different media, respectively. According to the
microbiological analysis results, the total bacteria counted on NA medium was in
the range of 2.20-7.70 and 3.53-7.39 log CFU/g for chickpea liquid starter and
dough samples, respectively. The control chickpea liquid starter and dough samples
were produced in duplicate under laboratory conditions. Chickpea liquid starter
fermentations were conducted at 32 and 37°C for 18 h. At the end of the
fermentation, the pH level at 32 and 37°C were 4.91 and 4.75, respectively. Total
acidity values were 1.95 and 2.95 mL 0.1 N NaOH/10 g sample for liquid starters
fermented at 32 and 37°C, respectively. Following chickpea liquid fermentations,
the fermented liquid starter was used in chickpea dough production. At the end of 4
hours of fermentation, the final pH values of both fermentations were close to each
other as 4.84 at 32°C and 4.81 at 37°C. Total acidity values were 4.80 and 5.00 mL
0.1 N NaOH/ 10 g dough in the doughs fermented at 32 and 37°C, respectively.
A total of 395 LAB and 238 yeast isolates were collected from chickpea
liquid starter and dough samples, including laboratory scale production. A total of
54 strains representing 149 isolates were confirmed to be members of the lactic
acid bacteria. Analysis by 16S rRNA gene sequencing grouped the strains into 12
LAB species, which belonged to six genera: Lactobacillus, Pediococcus,
Enterococcus, Leuconostoc, Weissella and Streptococcus. Weissella confusa
V
(44.6%) was the dominant species, followed by Enterococcus faecium (25.6%) and
Weissella cibaria (11.6%). Furthermore, Leuconostoc mesenteroides (5%),
Lactobacillus brevis (3.3%) and Streptococcus lutetiensis (2.5%) were found as
minor species. Conversely, Lactobacillus plantarum, Pediococcus acidilactici,
Streptococcus salivarius, Enterococcus lactis, Pediococcus pentosaceus and
Leuconostoc mesenteroides subsp. dextranium were only isolated from 1 or 2
samples.
A total of 59 isolates belonging to 5 species were identified by 26S rRNA
gene sequencing. Only one isolate was identified at the genus level as
Wickerhamiella spp. Saccharomyces cerevisiae (40.7%) was the dominant yeast
species among all isolated strains. Other isolated yeast species were Candida
parapsilosis (33.9%), Meyerozyma guilliermondii (20.3%), Pichia fermentans
(3.4%) and Cryptococcus albidosimilis (1.7%).
The most frequently isolated lactic acid bacteria species were investigated
for technological potential to be used as starter culture in sourdough and chickpea
fermentations. According to the technological potential, Lactobacillus plantarum
XL23 and Lactobacillus sanfranciscensis RL976 strains were used as mono- and
dual-culture in the production of experimental sourdoughs. Doughs inoculated with
mono- or dual-culture of Lactobacillus plantarum XL23 reached the pH values less
than 4.0 in 12 hours. Dough inoculated with Lactobacillus sanfranciscensis RL976
reached pH values less than 4.0 after 24 hours. After 48 hours, the control
sourdough exhibited the same patterns with the inoculated sourdoughs and reached
pH values below 4.0. Acidity values and LAB counts of the samples confirmed the
trend showed by pH. After 24 hours, acidity values of the inoculated sourdoughs
were in the range of 15.35-16.03 mL 0.1 N NaOH/10 g dough. At the last
refreshment, the highest acidity was determined in the sourdough produced with
dual-culture inoculum. As a result of the activities in sourdoughs, some VOC
compounds are generated. The SPME-GC-MS chromatographic analysis of the
VI
experimental sourdoughs revealed the presence of 37 VOC compounds belonged to
different chemical groups.
Weissella confusa RL1139 strain was used as mono-culture in the
production of experimental chickpea liquid starters at 37°C. The final pH and total
acidity values of the control and inoculated chickpea liquid starters were 4.92-4.82
and 4.4-4.1 mL 0.1 N NaOH/10 g sample respectively. The final pH values of the
control and inoculated chickpea doughs were 4.82 and 4.79, respectively. Final
acidity values of the control and inoculated doughs were 5.26 and 5.97 mL 0.1 N
NaOH/10 g sample, respectively. The SPME-GC-MS chromatographic analysis
revealed the presence of 32 VOC compounds in experimental chickpea
fermentations. Butanoic acid was found in all of the fermented chickpea liquid
starter and dough samples as the characteristic VOC compound.
In this study, different LAB and yeast species were identified in
sourdough, chickpea liquid starter and dough samples and some LAB strains were
used in the experimental sourdough and chickpea fermentations as starter culture.
Development of starter culture combinations is important to obtain products with
same characteristics during the industrial production since by starter culture
addition, large scale industrial production of standard sourdough and chickpea
breads will be possible everytime at the same quality.
VII
VIII
TÜRKÇE GENİŞLETİLMİŞ ÖZET
Bu çalışmada, ekşi hamur ve nohut mayası fermantasyonlarında etkili

laktik asit bakterileri ve mayalar farklı yerlerden iki farklı zamanda alınan
örneklerden izole edilerek moleküler yöntemlerle tanımlanmışlardır. Ayrıca
toplanan örneklerin kimyasal ve mikrobiyolojik özellikleri araştırılmıştır. Bazı
laktik asit bakteri suşlarının starter kültür olarak kullanılma potansiyelleri analiz
edilmiş ve seçilen suşlar ekşi hamur ve nohut mayası üretimlerinde kullanılmıştır.
Toplamda 20 adet ekşi hamur (8), nohut süzüntüsü mayası (6) ve nohut
mayası hamuru (6) örnekleri farklı fırınlardan iki farklı zamanda toplanmıştır.
Ayrıca laboratuvar koşullarında da ekşi hamur ve nohut mayası fermantasyonları
gerçekleştirilmiştir. Örneklerde mikrobiyolojik ve kimyasal analizler
gerçekleştirilmiş ve örneklerden 834 laktik asit bakterisi ve 473 maya izole
edilerek moleküler yöntemlerle tanımlanmışlardır.
Ekşi hamur örneklerinin pH ve toplam asitlik değerleri sırasıyla 3.71-3.96
ve 6.78-23.93 mL 0.1 N NaOH/10 g hamur olarak belirlenmiştir. HPLC
analizlerine göre maltoz+sakkaroz, glukoz, fruktoz, etanol, laktik asit ve asetik asit
miktarları sırasıyla <Tayin limiti-6.24, 0.81-2.30, 0.78-6.96, 4.39-14.94, 5.15-
14.12 ve 0.58-2.40 g/kg olarak hesaplanmıştır. Ekşi hamur örneklerinin
fermantasyon katsayısı 2.48-5.90 aralığında belirlenmiştir. Muhtemel laktik asit
bakterilerinin sayım sonuçları 4.78-11.96 log KOB/g aralığında bulunmuştur ve
mMRS besiyerinde sayılan en fazla koloni çavdar ekşi hamur örneğinde 11.67 log
KOB/g olarak tespit edilmiştir. Muhtemel toplam maya ve Saccharomyces
olmayan maya sayım sonuçları sırasıyla YPD besiyerinde 6.75-10.02 log KOB/g
ve L-lysine besiyerinde 2.70-8.22 log KOB/g aralığında belirlenmiştir. YPD
besiyerinde sayılan en fazla koloni çavdar ekşi hamur örneğinde tespit edilmiştir.
Laboratuvar koşullarında, 28°C sıcaklıkta 7 gün boyunca günlük tazeleme
yöntemiyle ekşi hamur üretimi gerçekleştirilmiştir. Son tazelemede pH ve toplam
IX
asitlik değerleri sırasıyla 3.60 ve 17.56 mL 0.1 N NaOH/10 g hamur olarak
belirlenmiştir. Laboratuvarda üretilen ekşi hamurun fermantasyon katsayısı 10.84
olarak hesaplanmıştır. Son tazelemedeki muhtemel laktik asit bakteri sayım
sonuçları mMRS besiyerinde 12 log KOB/g olarak belirlenmiştir.
Toplamda 439 laktik asit bakterisi ve 235 maya ekşi hamur
fermantasyonlarından izole edilmiştir. Toplamda 178 isolatı temsil eden 84 suş
laktik asit bakterisi olarak tanımlanmıştır. 16s rRNA sekans sonuçlarına göre
izolatlar 6 cinse ait 18 türe ayrılmıştır. Bu türler Lactobacillus, Pediococcus,
Enterococcus, Leuconostoc, Weissella and Lactococcus olarak belirlenmiştir.
Lactobacillus sanfranciscensis (%32.7), Lactobacillus plantarum (%18.6) ve
Lactobacillus paralimentarious (%15.9) en baskın türler olarak belirlenirken,
Lactobacillus paracasei (7.1 %), Leuconostoc mesenteroides (%4.4), Weissella
confusa (%3.5), Lactobacillus curvatus (%3.5) ve Lactobacillus brevis (%2.7) az
sayıda örnekte tespit edilen türlerdir. Diğer türler Lactobacillus pentosus,
Leuconostoc citreum, Lactobacillus paraplantarum, Lactobacillus acidophilus,
Enterococcus faecium, Pediococcus inopinatus, Lactobacillus parabrevis,
Lactococcus lactis subsp. cremoris, Weissella cibaria ve Pediococcus pentosaceus
sadece 1 veya 2 örnekten izole edilmiştir.
Toplamda 7 türe ait 153 maya izolatı 26S rRNA gen sekans sonuçlarına
göre tanımlanmışlardır. Saccharomyces cerevisiae (%72.5) ekşi hamur
fermantasyonlarındaki baskın tür olarak belirlenirken Kazachstania bulderi (%7.2),
Pichia fermentans (%5.9), Pichia membranifaciens (%5.2), Kazachstania servazzii
(%4.6), Kazachstania unispora (%2.6) ve Hanseniaspora valbyensis (%2) maya
türleri de ekşi hamur fermantasyonlarından izole edilmişlerdir.
Toplanan nohut süzüntü mayası örneklerinin pH ve TTA değerleri sırasıyla
4.82-5.67 ve 1.65-3.20 mL 0.1 N NaOH/10 g örnek olarak bulunurken, nohut
mayası hamurlarının pH ve toplam asitlik değerleri sırasıyla 5.12-5.53 ve 3.03-5.40
mL 0.1 N NaOH/10 g örnek olarak hesaplanmıştır. HPLC analizlerine göre nohut
mayası süzüntülerinde bulunan maltoz+sakkaroz, glukoz, fruktoz, etanol, laktik asit
X
ve asetik asit miktarları sırasıyla 1.25-4.50, 2.59-6.94, 2.18-6.44, 2.49-2.59, <Tayin
limiti-0.93 ve 0.86-1.23 g/kg olarak belirlenmiştir. Nohut mayası hamurlarındaki
maltoz+sakkaroz, glukoz, fruktoz, etanol ve laktik asit miktarları sırasıyla 20.38-
29.38, 5.54-9.80, 4.35-8.44, 2.45-2.81, <Tayin limiti-0.94 g/kg ve bütün örneklerde
asetik asit miktarları tayin limitinin altında belirlenmiştir. Toplanan nohut
süzüntüsü mayası örneklerinin mMRS besiyerinde belirlenen muhtemel laktik asit
bakteri sayım sonuçları 1.60-7.18 log KOB/g aralığında bulunmuştur. Toplanan
nohut mayası hamurlarının mMRS besiyerinde belirlenen muhtemel laktik asit
bakteri sayım sonuçları 4.30-6.89 log KOB/g aralığında bulunmuştur. Nohut
süzüntüsü mayası ve hamuru örneklerindeki muhtemel maya sayıları 0-5.85 ve
<1.00-6.83 log KOB/g aralığında belirlenmiştir. Mikrobiyolojik analiz sonuçlarına
göre, NA besiyerinde sayılan toplam bakteri sayısı sırasıyla nohut süzüntüsü
mayası ve hamur örneklerinde 2.20-7.70 and 3.53-7.39 log KOB/g aralığında
belirlenmiştir. Laboratuvar koşullarında nohut süzüntüsü fermantasyonları 32 ve
37°C'de 18 saat boyunca iki paralleli olarak yapılmıştır. Fermantasyon sonunda, 32
ve 37°C'de gerçekleştirilen fermantasyonlarda pH değerleri sırasıyla 4.91 ve 4.75
olarak belirlenmiştir. Toplam asitlik değerleri ise 32 ve 37°C'de sırasıyla 1.95 and
2.95 mL 0.1 N NaOH/10 g örnek olarak hesaplanmıştır. Nohut mayası süzüntüleri,
nohut mayası hamuru üretiminde kullanılmış ve 4 saatlik hamur fermantasyonu
sonucunda, 32°C'de pH değeri 4.84 olarak belirlenirken, 37°C'de 4.81 olarak
belirlenmiştir. Toplam asitlik ise 32 ve 37°C'de sırasıyla 4.80 ve 5.00 mL 0.1 N
NaOH/10 g örnek olarak belirlenmiştir.
Toplamda 395 laktik asit bakterisi ve 238 maya nohut mayası
fermantasyonlarından izole edilmiştir. 149 izolatı temsil eden 54 suş laktik asit
bakterisi olarak tanımlanmıştır. 16s rRNA sekans sonuçlarına göre izolatlar 6 cinse
ait 12 türe ayrılmıştır. Bu türler Lactobacillus, Pediococcus, Enterococcus,
Leuconostoc, Weissella ve Streptococcus olarak belirlenmiştir. Weissella confusa
(%44.6) en baskın tür olarak belirlenirken, Enterococcus faecium (%25.6) ve
Weissella cibaria (%11.6) türleri de en sık izole edilen türlerdir. Leuconostoc
XI
mesenteroides (%5), Lactobacillus brevis (%3.3) and Streptococcus lutetiensis
(%2.5) az sayıda örnekte tespit edilen türlerdir. Diğer türler Lactobacillus
plantarum, Pediococcus acidilactici, Streptococcus salivarius, Enterococcus lactis,
Pediococcus pentosaceus ve Leuconostoc mesenteroides subsp. dextranium sadece
1 veya 2 örnekten izole edilmiştir.
Toplamda 5 türe ait 59 maya izolatı 26S rRNA gen sekans sonuçlarına
göre tanımlanmışlardır. Sadece bir izolat Wickerhamiella spp. olarak cins
düzeyinde tanımlanmıştır. Saccharomyces cerevisiae (%40.7) nohut mayası
fermantasyonlarındaki tüm türler arasında en baskın tür olarak belirlenirken,
Candida parapsilosis (%33.9), Meyerozyma guilliermondii (%20.3), Pichia
fermentans (%3.4) ve Cryptococcus albidosimilis (%1.7) maya türleri de nohut
mayası fermantasyonlarından izole edilmişlerdir.
İzole edilen laktik asit bakterilerinin, ekşi hamur ve nohut mayası
fermantasyonlarında starter kültür olarak kullanılabilmeleri amacıyla en sık izole
edilen laktik asit bakterilerinin teknolojik potansiyelleri araştırılmıştır. Teknolojik
potansiyellerine göre, Lactobacillus plantarum XL23 ve Lactobacillus
sanfranciscensis RL976 suşları saf ve karışık kültür olarak laboratuvar koşullarında
ekşi hamur üretiminde kullanılmışlardır. Lactobacillus plantarum XL23 ile üretilen
hamurların pH düzeyleri 12 saat içinde 4.0'ın altına düşmüştür. Lactobacillus
sanfranciscensis RL976 ile üretilen hamurların pH değerleri 24 saat içinde 4.0'ın
altına düşmüştür. 48 saatin sonunda, kontrol ekşi hamuru inokule edilen hamurlarla
benzer özellikler göstermiş ve pH değeri 4.0'ün altına inmiştir. Asitlik değerleri ve
laktik asit bakteri sayıları da pH ile gözlemlenen benzer durumu yansıtmıştır. 24
saatin sonunda, starter kültürle üretilen ekşi hamurlardaki asitlik değerleri 15.35-
16.03 mL 0.1 N NaOH/10 g hamur aralığında belirlenmiştir. En son tazeleme
sonunda, en yüksek asitlik dual kültürle üretilen ekşi hamur örneğinde
gözlenmiştir. Ekşi hamurdaki mikrobiyal aktiviteler sonucunda bazı uçucu organik
bileşikler oluşmaktadır. SPME-GC-MS analiz sonuçlarına göre, laboratuvar
XII
koşullarında üretilen ekşi hamurlarda farklı gruplara ait 37 uçucu organik bileşik
tespit edilmiştir.
Laboratuvar koşullarında 37°C'de gerçekleştirilen nohut mayası
üretimlerinde Weissella confusa RL1139 izolatı saf kültür olarak kullanılmıştır.
Kontrol ve kültür inokule edilen süzüntülerdeki son pH ve asitlik değerleri sırasıyla
4.92-4.82 ve 4.4-4.1 mL 0.1 N NaOH/10 g örnek olarak belirlenmiştir. Kontrol ve
starter kültür içeren hamur örneklerinde ise son pH değerleri sırasıyla 4.82 ve 4.79
olarak hesaplanmıştır. Son asitlik değerleri ise kontrol ve starter kültür içeren
hamur örneklerinde sırasıyla 5.26 ve 5.97 mL 0.1 N NaOH/10 g örnek olarak
hesaplanmıştır. SPME-GC-MS analiz sonuçlarına göre 32 uçucu organik bileşik
tespit edilmiştir. Nohut mayası fermantasyonlarında tüm örneklerde tespit edilen
karakteristik bileşik bütirik asit olmuştur.
Bu çalışmada ekşi hamur, nohut süzüntüsü mayası ve nohut mayası
hamuru örneklerinde farklı laktik asit bakterileri ve maya türleri tanımlanmış ve
bazı laktik asit bakterileri türleri starter kültür olarak ekşi hamur ve nohut mayası
fermantasyonları denemelerinde kullanılmıştır. Starter kültür kombinasyonlarının
geliştirilmesi endüstriyel üretimde aynı kalitede son ürün eldesi açısından oldukça
önemlidir. Çünkü starter kültür eklenmesi sonucu endüstriyel olarak büyük ölçekli
ve her zaman aynı kalitede ekşi hamur ve nohut mayası ekmeklerinin üretimi
mümkün olabilecektir.
XIII
XIV
ACKNOWLEDGEMENTS
I would like to sincerely thank to my supervisor, Prof. Dr. Hüseyin

ERTEN, for his guidance, support and encouragement throughout the study. It was
a real privilege for me to share his exceptional scientific knowledge and also
extraordinary human qualities.
I would also like to thank the respectable committee members of my PhD Thesis,
Assoc. Prof. Dr. Luca SETTANNI, Prof. Dr. Filiz ÖZÇELİK, Prof. Dr. Turgut
CABAROĞLU and Prof. Dr. Haşim KELEBEK for their guidance and constructive
suggestions.
I would like to acknowledge to The Scientific and Technological Research
Council of Turkey (TÜBİTAK) for the financial support by the 2211-E National
Scholarship Programme for PhD students.
I am thankful to Pikan Bakery in Antalya, Gattini Bistro in Mersin, Canberk Food
Company in Ankara, Cumhuriyet Bakery in Soke/Aydın, Tokoğlu Bread in Birgi
Town/Odemis/Izmir and Yayla Bakery in Nevsehir for their help during the collection of
the samples.
I would like to acknowledge to the Scientific Research Projects of
Çukurova University (BAP) (Project Number: FBA-2017-9035) which gives
financial support for a part of my PhD thesis.
I am also so thankful to Res. Assist. Bilal AĞIRMAN, Prof. Dr. Hakan
ÖZKAN, Dr. Z. Dilan ÇELİK, Dr. Erdem ÇARŞANBA, Akram Ben GHORBAL,
Msc. students Hülya IŞIKALP, İslim GÖKALP and İnci ASLAN and other people
of the department for their help, support and friendly working atmosphere.
I am so deeply thankful to my sister Başak BOYACI, my father İsmail
BOYACI, my mother Fatma BOYACI, my husband İsmail GÜNDÜZ and my son
Mehmet Kıvanç GÜNDÜZ, for their big support. Thanks for their understanding,
patient and consolatory words. I thank them with all of my heart for their endless
love, patience and always believing in me.
XV
CONTENTS PAGE
ABSTRACT...............................................................................................................I
ÖZ ............................................................................................................................ II
EXTENDED SUMMARY...................................................................................... III
TÜRKÇE GENİŞLETİLMİŞ ÖZET ...................................................................... IX
ACKNOWLEDGEMENTS .................................................................................. XV
CONTENTS......................................................................................................... XVI
LIST OF TABLES ..............................................................................................XXII
LIST OF FIGURES .......................................................................................... XXVI
LIST OF ABBREVIATIONS .......................................................................... XXXII
1. INTRODUCTION ................................................................................................ 1
2. LITERATURE OVERVIEW................................................................................ 5
2.1. Fermentation .................................................................................................. 5
2.1.1. Biochemistry of the Fermentation Process .......................................... 6
2.1.2. Carbohydrate Catabolism .................................................................... 8
2.1.3. Lactic Acid Bacteria (LAB) ............................................................... 10
2.1.3.1. Homofermentative LAB ......................................................... 13
2.1.3.2. Heterofermentative LAB ........................................................ 16
2.1.4. Yeasts................................................................................................. 18
2.2. Sourdough .................................................................................................... 20
2.2.1. History of Sourdough......................................................................... 21
2.2.2. Sourdough Production ....................................................................... 22
2.2.2.1. Flour in Sourdough Production ............................................... 23
2.2.2.1.(1). Wheat Flour............................................................. 24
2.2.2.1.(2). Rye Flour................................................................. 25
2.2.2.1.(3). Other Flour Types ................................................... 26
2.2.2.2. Classification of Sourdough Production Methods................... 26
2.2.3. Sourdough Microflora........................................................................ 29
XVI
2.2.4. Beneficial Effects and Functional Aspects of Sourdough
Technology ........................................................................................ 40
2.2.4.1. Organic Acid Production ........................................................ 42
2.2.4.2. Antibacterial and Antifungal Activities .................................. 43
2.2.4.3. Phytase Activity ...................................................................... 45
2.2.4.4. Starch Digestibility ................................................................. 46
2.2.4.5. Protease Activity..................................................................... 47
2.2.4.6. Wheat Germ Stability ............................................................. 50
2.2.4.7. Exopolysaccharide Production ............................................... 50
2.3. Chickpea Fermentation ................................................................................ 51
2.3.1. Chickpea (Cicer arietinum L.) ........................................................... 51
2.3.2. Chickpea Bread .................................................................................. 53
2.3.3. Studies on Chickpea Bread ................................................................ 55
2.3.4. Chickpea Fermentation Microflora .................................................... 56
3. MATERIALS AND METHOD .......................................................................... 59
3.1. Materials and Sampling .............................................................................. 59
3.1.1. Sourdough Samples........................................................................... 59
3.1.2. Chickpea Liquid Starter and Dough Samples ................................... 60
3.2. Production of Sourdough and Chickpea Dough under Laboratory
Conditions ................................................................................................... 61
3.2.1. Laboratory Sourdough Production and Sampling ............................. 61
3.2.2. Laboratory Chickpea Liquid Starter and Dough Production and
Sampling ........................................................................................... 63
3.3. Determination of pH, Total Titratable Acidity, Carbohydrates, Organic
Acids and Ethanol ....................................................................................... 65
3.3.1. Determination of pH ......................................................................... 65
3.3.2. Determination of Total Titratable Acidity ........................................ 66
3.3.3. Assessment of Carbohydrates, Organic Acids and Ethanol Content
Using High Performance Liquid Chromatography (HPLC) ............. 66
XVII
3.3.3.1. Determination of Carbohydrates, Organic Acids and
Ethanol ................................................................................ 66
3.3.3.2. Preparation of the Standards ................................................ 66
3.3.3.3.Extraction Procedure ............................................................ 67
3.4. Microbiological Analyses ........................................................................... 67
3.4.1. Viable Counts of Presumptive LAB ................................................. 69
3.4.2. Viable Counts of Presumptive Yeasts ............................................... 70
3.4.3. Viable Counts of Presumptive Non-Saccharomyces Yeasts ............. 71
3.4.4. Viable Counts of Total Mesophilic Aerobic Bacteria ....................... 72
3.4.5. Viable Counts of Molds .................................................................... 72
3.4.6. Total Presumptive Coliform Count ................................................... 72
3.4.7. Viable Counts of Presumptive Bacillus spp. ..................................... 73
3.5. Molecular Identification of LAB Isolates ................................................... 73
3.5.1. DNA Extraction ....................................................................................... 73
3.5.2. Randomly amplified polymorphic DNA (RAPD) PCR analysis ...... 74
3.5.3. 16S rRNA Gene Sequence Analysis ................................................. 76
3.6. Molecular Identification of Yeast Isolates .................................................. 77
3.6.1. DNA Extraction ................................................................................ 77
3.6.2. ITS Region Amplification of the 5.8S rRNA Gene .......................... 78
3.6.3. RFLP (Restriction Fragment Length Polymorphism) Analysis ........ 79
3.6.4. 26S rRNA Gene Sequence Analysis ................................................. 79
3.7. Functional Characterization of Selected LAB Isolates ............................... 81
3.7.1. Acidification Activity ....................................................................... 81
3.7.2. Determination of Lactic and Acetic Acids ........................................ 81
3.7.3. Quantitative EPS Analysis ................................................................ 82
3.7.4. EPS Production on Agar Medium ..................................................... 82
3.7.5. Antimicrobial Activity Against Selected Species ............................. 83
3.7.6. Protease Capacity .............................................................................. 83
3.7.7. Growth at Different Conditions ........................................................ 83
XVIII
3.7.8. Enzyme Profile.................................................................................. 84
3.8. Production of Experimental Sourdoughs and Chickpea Liquid Starters with
Selected Strains .......................................................................................... 85
3.9. VOC Analysis by SPME-GC-MS in Experimental Samples...................... 88
3.10. Statistical Analysis .................................................................................... 89
4. RESULTS AND DISCUSSION ......................................................................... 91
4.1. Sourdough Samples .................................................................................... 91
4.1.1. Chemical Characteristics of Sourdough Samples ............................. 91
4.1.1.1. pH .......................................................................................... 91
4.1.1.2. Total Titratable Acidity ......................................................... 93
4.1.1.3. Carbohydrate, Organic Acid and Ethanol Contents of the
Sourdoughs ............................................................................ 97
4.1.2. Microbiological Characteristics of Sourdough Samples ................ 105
4.1.2.1. Presumptive LAB Counts .................................................... 106
4.1.2.2. Presumptive Yeast Counts ................................................... 107
4.1.2.3. Enumeration of other Microorganisms ................................ 108
4.1.3. Multivariate statistical analysis of the sourdough samples............. 112
4.1.4. Biodiversity of the LAB and Yeasts in Sourdough Samples .......... 117
4.1.4.1. LAB Identification ............................................................... 118
4.1.4.2. Phylogenetic Relation of the LAB Strains ........................... 135
4.1.4.3. Yeast Identification .............................................................. 139
4.1.4.4. Phylogenetic Relation of the Yeast Strains .......................... 148
4.2. Chickpea Liquid Starter and Dough Samples ........................................... 149
4.2.1. Chemical Characteristics of Chickpea Liquid Starter and Dough
Samples .......................................................................................... 149
4.2.1.1. pH ........................................................................................ 150
4.2.1.2. Total Titratable Acidity ....................................................... 153
4.2.1.3. Carbohydrate, Organic Acid and Ethanol Contents of the
Chickpea Liquid Starter and Dough Samples...................... 160
XIX
4.2.2. Microbiological Characteristics of Chickpea Fermentations.......... 164
4.2.2.1. Presumptive LAB Cell Counts ............................................ 165
4.2.2.2. Presumptive Yeast Cell Counts ........................................... 167
4.2.2.3. Enumeration of other Microorganisms ............................... 168
4.2.3. Multivariate Statistical Analysis of the Chickpea Liquid Starter and
Dough Samples .............................................................................. 173
4.2.4. Biodiversity of the LAB and Yeasts in Chickpea Fermentations ... 178
4.2.4.1. LAB Identification .............................................................. 178
4.2.4.2. Phylogenetic Relation of the LAB Strains .......................... 195
4.2.4.3. Yeast Identification ............................................................. 199
4.2.4.4. Phylogenetic Relation of the Yeast Strains ......................... 209
4.3. Evaluation of the Technological Attributes of Selected LAB .................. 210
4.3.1. LAB Strain Selection as Starter Culture in Sourdough and
Chickpea Fermentations ................................................................. 210
4.3.2. Investigaton of the some Properties of the Selected LAB
Strains ............................................................................................. 217
4.3.3. Production of Experimental Sourdoughs and Evaluation of
Chemical and Microbiological Properties ...................................... 222
4.3.4. Multivariate Statistical Analysis of Experimental Sourdoughs ...... 232
4.3.5. Production of Experimental Chickpea Liquid Starter and Dough
Samples and Evaluation of Chemical and Microbiological
Properties ........................................................................................ 234
4.3.6. Multivariate Statistical Analysis of Experimental Chickpea
Fermentations ................................................................................. 243
5. CONCLUSION ................................................................................................. 247
REFERENCES ..................................................................................................... 257
CURRICULUM VITAE ....................................................................................... 303
APPENDICES ...................................................................................................... 305
XX
XXI
LIST OF TABLES PAGE
Table 2.1. Fermentation metabolism of some LAB .............................................. 13

Table 2.2. LAB and yeasts isolated from various sourdoughs .............................. 37
Table 3.1. Sampling dates and locations ............................................................... 59
Table 3.2. Media used for the enumeration of microorganisms in the
sourdough, chickpea liquid starter and dough samples ....................... 69
Table 4.1. The pH levels of the samples ............................................................... 92
Table 4.2. Mean TTA levels of the sourdough samples ....................................... 94
Table 4.3. Mean carbohydrate and ethanol content (g/kg) of the sourdough
samples ................................................................................................ 99
Table 4.4. Mean organic acid content (g/kg) of the sourdough samples ............ 101
Table 4.5. Mean carbohydrate, organic acid and ethanol content (g/kg) of the
sourdough produced under laboratory conditions ............................. 101
Table 4.6. Mean values of cell counts (log CFU/g) of sourdough samples on
different media .................................................................................. 105
Table 4.7. Mold and coliform bacteria ................................................................ 112
Table 4.8. Correlation matrix (Pearson (n)) of the variables .............................. 116
Table 4.9. Identified LAB isolates at the species level (sequence length 1400
bp ≤) (98% ≤) in sourdough samples................................................. 120
Table 4.10. Identified LAB isolates at the genus (94%≤) or family level
(86%≤) in sourdough samples ........................................................... 122
Table 4.11. Percentage of the isolated LAB species in sourdoughs ..................... 123
Table 4.12. Number of LAB identified at the species level in sourdough
samples .............................................................................................. 125
Table 4.13. Restriction fragments of the identified yeast species from
sourdoughs......................................................................................... 141
XXII
Table 4.14. Accession numbers of the identified yeast species with their
closest relatives and type strains........................................................ 142
Table 4.15. Percentage of the isolated yeast species in sourdoughs ..................... 144
Table 4.16. Distribution of the yeast species among sourdough samples ............. 145
Table 4.17. pH levels of the chickpea liquid starter samples ................................ 150
Table 4.18. pH levels of the chickpea dough samples .......................................... 151
Table 4.19. TTA levels of the chickpea liquid samples ........................................ 154
Table 4.20. TTA levels of the chickpea dough samples ....................................... 155
Table 4.21. Mean carbohydrate and ethanol content (g/kg) of the chickpea
liquid starter and dough samples ....................................................... 160
Table 4.22. Mean organic acid content (g/kg) of the chickpea liquid starter
and dough samples ............................................................................ 162
Table 4.23. Mean values of cell counts (log CFU/g) of chickpea liquid starter
and dough samples on different media .............................................. 165
Table 4.24. Presumptive total coliform bacteria and indol test in chickpea
fermentations ..................................................................................... 171
Table 4.25. Correlation matrix (Pearson (n)) of the variables .............................. 177
Table 4.26. Identified LAB isolates (sequence length 1400 bp≤) at the species
level (98%≤) in chickpea fermentations ............................................ 181
Table 4.27. Identified LAB isolates at the genus (94%≤) or family level
(86%≤) in chickpea fermentations..................................................... 182
Table 4.28. Percentage of the isolated species in chickpea fermentations ............ 183
Table 4.29. Number of LAB identified at the species level in chickpea liquid
starter and dough samples ................................................................. 185
Table 4.30. Restriction fragments of the identified yeast species from
chickpea fermentations ...................................................................... 200
Table 4.31.. Accession numbers of the identified yeast species with their
closest relatives and type strains........................................................ 202
Table 4.32. Percentage of the isolated yeast species in chickpea fermentations .. 204
XXIII
Table 4.33. Number of yeasts identified at the species level in chickpea
fermentations ..................................................................................... 205
Table 4.34. Strains investigated for technological evaluation in sourdough
and chickpea fermentations ............................................................... 211
Table 4.35. Acidification kinetics of SFE by LAB strains ................................... 214
Table 4.36. Growth of the selected strains under different conditions ................. 220
Table 4.37. Enzyme activities of the strains ......................................................... 222
Table 4.38. Carbohydrate, ethanol and organic acid contents of the
experimental sourdoughs at the last refreshment .............................. 229
Table 4.39. VOCs in the experimental sourdough samples as relative peak
area (%) ............................................................................................. 231
Table 4.40. Mold and coliform counts during chickpea fermentations ................ 240
Table 4.41. Carbohydrate, ethanol and organic acid contents (g/kg) of the
experimental chickpea liquid starter and dough samples .................. 241
Table 4.42. VOCs in the experimental chickpea fermentations as relative
peak area (%) ..................................................................................... 243
XXIV
XXV
LIST OF FIGURES PAGE
Figure 2.1. Fermentation of glucose in homofermentatives ................................. 15

Figure 2.2. Heterofermentative metabolism ......................................................... 18
Figure 2.3. Sourdough types ................................................................................. 27
Figure 2.4. Chickpea breads from different regions ............................................. 53
Figure 2.5. Chickpeas and fermented liquid starter .............................................. 54
Figure 3.1. Sourdough samples were taken into sterile jars .................................. 60
Figure 3.2. Fermented chickpea liquid starter and obtained liquid after
separation of the chickpeas ................................................................. 60
Figure 3.3. Chickpea dough .................................................................................. 61
Figure 3.4. Sourdough production under laboratory conditions ........................... 62
Figure 3.5. Laboratory produced sourdough sample ............................................ 63
Figure 3.6. Chickpea liquid starter and dough production .................................... 64
Figure 3.7. Chickpea liquid starter production under laboratory conditions ........ 65
Figure 3.8. Centrifugation in the microcentrifuge to pellet the cells .................... 74
Figure 3.9. Gel Image System............................................................................... 76
Figure 3.10. Experimental sourdough production................................................... 86
Figure 3.11. Experimental chickpea liquid starter and dough production .............. 88
Figure 4.1. Acidity levels of the sourdough samples ............................................ 95
Figure 4.2. Changes in pH during a 7-day sourdough fermentation with daily
back-slopping...................................................................................... 96
Figure 4.3. Changes in carbohydrate, organic acid and ethanol contents under
laboratory conditions ........................................................................ 102
Figure 4.4. Presumptive LAB counts of sourdoughs on different media ............ 107
Figure 4.5. Presumptive yeast counts of sourdoughs on different media ........... 108
XXVI
Figure 4.6. Cell counts on mMRS, gM17, SDB, YPD, L-lysine and PCA
media of the laboratory scale sourdough sample .............................. 109
Figure 4.7. Dendrogram resulting from hierarchical cluster analysis on 13
variables determined on sourdoughs ................................................ 113
Figure 4.8. The eigenvalues and the corresponding factors by descending
order with the variability they represent ........................................... 114
Figure 4.9. Loading plot (A) and score plot (B) resulting from principal
component analysis of variables determined on sourdoughs ............ 115
Figure 4.10. Distribution of presumptive LAB and yeast isolates from
collected sourdoughs ........................................................................ 117
Figure 4.11. Distribution of presumptive LAB and yeasts isolates of
sourdough produced under laboratory conditions ............................ 118
Figure 4.12. Dendrogram obtained from RAPD-PCR (M13 primer) band
profiles of LAB isolates in sourdough fermentations ....................... 119
Figure 4.13. Species in SD-M sourdough samples at both sampling times .......... 126
Figure 4.14. Species in SD-T sourdough samples at both sampling times ........... 127
Figure 4.15. Species in SD-K sourdough samples at both sampling times ........... 127
Figure 4.16. Species in SD-R sourdough samples at both sampling times ........... 128
Figure 4.17. The number of identified LAB species in laboratory scale
production ......................................................................................... 129
Figure 4.18. Distribution of the LAB strains at the family level in the
sourdough fermentations .................................................................. 130
Figure 4.19. Frequency of the strains at the family level ...................................... 131
Figure 4.20. Distribution of the LAB strains at the genus level in the s
ourdough fermentations .................................................................... 132
Figure 4.21. Frequency of the strains at the genus level ....................................... 132
Figure 4.22. Distribution of species in the laboratory sourdough production....... 135
Figure 4.23. Evolutionary relationships of taxa by using the Minimum
Evolution method ............................................................................. 136
XXVII
Figure 4.24. Evolutionary relationships of taxa by using the UPMGA
method .............................................................................................. 138
Figure 4.25. Distribution of the genera of the identified yeast isolates ................ 139
Figure 4.26. Distribution of yeast species in sourdough samples ......................... 147
Figure 4.27. Evolutionary relationships of yeast with UPMGA method .............. 149
Figure 4.28. pH variation among chickpea liquid starter and dough samples ...... 152
Figure 4.29. TTA variation among chickpea liquid starter and dough samples ... 156
Figure 4.30. pH and TTA levels of the chickpea liquid starter and dough
samples produced under laboratory conditions ................................ 158
Figure 4.31. Carbohydrate, ethanol and organic acid contents of the chickpea
liquid starter and dough samples produced under laboratory
conditions.......................................................................................... 163
Figure 4.32. Presumptive LAB count of chickpea liquid starter and dough
samples on MRS and M17 agar ........................................................ 166
Figure 4.33. Presumptive yeast counts of chickpea liquid starter and dough
samples on YPD and L-lysine agar .................................................. 168
Figure 4.34. Cell counts in the laboratory scale chickpea fermentations .............. 172
Figure 4.35. Dendrogram resulting from hierarchical cluster analysis on
chickpea liquid starter and dough samples ....................................... 174
Figure 4.36. The Eigenvalues and the corresponding factors by descending
order withl the variability they represent .......................................... 175
component analysis of chickpea fermentations ................................ 176
Figure 4.38. Distribution of presumptive LAB and yeasts in the chickpea
fermentations .................................................................................... 178
Figure 4.39. Dendrogram obtained from RAPD-PCR(M13) band profiles of
LAB isolates in chickpea fermentations ........................................... 179
Figure 4.40. LAB species identified in Bakery A chickpea fermentations ........... 186
Figure 4.41. LAB species identified in Bakery B chickpea fermentations ........... 187
XXVIII
Figure 4.42. LAB species identified in Bakery N chickpea fermentations ........... 188
Figure 4.43. LAB species identified in the laboratory scale chickpea
fermentations .................................................................................... 189
Figure 4.44. Distribution of the LAB strains in the chickpea fermentations at
the family level ................................................................................. 190
Figure 4.45. Frequency of the LAB strains in the chickpea fermentations at
the family level ................................................................................. 191
Figure 4.46. Frequency of LAB strains in the chickpea fermentations at the
genus level ........................................................................................ 192
Figure 4.47. Distribution of LAB strains in the chickpea fermentations at the
genus level ........................................................................................ 193
Figure 4.48. The evolutionary history inferred using the Minimum Evolution
method .............................................................................................. 196
Figure 4.49. The evolutionary history inferred using the UPGMA method ......... 198
Figure 4.50. Distribution of the 6 genera in the identified yeast isolates.............. 201
Figure 4.51. Distribution of the yeast species in chickpea fermentations ............. 207
Figure 4.52. Evolutionary relationships of yeast with UPMGA method .............. 209
Figure 4.53. Acidification kinetics of SFE by the 21 strains ................................ 212
Figure 4.54. EPS production on agar media ......................................................... 216
Figure 4.55. Acid production and color change with different carbohydrate
sources .............................................................................................. 219
Figure 4.56. Investigation of the enzyme activities by API ZYM kit ............. 221
Figure 4.57. pH and TTA values of the experimental sourdoughs ....................... 223
Figure 4.58. Cell counts of LAB on the mMRS agar............................................ 225
Figure 4.59. mMRS petri dishes of the mono- and dual-culture inoculums at
the beginning of the fermentations ................................................... 226
Figure 4.60. Cell counts of yeasts and molds ....................................................... 226
Figure 4.61. Cell counts of total mesophilic aerobic counts ................................. 227
XXIX
component analysis of variables determined on sourdoughs ............ 233
Figure 4.63. pH and TTA values of the experimental chickpea liquid starter
and dough samples............................................................................ 235
Figure 4.64. pH values monitored during 10 hours .............................................. 236
Figure 4. 65. Cell counts on mMRS and YPD media ........................................... 237
Figure 4 66. Total bacteria counts on PCA and NA media ................................... 239
Figure 4.67. Total and spore forming bacteria during the first 10 hours of
chickpea liquid fermentation ............................................................ 240
component analysis of variables determined on chickpea
fermentations .................................................................................... 245
XXX
XXXI
LIST OF ABBREVIATIONS
µL : Microliter
µm : Mikrometer
µM : Mikromolar
µmol : Micromole
AHC : Agglomerative hierarchical clustering
ANOVA : Analysis of variance
ATP : Adenosine triphosphate
B. : Bacillus
BLAST : Basic local alignment search tool
bp : base pair
C. : Candida
CD : Chickpea dough
CFU : Colony forming unit
Cl. : Clostridium
CLS : Chickpea liquid starter
CO2 : Carbon dioxide
Cr. : Cryptococcus
d : Day
DAP : Dihydroxyacetone phosphate
DGGE : Denaturing gradient gel electrophoresis
E. : Enterococcus
EMP : The Embden–Meyerhof–Parnas
EPS : Exopolysaccharide
ev : Electronvolt
FQ : Fermentation quotient
g : Gram
GAP : Glyceraldehyde-3-phosphate
GC-MS : Gas chromatography-Mass spectrophotometry
GI : Glycaemic index
h : Hour
H2O : Water
HPLC : High performance lipid chromatography
H'spora. : Hanseniaspora
I. : Issatchenkia
XXXII
ITS : Internal transcribed spacer
K. : Kazachstania
Kg : Kilogram
L : Litre
LAB : Lactic acid bacteria
Lb. : Lactobacillus
Lc. : Lactococcus
Leu. : Leuconostoc
LOD : Limit of detection
LOQ : Limit of quantification
LST : Lauryl Sulfate Tryptose
mg : Milligram
min : Minute
mL : Milliliter
mM : Milimolar
mmol : Milimole
MPN : Most Probable Number
MRS : de Man Rogosa Sharpe
NA : Nutrient agar
NADH : Nicotinamide adenine dinucleotide
NCBI : National Center for Biotechnology Information
O2 : Oxygen
ºC : Centigrade degree
OD : Optical density
P. : Pichia
PCa : Principle component analySİ
PCA : Plate count agar
PCR : Polymerase chain reaction
Pd. : Pediococcus
R. : Rhodotorula
RAPD : Random Amplified Polymorphic DNA
rflp : Restriction Fragment Length Polymorphism
RID : Refractive Index Detector
rpm : Rotation per minute
s : Second
S. : Saccharomyces
XXXIII
SD : Sourdough
SDB : Sourdough bacteria medium
SFE : Sterile flour extract
SPME : Solid phase microextraction
St. : Streptococcus
T. : Torulaspora
TBE : Tris, boric acid and EDTA
TTA : Total titratable acidity
U/g : Units per gram
UPMGA : Unweighted pair group method with arithmetic average
USDA : US Department of Agriculture
UV : Ultra violet
VOC : Volitile organic compound
vol : volume
W. : Weisella
w/w : weight/weight
wt : Weight
YPD : Yeast Extract Peptone Dextrose
XXXIV
XXXV
1.INTRODUCTION Cennet Pelin BOYACI GÜNDÜZ
1. INTRODUCTION
Fermentation is one of the oldest known methods of food preservation and

production which has been used since ancient times. Fermented foods are produced
on a large scale in industry, in addition to traditional small-scale production.
Currently, there is an increasing interest into traditional fermented foods as a result
of the growing consumer demand for high quality, healthy and natural food
products. Nowadays, consumers demand natural food products with a long shelf
life. Bread is one of the main staple foods consumed by humans and
the trend towards healthy and natural breads with a long shelf life has been
increasing.
In recent years, traditional sourdough bread production has gained
importance due to increasing demand by consumers for more organic and healthy
foods (Arendt et al., 2007; Mariotti et al., 2014; Torrieri et al., 2014; Behera and
Ray, 2015). Actually, the use of the sourdough process for the production of
sourdough bread has a long tradition but recently there has been an increasing
demand for sourdough bread both globally and in Turkey. Sourdough is a mixture
of flour (mainly wheat or rye) and water that is fermented with lactic acid bacteria
(LAB) and yeasts (Gobbetti, 1998; Vogel et al., 1999; De Vuyst and Neysens,
2005). The use of the sourdough process as a form of leavening during the
production of sourdough bread is one of the oldest biotechnological processes in
food production and has been used for thousands of years and is "generally
regarded as safe". Via the sourdough process, sensorial properties, nutritional
values and the shelf life of bread are improved in a natural way. Chickpea bread is
a traditional bread produced using a chickpea dough, which is made by fermenting
chickpeas in hot water for 16-18 hours. Chickpea bread has a long history in this
country and is well-known in the Aegean Region, Thrace Region and also some
parts of the Middle Anatolia and Mediterranean Regions of Turkey; it is produced
in limited bakeries and, in addition to this country, it is also produced in Greece
1
and Macedonia (Hatzikamari et al., 2007a). In the literature, research on chickpea

fermentation is limited and studies mainly focus on the production of bakery
products using chickpea liquid starter and dough. Studies identifying the
microorganisms of chickpea fermentations are very scarce. In order to protect such
traditional foods, scientific research is essential.
Sensorial properties and the shelf life of breads are improved by
fermentation with different microorganisms. Chickpea dough is defined as a “sweet
dough” in some regions because of its taste. Sourdough has a lower pH and sour
taste compared with chickpea dough. In fact, sourdough and chickpea dough
contrast each other but they have a common feature, i.e., exhibiting differing aroma
profiles caused by different microorganisms. The above-mentioned bread types are
different from each other in many aspects but both are fermented traditional
products with great potential. They offer many advantages over bread produced
using baker’s yeast, i.e., sensorial properties and nutritional value are improved
with fermentation, and shelf life is increased. During the production of these breads
no additives or commercial yeasts are used and fermentation is conducted
spontaneously; therefore, microorganisms play an important role during the
fermentation of these foods. In particular, LAB and yeasts are the main
microorganisms that are responsible for this type of fermentation. The compounds
generated by LAB inhibit the growth of other microorganisms and contribute to the
taste and flavor of the final product. Identification of the microorganisms
responsible for this fermentation is important for the quality of the final product.
As a result of identifying these microorganisms, by the use of highly sensitive
methods, starter culture combinations can be developed and then industrial
products can be produced to achieve consistent quality of the final product. For this
purpose, microorganisms that are responsible for this fermentation must be isolated
and then identified using certain techniques. Nowadays, successful identification is
performed using genotypic methods and allows investigation of the microbial
diversity as a result of the sensitive and fast identification to species and strain
2
level. The interactions between LAB and yeasts that populate the sourdough and
chickpea doughs should be understood, as only then will it be possible to produce
these bread types industrially to reach consumers everywhere.
The objectives of the present study are:
 Isolation of LAB and yeasts from sourdough samples produced using

traditional method without adding any commercial yeasts collected
from three different bakeries in Mersin, Antalya and Ankara at two
different times.
 Isolation of the LAB and yeasts from the chickpea liquid starter and
dough samples collected from three different bakeries in Aydın, İzmir
and Nevşehir at two different times.
 Identification of isolated LAB and yeasts using molecular methods.
 Investigation of the technological properties of the identified LAB to
develop starter culture combinations for sourdough and chickpea
fermentations.
3
4
2. LITERATURE OVERVIEW Cennet Pelin BOYACI GÜNDÜZ
2. LITERATURE OVERVIEW
2.1. Fermentation
Fermentation is one of the oldest food processing and preservation
technique which can be traced back thousands of years. Organoleptic properties are
improved and shelf life is extended by fermentation (Smid and Hugenholtz, 2010;
Ray and Joshi, 2015). In fermented foods, microbial stability and safety is
improved even at ambient temperatures and sensorial properties such as taste,
flavor and aroma are developed. The common microorganisms involved in food
fermentations are bacteria, mainly LAB, yeasts and molds. LAB, in particular, and
then yeasts are the most commonly found microorganisms in fermented foods (Ray
and Montet, 2015).
Fermentation plays different roles in food processing as given below:
(Hutkins, 2006; Ray and Joshi, 2015).
1. Foods are preserved by fermentation as a result of the formation of

inhibitory compounds such as organic acids (lactic acid, acetic acid,
formic acid, and propionic acid), ethanol, carbon dioxide, diacetyl,
reutrin, bacteriocins, etc., and sometimes in combination with a
decrease of water activity by drying or using salt.
2. Food safety is improved by fermentation as a result of the inhibition of
pathogens and removal of toxic compounds (Adams and Nicolaides,
1997; Gaggia et al., 2011).
3. Nutritional value is increased (Poutanen et al., 2009).
4. Shelf life is extended (Van Boekel et al., 2010).
5. Functionality and sensory properties of end products are enhanced.
6. Technological aspects and overall quality of the food is developed.
5
A diverse range of fermented foods are found worldwide and a number of

them are globally distributed and produced on a large scale in industry, in addition
to small scale production at home (Smid and Hugenholtz, 2010). On the other
hand, some fermented foods are produced in specific regions or many regions with
different cultural practices. Global fermented foods include plant based fermented
foods such as table olives, pickles, sauerkraut, vinegar, cereal base fermented foods
such as bread, sourdough, alcoholic beverages such as wine, beer and fermented
meat and dairy products such as cheese, yoghurt etc., which are produced
worldwide on a large scale. Local fermented foods can be derived from different
sources such as cereals, fish, meat, milk, dairy products, vegetables and plants, and
are produced in many different parts of the world. Tarhana, şalgam, boza,
hardaliye, turşu (Turkey); kefir, koumiss (Caucasian, Central Asia); kimchi
(Korea); tempeh (Indonesia); brovada (Italy); gundruk (Indiana, nepal); Pak-Gard-
Dong (Thailand); khalpi (Nepal) and fufu (Nigeria) are a few examples of regional
fermented foods of the world (Erten and Tangüler, 2014; Erten et al., 2016; Erten
et al., 2017; Oguntoyinbo and Franz, 2017; Patra et al., 2017; Swain and
Ananadharaj, 2017; Wiander, 2017).
2.1.1. Biochemistry of the Fermentation Process

The word fermentation derives from the Latin verb fervere meaning "to
boil" as a result of the impression of boiling observed at the beginning of wine
fermentation, with the continous release of gas bubbles to the surface (Okafor,
2007). In the middle of the 19th century, Louis Pasteur established the role of
microorganisms during fermentation and showed that fermentation is a microbial
process; he also demonstrated that it occurs without oxygen (O2) with the words
“life without air” (la vie sans l’air) (Prajapati and Nair, 2008; Dufour et al., 2011;
Mckay et al., 2011; Madigan et al., 2012).
The word "fermentation" has different meanings (Okafor, 2007). From a
microbiological point of view, the term fermentation can be used for any process
6
produces a microbial product by the mass culture of microorganisms (Stanbury,

2000). However, biochemically, fermentation is defined as a metabolic process
involving a carbon source in which organic compounds act as both electron donors
and acceptors and energy is generated under anaerobic conditions (Madigan et al.,
2012; Erten et al., 2016). In industrial microbiology, the term "fermentation" is any
process in which microorganisms are grown on a large scale, even if the final
electron acceptor is not an organic compound (i.e., even if growth is carried out
under aerobic conditions) (Okafor, 2007).
Every cell needs energy for growth and maintenance and that energy is
generated by energetic pathways via metabolic processes (Dufour et al., 2011;
Erten et al., 2016). The released energy is conserved in cells by the simultaneous
synthesis of energy-rich compounds to drive future energy-requiring cell functions.
In living organisms, energy is primarily conserved in phosphorylated compounds
and the most important energy-rich phosphate compound in cells is adenosine
triphosphate (ATP).
Fermentation and cellular respiration are the two series of reactions that are
linked to energy conservation. Fermentation is a form of anaerobic catabolism in
which an organic compound functions as both an electron acceptor and an electron
donor. During fermentation, ATP is produced by substrate-level phosphorylation
directly from energy-rich intermediates via the steps of carbohydrate catabolism
(Madigan et al., 2012). The fermentable substrate in fermentation is both the
electron donor and electron acceptor and not all compounds can be fermented;
however sugars, especially hexoses are excellent fermentable substrates which are
preferred by many microorganisms and there are only a few organisms that cannot
utilize it (Waites et al., 2001). When the final acceptor is an inorganic compound,
the process is called respiration and respiration is referred to as aerobic if the final
acceptor is O2 or anaerobic if the final acceptor is an O2 substitute, i.e., some other
inorganic compound e.g., sulphate or nitrate (Okafor, 2007).
7
Fermentation and respiration are alternative metabolic choices available to

some microorganisms. For example, yeasts are organisms that can both ferment
and respire and fermentation is necessary under low level O2 conditions and when
terminal electron acceptors are absent. If sufficient O2 is available, respiration can
take place. Considerably more ATP is produced during respiration, compared with
fermentation, and is the preferred choice. However, in many microbial habitats that
lack O2 or other electron acceptors that can be a substitute for O2, fermentation is
the only option for energy conservation (Madigan et al., 2012).
In glycolysis, one molecule of glucose is converted into two molecules of
pyruvate and energy and reducing power is conserved in the form of ATP and
NADH, respectively. If O2 is present, glycolysis leads to aerobic respiration;
however, in the absence of O2 it leads to fermentation (Hardin et al., 2012). Under
anaerobic conditions, the electrons that are removed from NADH are transferred to
pyruvate and then pyruvate is reduced to various fermentation end products, which
vary depending on the microorganism (Tortora et al., 2010). The most common
end products of pyruvate reduction are lactic acid by LAB, and ethanol and carbon
dioxide by yeasts and some other microorganisms (Hardin et al., 2012). During
ethyl alcohol fermentation, pyruvate is reduced to acetaldehyde and then
acetaldehyde is converted to ethanol by yeasts and some other microorganisms. In
lactic acid fermentation, pyruvate is converted to lactic acid as the major end
product by LAB.
2.1.2. Carbohydrate Catabolism

Carbohydrate catabolism is the breakdown of carbohydrate molecules to
produce energy. In particular, the six carbon sugar, glucose (C6H12O6), is very
important for catabolism. In many vertebrates, including humans, glucose is the
main sugar in the blood and also the main energy source for most of the cells in the
body. In plants, glucose is the monosaccharide that is produced upon the
breakdown of starch. Also, it is one-half of the disaccharide sucrose (glucose+
8
fructose), the major sugar in the vascular system of most plants (Hardin et al.,
2012). As it can be seen, glucose is very important for metabolism and many
energy-rich substances are converted into the intermediates of the pathway for
glucose catabolism in plants, animals and microorganisms.
Energy is produced from carbohydrates via different pathways such as
Embden–Meyerhof–Parnas (Glycolysis), Pentose Phosphate, Entner–Doudoroff
and Phosphoketolase (Okafor, 2007; Tortora et al., 2010). Glycolysis, which is also
known as the Embden–Meyerhof–Parnas (EMP) pathway, named after its major
discoverers, is the most common route and is found in all major groups of
organisms, including filamentous fungi, yeasts and many bacteria (Waites et al.,
2001).
EMP pathway or Glycolysis is the first stage of carbohydrate catabolism
and it occurs in most living cells. The result of this pathway is the breakdown of
glucose into pyruvate to generate ATP. Whether glucose is fermented or respired, it
travels through this pathway. During glycolysis, two molecules of ATP are
consumed and four molecules of ATP are generated and the net energy yield in
glycolysis is two molecules of ATP per molecule of glucose (Tortora et al., 2010;
Dufour et al., 2011; Madigan et al., 2012).
During the first stage of glycolysis, glucose is phosphorylated and becomes
glucose-6-phosphate, is isomerized to fructose-6-phosphate and then 1,6-
bisphosphate is produced via phosphorylation. These stages of glucose breakdown
consume two molecules of ATP. Fructose 1,6-bisphosphate is then split into two 3-
carbon phosphates, glyceraldehyde-3-phosphate (GAP) and its isomer
dihydroxyacetone phosphate (DAP) (Wang et al., 2001; Dufour et al., 2011). DAP
is isomerized to GAP as only GAP is directly processed through the pathway.
Subsequently, a molecule of inorganic phosphate is added to GAP to generate 1,3-
bisphosphoglycerate along with the reduction of NAD+ to NADH (Nicotinamide
adenine dinucleotide) by glyceraldehyde-3-phosphate dehydrogenase (Madigan et
al., 2012). This redox reaction occurs twice as two molecules of GAP are produced
9
from glucose. Oxidation of the resultant one glucose molecule to two pyruvate
molecules as the end product generates energy in the form of four ATP molecules.
However, the net gain in glycolysis is two molecules of ATP per molecule of
glucose due to its consumption in the earlier reactions as shown in Equation 1
(Waites et al., 2001).
Eq. 1
The phosphoketolase (PK) pathway is characteristically observed in

heterolactic bacteria, Leuconostoc and some Lactobacillus species. In this pathway,
phosphoketolase is the key enzyme and coverts 5-carbon pentoses such as xylulose,
into a 2-carbon acetyl phosphate and 3-carbon glyceraldehyde 3-phosphate (Butler
et al., 2010). In this pathway, glucose fermentation yields lactic acid, ethanol and
CO2. It produces only half the yield of ATP compared with the EMP pathway but
also allows pentose formation from hexose sugars for nucleic acid synthesis and
the catabolism of pentoses (Waites et al., 2001; Okafor, 2007).
2.1.3. Lactic Acid Bacteria (LAB)

LAB produce lactic acid as the major end product of fermentation. LAB
comprises a diverse group of microorganisms that have a common metabolic
property, i.e., the production of lactic acid from the fermentation of carbohydrates
as the major end product (Mayo et al., 2010). The produced lactic acid may be in
the form of L (+) or D (-) or a mixture of both (Caplice and Fitzgerald, 1999).
The NAD+ used during glycolysis must be regenerated for the continuation
of glycolysis (Hames and Hooper, 2000). In lactic acid fermentation, NAD+ is
regenerated by the conversion of pyruvate to lactic acid as the end product via
10
lactate dehydrogenase under anaerobic conditions, as shown in Equation 2 on a

per-glucose basis:
Eq. 2
LAB are one of the most industrially important groups of bacteria used in
many processes including food production, health improvement, and the
production of macromolecules, enzymes and metabolites. They are found in many
foods including milk and dairy products, plant based foods, cereals, and also meat
and meat products. LAB are very important in food fermentations and have a very
long history of use in the production of many fermented food products such as
yoghurt, cheese, pickles, olives, sourdough etc. Many species are used for the
production and preservation of fermented foods; in addition, enzymatic activities of
LAB contribute to the final organoleptic, rheological and nutritional properties of
fermented products (Leroy and De Vuyst, 2004; Mayo et al., 2010).
LAB are Gram-positive, catalase-negative, facultatively anaerobic, usually
non-motile, non-respiring and non-spore-forming rods or cocci (Hammes and
Hertel, 2009). These unicellular prokaryotes are grouped in the order
Lactobacillales under the class Bacilli of the phylum Firmicutes. The order
includes 6 families as follows: Aerococcaceae, Carnobacteriaceae,
Enterococcaceae, Lactobacillaceae, Leuconostoccaceae and Streptococcaceae
(Garrity and Holt, 2001; Holzapfel and Wood, 2014). LAB are a rapidly expanding
group of bacteria with 6 families and 40 genera in their broad physiological
definition (Holzapfel and Wood, 2014), with Carnobacterium, Enterococcus (E.),
Lactobacillus (Lb.), Aerococcus, Lactococcus (Lc.), Leuconostoc (Leu.),
Oenococcus, Pediococcus (Pd.), Streptococcus (St.), Tetragenococcus, Vagococcus
and Weissella (W.) generally considered to be the principal LAB genera from a
food technology point of view (Axelsson, 2004). Also, the genus Bifidobacterium
11
is often considered to be the genuine LAB due to sharing some typical features but
it is phylogenetically unrelated and has a unique mode of sugar fermentation. The
genus belongs to the phylum Actinobacteria since the members of the genera are
phylogenetically more related to the Actinomycetaceae group of bacteria
(Axelsson, 2004). Bifidobacterium metabolizes glucose via the ‘Bifidus pathway’
(Scardovi, 1986) to yield lactic acid and acetic acid and this is a special pathway,
unique to the genus, which clearly separates them from LAB (Hammes and Hertel,
2009; Endo and Dicks, 2014).
From a biochemical perspective, LAB can be grouped according to their
ability to ferment glucose as homofermentative (homolactic) or heterofermentative
(heterolactic). Homofermentative bacteria produce lactic acid as the main
fermentation end product (Von Wright and Axelsson, 2012), whereas
heterofermentatives produce a variety of fermentation end products such as
ethanol, CO2, acetic acid, formic acid, acetaldehyde, diacetyl and acetoin, in
addition to lactic acid (Kleerebezem and Hugenholtz, 2003). Fermentation
metabolism of some LAB are shown in Table 2.1.
12
Table 2.1. Fermentation metabolism of some LAB

Species Obligately Facultatively Obligately
homofermentative heterofermentative heterofermentative
Lactococcus +
Pediococcus +
Streptococcus +
Enterococcus +
Weisella +
Leuconostoc +
Oenococcus +
Lactobacillus Lb. acidophilus Lb. paralimentarius Lb. brevis
Lb. amylovorus Lb. paracasei Lb. buchneri
Lb. farciminis Lb. pentosus Lb. fermentum
Lb. helveticus Lb. plantarum Lb. reuteri
Lb. mindensis Lb. curvatus Lb. sanfranciscensis
Lb. salivarius Lb. casei Lb. hammesii
Lb. amylolyticus Lb. sakei Lb. spicheri
Lb. manihotivorans Lb. alimentarius Lb. kefir
Lb. suntoryeus Lb. rhamnosus Lb. acidifarinae
Lb. satsumensis Lb. kimchii Lb. panis
Lb. kefiranofaciens Lb. graminis Lb. pontis
Lb. amylophilus Lb. coryniformis Lb. reuteri
Lb. delbrueckii Lb. cypricasei Lb. suebicus
subsp. delbrueckii Lb. versmoldensis Lb. zymae
Lb. delbrueckii Lb. zeae Lb. rossii
subsp. bulgaricus Lb. acidipiscis Lb. parakefiri
Lb. delbrueckii Lb. paracollinoides
subsp. lactis Lb. frumenti
Lb. delbrueckii Lb. ferintoshensis
subsp. indicus Lb. durianis
Lb. diolivorans
2.1.3.1. Homofermentative LAB

In homofermentatives, hexoses are fermented via the EPM as shown in
Figure 2.1. The families using this pathway are Enterococcaceae, Lactobacillaceae
and Streptococcaceae, except for one group in the genus Lactobacillus. The
members of these families use the glycolytic pathway and in this pathway, glucose
is converted into lactic acid as the end product. In theglycolytic pathway, fructose-
1,6-diphosphatase is the key enzyme and theoretically 2 moles of ATP are
generated per mole of glucose consumed (Endo and Dicks, 2014).
13
Homofermentative LAB include Lactococcus, Streptococcus, Pediococcus,

Enterococcus and also Vagococcus. In addition, some species of Lactobacillus
such as Lb. delbrueckii subsp. delbrueckii, Lb. delbrueckii subsp. bulgaricus, Lb.
delbrueckii subsp. lactis, Lb. delbrueckii subsp. indicus, Lb. acidophilus, Lb.
helveticus, Lb. salivarius and some others such as Lb. amylolyticus, Lb. mindensis,
Lb. manihotivorans, Lb. suntoryeus, Lb. satsumensis, Lb. kefiranofaciens, Lb.
farciminis, Lb. amylophilus and Lb. amylovorus use this pathway (Schleifer and
Ludwig, 1995; Von Wright and Axelsson, 2012).
14
Figure 2.1. Fermentation of glucose in homofermentatives adapted from Endo and

Dicks (2014) and Hames and Hooper (2000)
15
2.1.3.2. Heterofermentative LAB

During heterofermentative or heterolactic fermentation, hexoses are
metabolized through the pentose phosphoketolase pathway. Heterofermentatives
produce some other compounds such as CO2 and ethanol or acetic acid besides
lactic acid as shown in Figure 2.2 (Endo and Dicks, 2014). Heterofermentative
LAB include members of the Leuconostocaceae family including the genera
Leuconostoc, Oenococcus and Weissella. Also several species in the genus
Lactobacillus use heterolactic fermentation. Obligate heterofermentative
lactobacilli include the mainly isolated species Lb. brevis, Lb. buchneri, Lb.
fermentum, Lb. reuteri, Lb. sanfranciscensis, Lb. hammesii, Lb. spicheri, Lb. kefir,
Lb. panis, Lb. pontis, Lb. reuteri, Lb. suebicus, Lb. zymae Lb. rossii, Lb. parakefiri,
Lb. paracollinoides, Lb. frumenti, Lb. ferintoshensis, Lb. durianis, Lb. diolivorans
and Lb. acidifarinae (Schleifer and Ludwig, 1995; Von Wright and Axelsson,
2012).
Heterofermentative LAB cannot metabolize hexoses via the EMP pathway
due to the lack of the glycolytic enzyme fructose 1,6 bisphosphate aldolase;
therefore, they cannot break down fructose 1,6-bisphosphate into triose phosphate.
However, they oxidize glucose 6-phosphate to 6-phosphogluconate and then 6-
phosphogluconate is decarboxylated to pentose phosphate together with 1 mole of
CO2. This decarboxylation steps leads to CO2 gas production (Madigan et al.,
2012). Following decarboxylation step, the three carbon metabolite GAP and acetyl
phosphate are produced. The first metabolite, GAP, is then converted to lactic acid.
On the other hand, the second metabolite, acetyl phosphate, is converted into
ethanol or acetate (Mayo et al., 2010). The energetic yield of this pathway is lower
than homolactic fermentation and yields only 1 mole of ATP per mole of
consumed hexose. During the conversion of acetyl phosphate to acetic acid in the
presence of alternative electron acceptors, an extra ATP is generated (Sobczak and
Lolkema, 2005; Pessione, 2012). As a result of following a different pathway,
16
heterofermentative LAB produce more flavor and aroma compounds, such as

acetaldehyde and diacetyl, compared with homofermentatives (Jay et al., 2005).
Facultative heterofermentatives use both pathways and ferment hexoses
and also pentoses (Hammes et al., 1991). This group includes some species of
lactobacilli as follows Lb. casei, Lb. curvatus, Lb. plantarum, Lb. sakei and also
Lb. graminis, Lb. alimentarius, Lb. coryniformis, Lb. paracasei, Lb. pentosus, Lb.
rhamnosus, Lb. kimchii, Lb. cypricasei, Lb. versmoldensis, Lb. zeae, Lb.
paralimentarius and Lb. acidipiscis (Schleifer and Ludwig, 1995; Von Wright and
Axelsson, 2012).
17
Figure 2.2. Heterofermentative metabolism adapted from Butler et al. (2010) and
(Endo and Dicks, 2014)
2.1.4. Yeasts
Under anaerobic conditions, NAD+ is regenerated via alcoholic
fermentation, which is mainly conducted by yeasts. During alcoholic fermentation,
18
pyruvic acid produced via glycolysis is converted to acetaldehyde and CO2 by

pyruvate decarboxylase, as shown in Equation 3. Following that step, acetaldehyde
is reduced to produce ethanol, the alcohol for which the process is named, by
alcohol dehydrogenase and at the same time NADH is oxidized to NAD+, as
shown in Equation 4 (Hames and Hooper, 2000). It is a low-energy-yield process
because most of the energy contained in the initial glucose molecule remains in the
end product, ethanol (Tortora et al., 2010; Hardin et al., 2012). Equations of the
process are shown in Equations 5 and 6:
Eq. 3
Eq. 4
Eq. 5
Eq. 6
Yeasts are an important group of eukaryotic microorganisms (Tamang and

Fleet, 2009). They are classically defined as unicellular fungi that belong to
different taxonomic groups and among these, Saccharomyces (S.) sensu stricto
yeast species are widely known all over the world, especially in the production of
fermented beverages and foods (Sicard and Legras, 2011).
Alcoholic fermentation by yeast cells is a key process in the baking,
brewing and winemaking industries (Hardin et al., 2012). In dough fermentation,
yeast cells break down glucose to ethanol and CO2. Ethanol is driven off during
baking and CO2 is trapped in the dough and causes the bread dough to rise. The
CO2 produced is responsible for the bubbles in beer and in sparkling wines and
ethanol made by yeasts is the alcohol in alcoholic beverages. In brewing, both
ethanol and CO2 are essential as beer is an alcoholic carbonated beverage (Tortora
et al., 2010; Hardin et al., 2012).
Under anaerobic conditions, yeasts perform alcoholic fermentation and
produce ethanol and CO2 from each mole of glucose. On the other hand, under
19
highly aerobic conditions and at a low concentrations of sugar, yeast consume

glucose by the aerobic oxidative pathway to produce biomass. This is known as
Pasteur effect. However, under high glucose concentrations, yeasts do not shift the
metabolism from fermentative to a complete oxidative mode. Instead, the yeasts
continue to ferment the glucose and perform an aerobic fermentation. This
phenomenon is referred as the Crabtree effect (Reed and Nagodawithana, 1990).
The yeasts exhibiting this performance are referred to as Crabtree-positive yeasts
(Pronk et al., 1996; Johnston, 1999; Sicard and Legras, 2011).
2.2. Sourdough
The use of the sourdough process as a means of leavening is one of the
oldest biotechnological processes in cereal food production. Bread can be made
with either baker’s yeast or sourdough for dough leavening, and sourdough bread is
leavened with a sourdough starter (Catzeddu, 2011). The sourdough starter is a
mixture of flour and water that are spontaneously fermented with LAB and yeasts.
Sourdough microflora determine the bread characteristicsin terms of acid
production, aroma and leavening (Hammes and Ganzle, 1998; Vogel et al., 1999;
Moroni et al., 2009). Back-slopping, the addition of new flour and water to the
dough, allows a composite ecosystem of LAB and yeast to take place inside the
dough. The yeast is mainly responsible for the production of CO2, and LAB,
mainly heterofermantative, are responsible for the production of lactic and/or acetic
acid; both microorganisms are responsible for the production of aromatic
precursors of the bread (Catzeddu, 2011). During the production of sourdough, the
produced lactic and acetic acids in the flour and water mixture causes a typical
sour-tasting end product (Chavan and Chavan, 2011).
The use of sourdough in baking is an ancient craft that is currently
undergoing a revival of interest. Bread production has relied on the use of
sourdough as a leavening agent for most of human history as the primary form of
bread leavening, whereas the use of baker’s yeast as a leavening agent dates back
20
less than 150 years (Ganzle, 2014b). Nowadays, traditional sourdough bread is
mostly produced in retail and artisan bakeries, but has also started to be used in
industrial baking instead of baker’s yeast as the leavening agent (Catzeddu, 2011).
2.2.1. History of Sourdough

Bread is considered to be the oldest processed food as its history goes back
to ancient times. It is known that cereals were first cultivated in the Middle East
10,000 years ago. In ancient times, bread was unleavened since as there were no
raising agents (Fob, 2011). In its earliest form, bread would probably be equivalent
to today’s modern flat breads, i.e., the Indian "chapatti", Mexican "tortilla" and
Middle East's "pita"(Cauvain, 2001; Fob, 2011).
One of the oldest sourdough breads dates from 3,700 BC and was
excavated in Switzerland, but the start of the use of sourdough in bread leavening
can be traced back several thousand years earlier to the origin of agriculture in
ancient Egypt (Fob, 2011; Ganzle, 2014b). In early Egypt, as well as in the Roman
Empire, bread was produced on a large scale and it is known that back-slopping,
dough acidification and yeast from winemaking were used for sourdough
fermentations. It is believed that from there sourdough spread gradually to Europe,
throughout ancient Greece and the Roman Empire into the present. The Romans
learned this baking process due to their connection with the Greek civilization. In
the first century, some methods for dough leavening, such as sourdough that was
air-dried after 3 days of fermentation, the use of dried grapes as a starter culture
and the use of back-slopping of dough, were reported by the Romans (Catzeddu,
2011). In Europe, sourdough fermentation was the main process for dough
leavening, before the use of excess brewer’s yeast became common in the 15th
and
16th centuries (Ganzle, 2014b). In France, sourdough was used alone to ensure
fermentation of the dough and also wine, vinegar or rennet was added in some
French regions until the discovery of the use of brewer’s yeast for bread making
(Cappelle et al., 2013). After the use of brewer’s yeast became common, brewing
21
and baking were carried out in the same facility. In Germany, bakers and brewers
were often organised in the same place and in many cities bakers had the right to
brew (Krauß, 1994; Brandt, 2005; Cappelle et al., 2013).
In the United States, sourdough bread is usually associated with San
Francisco as sourdough bread from this city is the most famous type currently
produced in the United States. Sourdough was introduced to the San Francisco area
after the California gold rush and Canada after the Klondike gold rush in the 19th
century (Cappelle et al., 2013). The sourdough starter was relatively easy to
preserve for using as a leavening agent for baking by pioneers or gold prospectors
travelling in slow-moving wagon parties. If sourdough failed, another starter could
be prepared from flour and water during the journey (Catzeddu, 2011; Ganzle,
2014b).
Artisanal bread production relied on the use of sourdough as the main
leavening agent until the 20th century. However, in the second half of 19th century,
baker’s yeast started to be used as the leavening agent instead of sourdough.
Baker’s yeast was a rapid and simple leavening process that was suitable for the
adaptation to mechanized bread production in modern baking processes (Catzeddu,
2011). However, sourdough bread still continued to play a significant role in bread
production in some parts of Europe, particularly in countries where rye bread is
common, including Scandinavia, the Baltic States, Germany, Eastern Europe and
the former Soviet Union, as well as in parts of the Middle East (Ganzle, 2014b).
In recent years, traditional sourdough breads have again started to attract
consumers due to the high nutritional value, healthy properties, pronounced flavor,
prolonged shelf life and natural production, i.e., without the use of any additives.
2.2.2. Sourdough Production

Sourdough is defined as the mixture of wheat or rye flour and water,
fermented spontaneously by LAB and yeasts (Vogel et al., 1999; Corsetti, 2013).
The acidifying and leavening capacity of the dough is optimized by consecutive
22
refreshments, also known as re-buildings, replenishments, back-slopping etc.

(Corsetti and Settanni, 2007; Corsetti, 2013). For back-slopping, a flour and water
mixture is fermented for a certain time at a defined temperature and it is then added
as an inoculum to start the fermentation of a new mixture of flour and water
(Corsetti, 2013).
Back-slopping, the addition of a new flour and water to the dough, allows a
composite ecosystem of yeast and LAB to populate inside the dough, giving it its
typical sour taste. The technological performance of the dough and flavor, the
nutritional value, shelf life and overall quality of the bread are affected by the
metabolic activity of the sourdough microorganisms (Catzeddu, 2011).
Sourdough bread offers many advantages over bread produced by baker’s
yeast such as leavening of dough without using any commercial yeast, improving
the dough properties, as well as enhancing the flavor and taste of the bread. In
addition, the nutritional value of sourdough bread is improved due to the higher
bioavailability of minerals and lower glycaemic index (GI). Furthermore, the shelf
life is extended as a result of the longer mold-free period, anti-staling effect and
prevention of rope formation in bread (Hansen, 2012).
2.2.2.1. Flour in Sourdough Production

The history of sourdough started with the beginning of agriculture and
sourdough bread has been produced on at artisanal scale in different parts of the
world throughout the centuries, resulting in different types of knowledge from
agricultural practices and technological processes through to cultural heritage
(Cappelle et al., 2013). Flour and water are the raw materials in the production of
sourdough. In rye-growing areas, rye sourdough is very common and rye flour is
used for sourdough production. Besides rye flour, whole-meal wheat and white
flour are commonly used in sourdough production in many countries. Moreover,
due to gluten-free bread production, other flours are being started to be used in
sourdough production such as corn, sorghum or rice flours (Ganzle, 2014b).
23
2.2.2.1.(1). Wheat Flour

In bread making, flour is the most important ingredient as it impacts the
development of the specific characteristics of bakery products. It consists of
protein, starch and other carbohydrates, ash, fibres, lipids, water and small amounts
of vitamins, minerals and enzymes (Chavan and Chavan, 2011). Wheat flour is the
most common flour used and wheat-based products are a major source of nutrients
in many regions of the world (Fincher and Stone, 1986; Hoseney et al., 1988).
Therefore, wheat is one of the world’s most important grains and is primarily used
for human consumption, with almost 15% used for animal feed (Manley, 2000;
Morris and Bryce, 2000). In Turkey, climate and ecological properties are suitable
for agricultural activities and wheat is one of the most commonly produced cereal
crops. The total area of land that is suitable for agriculture is around 11.3 million
hectares and wheat surpasses other cereals in terms of the number of hectares
dedicated to its cultivation accounting for 67% in this country (Anonymous, 2013).
Wheat is used as the raw material for bread, bulgur, biscuits, pasta and breakfast
cereals, and provides bulk and structure to bakery products as it has the ability to
form dough after mixing with water (Cotton and Ponte, 1973). Glutenin and gliadin
are two proteins that form gluten in wheat flour. Gluten is very important in
leavened doughs for the development of dough strength, as the formation of gluten
creates an elastic and extensible matrix. In addition, the gas-holding capacity of
wheat dough is dependent upon gluten since as it entraps huge amounts of the gas
(CO2) produced by yeasts during fermentation or by chemical leavening, thus
yielding a leavened product (Hoseney, 1994; Lai and Lin, 2006; Salovaara and
Gänzle, 2012).
For the production of sourdough, wheat (Triticum durum or Triticum
aestivum) flour is commonly used. Wheat kernels are naturally contaminated by
microorganisms and the cereal based products produced from wheat, and wheat
flour, can be a source of viable LAB (Corsetti et al., 2007a; Alfonzo et al., 2013).
Besides the flour, LAB can originate from the equipment used in the flour milling
24
and then these microflora can contribute to the sourdough fermentation and/or
production process (Berghofer et al., 2003; Alfonzo et al., 2013).
2.2.2.1.(2). Rye Flour

Besides wheat products, sourdough fermentation is a traditional process
employed in rye (Secale cereale) baking (Gänzle et al., 2008). After baker’s yeast
began to be used as the leavening agent in modern baking processes, the use of
sourdough for bread production was reduced (Catzeddu, 2011). However,
sourdough bread still continued to play a significant role in bread production in
rye-growing areas, where rye bread has a major share of the bread market. Rye
sourdoughs have been characterized from Germany (Böcker et al., 1995),
Scandinavian countries including Finland (Salovaara and Katunpaa, 1984), Sweden
(Spicher and Lonner, 1985), Denmark (Knudsen, 1924; Rosenquist and Hansen,
2000) and also Poland, the Czech Republic, Austria, Portugal and the Baltic States
in Europe and Russia (Hansen, 2012; Ganzle, 2014b). The continued use of
sourdough in those countries relates to the use of rye flour in bread production as
sourdough was used for the acidification of rye dough to reach optimal rye bread
quality (Cappelle et al., 2013). After the introduction of baker’s yeast as a
leavening agent, the aim of using sourdough fermentation in rye baking shifted to
its use as an acidifying agent. Free sugars and amylolytic enzymes are higher in rye
than in other cereals and endogenous enzymes in rye can be used to break down the
starch to simple fermentable sugars (Salovaara and Gänzle, 2012). Rye flour
contains high levels of pentosan compared with wheat flour, and the pentosans
inhibit the formation of the gluten network in rye doughs and in the structure
forming process of a rye dough the proteins play a lesser role than in wheat doughs
(Cauvain, 1998). Sourdough fermentation promotes the solubilisation of rye
pentosans at the dough stage and enhances water binding and gas retention of the
dough resulting in conversion of the starch to gel which form a matrix during
baking (Martinez-Anaya and Devesa, 2000; Brandt, 2007; Catzeddu, 2011). The
25
solubility and swelling behaviour of pentosans increase at the low pH values

characteristic of sourdoughs (Hammes and Ganzle, 1998; Arendt et al., 2007).
Gluten is lacking in rye and the gas-holding capacity of rye dough is dependent
upon polymeric arabinoxylans (Salovaara and Gänzle, 2012). Acidification is also
very important in rye bread production for inhibiting the flour α-amylase thus
preventing excessive starch degradation (Catzeddu, 2011). Acidification is very
important in both flours, wheat and rye, since cereal phytases are activated due to
the acidificiation and more nutrients become available (Fretzdorff and Brummer,
1992; De Vuyst and Neysens, 2005). The acidification of rye doughs improves
their physical properties by making them more elastic and extensible (Arendt et al.,
2007).
2.2.2.1.(3). Other Flour Types

Other cereal and also legume flours can be used in sourdough production.
Studies have been conducted on sourdoughs produced with other cereal and
legume flours such as maize flour in combination with rye flour (Rocha and
Malcata, 2016), faba bean (Vicia faba L.) flour obtained from different cultivars
(Coda et al., 2017), quinoa flour (Ruiz Rodriguez et al., 2016a), chickpea, lentil
and bean flour combinations with wheat flour (Rizzello et al., 2014), hull-less
barley (Hordeum vulgare) flour with wheat flour (Mariotti et al., 2014), sorghum
flour (Schober et al., 2007), Teff (Eragrostis tef) together with wheat flour, rice
sourdough (Meroth et al., 2004), amaranth flour (Sterr et al., 2009; Ruiz Rodriguez
et al., 2016b) etc.
2.2.2.2. Classification of Sourdough Production Methods

Sourdoughs are grouped as shown below on the basis of the production
technology applied (Figure 2.3):
26
-Type I traditional sourdoughs

-Type II semi fluid sourdoughs
-Type III dried sourdoughs
Figure 2.3. Sourdough types adapted from Corsetti and Settanni (2007)
Type I-traditional sourdoughs are produced by using a sourdough that was

part of the previous fermentation (Chavan and Chavan, 2011). These types of
sourdoughs are characterized by continuous and daily refreshment/back-slopping to
maintain the microorganisms in an active state. Dough leavening is achieved
without the addition of baker’s yeast (Corsetti, 2013). Fermentation is performed
spontaneously at an ambient temperature of around 20–30°C and the pH is
approximately 4.0 (De Vuyst and Neysens, 2005). At the beginning, a sourdough
starter is prepared by mixing flour and water and the mixture is fermented in a
warm place. After 12-24 hours visible fermentation occurs accompanied by sour,
alcoholic odor. A portion of fermented sourdough is used to inoculate the next
27
batch and the inoculation of each new batch with sourdough containing active
fermenting yeasts and LAB results in more rapid fermentation. After a few
refreshments characterized with different dough yield, temperature and time, a
natural starter culture sourdough with a stable fermentation microbiota consisting
of heterofermentative LAB and yeasts is established (Hammes, 1991). In this type
of sourdoughs, sourdoughs are regularly refreshed for very long periods of time
with stable fermentation microbiota documented over a period of more than 20
years (Ganzle, 2014b).
Type I sourdoughs can be further classified into Type Ia, Ib and Ic (De
Vuyst and Neysens, 2005). Type Ia sourdoughs can be pure cultures from natural
sourdoughs of different origin, for example, San Francisco French bread (Gobbetti
and Corsetti, 1997; Tucker, 2016). Type Ib sourdoughs are spontaneously
developed, mixed culture sourdoughs prepared from wheat and rye, or their
mixtures, through multiple-stage fermentation processes. Traditional rye sourdough
is an example of that type. When the fermentation is completed, the fully
developed starter, mother dough, is used as the inoculum for future batches of
bread dough. Consecutive back-sloppings of a new batch from a previous batch
ensure the continuity of the microflora (De Vuyst and Neysens, 2005; Tucker,
2016), which has a very important role in the acidification and leavening of the
dough as well as aroma formation. According to environmental conditions,
different species of LAB dominate the fermentation. Type Ic sourdoughs, for
example, African sorghum sourdoughs, are fermented at high temperatures
(>35°C) in tropical regions (Stolz, 1999; De Vuyst and Neysens, 2005;
Paramithiotis and Drosinos, 2017).
Large-scale sourdough fermentation processes resulted in the development
of type II sourdoughs (De Vuyst and Neysens, 2005). Type II sourdoughs are an
industrial type of sourdough in the form of semi-fluid or liquid preperations so it is
easily pumpable in an industrial bakery (Chavan and Chavan, 2011). Adapted
strains are used to start the fermentation, which mainly serve as dough acidifiers
28
(Paramithiotis and Drosinos, 2017). These types of processes continue for 2-5 days
and are often performed at increased fermentation temperature (>30 °C) to speed-
up the process (Böcker et al., 1995; Hammes and Ganzle, 1998). The resulting
sourdough has a high acid content at a pH of 3.5 after 24 hours of fermentation.
The high dough yield (DY) values of Type II sourdoughs enable pumping of the
dough (De Vuyst and Neysens, 2005).
Type III sourdoughs are dried doughs in powder form and is often used by
industrial bakeries as the quality constant (Chavan and Chavan, 2011). They are
initiated by defined starter cultures and used as acidifier supplements and aroma
carriers during bread making. Drying leads to an increased shelf life of the
sourdough and also provides a stock product until further use. Spray or drum
drying processes are the most commonly used techniques and dried sourdoughs are
simple to be used in dough processing and also result in standardized end products.
This type of sourdoughs can be distinguished by color, aroma and acid content
(Stolz and Bocker, 1996; De Vuyst and Neysens, 2005).
In contrast to Type I preparations, doughs of Types II and III require the
addition of baker’s yeast (S. cerevisiae) for leavening (De Vuyst and Neysens,
2005).
2.2.3. Sourdough Microflora

From a microbiological point of view, the effect of sourdough fermentation
is related to the metabolic activities of two groups of microorganisms: yeasts and
LAB. Nevertheless, LAB are mainly responsible for all the nutritional and
functional advantages of sourdough fermentation, whereas yeasts are mostly
related to leavening and aroma formation (Rizzello et al. 2017). LAB and yeasts
isolated from some sourdough samples is shown in Table 2.2.
Fermented sourdoughs include Gram-negative aerobes (e.g.,
Pseudomonas) and facultative anaerobes (Enterobacteriaceae), as well as Gram-
positive LAB including mainly Lactobacillus species and also some Enterococcus,
29
Lactococcus, Pediococcus, Leuconostoc and Weissella species. Sourdough is rich

in fermentable carbohydrates and therefore allows the spontaneous development of
characteristic LAB species, as previously mentioned. These LAB can originate
from the cereals or flours and depend on the flour preparation and sourdough
production technology applied (De Vuyst and Neysens, 2005).
Alfonzo et al. (2013) investigated the wheat flour microflora used to
produce sourdough bread in Southern Italy using phenotypic characteristics and
genetic analysis and it was found that flours harbor LAB of high technological
potential with respect to sourdough bread production. Analysis by 16S rRNA gene
sequencing grouped the strains into 11 LAB species, which belonged to six genera:
Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and
Weissella. It was determined that W. cibaria, Lb. plantarum, Leu.
pseudomesenteroides and Leu. citreum were the most prevalent species
In the production of different types of sourdoughs, different LAB species
dominate the fermentation. Type Ia sourdoughs contain well-adapted microflora
with a stable composition, especially obligate heterofermentative Lb.
sanfranciscensis. Actually majority of type I sourdoughs contain Lb.
sanfranciscensis. Lb. sanfranciscensis (previously named Lb. sanfrancisco or Lb.
brevis subsp. lindneri), which is an obligate heterofermentative Lactobacillus
species that was first isolated from German rye sourdoughs and from the San
Francisco French bread processes (Sugihara et al., 1970; Kline and Sugihara, 1971;
Salovaara and Gänzle, 2012). For example, San Francisco French bread is
produced by using a Type Ia starter and Lb. sanfranciscensis is responsible for the
souring activity by producing large amounts of lactic and acetic acids, resulting in a
strong sour taste, and also helps dough leavening due to gas production (Gobbetti
and Corsetti, 1997; Tucker, 2016). Type Ib sourdoughs consist of obligate
heterofermentative strains of Lb. sanfranciscensis and also, depending on the
fermentation conditions, other species such as Lb. brevis and also Lb. buchneri, Lb.
fermentum, Lb. fructivorans, Lb. pontis, Lb. reuteri, W. cibaria, Lb. alimentarius,
30
Lb. casei, Lb. paralimentarius and Lb. plantarum, Lb. acidophilus, Lb. delbrueckii,
Lb. farciminis and Lb. mindensis occur in relevant cell counts (Hammes and
Ganzle, 1998; Vogel et al., 1999). Lb. sanfranciscensis is replaced by species that
are more adapted to higher temperatures in sourdoughs fermented at increased
temperatures (Meroth et al., 2003).Type Ic sourdoughs contain Lb. fermentum, Lb.
pontis and Lb. reuteri species, as well as Lb. amylovorus (Hamad et al., 1992).
Different process parameters of type II sourdough fermentations result in a
different LAB flora including Lb. acidophilus, Lb. delbrueckii, Lb. amylovorus, Lb.
farciminis, Lb. johnsonii, Lb. brevis, Lb. fermentum, Lb. frumenti, Lb. pontis, Lb.
panis, Lb. reuteri and W.confusa species (Vogel et al., 1999; Müller et al., 2001).
Type III sourdoughs predominantly contain LAB those are resistant to drying and
are able to survive in that form, for example, Lb. brevis, Pd. pentosaceus and Lb.
plantarum strains (Böcker et al., 1995; De Vuyst and Neysens, 2005).
The production of lactic acid and CO2 is the most prominent metabolic
activity of LAB in sourdough; whereas, gas production and aroma formation is
achieved by yeasts. Type Ia and Ib sourdoughs can contain Candida (C.) humilis
(Torulaspora (T.) holmii, C. milleri) and also S. exiguus. Type Ic sourdoughs can
contain Issatchenkia (I.) orientalis (C. krusei) (De Vuyst and Neysens, 2005).
Types II and III can contain S. cerevisiae as baker’s yeast if added, since as
normally this type does not contain yeasts at important levels. In addition,
Kazachstania (K.) exigua (formerly S. exiguus) can be found in the sourdough
environment which is tolerant to more acidic environments (Cappelle et al., 2013).
The microflora of sourdough varies according to the environment, type and
fermentation conditions of produced sourdoughs, as reported in many studies. LAB
and yeasts have been identified in sourdoughs, collected from different countries,
using phenotypic and molecular methods.
Corsetti et al. (2001) characterized the microflora of 25 wheat sourdoughs
from Italy. The number of LAB and yeasts ranged from 7.5 to 9.3 log CFU/g and
from 5.5 to 8.4 log CFU/g, respectively. Isolated LAB and yeasts were identified
31
by conventional physiological and biochemical tests and also confirmed with

molecular techniques 16S rDNA and 16S/23S rRNA spacer region PCR. The
microflora distribution was heterogeneous. Lb. sanfranciscensis (30%) and then
Lb. alimentarius (20%) were the most isolated species. Other isolated species
include Lb. brevis, Leu. citreum, Lb. plantarum, Lc. lactis subsp. lactis, Lb.
fermentum, Lb. acidophilus, W. confusa and Lb. delbrueckii subsp. delbrueckii. S.
cerevisiae was largely found in sourdoughs, and some of the sourdoughs also
contain S. exiguus and C. krusei.
Ricciardi et al. (2005) determined the composition of the LAB community
of sourdoughs produced in Southern Italy using a set of 29 phenotypic tests. Counts
of LAB ranged between 107–108 cfu/g and a total of 111 LAB strains were
randomly isolated. Most strains were identified as Lb. plantarum, Lb. paracasei,
Lb. casei, Lb. brevis and Leu. mesenteroides.
In another study conducted in Italy, yeasts were identified in collected
homemade sourdough samples. Results showed that S. cerevisiae was the dominant
species, followed by C. milleri, C. humilis, S. exiguus and I. orientalis (Pulvirenti
et al., 2004).
Randazzo et al. (2005) evaluated the LAB in 9 artisanal wheat sourdough
samples, that were collected in different areas of Sicily, using physiological and
biochemical methods along with molecular techniques. Restriction fragment length
polymorphism and 16s ribosomal DNA gene sequencing showed a variety of
species with the dominance of Lb. sanfranciscensis and Lb. pentosus in all tested
sourdoughs. In addition, Lb. casei, Lb. kimchii/Lb. alimentarius and Lb. plantarum
were identified.
Valmorri et al. (2006) characterized the lactobacilli community of 20
sourdoughs, collected from different cities in central Italy, using a novel polyphasic
approach consisting of a two-step multiplex PCR system, 16S rRNA gene sequence
analysis and physiological features. Yeast and LAB counts were in the range of
5.03-8.61 and 7.55-9.45 log CFU/g, respectively. Identified species included Lb.
32
plantarum, Lb. alimentarius, Lb. paralimentarius, Lb. sanfranciscensis, Lb. brevis,

Lb. fermentum, Lb. rossiae, W. cibaria and Lb. graminis/Lb.sakei/Lb. curvatus. In
another study, Valmorri et al. (2010) identified yeasts from different sourdough
samples using PCR-RFLP analysis and isolates were identified as mainly S.
cerevisiae, with the other dominant species being C. milleri, C. krusei and T.
delbrueckii.
In another study conducted in Italy, molecular techniqes, 16Sr DNA
sequencing and RAPD-PCR were used for the identification and typing of LAB
isolated from 25 samples of sourdoughs. Twelve different species of LAB were
identified, and most isolates were classified as facultative heterofermentative
lactobacilli. Lb. pentosus dominated the lactic microflora of many samples and
other frequently isolated species were Lb. plantarum, Lb. brevis, W. confusa and
Lb. sanfranciscensis. Other species were identified as Lb. casei, Lb. zeae, Pd.
pentosaceus, Lb. sakei, Lb. alimentarius, Lb. farciminis and Leu. citreum
(Catzeddu et al., 2006).
Minervini et al. (2012a) reported the microbiota of 19 Italian sourdoughs
used for the manufacture of traditional/typical breads through a culture-dependent
method and pyrosequencing. The most frequent LAB isolates were Lb.
sanfranciscensis, Lb. plantarum and Lb. paralimentarius. S. cerevisiae was
identified in many of the sourdoughs and along with C. humilis, K. barnettii and K.
exigua yeasts.
Gaglio et al. (2017) investigated the microbial community of Italian
sourdoughs using culture-dependent and culture-independent approaches. LAB and
yeast counts were approximately 109 CFU/g and 106 CFU/g, respectively.
Identified LAB species present in Sicilian sourdough were Lb. sanfranciscensis,
Lb. paralimentarius, Lb. brevis and Lb. coryniformis and yeasts were S. cerevisiae,
Pichia (P.) guiliermondii, P. segobiensis, Rhodotorula (R.) acuta and R.
mucilaginosa.
33
Another study conducted in Italy identified the LAB and yeast species Lb.
sanfranciscensis, C. milleri and S. cerevisiae as the microbiota characterizing the
sourdough of Italian PDO Tuscan bread (Palla et al., 2017).
Lattanzi et al. (2013) used a similar method, pyrosequencing and culture-
dependent methods, in the investigation of 18 sourdoughs used for the manufacture
of traditional/typical Italian breads and the results of pyrosequencing were in
agreement with the results of the culture-dependent method. Lb. sanfranciscensis
was identified in almost all the sourdoughs. Lb. plantarum and Leu. citreum were
also isolated with a relatively high frequency. S. cerevisiae was identified in many
samples and C. humilis were also identified in some of the sourdoughs, along with
Lc. lactis, Lb. brevis, Lb. casei and also Lb. curvatus, Lb. fermentum, Leu.
mesenteroides, Pd. acidilacticii and W. cibaria species.
From French wheat sourdough samples, a total of 20 morphologically
different strains were chosen and identified as Lb. plantarum, Lb. paralimentarius,
Lb. sanfranciscensis, Lb. spicher, Lb. sakei and also, two isolates belonging to a
novel Lactobacillus species, proposed in that work as Lb. hammesii (Valcheva et
al., 2005)
In another study involving 16 sourdoughs used for the manufacture of
traditional French breads, Lb. sanfranciscensis was determined as the dominant
species in French sourdoughs according to the results of genotypic analyses. The
median values of cell density of LAB was 9.2 log CFU/g and the ratio between
LAB and yeasts ranged from 10,000:1 to 10:1. Other species frequently
encountered were Lb. parabrevis/Lb. hammesii, Lb. plantarum and Leu.
mesenteroides and for the first time Lb. xiangfangensis and Lb. diolivorans were
found in sourdough. The yeast microbiota of French sourdoughs was dominated by
S. cerevisiae and also K. servazzii (formerly S. servazzii) was found as the
dominant or co-dominant yeast species in two samples (Lhomme et al., 2015).
Scheirlinck et al. (2009) investigated the predominant sourdough LAB
species during the production of two Belgian artisan sourdough by using molecular
34
methods, and sourdoughs were found to be mainly dominated by Lb. spicheri, Lb.
plantarum and Lb. sanfranciscensis.
Viiard et al. (2012) analysed the lyophilized starter and industrial
sourdough in Estonia and reported Lb. helveticus as the dominant LAB species in
the analysed samples. Furthermore, Lb. panis and Lb. pontis and also some other
species, Lb. vaginalis, Lb. reuteri, Lb casei/paracasei, Lb. fermentum and Lb.
paralimentarius were identified in the study.
Saeed et al. (2009) investigated the wheat sourdough samples that were
collected from different bakeries in Pakistan and isolates were identified as
phenotpic methods. LAB isolates were identified as Lb. brevis, Lb. fermentum and
Lb. plantarum. S. cerevisiae was also identified in the samples which is certainly
related to the addition of baker’s yeast to the doughs
Zhang et al. (2015) investigated Chinese traditional sourdoughs, collected
from different areas of China, using culture-dependent and DGGE methods. The
culture dependent method results showed that S. cerevisiae and Lb. plantarum were
the predominant species among the yeasts and LAB microflora. According to the
PCR-DGGE approach, S. cerevisiae was predominant, while the yeast C. tropicalis
represented the subdominant species of the yeast community. Among the LAB
community, Lb. sanfranciscensis was the predominant species, while Lc. qarvieae,
E. faecium, Lb. delbrueckii and E. cecorum were among the less dominant species.
In addition, some studies have investigated Turkish sourdoughs (Menteş et
al., 2004; Gül et al., 2005; Şimşek et al., 2006; Dertli et al., 2016; Yagmur et al.,
2016). Menteş et al. (2004) collected 20 sourdough samples from Ankara, Bursa
and Trabzon, and investigated LAB flora using phenotypic methods. The study
reported the dominant species as Lb. alimentarius (31 of 150 isolates) and Lb.
plantarum (21 of 150 isolates). Other species were Lb. sake, Lb. acidophilus, Lb.
fermentum, Lb. curvatus, Lb. delbrueckii subsp. delbrueckii, Lb. farciminis, Lb.
casei subsp. casei, Lb. helveticus, Lb. collinoides, Lb. buchneri, Lb. brevis, Lb.
35
amylophilus, Lb. reuteri, Lb. divergens, Lb. viridescens, Lb. amylovorus and Lb.
agilis
Gül et al. (2005) collected 14 sourdough samples from Isparta and reported
the LAB as Lb. divergens (6.1%), Lb. brevis (15.1%), Lb. amylophilus (6.1%), Lb.
sake (6.1%), Lb. acetotolerans (6.1%), Lb. plantarum (3%), Pd. pentosaceus
(6.1%) and Pd. acidilactici (6.1%) species and yeasts were S. cerevisiae (27%), T.
delbrueckii (2.7%), T. holmii (10.8%) and T. a unisporus (2.7%). LAB and yeast
counts were in the range of 5.28-9.66 and 6.33-9.96 log CFU/g, respectively.
Şimşek et al. (2006) analysed the LAB microflora in sourdough samples
collected from Usak and reported that Lb. brevis spp. lindneri, Lb. viridenscens,
Pediococcus sp. and Lb. delbrueckii are the strains with best potential as sourdough
starters.
Dertli et al. (2016) investigated the Turkish wheat sourdoughs from the
Eastern Black Sea region of Turkey and reported the presence of 47 distinct LAB
strains belonging to 11 different including: Lb. plantarum, Lb. paraplantarum, Lb.
curvatus, Lb. rossiae, Lb. sanfranciscensis, Lb. brevis, Lb. paralimentarius, W.
paramesenteroides, Leu. mesenteroides, Leu. pseudomesenteroides and W. cibaria.
The pH of the sourdoughs ranged from 3.37 to 3.95. The LAB and yeast counts of
these samples ranged between 8.35 and 8.91 log CFU/g and 6.70 and 6.96 CFU/g,
respectively.
Another study conducted in our country investigated the microbial flora in
different sourdough samples collected from Ankara, Trabzon, Kütahya, Isparta and
Adana. The main LAB species identified were Lb. sanfranciscensis, Pd.
pentosaceus, Lb. plantarum, Lb. namurencis, Lb. rossiae, Leu. mesenteroides and
Lb. zymae. Lb. spicheri, Lb. paralimentarius, Lb. mindensis, Lb. farciminis, Lb.
acetotolerans, Lb. casei, E. faecium and E. durans were also found in sourdoughs
at subdominant levels. Among yeasts, mainly S. cerevisiae and also P.
36
guiliermondii and T. delbrueckii were detected as the predominant yeast species in

sourdoughs (Yagmur et al., 2016).
Another study, conducted on the isolated bacteria and yeasts from rye,
maize flours and sourdoughs, reported the most frequently isolated yeasts as S.
cerevisiae and C. pelliculosa. The most frequently isolated LAB in their study were
Lb. brevis, Lb. curvatus, and Lb. lactis spp. lactis; Lc. lactis spp. lactis, E.
casseliflavus, E. durans, E. faecium, S. constellantus and S. equinus (Rocha and
Malcata, 1999).
Table 2.2. LAB and yeasts isolated from various sourdoughs

Place LAB Yeast Reference
Southern Lb. sanfranciscensis S. cerevisiae (Corsetti et al.,
Italy Lb. alimentarius S. exiguus 2001)
Lb. brevis C. krusei
Leu. citreum
Lb. plantarum
Lc. lactis subsp. lactis
Lb. fermentum
Lb. acidophilus
W. confusa
Lb. delbrueckii subsp.
delbrueckii
Italy S. cerevisiae (Pulvirenti et al.,
C. milleri 2004)
C. humilis
S. exiguus
I. orientalis
Italy Lb. plantarum (Ricciardi et al.,
Lb. paracasei 2005)
Lb. casei
Lb. brevis
Leu. mesenteroides
Italy Lb. sanfranciscensis (Randazzo et al.,
Lb. pentosus 2005)
Lb. casei
Lb. kimchii
L. alimentarius
Lb. plantarum
37
Table 2.2. Continued

Italy Lb. plantarum (Valmorri et al.,
Lb.alimentarius 2006)
Lb.paralimentarius
Lb. sanfranciscensis
Lb. brevis
Lb. fermentum
Lb. rossiae
W. cibaria
Lb. graminis/ Lb.sakei/Lb.
curvatus
Italy S. cerevisiae (Valmorri et al.,
C. milleri 2010)
C. krusei
T. delbrueckii
France Lb. plantarum (Valcheva et al.,
Lb. paralimentarius 2005)
Lb. spicher
Lb. sakei
Lb. hammesii
Italy Lb. pentosus (Catzeddu et al.,
Lb. plantarum 2006)
Lb. brevis
W. confusa
Lb. casei
Lb. zeae
Pd. pentosaceus
Lb. sakei
Lb. alimentarius
Lb. farciminis
Leu. citreum
Pakistan Lb. brevis S. cerevisiae (Saeed et al.,
L. fermentum 2009)
L. plantarum
Belgium Lb. spicheri (Scheirlinck et al.,
Lb. plantarum 2009)
Italy Lb. sanfranciscensis S.cerevisiae (Minervini et al.,
Lb.plantarum C. humilis 2012a)
Lb. paralimentarius K. barnettii
K. exigua
38

France Lb. sanfranciscensis S. cerevisiae (Lhomme et al.,
Lb. parabrevis/ K. servazzii 2015)
Lb. hammesii
Lb. plantarum
Leu. mesenteroides
Lb. xiangfangensis
Lb. diolivorans
Turkey Lb. brevis spp. lindneri (Şimşek et al.,
Lb. viridenscens 2006)
Pd. spp.
Lb. delbrueckii
Italy Lb. sanfranciscensis S. cerevisiae (Lattanzi et al.,
Lb. plantarum C. humilis 2013)
Leu. citreum
Lc. lactis,
Lb. brevis,
Lb. casei
Lb. curvatus,
Lb. fermentum,
Leu. mesenteroides,
Pd. acidilacticii
W. cibaria
Estonia Lb. helveticus (Viiard et al.,
Lb. pontis 2012)
Lb. vaginalis
Lb. reuteri
Lb casei/paracasei
Lb. fermentum
Lb. paralimentarius
China Lb. plantarum S. cerevisiae (Zhang et al.,
Lb. sanfranciscensis C.tropicalis 2015)
Lc. qarvieae
E. faecium
Lb. delbrueckii
E.cecorum
Turkey Lb. divergens S.cerevisiae (Gül et al., 2005)
Lb. brevis T.delbrueckii
Lb. amylophilus T. holmii
Lb. sake T.unisporus
Lb. acetotolerans
Lb. plantarum
Pd. pentosaceus
Pd. acidilactici
39
Table 2.2 Continued

Turkey Lb. plantarum (Dertli et al.,
Lb. paraplantarum 2016)
Lb. curvatus
Lb. rossiae
Lb. brevis
Lb. paralimentarius
W. paramesenteroides
Leu. mesenteroides
Leu. pseudomesenteroides
W. cibaria.
Turkey Lb. sanfranciscensis S. cerevisiae (Yagmur et al.,
Pd. pentosaceus, P. 2016)
Lb. plantarum, guiliermondii
Lb. namurencis, T. delbrueckii
Lb. rossiae,
Leu. mesenteroides
Lb. zymae.
Lb. spicheri,
Lb. paralimentarius,
Lb. mindensis,
Lb. farciminis,
Lb. acetotolerans,
Lb. casei,
E. faecium
E. durans
2.2.4. Beneficial Effects and Functional Aspects of Sourdough Technology

Sourdough fermentation is a traditional process for the production of wheat
and rye breads. At present it is also used for the production of other bread types
with different flours and for the manufacture of various cereal-based products such
as breads, cakes and crackers. The use of sourdough technology improves the
quality of cereal products which is characterized by its flavor, nutritional value
texture and shelf life (Arendt et al., 2007). In addition, it confers a natural image to
the product from a consumer perspective (Salovaara, 1998). The typical
characteristic of sourdough mainly relies on the metabolic activities of its active
microflora, basically represented by LAB and yeasts. As a result of the metabolic
activities of LAB and yeasts, biochemical changes occur during sourdough
fermentation and the quality of the dough and bread are affected (Galle, 2013).
40
Various components in flours (carbohydrates, nitrogen sources, minerals, lipids and

free fatty acids and enzyme activity) and process parameters of the dough
(temperature, dough yield, O2, fermentation time, and number of sourdough
propagation steps) influence the microflora of sourdough and the propeties of the
leavened product, as during fermentation, the action of microbial and indigenous
enzymes causes the biochemical changes in the components of the flour (Hammes
and Ganzle, 1998; Chavan and Chavan, 2011). The rate and extent of these changes
greatly influence the properties of the sourdough and the quality of the resulting
baked product (Arendt et al., 2007). Organic acid production, volatile compound
synthesis, proteolytic and amylolytic activities, improvement of the texture and
sensorial properties, delaying microbial spoilage and EPS production occur during
sourdough fermentation (Hammes and Ganzle, 1998; Gobbetti et al., 1999).
The microflora involved in fermented foods contribute to the improvement
of the organoleptic properties, the shelf life of the final products and their
nutritional profile (Kotzekidou and Tsakalidou, 2006), due to the metabolic activity
of the microorganisms which is governed by the interaction with the grain
constituents. LAB produce lactic and acetic acids and the pH is typically decreased
below pH 5 with acidification contributing to the activation of certain enzymes
such as proteases, amylases, hemicellulases and phytases. It has been reported that
the activity of enzymes and microbial metabolites affect the nutritional quality of
bread, including proteins, starches, lipids, dietary fibres, vitamins, minerals and
phenolics, via different mechanisms (Poutanen et al., 2009). Sourdough
fermentation can influence the nutritional quality by decreasing or increasing level
of these compounds and enhancing or retarding the bioavailability of nutrients. In
particular, enhanced mineral bioavailability, fibre solubilisation, the production of
bioactive peptides and reduction of starch digestibility are important potential
mechanisms in sourdough fermentation (Poutanen et al., 2009).
41
2.2.4.1. Organic Acid Production

During sourdough fermentation, LAB produce lactic and acetic acids and
pH is typically reduced to below 5. Organic acid production enhances flavor,
improves texture and also inhibits pathogenic and spoilage organisms as a result of
the low pH levels and inhibitory effect of some organic acids (Salovaara and
Gänzle, 2012). During the sourdough process, lactic acid and acetic acid in
particular exert an inhibitory effect (Hansen, 2012). The inhibitory effect of
organic acids are related to impacting cell homeostatic systems since, as the
decreasing pH acidifies the cell and then the cell consumes a great amount of
energy to maintain intracellular pH homeostasis (Kang et al., 2003; Hassan et al.,
2015; Erkmen and Bozoglu, 2016). Organic acids dissociate depending on their
dissociation constants (pKa), temperature and certain other factors, and produce
protons. However, the cytoplasmic membrane is impermeable to protons. On the
other hand, organic acids are mainly lipophilic and undissociated molecules of
organic acids easily enter through the cell membrane. In the cell, a higher internal
pH than pKa causes organic acids to dissociate when entering the cytoplasm, which
decreases the intracellular pH by releasing the proton. Protons acidify the
cytoplasm and cells try to overcome this problem by pumping out the protons to
the external environment. The cell uses the main part of its energy content to
remove newly formed protons, which results in slower growth kinetics (León
Peláez et al., 2012; Hassan et al., 2015). At very low pH levels (4.5 or below), it is
difficult to remove all the protons from the cell and it cannot retain its internal pH.
Bacteria maintain internal pH near neutrality to prevent denaturation of structural
proteins, enzymes, nucleic acids and phospholipids. Organic acids can also affect
membrane permeability. A low pH and high proton concentration in the cell
denature proteins, reduces the membrane proton gradient, neutralizizing the proton-
motive force. The exposure of cellular compounds to protons can affect the ionic
bonds of macromolecules and structure. A low pH may damage cellular
42
macromolecules in the cell wall, cell membrane, metabolic enzymes, protein

synthesis and genetic material (Erkmen and Bozoglu, 2016).
Furthermore, the acetic acid formed is of major importance for the
development of flavor and contribute to the aroma of bread dough. Therefore, the
content of lactic and acetic acids in sourdoughs is very important for the taste and
flavor of sourdough bread (Hansen and Hansen, 1996). The molar ratio between
lactic and acetic acid is defined as the fermentation quotient (FQ) and the FQ
should be around 4 in sourdoughs to obtain a balanced bread taste (Hansen, 2012);
with the optimum considered to be in the range of 2.0-2.7 (Hammes and Ganzle,
1998). Regarding rye sourdough bread, the optimal FQ was reported to be in the
range of 1.5-4.0 (Spicher, 1983).
A drop of pH causes some modifications by enhancing the performance of
certain enzymes, such as amylases, proteases, hemicellulases and phytases.
Differences in pH and enzyme activity affect the nutritional quality of the
structure-forming components; i.e., protein, starch, lipid, dietary fibre, vitamins,
minerals, sterols and phenolics, via different mechanisms (Gänzle et al., 2008;
Poutanen et al., 2009).
In rye sourdoughs, acidification is also important as the acidification of rye
doughs improves the physical properties of the dough by making them more elastic
and extensible and confers the acid flavor notes so characteristic of rye breads
(Cauvain, 1998).
2.2.4.2. Antibacterial and Antifungal Activities

During sourdough fermentation, the rapid consumption of fermentable
carbohydrates by LAB and the formation of lactic acid, accompanied by a
reduction of the pH, have an inhibitory effect on other microorganisms. Besides
lowering the pH, a wide range of antimicrobial compounds, for example, diacetyl,
hydrogen peroxide, acetic acid and other short chain fatty acids, are synthesised by
43
LAB and some of them effectively for the inhibite pathogen bacteria and fungi
(Gänzle and Gobbetti, 2013).
Larsen et al. (1993) screened 335 LAB strains isolated from sourdoughs
and 18 isolates that belonged to three different Lactobacillus species, Lb. sakei
(formerly Lb. bavaricus), Lb. curvatus and Lb. plantarum showed antimicrobial
activity indicated by a proteinaceous compound. Pepe et al. (2003) reported
ropiness development in breads was inhibited for more than 15 days with LAB and
inhibition of rope symptoms increased at a low pH (3.7 to 4.3). Lb. plantarum E5
and Leu. mesenteroides A27 showed the most effective antirope activity in their
study. Corsetti et al. (1996) reported antimicrobial activity by Lactobacilli isolated
from wheat sourdoughs belonging to the species Lb. sanfranciscensis, Lb. brevis,
Lb. fructivorans, Lb. fermentum, Lb. plantarum, Lb. farciminis, Lb. acidophilus,
Lb. alimentarius and Lb. hilgardii. Lb. sanfranciscensis and Lb. plantarum strains
showed the largest spectrum of inhibition among the strains. On the other hand, Lb.
fermentum and Lb. alimentarius strains had the narrowest inhibition spectrum.
Furthermore, a bacteriocin-like inhibitory substance from Lb.sanfranciscensis C57
has been characterized. Antimicrobial activity of Lb. reuteri LTH2584, LTH3566
and Lb. sanfranciscensis LTH2594 isolated from wheat and rye sourdoughs was
reported previously and the antimicrobial compound produced by Lb. reuteri
LTH2584 exhibited the broadest inhibitory spectrum (Ganzle, 1998; Messens and
De, 2002).
Inhibitory activity of certain antimicrobial compounds produced under
bread-making conditions can be changed and the assessment of certain compounds
under sourdough bread production conditions is necessary to elucidate any
antimicrobial effects (Coda et al., 2011). A total of 437 Lactobacillus strains
isolated from sourdoughs were screened for their antimicrobial compound
production against four indicator strains (Lb. farciminis CC10, Lb. sakei LMG
2313, Lb. delbrueckii spp. bulgaricus B397 and Listeria innocua 4202) and 85
strains produced an inhibition zone against one or more indicators. It was reported
44
that a bacteriocin-like inhibitory substance was produced by Lb. pentosus isolated

from sourdough, which was also active under sourdough conditions (Corsetti et al.,
2004). In another study, in situ bacteriocinogenic activity of lacticin 3147-like
bacteriocin from Lc. lactis M30 was reported (Settanni et al., 2005). In another
study, prevention of visual rope generation caused by B. subtilis and B.
licheniformis in sourdoughs at low pH values was reported. However, higher pH
values were not effective in preventing rope since bacteriocins produced by
Lactobacillus strains have optimal activities at pH 3.0-4.0 (Menteş et al., 2005;
Menteş et al., 2007; Settanni and Corsetti, 2008). Corsetti et al. (1998) reported the
antimold activity of some Lactobacillus spp. Among the species, Lb.
sanfranciscensis had the largest spectrum and inhibited molds related to bread
spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. Caproic acid and
also acetic, formic, propionic, butyric and n-valeric acids, in particular, were
responsible for the antimold activity. Another study investigated the antifungal
activity of several sourdough LAB. Lb. plantarum 21B showed a very broad
spectrum of activity and inhibited many fungal species belonging to Eurotium,
Penicillium, Endomyces, Aspergillus, Monilia and Fusarium that are most
commonly isolated from contaminated baked goods (Lavermicocca et al., 2000).
2.2.4.3. Phytase Activity

Whole meal cereals are good sources of minerals but the bioavailability of
minerals may be limited due to the presence of phytate, the salt form of phytic acid.
Phytic acid (myoinositol hexakisphosphate) is a compound found in most cereal
grains, legumes and nuts and it strongly binds minerals like iron and zinc (Lopez et
al., 2002; Rizzello et al., 2017). By forming insoluble complexes with dietary
cations, it impairs mineral absorption in humans (Poutanen et al., 2009; Hansen,
2012). The low pH values associated with sourdough fermentation lead to the
solubilisation of the phytic acid complex as a result of the phytase activity of grain
raw materials, LAB and yeasts; therefore, mineral bioavailability is increased
45
(Chavan and Chavan, 2011) as phytase activity is accelerated in the acidic

environment of sourdough fermentation (Leenhardt et al., 2005). Acid production
and lowering the pH is the major mechanism for LAB to improve mineral
bioavailability (Poutanen et al., 2009). A wide variation in phytase activity has
been detected in some yeasts and LAB isolated from sourdoughs (Chaoui et al.,
2003; De Angelis et al., 2003; Reale et al., 2004).
2.2.4.4. Starch Digestibility

Sourdough has also been shown to be useful in the production of breads
with slow starch digestibility and hence low glyceamic responses (Chavan and
Chavan, 2011). Dietary carbohydrate represents a major source of plasma glucose
and an increase in the amount of rapidly digestible carbohydrate in the diet
increases blood glucose levels (GI). The level of starch digestibility is generally
characterized by the rate and duration of the glyceamic response (Singh et al.,
2010), and the GI is an important indicator of starch digestibility. Various
physiological factors such as binding of α-amylase to substrates, gastric emptying,
enzyme inhibitors, properties of digestive enzymes and viscosity within the
digestive tract affect starch digestibility (Zhang et al., 2008; Zhang and Hamaker,
2009). The use of sourdough fermentation technology, especially in low pH levels,
leads to a significant reduction in the glyceamic response (GI about 50) in
comparison with usual yeast leavened white bread (white-wheat flour; GI 100)
(Adam et al., 2003; Ostman, 2003; Fardet et al., 2006; De Angelis et al., 2007;
Maioli et al., 2008; De Angelis et al., 2009). The effect of the sourdough process
on the starch digestibility can be related to the formation of organic acids. It was
reported that response of glucose and insulin are reduced in subjects who
consumed sourdough compared with whole-meal bread alone and concluded that
lactic acid lowers the rate of starch digestion in bread (Liljeberg et al., 1995). In
addition, the chemical in sourdough fermentation can affect starch gelatinisation
and promote the formation of resistant starch that is less digestible (Ostman, 2003;
46
Gobbetti et al., 2014). Moreover, pH-dependent proteolysis produces significant

amount of peptides and amino acids (Nilsson et al., 2007; Gänzle et al., 2008), and
increased levels of free phenolic compounds in fermented cereals may have a role
in regulating glucose metabolism to decrease the GI (Katina et al., 2007; Solomon
and Blannin, 2007; Poutanen et al., 2009).
2.2.4.5. Protease Activity

Proteolytic enzymes, proteases, are grouped into proteinases and
peptidases. Proteinases catalyse protein degradation into smaller peptide fractions
and peptidases hydrolyse specific peptide bonds or completely breakdown peptides
to amino acids (Gänzle et al., 2008). The gluten protein network in wheat doughs
determines dough rheology, gas retention and thus bread volume and texture. The
viscoelastic properties of the gluten network allow the entrapment of CO2 released
during fermentation and result in breads with a light, porous crumb structure.
Gluten is divided into two fractions according to the solubility in alcohol-water
solutions namely, the soluble monomeric gliadins (50-60%) and insoluble
polymeric glutenins (40-50%) (Osborne, 1907; Payne et al., 1984). They are
regarded as gluten proteins and show different structure and functionality. The
viscous properties of doughs are associated with the gliadins and low molecular
weight glutenins. On the other hand, high molecular weight glutenins provide
strength and elasticity to dough (Loponen, 2006; Gänzle et al., 2008). Their most
important function is the formation of the gluten network during the preparation of
doughs (Wang et al., 2015).
In sourdough production, acidification causes increased gluten solubility
and endogenous cereal proteinase activity. Primary proteolysis, the break down of
proteins to peptides, is mainly attributable to endogenous cereal proteases in wheat
and rye sourdoughs (Thiele et al., 2002; Loponen et al., 2004; Tuukkanen et al.,
2005). The flour endogenous proteinases of flour have an optimum pH at 3.0 to 4.0
and they are considered to be important for proteolysis in sourdough fermentations.
47
Besides primary proteolysis by cereal proteases, strain-specific proteolytic activity

of LAB also contributes to proteolysis. Microbial reduction of disulfide bonds in
gluten proteins by heterofermentative lactobacilli increase the solubility of gluten
proteins and make them more susceptible to proteolytic degradation. The
degradation of wheat and rye proteins is very important for bread flavor, volume
and texture (Gänzle et al., 2008).
The partial hydrolysis of glutenins during sourdough fermentation results
in disruption of the gluten network and increases the solubility of gluten proteins
(Gänzle et al., 2008). The increasing solubility of gluten proteins promotes
swelling and increased water uptake (Schober et al., 2003). Besides gluten, the
partial acid hydrolysis of starch also leads to increased water binding capacity
(Galle, 2013). Degradation of gluten protein structures in sourdoughs affects the
viscoelastic properties of the final dough depending on the extent of the protein
degradation. The rheological consequence of gluten degradation is the reduction of
elasticity and firmness of the sourdough and subsequent bread dough. A weaker
gluten network increases the expansion of dough, but also decreases gas retention.
Therefore, the acidity level of sourdough and subsequent bread dough must be
carefully controlled to attain increased volume (Galle, 2013). It is generally
observed that a limited degree of proteolysis during sourdough fermentation is
beneficial and improves the bread flavor without adverse effects on texture and
volume (Thiele et al., 2002; Gänzle et al., 2008).
The gluten proteins of wheat and secalins of rye belong to prolamins,
which are responsible for coeliac disease (gluten-sensitive enteropathy), a chronic
gastrointestinal tract disorder where the ingestion of gluten from wheat, rye and
barley and their crossbred varieties, leads to damage of the small intestinal mucosa
by an autoimmune mechanism in genetically susceptible individuals (Green and
Cellier 2007; Tye-Din and Anderson, 2008). The current treatment for coeliac
disease is a life-long gluten-free diet. However, controlled proteolysis in wheat and
rye sourdoughs may be used as a tool to reduce gluten levels to such an extent that
48
the products can be tolerated by coeliac patients (Rizzello et al., 2007; Katina and
Poutanen, 2013).
Proteolytic degradation during fermentation provides the substrates for
microbial growth and conversion of amino acids to flavor precursor compounds
(Thiele et al., 2002) and antifungal metabolites (Lavermicocca et al., 2000).
Therefore, protein hydrolysis and amino acid metabolism affect the flavor of
sourdough and contribute to the beneficial effects of sourdough fermentation on
bread quality; as proteins are degraded to free amino acids that may be converted to
flavor compounds; i.e. the liberated amino acids act as flavour precursors during
sourdough fermentation. The hydrolysis of peptides (secondary proteolysis) by
sourdough lactobacilli leads to the accumulation of aminoacids in the dough in a
strain dependent manner. On the other hand, yeasts decrease amino acids levels in
dough (Gänzle et al., 2008). As a result, both the sourdough yeasts and LAB may
facilitate the flavour formation, either directly via metabolizing amino acids to
flavor compounds or indirectly by transforming them into secondary compounds
that can serve as new precursors for further conversions (Loponen, 2006).
Different strains of LAB exhibit proteolytic activity during sourdough
fermentation. Among LAB, Lb. sanfranciscensis has been shown to be particularly
capable of degrading proteins or peptides, and proteinase, dipeptidase and
aminopeptidase are the main enzymes that characterize the proteolytic system of
this bacterium (Gobbetti et al., 1994; Gobbetti et al., 1996a). Gobbetti et al.
(1996b) reported high proteolytic activity on gluten and especially high peptidase
activities of Lb. brevis subsp. lindneri, Lb. plantarum and Lb. farciminis strains
during sourdough fermentation. In particular, aminopeptidase, dipeptidase,
tripeptidase and iminopeptidase activities were the highest in Lb. brevis subsp.
lindneri CBI and A79 strains. In another study, gluten breakdown activities of
Lactobacilli and Pediococci strains isolated from sourdough were investigated and
besides Lactobacillus species, Pd. pentosaceus showed high proteolytic acitivity on
gluten (Gerez et al., 2006). In addition, the presence of proteolytic Lb. casei strains
49
with the capacity to individually metabolize the coeliac-disease-related 33-mer

peptide in sourdough was reported (Alvarez-Sieiro et al., 2016). Rollán et al.
(2005) reported that Lb. plantarum CRL 759 and CRL 778 have an active
proteolytic system, which is responsible for the high amino acid release during
sourdough fermentation and the hydrolysis of the 31–43 α-gliadin-like fragment.
2.2.4.6. Wheat Germ Stability

Wheat germ is particularly rich in vitamins, lipids, high quality proteins
and contains a significant amount of dietary fibre. However, the use of wheat germ
in bread making is still moderate because of its poor shelf life stability; high lipase
and lipoxygenase activities lead to the release of free fatty acids resulting in
rancidity of baked goods(Gobbetti et al., 2014). Acidification with sourdough
fermentation technology can affect wheat germ stability, chemical and nutritional
characteristics, and also the texture and sensory characteristics of the white bread
and it was reported that sourdough fermented wheat germ is an ingredient able to
enhance the nutritional, texture and sensory properties of bread (Rizzello et al.,
2010). It was also reported that sourdough fermentation partially inhibits the
endogenous lipase activity of wheat germ and also increases the shelf life
(Minervini et al., 2010).
2.2.4.7. Exopolysaccharide Production

Microbial exopolysaccharides (EPS), long chain sugar polymers, are
metabolites produced by bacteria, microalgae and to a lesser extent, yeasts and
fungi (Sutherland, 1972; De Vuyst and Degeest, 1999). Extracellular
polysaccharides are secreted into the extracellular environment in the form of slime
or associated with the cell surface in the form of capsules. Many food-grade
microorganisms produce EPS especially LAB, propionibacteria and bifidobacteria
(Cerning, 1990; Abbad Andaloussi et al., 1995; Cerning, 1995). Some LAB species
are a good source of EPS which are also recognized for their contribution to the
50
texture, mouth feel, taste perception and stability of the final food product (Jolly et
al., 2002; Gonzalez, 2006). Most of the EPS-producing LAB strains studied were
isolated from dairy products but it is known that some LAB species produce EPS
and links between specific metabolic activities of sourdough cultures and product
quality are well-described for traditional sourdoughs (Galle et al., 2010). Tieking et
al. (2003) analysed a total of 111 LAB and found EPS production by sourdough
origining LAB such as Lb. sanfranciscensis, Lb. frumenti, Lb. pontis, Lb.
reuteri, Lb. panis and W. confusa. They reported the production of EPS from
sucrose as a metabolic activity which is common among sourdough LAB. In
another study, production of linear dextrans from sucrose by W. confusa and W.
cibaria isolated from wheat sourdoughs was reported (Amari et al., 2013). In
another study, a strain of W. confusa produced dextrans and isomalto-
oligosaccharides in sourdoughs without strong acidification. It was reported that
the dextran significantly increased the viscosity of the sourdoughs (Katina et al.,
2009). The application of dextran-enriched sourdoughs in bread baking has been
reported to provide mildly acidic wheat bread with improved volume (up to 10%)
and crumb softness (25–40%) (Di Cagno et al., 2006; Lacaze et al., 2007; Katina et
al., 2009). Di Cagno et al. (2006) reported EPS synthesized from sucrose by
sourdough W. cibaria, Lb. plantarum and Pd. pentosaceus strains. Another study
reported, EPS production by Lb. sanfranciscensis during sourdough fermentation
(Korakli et al., 2001).
2.3. Chickpea Fermentation

2.3.1. Chickpea (Cicer arietinum L.)
Chickpea is a legume of the family Fabaceae, in the Plantae Kingdom. It
belongs to the Faboideae subfamily and Cicer genus. Cultivated chickpea (Cicer
arietinum L.) is reported to be one of the first grain legumes domesticated in the
world and ranks as the third most important legume worldwide (Hannan et al.,
51
2001). It is also a very important legume in Turkey and has been grown for nearly
7,400 years (Bhardwaj et al., 1999).
The history of chickpea dates back to around 7,000 BC and has been found
in prehistoric sites in the east Mediterranean area. Helbaek (1970) reported the
oldest known occurrence of chickpea in Hacilar near Burdur in Turkey, dated to
about 5,450 BC (Helbaek, 1970). Chickpea has its origin in southeastern Turkey,
and after its domestication in the Middle East, reached the Mediterranean region,
India and Ethiopia (Ladizinsky, 1975; Varshney et al., 2017).
The mean annual production of chickpea was reported to be 10.16 million
tons from 2004 to 2013. Chickpeas are produced in over 50 countries with India
having the largest production and accounting for over 70% of total global
production. Pakistan, Australia and Turkey are the next most important producers.
Turkey accounts for 4 % of the world’s production (Muehlbauer and Sarker, 2017).
In Turkey, the mean yields of chickpea, harvest area and total production were
reported to be 1191 (kg/ha), 437472 ha and 520935 tonnes production, respectively
(Muehlbauer and Sarker, 2017).
The chickpea is the most cultivated legume in Turkey with 45% of the total
among 8 legumes (Anonymous, 2013). It is an important source of protein and
carbohydrates and the quality of protein is considered to be better than other pulses.
Moreover, it contains significant amounts of all the essential amino acids except
sulphur-containing amino acids, minerals (Ca, Mg, P and K), vitamins (riboflavin,
niacin, thiamin, folate and the vitamin A precursor β-caroten) and dieatary fibre
(Jukanti et al., 2012; Bidyarani et al., 2016). The nutritional composition of
chickpea changes according to the growth conditions and variety. According to the
USDA Food Composition Database, the macronutrient content of raw chickpeas
are: 62.95% carbohydrates, 20.47% proteins and 6.04% fat. Carbohydrates include
dietary fibre, oligosaccharides, starch and simple sugars, and the total dietary fibre
composition in chickpea was reported to be 12.2 % (Wallace et al., 2016).
Although lipids are present in low amounts, the chickpea is rich in nutritionally
52
important unsaturated fatty acids such as linoleic and oleic acids (Jukanti et al.,
2012).
The chickpea is consumed to a significant degree in the Middle Eastern
diet. Foods based on chickpeas are prepared by a wide range of recipes and
preparation methods include soaking, grinding, sprouting, fermentation, boiling,
mashing, roasting, frying and steaming treatments (Deshpande and Damodaran,
1990; Köksel et al., 1998). Chickpea flour addition to bread formulations improves
the protein nutritional quality of produced bread (Estevez et al., 1987; Mohammed
et al., 2012; Pathania et al., 2017). Moreover, in some Mediterranean countries,
fermented chickpea is used as a leavening agent for the production of traditional
breads and rusks (Hatzikamari et al., 2007a).
2.3.2. Chickpea Bread

Chickpea bread is produced in some Mediterranean and Balkan countries.
Fermented chickpea liquid starter and dough are produced by the fermentation of
chickpeas and used as a leavening agent for the production of chickpea bread. In
Turkey, this bread type has been known especially in the Aegean and Thrace
Regions and also some parts of the Middle Anatolia and Mediterranean Regions for
a long time (Figure 2.4). Chickpea bread is traditionally produced at homes in
those regions and also in small-scale bakeries (Hatzikamari et al., 2007a).
Figure 2.4. Chickpea breads from different regions
53
For the production of the chickpea liquid starter, coarsely ground chickpeas
are put into a jar or bottle and then hot water is added. Chickpea fermentation is
conducted in a hot location for around 16-18 hours. Bakeries put chickpea liquid
jars in a hot place in the bakery and normally use blankets to prevent them cooling
down. After 16-18 hours, a thick foam layer and the smell of chickpea liquid
indicate the end of fermentation (Figure 2.5). This liquid is then used completely,
or in some bakeries used after seperating the chickpeas for dough production. For
that purpose, chickpea liquid is mixed with wheat flour and hot water. In some
bakeries, boiled water is used instead of hot water. The resulting chickpea dough is
kept in a warm place for a few hours and then used as a leaving agent in bread
production.
Figure 2.5. Chickpeas and fermented liquid starter
Sensorial properties and shelf life are improved by using chickpea dough
as the leavening agent; however, traditional chickpea bread is only known in some
regions. As a result, studies on chickpea bread and fermentation are very limited.
54
2.3.3. Studies on Chickpea Bread

Chickpea dough is used in the production of some bread types and also
different traditional bakery products. For example, chickpea leavened simit bread is
well known in the Aegen region. In a study, the effect of chickpea type on the
chemical, physical and microbial properties of the commercially produced
chickpea fermentation liquid and simit bread were investigated (Kasım, 2014). The
study optimized the production conditions and reported that different types of
chickpeas resulted in end products with differing properties.
Chickpea bread has a different aroma as a result of the fermentation of
different microorganisms (Özkaya, 1992). Hancıoğlu-Sıkılı (2003) investigated the
aroma profile of chickpea breads produced using starters under laboratory
conditions and reported higher levels of some carbonil compounds in chickpea
breads than any other bread types (Hancıoglu-Sıkılı, 2003). Özkaya (1992)
produced breads with different types of leaveners and reported that chickpea bread
has a distinct aroma when it was compared with the bread produced using
commercial instant yeast (Özkaya, 1992).
In the study of Baykara (2006), similar results were obtained. In this study,
bread was manufactured using three different bread leaveners including
commercial press yeast (S. cerevisiae), traditional leavener prepared with chickpea,
and commercial press yeast (0.5%) + chickpea leavener. Sensorial evaluation
results showed that bread samples made with press yeast (0.5%) + chickpea
leavener were preferred to breads made with the other two leaveners (Baykara,
2006). Similar sensorial results were obtained in the study of Narlıoğlu (2013). In
that study, a commercial yeast, a chickpea dough and a combination of them were
used in the production and from sensorial point of view, a combination of two
leaveners were preferred. In addition, results showed that during storage, water loss
was the lowest in the chickpea doughs (Narlıoglu, 2013).
Çebi (2014) investigated the effects of different strains isolated from
chickpea fermentations on the volatile profile, texture and color properties of
55
chickpea bread. It was reported that using a starter culture had statistically
significant effects on crumb hardness, cohesiveness and chewiness values of the
breads. 58 volatile compounds belonging to different chemical groups were
determined in the dough, crumb and crust of bread produced with chickpea dough
with the selected strains
2.3.4. Chickpea Fermentation Microflora

Studies on chickpea fermentations identified the microflora as some LAB,
yeasts, Bacillus and Clostridium spp. (Katsaboxakis and Mallidis, 1996;
Hancıoglu-Sıkılı, 2003; Hatzikamari et al., 2007b). Katsaboxakis and Mallidis
(1996) isolated species belonging to Lactobacillus, Corynebacterium,
Micrococcus, Pediococcus, Bacillus and Clostridium genera during the
fermentation of coarsely ground chickpeas in water at 32, 37 and 42°C.
Hatzikamari et al. (2007b) indicated that Bacillus and Clostridium species have an
effect on the enzymatic and chemical changes observed during chickpea
fermentations. These species degrade the compound into chickpea water and
produce gas. In that study, at the beginning of the fermentation, Bacillus species, B.
cereus, B. thuringiensis and B. licheniformis and then Clostridium species, Cl.
perfringens and Cl. beijerinckii were identified. It was reported that B. cereus and
Cl. perfringens grew predominantly during fermentation and did not seem to form
toxins, hence any health hazard after consumption of the bread, properly baked,
seems improbable.
In another study conducted in Turkey, chickpea liquid starters and doughs
collected from bakeries were investigated for their microflora. E. mundtii, E.
gallinarum, E. casseliflavus, Lb. plantarum, Lb. pentosus, Lb. sanfranciscensis, Lb.
viridescens, Lb. bifermentans, Pd. urinaeequi, St. thermophilus and Lc. lactis
subsp. cremoris and yeast S. cerevisiae were identified. The chickpea dough was
then produced by using selected LAB starters (Hancıoglu-Sıkılı, 2003).
56
Çebi (2009) produced chickpea dough using traditional procedure under

laboratory conditions and isolated and identified the LAB devoloped during the
fermentation of chickpea liquid starter and the dough. The isolated LAB strains
belonged to Lactobacillus, Lactococcus and Weissella genera. Species isolated
from the chickpea liquid starter were identified as Lc. spp. lactis, Lb. brevis and
Lb.plantarum, and those isolated from chickpea dough were identified as Lc. lactis,
Lb. brevis, Lb. plantarum, Lb. pentosus and W. confusa.
Erginkaya et al. (2016) reported the dominant microflora in chickpea
fermentations to be LAB, yeasts, aerobic and anaerobic spore-forming bacteria.
They produced chickpea bread under laboratory conditions and supported Bacillus
and Clostridium species in chickpea fermentations.
57
58
3. MATERIAL AND METHOD Cennet Pelin BOYACI GÜNDÜZ
3. MATERIALS AND METHOD
3.1. Materials and Sampling

In the present study, six commercial bakeries in different cities were
selected based on their traditional production of natural sourdough and chickpea
dough. A total of 20 samples were collected including sourdough (8), chickpea
liquid starter (6) and chickpea dough (6) samples at two different times as shown in
Table 3.1.
Table 3.1. Sampling dates and locations

City Type of the sample 1. sampling 2. sampling
Mersin Sourdough 26/04/2016 08/11/2016
Antalya Sourdough 02/05/2016 02/01/2017
Ankara Sourdough 09/06/2016 10/02/2017
Birgi Chickpea liquid starter
25/05/2016 23/02/2017
Town/Ödemis/İzmir and dough
Chickpea liquid starter
Söke/Aydın 25/05/2016 23/02/2017
and dough
Chickpea liquid starter
Nevşehir 01/04/2016 14/11/2016
and dough
3.1.1. Sourdough Samples

Whole-meal wheat sourdough samples were collected from Pikan Bakery
in Antalya, Gattini Bistro in Mersin and Canberk Food Company in Ankara. In
addition, a rye sourdough sample was taken from one of the locations together with
whole-meal wheat sourdough. Collected sourdoughs were Type I sourdoughs
produced without baker’s yeast. The sourdough sample was taken aseptically
before the daily refreshment step, and put into sterile jars (Figure 3.1). Samples
were kept at 4°C until analyses. All samples were subjected to chemical and
microbiological analyses in the Industrial Microbiology Laboratory at the Food
Engineering Department in Çukurova University within 24 hours.
59
Figure 3.1. Sourdough samples were taken into sterile jars
3.1.2. Chickpea Liquid Starter and Dough Samples

The chickpea liquid starter and dough samples were collected from
Cumhuriyet Bakery in Soke/Aydın, Tokoglu Bread in Birgi Town/Odemis/Izmir
and Yayla Bakery in Nevsehir. All of the bakeries have been producing chickpea
bread for years and are well-known in their regions. Chickpea liquid starter
samples were obtained by seperating chickpeas from the fermentation liquid at the
end of the fermentation (Figure 3.2). Chickpea dough samples were collected by
taking a piece of a final leavened dough (Figure 3.3).
Figure 3.2. Fermented chickpea liquid starter and obtained liquid after separation
of the chickpeas
60
Figure 3.3. Chickpea dough
Samples were taken aseptically, placed into sterile jars and kept at 4°C
until analyses. All samples were subjected to chemical and microbiological
analyses in the Industrial Microbiology Laboratory at the Food Engineering
Department in Çukurova University (within 24 hours).
3.2. Production of Sourdough and Chickpea Dough under Laboratory

Conditions
Sourdough and chickpea dough samples were produced as controls on a
laboratory scale and chemical and microbiological analyses were also performed
on the control samples. All analyses were performed in duplicate.
3.2.1. Laboratory Sourdough Production and Sampling

Sourdough production under laboratory conditions proceeded according to
the traditional (sourdough Type I) protocol without using starter culture or baker’s
yeast (Figure 3.4.). Doughs were prepared with boiled and cooled tap water and
whole-meal wheat flour from local company belonging to the same production
batch. For dough preparation, whole-meal wheat flour (216.21 g) and boiled and
cooled tap water (183.79 mL) were mixed manually to produce 400 g of dough
61
with a dough yield [(dough weight/flour weight)×100] of 185. Each sourdough was
fermented at 28°C for 24 h in glass jars covered with a lid. The resulting
sourdoughs were propagated over a period of 7 days according to the daily back-
slopping (refreshment) procedure and the sourdough from the previous day's
fermentation was used as the starter (20% [wt/wt] of inoculum) to ferment a new
mixture of flour (172.98 g) and tap water (147.02 mL), resulting in a dough yield
of 185 (Figure 3.5). Sourdough production was carried out in duplicate.
Figure 3.4. Sourdough production under laboratory conditions
The first sample (0 h) was taken from the flour and water mixture,
unfermented dough, after mixing. During the back-slopping procedure, sampling
was performed on the sourdoughs immediately before the daily refreshment step.
Both total titratable acidity (TTA) and pH measurements were carried out on all
samples collected after 4 (4 h), 8 (8 h) and 12 (12 h) hours of the experiment and
once every 24 hours of until the last refreshment of the sourdough production. The
samples collected at 0 h, 1 (1d), 2 (2d), 4 (4d) and 7 (7d) days of daily back-
slopping were also subjected to plate counting and isolation of presumptive LAB
and yeasts in addition to TTA and pH measurements. Furthermore, carbohydrate,
organic acid and ethanol analyses were conducted on these samples. Samples were
analysed in duplicate.
62
Figure 3.5. Laboratory produced sourdough sample
3.2.2. Laboratory Chickpea Liquid Starter and Dough Production and

Sampling
Production of chickpea liquid starter and dough is shown in Figure 3.6.
Chickpeas (Koçbaşı variety) were ground for the production of chickpea liquid.
Then 50 g of chickpeas were put into sterile glass jars and mixed with 400 mL of
boiled and cooled tap water (50°C). Fermentations were conducted in glass jars
covered with a lid during 18 hours at two different temperatures, 32 and 37°C.
After the typical smell of the chickpea liquid and foam formation occured,
fermentation was terminated. At the end of the fermentation, chickpeas were
seperated and the liquid was used for the production of the chickpea dough. 150
mL of liquid is mixed with 200 g of flour (DY 175) from local company and
fermented at 32 and 37 °C for 4 hours. Chickpea liquid starter and dough
production were carried out in duplicate. Chickpea liquid starter production is
shown in Figure 3.7. Chemical and microbiological analyses were performed in
duplicate on the chickpea liquid starter (CLS) and chickpea dough (CD) samples
fermented at two different temperatures. Samples were collected at the beginning
and end of the fermentations, 0 and 18 h, in chickpea liquid starter and 0 and 4h in
chickpea dough. The collected samples were subjected to plate counting and
isolation of presumptive LAB and yeasts besides the TTA and pH measurements
63
and also carbohydrate, organic acid and ethanol analyses. Samples were analyzed
in duplicate.
Figure 3.6. Chickpea liquid starter and dough production
64
Figure 3.7. Chickpea liquid starter production under laboratory conditions
3.3. Determination of pH, Total Titratable Acidity, Carbohydrates, Organic

Acids and Ethanol
Chemical analyses were conducted on the sourdough, chickpea liquid
starter and dough samples that were collected from different bakeries and also
samples produced on laboratory scale. For each sample pH, TTA, carbohydrate,
organic acid and ethanol analyses were conducted. All analyses were performed at
least in duplicate.
3.3.1. Determination of pH
The pH measurement of the samples was performed using a digital glass
pH meter (Mettler Toledo, SevenCompact™ pH Ion S220, Switzerland) previously
calibrated with 3 standard solutions at pH 4, 7 and 11. For the determination of pH,
10 g of sample was homogenized with 90 mL of distilled water, using a magnetic
plate stirrer for 3 min, and the pH was measured by inserting the probe into the
mixture (Lopez et al., 2001).
65
3.3.2. Determination of Total Titratable Acidity

Total titratable acidity of the samples was determined after homogenization
of 10 g of sample with 90 mL of distilled water on a magnetic plate stirrer. The
mixture was then titrated with 0.1 N NaOH to a final pH of 8.5. The TTA was
expressed as the amount (mL) of 0.1 M NaOH needed to achieve the pH of 8.5
(Lopez et al., 2001).
3.3.3. Assessment of Carbohydrates, Organic Acids and Ethanol Content

Using High Performance Liquid Chromatography (HPLC)
3.3.3.1. Determination of Carbohydrates, Organic Acids and Ethanol
Maltose, sucrose, glucose, fructose, ethanol and lactic and acetic acids
were determined using HPLC. The liquid chromatographic apparatus (LC-20 AD,
Shimadzu, Kyoto, Japan) consisted of a pump system, an on-line degasser (DGU-
20A5), a column oven (CTO-10ASVP), a refractive index detector (RID-10A) for
sugar and ethanol analysis and a UV/Vis detector (SPD-20A) monitored at 210 nm
for the analysis of organic acids. The injection volume was 20 µL.
Chromatographic separation was performed using an Aminex HPX-87H column
(300 x 7.8 mm, Bio-Rad, Hercules, CA, USA) under the following conditions: flow
rate 0.5 mL/min and column temperature 50°C. The mobile phase was 5 mM
H2SO4. Specific Shimadzu software was used for data evaluation. All of the
analyses were performed in duplicate and the results are expressed as means ±
standard deviation.
3.3.3.2. Preparation of the Standards

Stock standard solutions were prepared individually from HPLC grade
standards obtained from Sigma-Aldrich. Seven-point standard curves were
constructed from standard solutions. The method’s limit of detection (LOD), limit
of quantification (LOQ) and recovery were determined for maltose, sucrose,
glucose, fructose, lactic acid, acetic acid and ethanol. The LOD and LOQ values
66
were estimated as 3 and 10 times the standard deviation derived from analyses of
10 injections at the lowest calibration levels, respectively. For the recovery test,
the dough sample was spiked with standards during the homogenization step at
final concentrations in the linear range of the calibration curves. Spiked and
unspiked samples of the dough were analysed under the same conditions. Six
replicates were used for the determination of recovery and results were calculated
for each standard based on the following formula:
Cs= Concentration of the analyte in spiked sample

Cu= Concentration of the analyte in unspiked sample
Cs= Concentration of the analyte added
3.3.3.3. Extraction Procedure

Ten g of sample was homogenized with 90 mL 25 mM phosphate buffer
(pH 5.6) according to the extraction method of Paramithiotis et al. (2006). The
mixture was centrifuged (12,000 rpm, 10 min, at 4°C; Kubota 7780, Japan ). One
mL of the supernatant was mixed with 50 µl of perchloric acid and kept at 4°C for
24 h. Protein agglomerates were removed by centrifugation (12,000 rpm, 60 min,
4°C; Hettich® Universal 320R, Germany) and the supernatant was filtered through
a 0.45 µm PVDF Syringe Filter (Isolab) and injected into the HPLC system.
3.4. Microbiological Analyses

Sourdough (SD) and chickpea dough (CD) samples (10 g) were suspended
with 90 mL of sterile 0.85% (wt/vol) NaCl solution in a sterile stomacher bag and
homogenized for 3 min using a bag mixer (Interscience, model 400 P, France) and
a 10-fold dilution series of the samples was made by using 9 mL of a sterile 0.85%
(wt/vol) NaCl solution. Chickpea liquid starter (CLS) samples were serially diluted
67
by using 9 mL of a sterile 0.85% (wt/vol) NaCl solution. From each homogenate,

10-fold dilution series were prepared and aliquots (0.1 mL) of decimal dilutions
were spread onto selected agar media, as shown in Table 3.2 by the spreading plate
method for microbial counts of LAB, yeasts, total mesophilic aerobic bacteria,
molds (filamentous fungi) (Madigan et al., 2012). For coliform group bacteria,
liquid media (broth) was used to conduct the Most Probable Number (MPN)
method instead of the spreading plate method (Clesceri et al., 1998). Selected
media were supplemented with different sterile filtered (Millex-GS, 0.22 µm filter)
antibiotics according to the target organism. For that purpose, cycloheximide (0.1
g/L, Sigma), sodium propionate (2 g/L, Sigma-Aldrich) and oxytetracycline (0.1
g/L, Sigma) antibiotics were used to prevent yeast, mold and bacteria growth,
respectively.
68
Table 3.2. Media used for the enumeration of microorganisms in the sourdough,
chickpea liquid starter and dough samples
Sample Presumptive group Media Incubation
of microorganism conditions
Sourdough LAB mMRS agar 30°C,
Chickpea liquid 48-72 hours
starter anaerobic
Chickpea dough
Sourdough LAB gM17 agar 30°C,
starter anaerobic
Chickpea dough
Sourdough LAB SDB agar 30°C,
48-72 hours
anaerobic
Sourdough Total yeasts YPD agar 28°C,
starter
Chickpea dough
Sourdough non- Saccharomyces L-lysine agar 28°C,
Chickpea liquid yeasts 48-72 hours
starter
Chickpea dough
Sourdough Total mesophilic PCA agar 30°C,
Chickpea liquid aerobic bacteria 72 hours
starter
Chickpea dough
Sourdough Mold MEA agar 28°C,
Chickpea liquid 3-5 days
starter
Chickpea dough
Sourdough Presumptive coliform LST broth 37°C,
starter
Chickpea dough
Chickpea liquid Total mesophilic Nutrient agar 37°C,
starter aerobic bacteria 18 hours
Chickpea dough (Presumptive Bacillus
spp.)
3.4.1. Viable Counts of Presumptive LAB

Enumeration of presumptive LAB was estimated by plating serially diluted
SD, CD and CLS samples onto different media including modified de Man Rogosa
Sharpe (mMRS) (Merck) agar containing 1% maltose (w/v) and 5% fresh yeast
69
extract solution (v/v) and modified glucose M17 (gM17) (Merck) agar containing
0.5% glucose. In additon, the microbial suspensions of sourdough samples were
plated on sourdough bacteria agar (SDB) (Obis et al., 2001; Valmorri et al., 2006;
Settanni et al., 2011). SDB agar medium contained 2% maltose (w/v), 0.6%
pancreatic digest of casein (w/v), 0.3% yeast extract (w/v), 10% fresh yeast extract
solution (v/v), 0.03% Tween 80 (v/v) and 1.5% agar (w/v) (Kline and Sugihara,
1971). All media were supplemented with cycloheximide (0.1 g/L) and sodium
propionate (2 g/L) to prevent growth of yeasts and molds, respectively. Incubation
of plates was performed anaerobically by using Anaerocoult A (Merck 1.13829) in
sealed jars at 30 °C for 48-72 hours. Each plate (mMRS, M17 and also SDB for
sourdoughs) was counted and results are expressed as CFU/g or mL (colony
forming units per gram or mL sample).
From the selected plates, 10-15 colonies/plate were randomly picked and
streaked onto a single agar plate containing appropriate agar media for isolation by
the plate-streaking technique. Streaked plates were incubated at 30°C for 48 hours
anaerobically and then colonies were examined. When all of the colonies on the
plate had the same general appearance, a colony was picked and subsequently
transferred into the corresponding broth media and incubated at 30°C for 48 hours.
Each colony that had a different appearence on a plate was streaked again onto a
separate plate until a pure culture was obtained. Then, isolated LAB colonies were
further subjected to Gram stain and catalase tests, and Gram (+) and catalase (-)
isolates were transferred into the corresponding broth media containing 40% (v/v)
sterile glycerol solution and stored at -25 °C.
3.4.2. Viable Counts of Presumptive Yeasts

Cell densities of total yeasts were estimated by plating serially diluted
samples on Yeast Extract Peptone Dextrose (YPD) (Sigma) agar medium. YPD
agar medium was supplemented with oxytetracycline (0.1 g/L) and sodium
propionate (2 g/L) to prevent the growth of bacteria and molds, respectively.
70
Incubation of plates was performed at 28 °C for 48-72 h. Each plate was counted
and results are expressed as CFU/g or mL (colony forming units per gram or mL
sample).
streaked onto YPD agar media for isolation by the plate-streaking technique.
Streaked plates were incubated at 28°C for 48 hours and then colonies were
examined. When all of the colonies on the plate had the same general appearance, a
colony was picked and subsequently transferred into the corresponding broth media
and incubated at 28°C for 48 hours. Each colony that had a different appearence on
a plate was streaked again onto a separate plate until a pure culture was obtained.
Pure cultures were transferred into the corresponding broth media containing 40%
(v/v) sterile glycerol solution and stored at -25 °C.
3.4.3. Viable Counts of Presumptive Non-Saccharomyces Yeasts

Enumeration of non-Saccharomyces yeasts were estimated by plating
serially diluted samples on L-lysine agar medium supplemented with
oxytetracycline (0.1 g/L) and sodium propionate (2 g/L) to prevent the growth of
bacteria and molds, respectively. Incubation of plates was performed at 28 °C for
48-72 h. Each plate was counted and results are expressed as CFU/g or mL (colony
streaked onto YPD agar media for isolation by the plate-streaking technique.
Streaked plates were incubated at 28°C for 48 hours and then colonies were
examined. When all of the colonies on the plate had the same general appearance, a
colony was picked and subsequently transferred into the corresponding broth media
and incubated at 28°C for 48 hours. Each colony that had a different appearence on
a plate was streaked again onto a separate plate until a pure culture was obtained.
Pure cultures were transferred into the corresponding broth media containing 40%
(v/v) sterile glycerol solution and stored at -25°C.
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3.4.4. Viable Counts of Total Mesophilic Aerobic Bacteria

Total mesophilic aerobic bacteria were enumerated by plating serially
diluted samples on Plate Count Agar (PCA) (Merck) supplemented with
cycloheximide (0.1 g/L) and sodium propionate (2 g/L) to prevent the growth of
yeasts and molds, respectively. Incubation of plates was performed at 30 °C for 72
hours. Each plate was counted and results are expressed as CFU/g or mL (colony
3.4.5. Viable Counts of Molds

Molds were enumerated by plating serially diluted samples on Malt Extract
agar (MEA) (Merck) medium supplemented with oxytetracycline (0.1 g/l) and
cycloheximide (0.1 g/l) to prevent growth of bacteria and yeasts,
respectively.Incubation of plates was performed at 28 °C for 3-5 days. Then plates
were counted and results were expressed as CFU/g or ml (colony forming units per
gram or ml sample).
3.4.6. Total Presumptive Coliform Count

Total coliform counts were performed using the MPN technique. One mL
aliquots of decimal dilutions of the samples were inoculated into 3 tubes containing
10 mL of Lauryl Sulfate Tryptose (LST) broth (Merck) with Durham tube. The
tubes were incubated at 37°C for 24 h. After 24 hours, the tubes were removed
from the incubator and the inner durham tubes were examined for gas production.
Growth and gas production in the tubes showed presumptive coliforms and these
tubes were recorded as positive and estimated using the MPN method. Gas-
negative tubes were re-incubated for an additional 24 h and then examined again at
48 h (Feng et al., 2002). The indole test was conducted by adding 0.2-0.3 mL of
Kovacs' indole reagent (Merck) to the gas-positive tubes and development of a
distinct red color in the upper layer was recorded as positive showing the growth of
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an indole positive culture. Indole-positive tubes were reported as presumptive

Escherichia coli and evaluated using the MPN method (Halkman, 2005).
3.4.7. Viable Counts of Presumptive Bacillus spp.

Presumptive Bacillus spp. were enumerated by plating serially diluted
samples onto Nutrient agar (NA) (Merck) and incubated at 37°C for 18 hours
(Hatzikamari et al., 2007b). Then plates were counted and results were expressed
as CFU/g or mL (colony forming units per gram or mL sample).
In laboratory produced samples, heat treatment was applied to samples for
the enumeration of spore-forming bacteria. Before inoculation, 1:10 dilutions of the
samples were heated to 80°C for 10 minutes. Dilutions were then cooled down to
37°C and inoculated onto petri dishes using the spread plating method. Heat
application enabled survival of only spore-forming bacteria, resulting in the
enumeration of aerobic spore-forming bacteria, most probably Bacillus spp., as
reported by other researchers (Halkman, 2005; Erginkaya et al., 2016).
3.5. Molecular Identification of LAB Isolates

Potential LAB isolates were subjected to genotypic characterization by
RAPD-PCR analysis and identification by sequence analysis of 16S rRNA genes.
LAB isolates were grown overnight in corresponding broth media at 30°C, cells
were havested and genomic DNA was extracted using an InstaGene Matrix kit
(Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.
3.5.1. DNA Extraction

Stored LAB isolates were activated and genomic DNA was prepared from
LAB isolates after overnight growth at 30°C in a microcentrifuge tube containing
broth media. For the genomic DNA extraction, overnight grown LAB culture was
centrifuged (Thermo Scientific™ MicroCL 17 microcentrifuge, Germany) at
13,300 rpm for 3 min to pellet the cells (Figure 3.8). The supernatant was discarded
73
without disturbing the cell pellet. The pellets were then resuspended in sterile ultra-
distilled water, vortexed at high speed for 10 s and centrifuged again. This step was
repeated three times and the supernatant was removed. Then 150-200 μL of
InstaGene matrix kit was added to the pellet using 1,000 μL pipette tip, vortexed
for 10 s and incubated at 56 °C for 30 min. Following the incubation, the tubes
were vortexed for 10 s and placed in a 100°C boiling waterbath for 8 min. The
tubes were then vortexed at high speed for 10 s and centrifuged at 13,300 rpm for 3
min. The resulting supernatant including crude cell extract was stored at -20 °C.
The stored DNA extract was used for further PCR assays.
Figure 3.8. Centrifugation in the microcentrifuge to pellet the cells
3.5.2. Randomly amplified polymorphic DNA (RAPD) PCR analysis

Differentiation of the LAB isolates was performed using RAPD-PCR
analysis in a 25-µL reaction mix using the M13 primer with the sequence 5'-
GAGGGTGGCGGT TCT-3' (Stenlid et al., 1994; Settanni et al., 2012). The PCR
mix for the M13 primer was prepared as follows:1.25 µL (50 ng) DNA, 2.5 µL
Dream Taq buffer (10x +20mM MgCl2Thermo Scientific), 2 µL dNTP (2.5 mM,
Thermo Scientific), 1 µL MgCl2 (25 mM, Thermo Scientific), 0.2 µL M13 primer
(100 µM, Thermo Scientific), 0.2 µL Dream Taq DNApolymerase (5 U/µL,
Thermo Scientific) and sterile distilled water in 25-µL mixture.
74
The concentrations of DNA extracts were determined using a Qubit 3.0

Fluorometer using Qubit® dsDNA BR Assay Kit (Life Technologies, Invitrogen).
DNA amplifications with the M13 primer were performed in the thermocycler
(Techne TC-Plus 02, UK) programmed as follows: initial denaturation at 94°C for
2 min; 40 cycles of denaturation at 94°C for 1 min; annealing at 42°C for 20 sec
and extension at 72°C for 2 min; plus a final extension step at 72°C for 10 min.
RAPD fragments were separated using 1.2% (w/v) agarose (Sigma) gel
electrophoresis prepared with 1 x TBE diluted from 5 x TBE that contained 54 g/L
(w/v) Trisma base (Sigma), 27.5 g/L (w/v) boric acid (Merck) and 7.44 g/L (w/v)
EDTA (Titriplex® III, ethylenedinitrilotetraacetic acid disodium salt dihydrate,
Merck). SYBR SafeTM DNA gel stain (Invitrogen) was used for visualization of
DNA bands under UV light. DNA samples were loaded onto the agarose gel with
DNA loading dye (6X LD, Thermo Scientific). One kb Gene ruler (Thermo
Scientific) and O'Gene Ruler mix (Thermo Scientific) DNA ladders were used as
the molecular size markers to determine the size of the amplified DNA fragment.
The electrophoresis was run in 1 x TBE at 120 V and then visualized (Vilber
Lourmat Infinity V X 2, France) in the gel Image system as shown in Figure 3.9.
RAPD-PCR profiles were analysed using band pattern analysis employing the
software package (Infinity V X 2). Images of amplification fragments were scored
as band absent (0) or present (1) and data were entered into a binary matrix.
Similarity indices of band profiles were calculated on the basis of the Jaccard
coefficient. Dendrograms were constructed by means of the unweighted pair group
method with arithmetic average (UPGMA) and 1 or 2 LAB isolates in each cluster
were identified by 16S rRNA gene sequencing.
75
Figure 3.9. Gel Image System (Vilber Lourmat Infinity)
3.5.3. 16S rRNA Gene Sequence Analysis

Molecular identification of LAB with different RAPD-PCR profiles was
carried out using 16S rRNA gene sequencing. PCR amplification was performed
using primers fD1(5'- AGAGTTTGATCCTGGCTC AG-3’) and rD1 (5'-
AAGGAGGTGATCCAG CC-3') (Weisburg et al., 1991). The PCR mix was
prepared as follows: 5 µL DNA, 6 µL Dream Taq buffer (10x +20 mM MgCl 2,
Thermo Scientific), 5 µL dNTP (2.5 mM, Thermo Scientific), 1.2 µL MgCl2 (25
mM, Thermo Scientific), 0.08 µL fD1 primer (100 µM, Thermo Scientific), 0.08
µL rD1 primer(100 µM, Thermo Scientific) and 0.5 µL Dream Taq DNA
polymerase (5 U/µL, Thermo Scientific) in a 50-µL mixture. Amplification was
performed in the thermocycler (Techne TC-Plus 02, UK) which was programmed
as follows: initial denaturation at 95°C for 3 min; 30 cycles of denaturation at 94°C
for 1 min; annealing at 54°C for 45sec and extension at 72°C for 2 min; plus a final
extension step at 72°C for 7 min.
PCR products were separated by electrophoresis on a 1.5% (w/v) agarose
(Sigma) gel stained with SYBR SafeTM DNA gel stain (Invitrogen) and
subsequently visualized by Vilber Lourmat Infinity (V X 2, France). PCR
amplicons were sent to BM Laboratuvar Sistemleri (Ankara) for sequencing. The
76
ABI chromatograms of the sequences were examined, multiple alignments were

performed using ClustalW Multiple alignment (Bioedit version 7.0.9) and then
resultant sequences were compared by Basic Local Alignment Search Tool
(BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi ) with nucleotide sequences
deposited at the database National Center for Biotechnology Information (NCBI)
(Altschul et al., 1997). Species identity was determined by comparison to reference
sequences of the 16S rRNA gene sequences with a threshold of 98% (Yarza et al.,
2014).
Phylogenetic analyses were performed using the sequences from at least
1,400 bp DNA fragments that were generated using the fD1 and rD1 primers.
Phylogenetic trees were constructed based on aligned sequences using the two
possible tree reconstruction methods, i.e., UPMGA and Minimum evolution, with
MEGA 7.0 software.
3.6. Molecular Identification of Yeast Isolates

Potential yeast isolates were subjected to genotypic characterization by ITS
region amplification of the 5.8S rRNA gene, its Restriction Fragment Length
Polymorphism (RFLP) analysis and identification by sequence analysis of the
D1/D2 domain of the 26S rDNA gene. Yeast isolates were grown for 24-36 h in
YPD broth media at 28°C, cells were harvested and genomic DNA was extracted
using the InstaGene Matrix kit (Bio-Rad, Hercules, CA, USA) according to the
manufacturer’s protocol.
3.6.1. DNA Extraction

Stored yeast isolates were activated and genomic DNA was prepared from
yeast isolates after 24-36 h growth in YPD broth media at 28°C. For the genomic
DNA extraction, yeast culture grown in a microcentrifuge tube was centrifuged
(Thermo Scientific™ MicroCL 17 microcentrifuge, Germany) at 13,300 rpm for 3
min to pellet the cells. The supernatant was discarded without disturbing the cell
77
pellet. The pellets were then resuspended in sterile ultra-distilled water, vortexed at
high speed for 10 s and centrifuged again. This step was repeated three times and
the supernatant was removed. Then, 50 µL of freshly prepared lyticase (L4025,
≥200 units/mg solid, Sigma-Aldrich, MO, USA) solution (4 U/µL) was added to
the pellet and incubated at 37°C for 1 h to digest the yeast cells. The pellets were
then centrifuged at 13,300 rpm for 3 min. Supernatant was discarded and the
pellets were resuspended in sterile ultra-distilled water, vortexed at high speed for
10 s and centrifuged again. This step was repeated twice. The supernatant was
removed and 120-200 μL of InstaGene matrix kit (Bio-Rad Laboratories, USA)
was added to the pellet using 1,000 μL pipette, vortexed 10 s and incubated at 56°C
for 30 min. Following incubation, the tubes were vortexed for 10 s and placed in a
100°C boiling waterbath for 8 min. The tubes were then vortexed at high speed for
10 s and centrifuged at 13,300 rpm for 3 min. The resulting supernatant including
crude cell extract was stored at -20°C. The stored DNA extract was used for further
PCR assays.
3.6.2. ITS Region Amplification of the 5.8S rRNA Gene

Differentiation of the yeasts was performed via internal transcribed spacer
(ITS) region amplification of the 5.8S ITS rRNA region using primers ITS1 (5'-
TCCGTAGGTGAACCTGCG G-3') and ITS4 (5′-
TCCTCCGCTTATTGATATGC-3′) as described previously (Esteve-Zarzoso et al.,
1999). The PCR mix for ITS region amplification was prepared as follows: 2.5 µL
(50-100 ng) DNA, 5 µL Dream Taq buffer (10x +20 mM MgCl2, Thermo
Scientific), 5 µL dNTP (2.5 mM, Thermo Scientific), 2.5 µL MgCl2 (25 mM,
Thermo Scientific), 0.126 µL ITS1 primer (100 µM, Thermo Scientific), 0.126 µL
ITS4 primer (100 µM, Thermo Scientific) and 0.150 µL Dream Taq DNA
polymerase (5 U/μl) and sterile distilled water in a 50-µL mixture. 5.8S ITS rRNA
regionamplifications were performed in the thermocycler (Techne TC-Plus 02,
UK) which was programmed as follows: initial denaturation at 95°C for 5 min; 35
78
cycles of denaturation at 94°C for 1 min; annealing at 55°C for 2 min and
extension at 72°C for 2 min; plus a final extension step at 72°C for 10 min.
PCR products were separated on a 1.5% w/v agarose gel stained with the
SYBR SafeTM DNA gel stain (Invitrogen). The electrophoresis was run in 1× TBE
at 110 V and subsequently visualized (Vilber Lourmat Infinity V X 2, France). The
sizes of the fragmentswere determined using a standard molecular weight marker
(100 bp/plus ladder, Thermo Scientific) (Settanni et al., 2011).
3.6.3. RFLP (Restriction Fragment Length Polymorphism) Analysis

PCR products of the 5.8S ITS region were digested using the restriction
endonucleases Hae III, Hha I and Hinf I (Thermo Scientific). Digestions with three
different endonucleases were performed seperately by adding 10 µL of the
amplified DNA to 15 µL of the restriction enzyme mixture including 2.5 µL
restriction enzyme buffer (Buffer R for Hae III and Hinf I and Buffer Tango for
Hha I), 11.5 µL sterile distilled water and 1 µL (10 U/µL) restriction enzyme. The
mixtures were then put into a water bath at 37°C for 10-16 hours.
Restriction fragments were analysed through 2% (w/v) agarose gel in 1 x
TBE buffer and stained with SYBR SafeTM DNA gel stain (Invitrogen). The
electrophoresis was run in 1 x TBE at 120 V and subsequently visualized (Vilber
Lourmat Infinity V X 2, France).The sizes of the fragments were determined using
a standard molecular weight marker (50 bp ladder, Thermo Scientific) (Settanni et
al., 2011).
Yeast sharing identical restriction patterns were classified into groups and
1 or 2 isolates were chosen as a representative of each group for sequence analysis
of the D1/D2 domains of the 26S rRNA gene.
3.6.4. 26S rRNA Gene Sequence Analysis

Sequence analysis of the D1/D2 domain of the 26S rDNA gene was used to
differentiate yeast isolates. Amplification of the D1/D2 domains of 26S rRNA was
79
carried out using NL1 (5'-GCA TAT CAATAAGCGGAGGAA AAG-3') and NL4
(5'-GGTCCGTGTTTCAAGACG G-3') primers as described previously (Kurtzman
and Robnett, 1998). The PCR mix for D1/D2 domain of the 26S rDNA gene
sequence analysis was prepared as follows: 5 µL (50-100 ng) DNA, 5 µL Dream
Taq buffer (10x +20 mM MgCl2, Thermo Scientific), 0.5 µL dNTP (2.5 mM,
Thermo Scientific), 2.5 µL MgCl2 ( 25 mM), 0.1 µL NL1 primer (100 µm, Thermo
Scientific), 0.1 µL NL4 primer (100 µm, Thermo Scientific) and 0.5 µL Dream
Taq DNApolymerase (5 U/μl, Thermo Scientific) and sterile distilled water in 50-
µL mixture. Amplifications of the D1/D2 domains of 26S rRNA were performed
in the thermocycler (Techne TC-Plus 02, UK) which was programmed as follows:
initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 1 min;
annealing at 52°C for 45 sec and extension at 72°C for 1 min; plus a final extension
step at 72°C for 7 min.
PCR products were separated by electrophoresis on a 1.5% (w/v) agarose
(Sigma) gel stained with the SYBR SafeTM DNA gel stain (Invitrogen) and
subsequently visualized (Vilber Lourmat Infinity V X 2, France). PCR amplicons
were sent to BM Laboratuvar Sistemleri (Ankara) for sequencing. Resultant
sequences were compared using the BLAST
(https://blast.ncbi.nlm.nih.gov/Blast.cgi ) with nucleotide sequences deposited at
the database National Center for Biotechnology Information (NCBI) (Altschul et
al., 1997). The sequence alignments were evaluated using ClustalW with type
strains and their closest relatives (Bioedit version 7.0.9) (Thompson et al., 1997;
Francesca et al., 2014). Phylogenetic analyses were performed using the sequences
obtained from the 26S rRNA gene sequence analysis. Phylogenetic trees were
constructed based on aligned sequences using the phlogenetic tree reconstruction
method, UPMGA, with MEGA 7.0 software.
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3.7. Functional Characterization of Selected LAB Isolates

Strains of the LAB species frequently isolated in sourdough and chickpea
fermentations were selected for the evaluation of the functional properties to be
used as a starter culture in sourdough and chickpea fermentations. Functional
analysis were performed on selected strains at least in duplicate.
3.7.1. Acidification Activity

The acidification test of selected LAB strains was performed in sterile flour
extract (SFE) liquid broth according to a previously described method (Alfonzo et
al., 2013). For the preperation of SFE, 200 g of white wheat flour was suspended in
1 L of distilled H2O and sterilized at 121°C for 20 min. The supernatant was then
used as the liquid media in subsequent experiments.
Overnight grown LAB cultures in MRS broth were harvested by
centrifugation at 13,300 rpm for 3 min (Thermo Scientific MicroCL 17, Germany),
washed with Ringer’s solution and resuspended in the same solution to an optical
density at 600 nm of 1.00 (Shimadzu UV-1700, Japan) to standardize bacterial
inocula. Twenty mL of SFE was inoculated with 1% (v/v) of the solution
consisting of the cell suspension and incubated at 30°C. The acidifying capacity of
LAB was monitored during their incubation by pH measurements taken at 2 h
intervals for the first 8 h of incubation and then at 24 ,48 ,72 h and 7 d after
inoculation. Uninoculated SFE was used as the control.
3.7.2. Determination of Lactic and Acetic Acids

According to the acidification capability, some strains were selected and
also analysed for their ability to produce lactic and acetic acids following 8 h of
fermentation in sterile flour extract. For that purpose, acidified SFE (aSFE) of the
selected strains were used for further analysis. Perchloric acid was used for protein
precipitation in the samples following storage at 4°C for 24 h. Protein agglomerates
were removed by centrifugation (13,300 rpm, 60 min, 4°C; Hettich® Universal
81
320R, Germany) and the supernatant was filtered through a 0.45 µm PVDF
Syringe Filter (Isolab) and injected to the HPLC system.
3.7.3. Quantitative EPS Analysis

Overnight grown LAB cultures in MRS broth were harvested by
centrifugation at 13,300 rpm for 3 min (Hettich® Universal 320R, Germany),
washed with Ringer’s solution and resuspended in the same solution to an optical
density at 600 nm of 1.00 (Shimadzu UV-1700, Japan) to standardize bacterial
inocula. For EPS yield determination, MRS broth supplemented with 50 g/L
sucrose was inoculated with the cell suspension solution 1% (v/v) and incubated at
30°C (Tayuan et al., 2011). Sucrose was separately autoclaved and then added to
the sterilized MRS medium (Malik et al., 2015). EPS were extracted from the 72
hour-old bacteria culture and boiled at 100°C for 10 min to inactivate EPS
degrading enzymes (Kusmiati et al., 2016). After cooling, 1 mL of the cell culture
was treated with 17% (v/v) of 85% trichloracetic acid solution. The solution was
incubated at 4°C overnight and then centrifuged (14,000 rpm, 4°C, 30 min) to
remove cells and protein (Frengova et al., 2000; Onbasli and Aslim, 2008). Each
supernatant was treated with three-volumes of ethanol (95%) and left overnight at
4°C (Joshi and Koijam, 2014). The precipitated EPS was centrifuged at 14,000 rpm
at 4°C for 20 min and the supernatant was discarded (Abdelnasser et al., 2017).
The precipitate of pure EPS was dried at 60°C for 24 h in the same centrifuge tubes
to minimize EPS loss and total EPS yields were determined gravimetrically by
measuring the dry mass (Osińska-Jaroszuk et al., 2014; Huang et al., 2017).
3.7.4. EPS Production on Agar Medium

Selected bacteria cultures were streaked onto MRS agar medium
supplemented with 50 g/L sucrose and incubated at 30°C for 72 hours. The
formation of mucoid or viscous colonies on the agar was considered to be EPS
production (Lule et al., 2015).
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3.7.5. Antimicrobial Activity Against Selected Species

Selected strains were evaluated for their antimicrobial activity using the
dual culture overlay technique against B. subtilis, B. lincheniformis, Escherichia
coli, Penicillium expansum and Penicillium digitatum. Firstly, the selected LAB
strains to be tested for bacteriocin production were grown on the surface of mMRS
containing 1.5% agar at 30°C for 24 h anaerobically. The indicator strains, B.
subtilis and B. lincheniformis were grown in Nutrient broth at 37°C, Escherichia
coli in Brain Heart Infusion broth at 37°C and the molds Penicillium expansum and
Penicillium digitatum in Malt extract broth at 28°C until reaching OD600=1.0.
Indicator strains were inoculated (1%) onto soft agar medium (containing 0.75%
agar) specific for each strain and the soft media were poured onto the plates where
growth of the producers had occurred and the plates were incubated at the optimal
growth temperature and time for the indicator strains. After incubation, the plates
were controlled for zone formation. A detectable clear zone around the colonies of
the producer strain was scored as positive inhibition (Schillinger and Lücke, 1989;
Corsetti et al., 2004).
3.7.6. Protease Capacity

Protease activity of selected LAB strains was assessed on mMRS agar
containing 2% skim milk powder. The medium was sterilized by autoclaving at
115°C for 10 min. Isolates were incubated at 28°C for 8 days. After incubation, the
formation of halo zones around microbial colonies indicated protease capacity
(Palla et al., 2017).
3.7.7. Growth at Different Conditions

Selected strains were evaluated for growth at different temperatures, pH
levels, salt concentrations and carbohydrate sources. For growth at different
temperatures, strains (OD=1) were inoculated onto mMRS broths (1%). For the
groth at differnt temperatures, inoculated mMRS broths were incubated at 15, 28,
83
37 and 45°C for 2-7 days. For tolerance to different pH values, strains (OD=1)
were inoculated onto mMRS broth (1%) prepared at pH 3.5, 4.5 and 6.5 with filter
sterilized 5 N HCl and 2 N NaOH solutions and incubated at 30°C for 3 days. For
tolerance to different salt concentrations, strains (OD=1) were inoculated onto
mMRS broth (1%) containing 4, 6 and 8% NaCl (w/v) and incubated at 30°C for 3
days. Precipitation and turbidity in the broth media was accepted as growth of the
strain at the condition.
The ability to ferment various carbohydrates was evaluated using MRS
broth prepared without glucose and meat extract. Each sugar solution (1%, w/v)
was added to MRS broth media via filter sterilization. Tested carbohydrates were D
(+) glucose monohydrate (Sigma-Aldrich), D (-) fructose (Merck), D (+) galactose
(Fluka), lactose monohydrate (Merck), sucrose (Merck), maltose monohydrate
(Merck), L (+) rhamnose monohydrate, raffinose (Difco), D (-) mannitol (Merck),
D (+) mannose (Fluka), D (-) arabinose (Fluka) and D (+) xylose (Sigma Aldrich).
The control broth lacked sugar addition. Chlorophenol red (0.004 %, w/v) was
added as the indicator and conversion of the color from red-purple to yellow
indicated low pH values due to the growth and production of lactic acid
(Schillinger and Lücke, 1987).
3.7.8. Enzyme Profile

For the enzyme profile assessment, the API ZYM enzyme (Biomerieux,
France) testing system was used according to the manufacturer's instructions. First
of all, 5 mL of distilled water was distributed into the incubation wells of the
incubation tray to create a humid atmosphere. Then selected bacterial cell
suspensions (5-6 McFarland turbidity) were inoculated into cupules of the
incubation tray and incubated at 37°C for 4-4.5 h. After incubation, 1 drop of ZYM
A reagent and 1 drop of ZYM B reagent was added to each cupule, and color
changes were recorded and evaluated.
84
3.8. Production of Experimental Sourdoughs and Chickpea Liquid Starters

with Selected Strains
Among mostly isolated LAB species, two and one strains were
characterized for use in sourdough and chickpea fermentations, respectively.
Sourdough production under laboratory conditions was performed
according to the traditional (sourdough Type I) protocol using selected strains
individually and also in combination. A control sourdough was produced without
using the selected starter culture. For dough preparation, whole-meal wheat flour
(216.21 g) and boiled and cooled tap water (183.79 mL) were mixed manually to
produce 400 g of dough with a dough yield [(dough weight/flour weight)×100] of
185. Strains (OD=1) were inoculated at a concentration of 1% (v/w) to the dough.
Each sourdough was fermented at 28°C for 24 h in glass jars covered with a lid.
The resulting sourdoughs were propagated until reaching constant acidity by a
daily back-slopping (refreshment) procedure and the sourdough from the previous
day's fermentation was used as the starter (20% [wt/wt] of inoculum) to ferment a
new mixture of flour (172.98 g) and tap water (147.02 mL), resulting in a dough
yield of 185. Sourdough productions were carried out in duplicate. Experimental
sourdough productions are shown in Figure 3.10.
85
Figure 3.10. Experimental sourdough production

A) Control B) Selected strain 1 C) Selected strain 2 D) Dual-combination of
selected strains
The first sample (0 h) was taken from the flour and water mixture,
unfermented dough, after mixing. Both TTAand pH measurements were carried out
86
on all samples collected after 4, 8 h and 12 h of the experiment and once every 24
hours of until the last refreshment of the sourdough production. During the back-
slopping procedure, sampling was performed on the sourdoughs immediately
before the daily refreshment step and the samples were subjected to plate counting,
TTA, pH measurements, carbohydrate, organic acid and ethanol analyses. Samples
were analysed in duplicate. Also sourdoughs were examined for their generation of
volatile organic compounds (VOCs) by SPME-GC-MS at the beginning and end of
the fermentations. Samples were analyzed in duplicate.
Chickpeas (Koçbaşı variety) were ground for the production of chickpea
liquid. Then 50 g of chickpeas were put into sterile glass jars and mixed with 400
mL of boiled and cooled tap water at 37°C as 50°C is very high for the strain
inoculation. The selected strain (OD= 1) was inoculated at a concentration of 1%
(v/w) into the chickpea liquid starter under aseptic conditions. The beginning of the
fermentation for the selected strain was immeadiately after starter inoculation. The
control chickpea liquid starter was produced without inoculating a starter culture.
Fermentations were conducted in glass jars covered with a lid at 37 °C for 18 h. At
the end of the fermentation, chickpeas were seperated and the liquid was used for
the production of the chickpea dough. 150 mL of liquid is mixed with 200 g of
flour (DY 175) and fermented at 37 °C for 4 hours. Productions were carried out in
duplicate. Experimental chickpea liquid starter and dough productions are shown in
Figure 3.11.
87
Figure 3.11. Experimental chickpea liquid starter and dough production

A) control production B) production with selected strain
The samples were taken at the beginning (0 h) and end of the fermentations (18 h
for chickpea liquid starters, 4 h for chickpea doughs). Samples were subjected to plate
counting, TTA, pH, carbohydrate, organic acid, ethanol and VOC analyses at the beginning
and end of the fermentations. Samples were analyzed in duplicate. For chickpea liquid
starter samples, pH, spore-forming bacteria and bacteria grown on NA were also monitored
every 2 hours during 10. Samples were analyzed in duplicate.
3.9. VOC Analysis by SPME-GC-MS in Experimental Samples

Experimental sourdough, chickpea liquid starter and dough samples were
examined for their generation of VOCs with the modified method of Settanni and
others (2013). VOCs were determined applying the solid phase micro extraction
(SPME) isolation technique. Each sample (3 g) was heated to 30°C in a vial for 30
min. Then headspace was collected by a fiber (85 µm Carboxen\PDMS) at 30°C
for 30 min. The SPME fibre was directly inserted into the GC/MS (Agilent 7000
88
Series Triple Quad) equipped with a HP - 5MS capillary column (30 m, 0.250 mm
i.d., film thickness 0.25 mm, %5 phenyl methyl-poly-cyloxane). Separation was
achieved by using the following temperature program: initial 40°C with a 4 min
hold and ramped to 90°C at 3°C/min, 130°C at 4°C/min, 240°C at 5°C/min and
held for 8 min. Helium was used as the carrier gas at a constant flow rate of 1.0
mL/min. Ionizing energy was 70 eV and MS were at the full-scan mode with scan
range of 50–600 m/z. The identification of VOCs was achieved by using the
National Institute of Standards and Technology (NIST 14L) reference library and
VOCs were expressed as relative peak areas (peak area of each compound/total
area*100).
3.10. Statistical Analysis

Data of the analysis were subjected to one-way analysis of variance
(ANOVA) and multiple comparison of means by Duncan’s procedure at a p<0.05
using the IBM SPSS 20 software. Multivariate statistical analyses were carried out
to investigate the correlations between the characteristics and the samples.
XLSTAT 2018 software for excel was used for data processing and graphic
construction. Dissimilarity index calculation and dendrogram construction were
carried out using DARwin (6.0.15) software package. Phylogenetic trees were
constructed from molecular sequences to investigate the phylogenetic relations
between strains by Mega 7.0 software.
89
90
4. RESULTS AND DISCUSSION Cennet Pelin BOYACI GÜNDÜZ
4. RESULTS AND DISCUSSION
4.1. Sourdough Samples

4.1.1. Chemical Characteristics of Sourdough Samples
Whole-meal wheat and rye sourdough samples were collected from three
different bakeries located in different cities at two different times. Codes were
assigned to each collected sample as letters and numbers without expressing the
bakery names due to the special request by the bakeries. Sourdough samples were
coded as SD-M1, SD-M2, SD-T1, SD-T2, SD-K1, SD-K2, SD-R1 and SD-R2,
with SD denoting sourdough, and a randomly chosen letter, and 1 or 2 indicating
the first or second sampling. R coded sourdough was rye sourdough and the other
were wheat sourdough samples. The first sourdough samples (coded as 1) were
collected from bakeries in the spring or at the beginning of summer and the second
samples (coded as 2) were collected at the end of the autumn or in winter, resulting
in sourdoughs with different characteristics.
4.1.1.1. pH
Results of the pH measurements of the 8 sourdoughs, including the two
sampling are shown in Table 4.1. The pH levels of the collected sourdough
samples ranged from 3.71 to 3.96 and the pH exhibited a mean value of 3.87. The
lowest pH level was measured in the SD-T2 as 3.71, on the other hand, the pH of
the first sampling of this sourdough, SD-T1, was 3.93. The highest pH value was
measured as 3.96 in the rye sourdough sample. The pH of the rye sourdough of the
second sampling, SD-R2, was 3.91. As it can be seen, pH values showed
differences among sourdoughs and sampling times.
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Table 4.1. The pH levels of the samples

Sample pH Std. deviation
SD-M1 3.86bc 0.04
SD-M2 3.85bc 0.01
SD-T1 3.93d 0.03
SD-T2 3.71a 0.04
SD-K1 3.91cd 0.05
SD-K2 3.82b 0.01
SD-R1 3.96d 0.02
SD-R2 3.91cd 0.03
a-dDifferent superscript letters within a column
indicate a significant difference (Duncan p<0.05)
Testing of the homogenity of variances showed that variances can be treated as

equal (p> 0.05) and a parametric ANOVA test was conducted. According to the statistical
results, the differences between the samples collected from different bakeries were
significant (p<0.05). The pH differences between samples could be due to the different
microflora in the sourdough samples, and different production methods and incubation
conditions since the samples were collected from different cities. These parameters directly
affect the pH of the end product and therefore sourdoughs produced in different places
exhibited different biochemical patterns. On the other hand, differences were observed
between some samples collected from the same bakery at two different times. There
weren’t any significant differences in SD-M and SD-R sourdough samples collected at two
different times. pH values of the SD-M1 and SD-M2 coded sourdough samples were found
as 3.86 and 3.85, respectively. On the other hand, differences in the pH levels of SD-T and
SD-K sourdough samples were statistically significant. pH values of the SD-K1 and SD-K2
coded sourdough samples were found to be 3.91 and 3.82, respectively. SD-T samples,
including the first and second sampling as SD-T1 and SD-T2, showed differences among
pH values. The fermentation conditions changed in that bakery, as mentioned by the owner,
between the time interval of the first and second sampling. They tried to stabilize the
conditions and for that purpose made some modifications during the fermentation; hence
the differences observed between the first and second sampling could be a result of the
modifications.
92
In the present study, pH results of the sourdough samples were in

consistent with the pH values determined in other studies. Actually, sourdough
shows great variation due to the artisan and region-dependent handling, including
production method, flour that is used in the production, type of sourdough, amount
of the sourdough inoculum, fermentation conditions, back-slopping times etc.
Therefore, pH values, TTA and microbiological flora might be differ among
different sourdough samples.
In the study of 19 Italian sourdoughs used for the manufacture of
traditional breads, pH values were ranged from 3.70 to 4.28 (Minervini et al.,
2012a). It was indicated that 13 of the 19 sourdoughs had pH values of less than
4.0, therefore, the pH values of many sourdoughs were in consistent with our
results. In another study, conducted in 18 Italian sourdoughs, pH values were in the
range of 3.90 and 5.01. Not all of them but many of the values were higher than the
pH values in the present study (Lattanzi et al., 2013). In another study, 20 wheat
sourdough samples were collected from central Italy, and exhibited a wide range of
pH values, 3.46-5.23 (Valmorri et al., 2010). Ventimiglia et al. (2015) analysed 15
sourdoughs produced in southern Italy and reported pH levels in the range of 3.81-
4.77. Lhomme et al. (2015) collected 16 sourdoughs from different regions of
France and reported pH values in the range of 3.23- 4.01 and the highest pH was
observed in the rye sourdough as it was detected in our study. Some studies have
reported higher pH values. In the study of Zhang et al. (2015), 25 traditional
sourdough samples were aseptically collected from China and mean pH values
were in the range of 3.76 and 5.51. As it can be seen, pH values exhibit a wide
variation among sourdoughs in different regions due to differences in the
production methods and conditions.
4.1.1.2. Total Titratable Acidity

Total titratable acidity is given as mL of 0.1 N NaOH consumed and results
of the 8 sourdoughs including both samplings are shown in Table 4.2. Acidity
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levels of the collected sourdough samples ranged from 6.78 to 23.93 mL 0.1 N
NaOH /10 g dough. The lowest and highest acidity values were calculated in the
SD-M2 and SD-K1 samples, respectively. As expected, acidity content was
significantly (p<0.05) different between sourdough samples.
Table 4.2. Mean TTA levels of the sourdough samples

Sample TTA (mL 0.1 N Std. deviation
NaOH /10 g
dough)
SD-M1 8.35a 0.05
SD-M2 6.78b 0.07
SD-T1 8.30a 0.1
SD-T2 10.98e 0.43
SD-K1 23.93f 1.11
SD-K2 18.05g 0.35
SD-R1 16.15c 0.15
SD-R2 13.70d 0.3
a-gDifferent superscript letters within a column
As it can be seen, acidity values exhibited differences among sourdoughs and

sampling times. Testing the homogenity of variances showed that variances were unequal
and the differences between the samples were significant (p<0.05).
Acidity levels of sourdoughs collected from the same bakery at two different times
were significantly different. However, among the samples the highest acidity was observed
in SD-K sourdough at both sampling times. SD-K samples were characterized by higher
acidity than other samples. Rye sourdough followed SD-K in terms of acidity. SD-M
sourdough samples showed the lowest acidity level at both sampling times. The acidity
content of the sourdoughs, except SD-T, decreased in the second sampling that was
collected in winter, as can be seen in Figure 4.1. As expected, a decreasing temperature
affected the fermentation of the sourdough resulting in lower acidity end products.
94
Figure 4.1. Acidity levels of the sourdough samples
Total acidity values of the sourdoughs showed a wide variation and were in
the range of 6.78-23.93 mL 0.1 N NaOH. The median value for the acidity was
determined to be 12.42 mL 0.1 N NaOH. In the study of Lhomme et al. (2015),
TTA exhibited a median value of 16.2 mL 0.1 N NaOH. In another study, an
acidity range of 12.3-13.0 mL NaOH was reported (Tamani et al., 2013). Viiard et
al. (2012) reported the pH and acidity values to be between 3.5-3.7 and 19.0-22.0
mL 0.1 N NaOH in the rye sourdough samples. In another study, final acidity
values of the rye sourdoughs fermented at 25°C, refreshed 12 times and fermented
at 30°C, refreshed 24 times were reported to be in the range of 15.2-17.7 and 20.3-
26.4 mL 0.1 N NaOH, respectively (Meroth et al., 2003). Ventimiglia et al. (2015)
reported the acidity values of 15 sourdoughs produced in southern Italy to be in the
range of 6.0-14.7 mL 0.1 N NaOH.
Under laboratory conditions, sourdough was produced at 28°C by propagating
over a period of 7 days using the daily back-slopping (refreshment) procedure. The first
sample (0 h) was taken from the flour and water mixture, unfermented sourdough,
95
immediately after mixing. During the back-slopping procedure, sampling was performed on
the sourdoughs before each daily refreshment step. pH and TTA of the sourdough samples
were determined at 4, 8 and 12 h of the fermentation and then once every 24 hours for 7
days.
The pH of the prepared sourdough did not change during the first 12 h of
fermentation, as shown in Figure 4.2. The pH started to drop slowly, ending at pH
4.58 after the first day of fermentation. On the second day, it decresed to 3.99 and
at the end of the fermentation, it was determined to be 3.60. TTA data had a
reverse relationship with pH and were correlated linearly with pH values. TTA was
stable during the first 12 hours, but then started to increase and reached 8.72 mL
0.1 N NaOH/10 g dough on the first day. The acidity level continued to increase
the next day and was determined to be 14.74 mL. During the following days, the
acidity continue to increase, but not greatly, reaching at final value of 17.56 mL 0.1
N NaOH.
Figure 4.2. Changes in pH during a 7-day sourdough fermentation with daily back-
slopping (Symbols: × shows changes in pH, ♦ shows changes in acidity
levels)
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During sourdough fermentation, TTA increases and pH decreases as a

result of the produced organic acids. Depending on the applied fermentation
conditions such as refreshment time, type of flour, temperature, dough yield and
starter culture addition, the pH and TTA values can differ in sourdoughs. In the
present study, sourdough produced under laboratory conditions showed pH and
acidity patterns that are similar to some other studies (Van Der Meulen et al., 2007;
Taccari et al., 2016; Fujimoto et al., 2018). Fujimoto et al. (2018) produced
sourdough by back-slopping at 28°C for 5 days. In their study, the pH declined to
less than 4.0 on day 2. On the 5th day, pH values of 3.62 and 3.61 for the two
produced sourdoughs are in agreement with our results. Some studies have
investigated sourdough production under laboratory conditions with starter
additions. Paramithiotis et al. (2005) prepared sourdoughs with various starter
culture combinations via propagation every 24 hours at 25 and then 30 °C. After 24
hours, pH and acidity levels of the sourdoughs were reported in the range of 3.57-
3.85 and 10.1-12.5 mL 0.1 N NaOH, respectively.
The flour type is important and directly affects the properties of the
sourdough. Taccari et al. (2016) studied the fermentation of Type I sourdough
propagated for 20 days with daily back-slopping under laboratory and artisan
conditions at room temperature with three different flour types. They reported the
pH and TTA values in the ranges of 3.72 to 4.02 and 9.40 to 18.10 ml, respectively.
They determined the highest acidity in the sourdough samples produced using
whole-meal flour.
4.1.1.3. Carbohydrate, Organic Acid and Ethanol Contents of the Sourdoughs

The content of maltose, sucrose, glucose, fructose and ethanol was
determined by HPLC using an RID detector. Lactic and acetic acid contents were
determined by HPLC with a UV detector. Retention times detected by the RID
detector were 9.393, 9.470, 11.168, 12.591 and 24.056 for maltose, sucrose,
glucose, fructose and ethanol, respectively. Retention times detected by the UV
97
detector were 16.291 and 17.902 for lactic and acetic acid, respectively. Standards
are shown with their chromatogram images in Appendix 1, 2 and 3. Discrimination
of the maltose and sucrose peaks was difficult as their retention times were close to
each other and they were eluted together. Therefore, the results of these compounds
are given together as maltose+sucrose.
The concentration ranges of the standards were 0.081-2.512, 0.082-2.543,
0.083-2.554, 0.079-2.445, 0.091-2.605, 0.113-3.257 and 0.30-9.80 g/L for maltose,
sucrose, glucose, fuctose, lactic acid, acetic acid and ethanol, respecively. Seven
point linear calibration curves (R2> 0.998) were constructed for all standards
according to the mean of three consecutive injections versus mean area. Calibration
curves are given in Appendix 4.
The LOD-LOQ values were calculated by multiplying 3 and 10 times the
standard deviation of the 10 standard injections and determined to be 0.032-0.108,
0.028-0.110, 0.023- 0.075, 0.029-0.096, 0.031-0.103, 0.030-0.100 and 0.095-0.316
for maltose, sucrose, glucose, fructose, lactic acid, acetic acid and ethanol,
respectively. A recovery test was conducted for each standard based on the six
injections of the spiked and unspiked samples. Recovery levels of the samples were
104.79, 101.66, 116.94, 92.73, 99.55 and 84.60 % for maltose+sucrose, glucose,
fructose, ethanol, lactic acid and acetic acid, respectively.
Mean values of the carbohydrate and ethanol content of the collected
sourdough samples are given in Table 4.3. Mean-median values of the content of
maltose+sucrose, glucose, fructose and ethanol were 2.43-1.62, 1.57-1.70, 2.67-
2.13 and 9.59-10.16 g/kg, respectively. A sourdough sample chromatogram image
is shown in Appendix 5.
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Table 4.3. Mean carbohydrate and ethanol content (g/kg) of the sourdough samples
Sourdoughs Maltose+sucrose Glucose Fructose Ethanol
SD-M1 5.47d±1.13 2.30e±0.14 2.30c±0.14 14.70d±1.05
SD-M2 2.32c±0.02 1.39b±0.05 1.96b±0.08 14.94d±1.63
SD-T1 6.24e±0.02 2.16de±0.06 2.79d±0.21 9.97c±0.04
SD-T2 1.37ab±0.09 0.84a±0.17 0.98a±0.13 10.80c±0.49
SD-K1 1.28ab±0.06 1.54b±0.04 0.78a±0.02 4.39a±1.13
SD-K2 <LOQ 0.81a±0.16 0.78a±0.06 10.35c±0.08
SD-R1 0.91a±0.19 2.00cd±0.23 6.96f±0.14 6.66b±1.89
SD-R2 1.87bc±0.02 1.86c±0.01 4.84e±0.25 4.87ab±0.075
Results indicate mean values± SD, Different superscript letters within a column
In the SD-K2 sample, the maltose+sucrose content was below the

quantification limit. This sample also had the lowest glucose and fructose content.
In the other samples, maltose+sucrose, glucose and fructose contents ranged from
0.91 to 4.47, 0.84 to 2.30 and 0.78 to 6.96 g/kg, respectively. Median values of
maltose+sucrose, glucose, fructose, lactic acid and acetic acid were determined to
be 0.83, 1.67, 2.09, 6.27 and 1.35 g/L, respectively. According to the statistical
results, the differences in sugar content between the samples collected from the
different bakeries were significant (p<0.05). The carbohydrate and organic acid
differences among samples could be due to the different flours used in the
production of sourdoughs together with different microflora and fermentation
conditions as the samples were collected from different cities. In the samples
collected from the same bakery at two different sampling times, the differences in
carbohydrate content were significant, except glucose in SD-R and fructose in SD-
K samples. SD-T samples, including the first and second sampling as SD-T1 and
SD-T2, showed a big difference in sugar content and this could be related to the
changing conditions during the production, as indicated previously. In the study of
Lattanzi et al. (2013), maltose, glucose and fructose concentrations in sourdoughs
used for the manufacture of traditional Italian breads were in the range of 0.3-28.7,
99
0.4-25.8 and 0.3-10.1 g/L, respectively. Median values of the sourdoughs were
reported to be 5.6, 3.4 and 3.5 g/L, respectively.
The sugar content changes according to bacterial consumption and
hydrolysis by flour enzymes; hence flour used for production and the
microorganisms directly affect the content in the dough (Paramithiotis et al., 2006;
Hansen, 2012). Sugars levels in the samples can decrease and increase as a result of
bacterial consumption and hydrolisation by flour enzymes, respectively. Therefore,
it is difficult to discuss the consumption ratio of sugars by microorganisms. It has
been reported previously that carbohydrates are continuously liberated during
fermentation, especially by endogenous flour enzymes, and it was not possible to
estimate their consumption at the end of fermentation (Lattanzi et al., 2013).
Korakli et al. (2001) reported maltose and glucose accumulation after 24 h
fermentation in doughs because of the amylase and α-glucosidase activities of
flour.
The mean-median content of lactic acid and acetic acid of the collected
sourdoughs were 7.3-6.16 and 1.40-1.42 g/kg, respectively. In terms of mmol/L,
lactic and acetic acids varied from 57 to 156 and 9 to 39 mM, respectively. The
lowest and highest lactic and acetic acid concentrations were determined in the SD-
M1 and SD-K1 sourdough samples, respectively. According to the statistical
results, the differences between the content of lactic and acetic acid in the samples
collected from different bakeries were significant (p<0.05). Lactic acid
concentrations in SD-K and SD-T and acetic acid concentrations in SD-T samples
at two different sampling times were not significant. Conversely, in other
sourdough samples, differences in the two sampling times were significant. The
mean organic acid content (g/kg) of the sourdough samples is shown in Table 4.4.
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Table 4. 4. Mean organic acid content (g/kg) of the sourdough samples

Sourdoughs Lactic acid Acetic acid FQ
SD-M1 5.15a±0.85 0.58a±0.09 5.90
SD-M2 6.25ab±0.22 1.53bc±0.51 2.73
SD-T1 5.89ab±0.31 1.20b±0.18 3.28
SD-T2 6.07ab±0.40 1.63bc±0.38 2.48
SD-K1 14.12d±0.98 2.40d±0.06 3.92
SD-K2 6.90b±0.27 1.80c±0.06 2.56
SD-R1 5.31a±0.18 0.76a±0.20 4.66
SD-R2 9.12c±0.13 1.32b±0.12 4.61
Results indicate mean values± SD, Different
superscript letters within a column indicate
asignificant difference (Duncan p<0.05)
In the laboratory poduced sourdough, the concentration of carbohydrate,

organic acids and ethanol cotents were determined in the unfermented dough and
also 1, 2, 4 and 7 refreshment steps of the backslopped sourdough during 7 days
(Table 4.5).
Table 4.5. Mean carbohydrate, organic acid and ethanol content (g/kg) of the
sourdough produced under laboratory conditions
Day Maltose+ Glucose Fructose Lactic Acetic Ethanol
Sucrose acid acid
0d 18.46±0.69 6.86±0.11 8.76±0.21 <LOQ <LOQ <LOQ
1d 18.46±0.43 6.82±0.21 3.60±0.08 3.68±0.35 2.45±0.11 3.09±0.35
2d 18.76±0.75 15.26±0.26 2.06±0.10 8.62±0.42 3.30±0.01 3.14±0.30
4d 17.08±0.18 11.77±0.16 2.25±0.10 11.31±0.40 2.22±0.06 3.11±0.17
7d <LOQ 1.30±0.08 1.34±0.07 14.96±0.49 0.91±0.14 15.01±1.08
Results indicate mean values± SD
In the laboratory produced sample, maltose+sucrose, glucose and fructose

concentrations were determined to be 18.46, 6.86 and 8.6 g/kg in the unfermented
dough. The maltose+sucrose concentration did not change over 4 days and then
decreased below the LOQ at the 7th backslopping. The fructose concentration
decreased during fermentation and was determined to be 1.34 g/kg at the last
101
refreshment. On the other hand, the gluose concentration increased after the first
day and then decreased to 1.30 g/kg at the last refreshment. Lactic acid increased
and reached up to 14.96 g/kg. Acetic acid increased until 2nd day of backsloping
and then decreased. Ethanol was determined to be 15.01 g/kg at the last
refreshment. Figure 4.3. shows the analyte concentrations at different backslopping
during a 7-day sourdough fermentation with daily back-slopping times in terms of
mol/g.
Figure 4.3. Changes in carbohydrate, organic acid and ethanol contents under
laboratory conditions
According to the carbohydrate analysis, the maltose+sucrose content was

around 53 mM for 4 days and was below the quantification limit at the final
refreshment. Glucose and fructose were found at residual concentrations of less
than 0.008 mmol/g (<8 mM) on the final refreshment day. In a study, the occurence
102
of maltose at concentrations of more than 60 mM was reported at the first

refreshment step. In the same study, maltose concentrations were determined to be
less than 20 and 40 mM on the 7th day refreshment at 23 and 30°C fermentations,
respectively (Vrancken et al., 2011).
Final metabolite content of the sourdoughs change according to the
metabolism of the flora in the fermentations. In the present study, metabolite
contents were started to increase following fermentation. Ethanol is produced as
the metabolite of yeast metabolism and its content was determined to be high at the
final refreshment of the laboratory produced sourdough in this study.
The results of the present study are in agreement with other studies. In a
study, the content of lactic and acetic acid in traditional organic sourdoughs
collected from French bakeries were reported to be in the range of 3.29-11.44 and
0.66-2.03 g/kg, respectively (Lhomme et al., 2016). In another study conducted on
sourdough samples prepared under laboratory conditions, lactic and acetic acid
levels at the 10th day of propagation were reported to be 88 mM for rye and 24 and
30 mM for wheat sourdoughs (Ercolini et al., 2013). Minervini et al. (2012a)
reported lactic and acetic acid contents of sourdoughs used for traditional Italian
breads to be in the range of 63.7-94 mM and 6-20.6 mM, respectively. Median
values were reported as 80 mM for lactic acid and 18 mM for acetic acid
(Minervini et al., 2012a). In this study, median values were calculated as 71 mM
for lactic acid and 22.5 mM for acetic acid. Paramithiotis et al. (2006) reported the
lactic and acetic acid contents in terms of mmol/g to be in the range of 0.04-0.11
and nd-0.02 in the sourdoughs produced with different yeast and LAB cultures. In
this study, the content of lactic and acetic acids were in the range of 0.057-0.156
and 0.009-0.039 mmol/g, respectively.
The molar ratio between lactic and acetic acids, known as the fermentation
quotient (FQ), represents an important parameter as it affects the aroma of the
sourdoughs (Corsetti and Settanni, 2007). In the present study, FQ levels were
determined in the range of 2.48-5.90 as shown in Table 4.4. The optimumin FQ
103
range is considered to be 2.0–2.7 (Hammes and Ganzle, 1998). For rye sourdough,
the optimal FQ was reported to be in the range of 1.5–4.0 (Spicher, 1983). In the
present study, rye sourdough samples at both sampling times exhibited the
optimum FQ value. Among wheat sourdoughs, the SD-M1 sample displayed the
highest FQ, which was above 4.00, indicating a low concentration of acetic acid
with respect to lactic acid. The second sampling of the wheat sourdoughs, SD-M2,
SD-T2 and SD-K2 showed optimum FQ levels. Corona et al. (2016) determined
lactic acid, acetic acid and also FQ of the sourdough after 16 hours fermentation as
7.01 mg/g, 0.72 mg/g and 6.49, respectively. Another study of the same research
group reported the lactic acid, acetic acid and FQ levels of the 15 collected
sourdoughs produced in southern Italy in the range of 1.97-9.41 mg/g, 0.36-1.46
mg/g and 0.91-6.80, respectively (Ventimiglia et al., 2015). On the other hand, the
FQ of the laboratory produced sourdough was high and determined as 10.84.
Suitable conditions such as propagation ratio, fermentation time and temperature
enables the growth of LAB resulting in increased metabolite production.
The mean ethanol content of the sourdough samples ranged from 4.87 to
14.94 g/kg. The highest ethanol content was determined in the SD-M samples at
both sampling times; whereas the lowest ethanol content was determined in the
SD-K1 sample. On the other hand, the first sampling of SD-K was 10.80 g/kg.
Ethanol concentrations in the samples at both sampling times were not significant
(p>0.05); except for the SD-K sample. Paramithiotis et al. (2006) reported the
ethanol contents to be nd-0.41 mmol/g in the sourdoughs produced with different
yeast and LAB cultures and the highest ethanol contents were observed in the
sourdoughs produced with yeast mono-cultures, as ethanol is the main yeast
metabolite in addition to being a product of heterofermentative metabolism.
Minervini et al. (2012a) reported the ethanol content of the sourdoughs used for
traditional Italian breads in the range of 0.05-0.50 M. In this study, the ethanol
content ranged between 0.10-0.32 M, in terms of molarity, which was in agreement
with previous studies.
104
The carbohydrate concentration in dough fermentations varies depending

on the activity of cereal enzymes and the flora. Flour contains different sources of
fermentable carbohydrates such as maltose, sucrose, glucose and fructose (Chavan
and Chavan, 2011). During dough fermentation, sucrose is rapidly coverted into
glucose and fructose, and maltose is hydrolysed to glucose by the endogenous
enzymes in the flour and microbial enzyme activity (Swanson, 2008). Although the
concentration of fermentable carbohydrates in wheat and rye flours is relatively
low, wheat and rye contain about 60–70% starch and starch degradation at the
dough stage is the predominant source of fermentable carbohydrates and reducing
sugars (Ganzle, 2014a; Struyf et al., 2017). therefore, higher levels of glucose,
fructose and maltose are detected compared with wheat flour (Codina et al., 2013).
During fermentation, fermentable carbohydrates are consumed and their content is
decreased. As it can be seen, the carbohydrate content in doughs is directly related
to the metabolism of the sourdough microbiota and activity of cereal enzymes.
4.1.2. Microbiological Characteristics of Sourdough Samples

Cell densities of LAB, yeasts, total mesophilic aerobic bacteria and molds
in the 8 sourdough samples studied are shown in Table 4.6.
Table 4.6. Mean values of cell counts (log CFU/g) of sourdough samples on
different media
LAB Yeasts TMAB Molds
Samples mMRS gM17 SDB YPD L- lysine LAB/ PCA MXA
yeasts*
SD-M1 9.15 8.93 9.15 7.58 7.41 37:1 9.08 <1
SD-M2 10.99 10.48 10.87 6.75 6.97 8313:1 10.37 <1
SD-T1 7.39 6.83 6.73 8.87 8.22 0.02:1 6.90 <1
SD-T2 10.89 4.78 10.82 7.54 7.85 13:1 7.78 <1
SD-K1 6.47 7.23 11.84 6.97 3.59 1693:1 6.83 <1
SD-K2 5.60 5.32 11.96 7.69 2.70 271:1 4.54 2.2
SD-R1 11.67 10.29 11.59 10.02 8.02 145:1 11.48 <1
SD-R2 9.42 7.22 9.99 6.97 6.78 100:1 7.16 <1
*LAB and yeasts ratio was calculated according to the mean counts of three and two
different media for LAB and yeasts, respectively.
105
4.1.2.1. Presumptive LAB Counts

The cell counts of presumptive LAB varied from 4.78 to 11.96 log CFU/g
on different media. The highest cell density on mMRS agar was 11.67 log CFU/g
in the rye sourdough sample, whereas the lowest cell density on mMRS agar was
5.60 in the SD-K2 sample. On the other hand, the highest cell count on SDB agar
was in that sample at 11.96 log CFU/g. The lowest and highest cell counts on
gM17 medium were 4.78 and 10.48 log CFU/g in the SD-T2 and SD-M2,
respectively. Plate counts using three culture media showed different values of
presumptive LAB, as shown in Figure 4.4. In the present study, the highest cell
densities were counted on the SDB agar medium. Furthermore, other studies have
reported higher cell counts on SDB medium (Minervini et al., 2012a; Lhomme et
al., 2015; Ventimiglia et al., 2015). Apart from the culture medium, the 25th,
median and 75th percentiles of the plate counts were determined to be 6.93, 9.29
and 10.88 log CFU/g, respectively. According to the culture medium, median and
mean cell densities were 9.28-8.95, 7.23-7.63 and 10.85-10.37 log CFU/g as
enumerated on mMRS, gM17 and SDB, respectively. These media contain
different nutrients and the differences in cell densities on different media can be
related to the qualitative and quantitative differences among media in terms of
nutrients and also different metabolic capacities among strains harboured in each
sourdough (Minervini et al., 2012a; Lattanzi et al., 2013). Minervini et al. (2012a)
reported median plate counts of presumptive LAB in four culture media as 9.01 log
CFU/g. Lhomme et al. (2015) determined median values of LAB cell densities in
French sourdoughs to be 6.2, 9.2 and 8.2 log CFU/g as enumerated on mMRS,
SDB, and MRS5, respectively. In another study on Italian sourdoughs, the cell
density of LAB varied from 6.3 to 9.2 log CFU/g with the median value of 8.05 log
CFU/g (Lattanzi et al., 2013). Ventimiglia et al. (2015) reported the cell counts of
15 sourdoughs produced in southern Italy in the range of 4.9-8.9, 4-8.5 and 4.5-9
log CFU/g as enumerated on MRS, M17 and SDB media, respectively. Palla et al.
106
(2017) determined the LAB counts on mMRS and SDB media to be 9.37 and 9.01
log CFU/g, respectively.
Figure 4.4. Presumptive LAB counts of sourdoughs on different media
4.1.2.2. Presumptive Yeast Counts

The cell counts of presumptive yeasts varied from 6.75 to 10.02 log CFU/g
and 2.70 to 8.22 log CFU/g on YPD and L-lysine media, respectively. The highest
cell density on YPD agar was counted in the rye sourdough sample, whereas the
second sampling of rye sourdough showed lower yeast counts. Plate counts using
two culture media showed different values of presumptive yeasts due to the
qualitative and quantitative differences among media in terms of nutrients (Figure
4.5). Regardless of the culture medium, median and mean yeast counts were
determined to be 7.48 and 7.12 log CFU/g.
In the study of Minervini et al. (2012a), malt extract and Sabouraud
dextrose agar counts showed difference and regardless of the culture medium, the
median value was reported as 7.30 log CFU/g. In another study, cell density of
yeasts in traditional French sourdoughs was reported between 4.7-7.6 log CFU/g
107
with the median value of 6.5 log CFU/g (Lhomme et al., 2015). The median value
of the cell density of yeasts was 7.03 log CFU/g in a study conducted on Italian
sourdoughs (Lattanzi et al., 2013). Yeast cells were present at concentrations
ranging from 5.03 to 8.61 CFU/g in traditional Italian wheat sourdoughs (Valmorri
et al., 2010).
Figure 4.5. Presumptive yeast counts of sourdoughs on different media
The ratio between LAB and yeasts of the sourdough exhibited a wide
variation. The LAB/yeast ratio was in the range of 0.02:1-8313:1. Similar results
were obtained in other studies (Valmorri et al., 2010; Minervini et al., 2012a).
4.1.2.3. Enumeration of other Microorganisms

In the present study, mold, total mesophilic aerobic bacteria and
presumptive coliform counts of the collected sourdough samples were investigated.
Total mesophilic aerobic bacteria counted on PCA were in the range of 4.54-11.48
log CFU/g with the median value 7.47 log CFU/g as shown in Table 4.5. The rye
108
sourdough sample showed the highest bacterial count on PCA, which is in

agreement with other counts of this sample. Total bacteria counts were in
agreement with other studies (Saeed et al., 2009; Yagmur et al., 2016). Except SD-
K2 sample, mold was not detected on the plates. For coliform, all of the tubes gave
negative results (<3MPN/g).
Cell densities of LAB, yeasts, total mesophilic aerobic bacteria, molds and
coliform bacteria were counted in the laboratory produced sourdough sample with
daily back-slopping at 28°C. Microbial counts were determined at 0,1, 2, 4 and 7 d
back-slopping days. Counts on mMRS, gM17, SDB, YPD, L-lysine and PCA
media are shown in Figure 4.6.
Figure 4.6. Cell counts on mMRS, gM17, SDB, YPD, L-lysine and PCA media of
the laboratory scale sourdough sample
109
Presumptive LAB counts on three different media, namely mMRS, gM17

and SDB, at the first sampling in unfermented dough were determined to be <1,
3.11 and 2.47 log CFU/g, respectively. LAB counts increased rapidly following the
next refreshment step in all three media. LAB counts on three different media on
the following days were in consistent with each other and reached 12, 11.97 and
9.85 log CFU/g at the end of the fermentation on mMRS, gM17 and SDB,
respectively. LAB counts of the present study were higher than previously reported
studies. High LAB counts in the present study could be related to the high
inoculum level, incubation temperature and refreshment time compared with other
studies. In a study, LAB counts of a sourdough fermentation produced with
backslopping during 10 days at different temperatures were reported (Vrancken et
al., 2011). The backslopping procedure was repeated during 10 days by inoculating
800 grams of ripe sourdough into a 7 kg water-flour mixture. LAB counts at 23 and
30°C with backslopping every 24 h were determined to be around 9 log CFU/g at
the last refreshment. Van der Meulen et al. (2007) investigated the microbial counts
of laboratory sourdough fermentations (30°C) propagated over a period of 10 days
with daily backslopping, without the addition of a starter culture, and reported low
LAB and yeast counts at the beginning of the fermentation that were in agreement
with our study. In their study, LAB counts rapidly increased, as shown in the
present study, and reached 8–9 log CFU/g at the last backslopping; however, final
LAB counts were less than the counts reported in this study (Van Der Meulen et
al., 2007). The backslopping procedure was repeated by inoculating 800 grams of
ripe sourdough into a 7.2 kg water-flour mixture. As it can be seen, the studies used
a sourdough inoculum of around 10% for the refreshment of the next batch.
However, in our study, a 20% inoculum was used for the refreshment, which
resulted in higher LAB counts. Minervini et al. (2012b) produced sourdoughs at
artisan and bakery levels by daily backslopping. Inoculum ratios were around 6–
30%. LAB counts were between 6.5–8.5 log CFU/g at the 20th and 80th day of
backslopping. The backslopping times were between 3-13 hours at 24–28°C and
110
less backslopping resulted in less LAB counts when compared to this study. In
another study, rye and wheat sourdoughs were propagated with a 25% inoculum
and cell densities of presumptive LAB reached values of more than 9 log CFU/g
for wheat sourdoughs (Ercolini et al., 2013). The inoculum of the study was close
to the inoculum level in the present study; however, the propagation time and
temperature was 5 h at 25°C, which was less than used in this study.
In the present study, total presumptive yeast counts on YPD medium were
below 4 log CFU/g at the beginning and showed fluctuations on the following
days. The yeast population decreased at the first refreshment day but then increased
again on the following days. At the last refreshment day, the yeast count was
determined to be 6.6 log CFU/g. For non-Saccharomyces yeasts, the microbial
count patterns were different. Until the last refreshment step, L-lysine counts were
<1 log CFU/g and 7th backslopping day the count was determined as 5.85 log
CFU/g. Van Der Meulen et al. (2007) reported the slow growth of yeasts compared
with LAB. In some fermentations, the yeast population started to develop after 8
days of back-slopping. Final yeast counts in terms of logaritmic colony forming
units were reported to be in the range of 5.95 and 7.53 log CFU/g (Van Der Meulen
et al., 2007). Vrancken et al. (2011) reported the yeast counts in a sourdough
fermentation with back-slopping during 10 days at different temperatures. Yeast
counts at 23 and 30°C with back-slopping every 24 h were determined to be around
7 and 8 log CFU/g at the last refreshment, respectively. Yeast counts were around 4
log CFU/g at the first refreshment and then increased (Vrancken et al., 2011).
Ercolini et al. (2013) reported the yeast count of wheat sourdough to be more than
4 log CFU/g at the beginning of sourdough production. It decreased on the 2nd day
of refreshment, but then continued to increase and reached more than 6 log CFU/g
on the 11th day of refreshment.
Total mesophilic aerobic bacteria counts on PCA medium were determined
to be 4.78 log CFU/g in the unfermented dough and then increased to 11.58 log
CFU/g at the 4th refreshment step of the sourdough On the other hand, counts
111
decreased to 9.6 log cfu/g on the last refreshment day. Mold and coliform bacteria
counts are shown in Table 4.7. As it can be seen, no molds and presumptive
coliform group bacteria were detected after the 2nd and 3rd refreshment steps,
respectively. Indole testing of the coliform bacteria were negative.
Table 4.7. Mold and coliform bacteria

Refreshment day Mold counts Presumptive total coliform
(log CFU/g) (MPN/g)/Indole test
0 day 2 23/-
1 day 1.5 20/-
2 day <1 9.2/-
4 day <1 <3
7 day <1 <3
4.1.3. Multivariate statistical analysis of the sourdough samples

The microbiological and chemical parameters of sourdoughs were
subjected to the multivariate analysis to evaluate the differences/variabilities
among the samples. Agglomerative hierarchical clustering (AHC) classified the
trials in accordance to their mutual dissimilarity and relationship and two main
mega-clusters were generated basically with the cut-off line 0.789111 as shown in
Figure 4.7. According to the AHC analysis, M1, M2 and T1 samples were included
in a group. Laboratory produced sourdough, SD-L7, and SD-T2 samples were in
another group. On the other hand, R and K sourdoughs were included as a one
group and clustered separately from the other samples. Both sampling of the each
sourdough belong to R, K and M bakeries were sorted into the same group as
expected. On the other hand, SD-T1 and SD-T2 sourdoughs were clustered in
different roots. As reported in the previous sections, properties of the SD-T
samples were different in the second sampling since processing conditions
changed. Laboratory produced sourdough was clustered with SD-T2 sample.
112
Figure 4.7. Dendrogram resulting from hierarchical cluster analysis on 13 variables

determined on sourdoughs
Data of the microbiological and chemical inputs of sourdough samples

were also subjected to principle component analysis (PCa) to expresss the
important information as principal components. Four eigen-values were higher than
1 and correspond to 88.59% of the variance. The eigenvalues and the
corresponding factors by descending order with the variability they represent is
shown in Figure 4.8.
Factor 1 and Factor 2 explained 35.50 and 24.48% of total variability,
respectively. A large part of the variability was taken into account by the two first
axes since the percentage of variability represented by these two factors was
59.99% of total variability as shown in Figure 4.8. Correlation matrix (Pearson) is
shown in Table 4.8.
113
Figure 4.8. The eigenvalues and the corresponding factors by descending order
with the variability they represent
Correlation circle (Figure 4.9A) composed of two distinct groups with

regards to F1. The first group include acetic acid, TTA, lactic acid and SDB which
were positively correlated. The second group contained other variables. Factor 2
has a lower incidence (24.48%) than Factor 1 but it was possible to explain rest of
the variables as two district groups with regards to F2. The score plot (Fig. 4.9B)
clearly shows the far distance among the sourdough samples collected from
different bakeries. As it can be seen, close relation was found between SD-K1 and
SD-K2 sourdoughs which were mainly characterized by acetic acid. Along Factor
1, SD-K, SD-T2 and SD-L7 sourdoughs differed from first sampling of the other
sourdoughs. SD-L7, laboratory produced sourdough, was differed from the
sourdoughs mainly along Factor 2 which has a lower incidence (24.48%) than
Factor 1.
114
Figure 4.9. Loading plot (A) and score plot (B) resulting from principal component
analysis of variables determined on sourdoughs
115
Table 4.8. Correlation matrix (Pearson (n)) of the variables

maltose+ lactic acetic
Variables MRS YPD M17 SDB PCA pH TTA sucrose glucose fructose acid acid ethanol
MRS 1 0.022 0.640 0.003 0.857 -0.389 -0.318 -0.182 0.046 0.384 -0.032 -0.573 0.364
YPD 0.022 1 -0.054 -0.142 0.223 0.535 -0.093 0.236 0.416 0.656 -0.570 -0.382 -0.288
M17 0.640 -0.054 1 -0.043 0.809 -0.165 -0.051 -0.080 0.337 0.309 0.274 -0.547 0.407
SDB 0.003 -0.142 -0.043 1 0.014 -0.059 0.594 -0.820 -0.582 -0.067 0.189 0.465 -0.273
PCA 0.857 0.223 0.809 0.014 1 -0.031 -0.290 0.015 0.355 0.505 -0.114 -0.601 0.312
pH -0.389 0.535 -0.165 -0.059 -0.031 1 -0.059 0.418 0.617 0.567 -0.420 0.064 -0.596
TTA -0.318 -0.093 -0.051 0.594 -0.290 -0.059 1 -0.675 -0.303 -0.118 0.710 0.485 -0.557
Maltose+sucrose -0.182 0.236 -0.080 -0.820 0.015 0.418 -0.675 1 0.722 0.083 -0.470 -0.350 0.194
Glucose 0.046 0.416 0.337 -0.582 0.355 0.617 -0.303 0.722 1 0.600 -0.226 -0.568 -0.134
Fructose 0.384 0.656 0.309 -0.067 0.505 0.567 -0.118 0.083 0.600 1 -0.350 -0.543 -0.405
Lactic acid -0.032 -0.570 0.274 0.189 -0.114 -0.420 0.710 -0.470 -0.226 -0.350 1 0.354 -0.161
Acetic acid -0.573 -0.382 -0.547 0.465 -0.601 0.064 0.485 -0.350 -0.568 -0.543 0.354 1 -0.436
Ethanol 0.364 -0.288 0.407 -0.273 0.312 -0.596 -0.557 0.194 -0.134 -0.405 -0.161 -0.436 1
Values in bold are different from 0 with a significance level alpha=0
116
4.1.4. Biodiversity of the LAB and Yeasts in Sourdough Samples

A total of 439 LAB and 235 yeast isolates were collected from sourdough
samples including the laboratory-scale produced sourdough. 305 LAB and 181
yeast colonies were collected from bakeries and their distribution according to the
sourdough is shown in Figure 4.10. Number of LAB isolates from SD-M and also
SD-T samples were almost same at both sampling times.
Figure 4.10. Distribution of presumptive LAB and yeast isolates from collected
sourdoughs
In addition, 134 LAB and 54 yeast colonies were randomly picked from
different days of the laboratory scale production as shown in Figure 4.11. The
number of the collected LAB isolates was higher on the 4th and last day of the
refreshment of the sourdough than on the initial days.
117
Figure 4.11. Distribution of presumptive LAB and yeasts isolates of sourdough

produced under laboratory conditions
4.1.4.1. LAB Identification

A total of 305 colonies were picked from mMRS, gM17 and SDB media of
8 sourdough samples collected from bakeries. Furthermore, 134 colonies were
collected from different days of the fermentation of the sourdough sample
produced under laboratory conditions. All of the 439 presumptive LAB cultures
were subjected to microscopic inspection and Gram-stain and catalase tests. After
Gram-stain characterization and catalase testing, 389 strains were still considered
putative LAB cultures (Gram-positive and catalase-negative). All of the LAB
cultures were grown in the MRS or M17 broth 12-24 hours and subjected to DNA
extraction by using Instagene matrix kit. Then genomic DNA of the isolates were
subjected to RAPD analysis using M13 primer. Some strains showed weak band
profile and were eliminated for further analysis. Bands were evaluated according to
the DNA marker by using the Infinity gel documentation imaging system software.
Band patterns of RAPD-PCR profiles of 299 strains were scored as band absent (0)
or present (1) and data were entered into a binary matrix. The dissimilarity index
was calculated on the basis of the Jaccard coefficient generated with the DARwin
(6.0.15) software package. A dendrogram was also constructed based on the
118
genetic distances with the UPGMA method as shown in Figure 4.12. According to
the calculated genetic distance matrix, a total of 102 strains were chosen for
sequence analysis that had a genetic distance at the level 0.4≤. A gel image of the
RAPD-PCR analysis with M13 primer of the strains is attached to Appendix 8.
Figure 4.12. Dendrogram obtained from RAPD-PCR (M13 primer) band profiles of
LAB isolates in sourdough fermentations
Selected strains were subjected to 16s rRNA gene sequencing analysis.

Obtained sequences and their ABI chromatograms were examined using Bioedit
Sequence Alignment Editor 7.2.6. (Hall, 1999). The sequences more than 1400 bp
were compared by Basic Local Alignment Search Tool (BLAST,
https://blast.ncbi.nlm.nih.gov/Blast.cgi) with nucleotide sequences deposited at the
database National Center for Biotechnology Information (NCBI) (Altschul et al.,
1990). Sequences with at least 98% identity to the sequences of the closest relative
available within the NCBI database showed strains belonging to the same species.
Strains with less than 98% identity were identified at the genus (94%<) and family
(86%<) level (Yarza et al., 2014).
119
A total of 84 strains representing 178 isolates were confirmed to be

members of the LAB group with a sequence length of more than 1250 bp. Based on
the 16s rRNA sequence analysis, a total of 52 strains (1400 bp≤) representing 113
isolates were identified at the species level (98%≤). The identified strains along
with their accession numbers are given in Table 4.9.
Table 4.9. Identified LAB isolates at the species level (sequence length 1400 bp ≤)
(98% ≤) in sourdough samples
Similarity %
Number (accession number
of of closest relative by Sequence Accession
Strain isolates Species GenBank) length (bp) number
RL17 3 Lb. paralimentarius 99 (NR_114844.1) 1497 MH704092
XL23 1 Lb. plantarum 99 (NR_104573.1) 1485 MH704093
BL45 2 Lb. paraplantarum 98 (NR_025447.1) 1490 MH704095
RL164 1 Lb. plantarum 98 (NR_104573.1) 1558 MH704096
RL177 2 Lb. brevis 99 (NR_116238.1) 1489 MH704098
RL214 1 Lb. acidophilus 98 (NR_117062.1) 1566 MH704099
RL227 2 Lb. paracasei 98 (NR_025880.1) 1479 MH704100
BL631 5 Lb. sanfranciscensis 99 (NR_116285.1) 1521 MH704102
BL635 1 Lb. plantarum 98 (NR_104573.1) 1523 MH704103
XL640 2 Lb. pentosus 98 (NR_029133.1) 1508 MH704104
RL658 10 Lb. sanfranciscensis 98 (NR_029261.2) 1527 MH704105
RL670 1 Pd. inopinatus 98 (NR_025388.1) 1499 MH704106
BL734 1 Lb. paralimentarius 98 (NR_114844.1) 1548 MH704107
BL740 2 Lb. paralimentarius 98 (NR_114844.1) 1506 MH704109
BL741 4 W. confusa 98 (NR_113258.1) 1485 MH704110
RL826 1 Lb. brevis 98 (NR_044704.2) 1544 MH704113
XL958 6 Lb. paracasei 98 (NR_025880.1) 1483 MH704117
XL959 1 E. faecium 98 (NR_114742.1) 1505 MH704118
BL969 1 Leu. citreum 99 (NR_041727.1) 1477 MH704120
RL975 1 Leu. citreum 98 (NR_041727.1) 1554 MH704122
120

RL1042 1 Lb. parabrevis 98 (NR_042456.1) 1499 MH704128
XL1542 1 Lc. lactis subsp. cremoris 98 (NR_040954.1) 1411 MH704130
RL1545 1 Leu. mesenteroides 98 (NR_074957.1) 1414 MH704131
RL1546 1 W.cibaria 99 (NR_036924.1) 1403 MH704132
RL1551 2 Lb. curvatus 98 (NR_042437.1) 1400 MH704133
XL1558 1 Leu. mesenteroides 98 (NR_074957.1) 1404 MH704134
BL1577 1 Leu. mesenteroides 98 (NR_074957.1) 1512 MH704135
BL1578 1 Lb. curvatus 98 (NR_113334.1) 1587 MH704136
RL1617 1 Lb. curvatus 98 (NR_113334.1) 1450 MH704138
RL1633 1 Leu. mesenteroides 98 (NR_074957.1) 1528 MH704141
BL1649 1 Pd. pentosaceus 98 (NR_042058.1) 1526 MH704143
32 strains representing 65 isolates were identified only at the genus (94%≤)

or family (86%≤) level as shown in Table 4.10.
121
Table 4.10. Identified LAB isolates at the genus (94%≤) or family level (86%≤) in
sourdough samples
Similarity %
(accession number
Number of of closest relative by Sequence Accession
Strain isolates Family/Genus GenBank) length (bp) Number
XL21 2 Lactobacillus spp. 97 (NR_104573.1) 1512 MH704197
BL47 3 Lactobacillus spp. 95 (NR_114844.1) 1489 MH704199
XL74 1 Enterococcus spp. 95 (NR_113933.1) 1489 MH704200
BL84 2 Lactobacillaceae 92 (NR_114844.1) 1562 MH704201
BL628 1 Lactobacillaceae 86 (NR_029261.2) 1472 MH704206
RL829 1 Lactobacillus spp. 94 (NR_044704.2) 1555 MH704207
RL835 1 Lactobacillus spp. 95 (NR_029261.2) 1437 -
RL839 1 Weissella spp. 96 (NR_113258.1) 1472 MH704209
RL1043 1 Lactobacillaceae 92 (NR_042456.1) 1501 MH704216
XL1531 4 Enterococcaceae 87 (NR_114742.1) 1255 MH704218
XL1532 2 Enterococcaceae 91 (NR_114742.1) 1406 -
RL1570 2 Weisella spp. 98 (NR_113258.1) 1362 MH704220
RL1622 2 Pediococcus spp. 95 (NR_042058.1) 1558 MH704222
In the present study, 113 strains belonging to 18 LAB species were

identified, as shown in Table 4.11. Lb. sanfranciscensis (32.7%) was the dominant
species, followed by Lb. plantarum (18.6%) and Lb. paralimentarious (15.9%). In
addition, Lb. paracasei (7.1%), Leu. mesenteroides (4.4%), W. confusa (3.5%), Lb.
curvatus (3.5%) and Lb. brevis (2.7%) were found to be minor species.
122
Furthermore, Lb. pentosus, Leu. citreum, Lb. paraplantarum, Lb. acidophilus, E.

faecium, Pd. inopinatus, Lb. parabrevis, Lc. lactis subsp. cremoris, W. cibaria and
Pd. pentosaceus were only isolated from 1 or 2 samples.
Table 4. 11. Percentage of the isolated LAB species in sourdoughs

Species Number of species %
Lb. sanfranciscensis 37 32.7
Lb. plantarum 21 18.6
Lb. paralimentarious 18 15.9
Lb. paracasei 8 7.1
Leu. mesenteroides 5 4.4
W. confusa 4 3.5
Lb. curvatus 4 3.5
Lb. brevis 3 2.7
Lb. pentosus 2 1.8
Leu. citreum 2 1.8
Lb. paraplantarum 2 1.8
Lb. acidophilus 1 0.9
E. faecium 1 0.9
Pd. inopinatus 1 0.9
Lb. parabrevis 1 0.9
Lc. lactis subsp. cremoris 1 0.9
W. cibaria 1 0.9
Pd. pentosaceus 1 0.9
Total 113 100%
The number of Lb. sanfranciscensis was 37 out of 113 strains, i.e. 1/3 of
the isolates were allotted to this species. Lb. sanfranciscensis was identified in all
of the sourdoughs, except SD-M1 and SD-R1. Lb. sanfranciscensis was detected in
T and K sourdough samples at both sampling times. On the other hand, it was only
isolated at the 2nd sampling from M and R sourdoughs as shown in Table 4.12. In
the laboratory produced sourdough, Lb. sanfranciscensis was not isolated during 7
days of fermentation. Lb. plantarum was detected in all collected sourdough
samples except SD-T1 and SD-R2 samples. In addition, it was determined on the
4th day of the laboratory-produced sourdough. Lb. paralimentarious was isolated
from all sourdoughs except the SD-K sample. It was also isolated at the 4th and 7th
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day of the laboratory-produced sourdough. Lb. paracasei was only found in the
SD-T samples at both sampling times. W. confusa was only identified in the SD-
M2 sample. Lb. brevis was isolated from both sampling of the SD-R sourdough.
Other isolated strains, Lb. pentosus, Leu citreum, Lb. paraplantarum, Lb.
acidophilus, E. faecium, Pd. inopinatus and Lb. parabrevis were minor species
isolated from different bakeries. Leu. mesenteroides, Lb. curvatus, Lc. lactis subsp.
cremoris, W. cibaria and Pd. pentosaceus strains were not isolated from the
collected samples and were only identified in the laboratory scale sourdough
production.
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Table 4.12. Number of LAB identified at the species level in sourdough samples
Species M1 M2 T1 T2 K1 K2 R1 R2 L1 L2 L4 L7
Lb. sanfranciscensis 2/12 1/7 10/23 18/22 4/14 2/6
Lb. plantarum 5/8 2/12 2/23 1/22 9/14 1/5 1/3
Lb. paralimentarious 1/8 4/12 3/7 2/23 2/5 3/6 1/3 2/3
Lb. paracasei 2/7 6/23
Leu. mesenteroides 1/4 3/6 1/3
W. confusa 4/12
Lb. brevis 2/5 1/6
Lb. curvatus 1/4 2/6 1/3
Lb. pentosus 2/22
Leu. citreum 2/23
Lb. paraplantarum 2/8
Lb. acidophilus 1/7
E. faecium 1/23
Pd. inopinatus 1/22
Lb. parabrevis 1/14
Lc. lactis subsp. 1/4
cremoris
W. cibaria 1/4
Pd. pentosaceus 1/6
Total LAB 8 12 7 23 22 14 5 6 4 6 3 3
125
The SD-M sample contained different species at both sampling times as

shown in Figure 4.13. In the SD-M1 sample, Lb. plantarum was detected as the
predominant species and Lb. paralimentarious and Lb. paraplantarum were
detected as minor species. On the other hand, Lb. paralimentarious and W. confusa
were co-dominant species in the SD-M2 sample and Lb. sanfranciscensis and Lb.
plantarum were found as minor species.
Figure 4.13. Species in SD-M sourdough samples at both sampling times
The SD-T sample contained Lb. sanfranciscensis, Lb. paralimentarious

and Lb. paracasei at both sampling times as shown in Figure 4.14. In the SD-T1
sample, Lb. paralimentarious and Lb. paracasei were detected as the predominant
species and Lb. sanfranciscensis and Lb. acidophilus were detected as minor
species. On the other hand, Lb. sanfranciscensis was the dominant species at the
second sampling. Lb. paracasei was also detected as the dominant species. The
minor species of the SD-T2 sourdough was Lb. plantarum, Lb. paralimentarious,
Leu. citreum and E. faecium.
126
Figure 4.14. Species in SD-T sourdough samples at both sampling times
The SD-K sample contained Lb. sanfranciscensis as the dominant species

at the first sampling as shown in Figure 4.15. In the SD-K1 sample, Lb.
sanfranciscensis was the predominant species and Lb. plantarum, Lb. pentosus and
Pd. inopinatus were detected as minor species. On the other hand, Lb. plantarum
was the dominant species at the second sampling. Lb. sanfranciscensis and Lb.
parabrevis were also detected in the SD-K2 sample.
Figure 4.15. Species in SD-K sourdough samples at both sampling times
Figure 4.16 shows the species of the rye sample at two different sampling
times. Rye sourdough contained Lb. paralimentarious as the dominant species at
127
both sampling. Lb. paralimentarious and Lb. brevis were co-dominant species and
Lb. plantarum was the minor species in the SD-R1 sample. In the SD-R2 sample,
the dominant species were Lb. paralimentarious followed by Lb. sanfranciscensis
and Lb. brevis.
Figure 4.16. Species in SD-R sourdough samples at both sampling times
In laboratory produced sourdough, LAB identified at the species level were

Leu. mesenteroides, Lb. curvatus, Lc. lactis subsp. cremoris and W. cibaria in the
first refreshment of the sourdough. Following refreshment, Leu. mesenteroides and
Lb. curvatus were detected but Lc. lactis subsp. cremoris and W. cibaria were not
isolated. Pd. pentosaceus was also detected at the second refreshment. At the 4th
refreshment, the LAB detected at the species level were Lb. plantarum, Lb.
paralimentarious and Lb. curvatus. On the last day of refreshment, Lb.
paralimentarious and Leu. mesenteroides were isolated. The number of identified
species is shown in Figure 4.17. In the sourdough sample produced on laboratory
scale, the well-known wheat sourdough LAB, Lb. sanfranciscensi, was not
detected.
128
Figure 4.17. The number of identified LAB species in laboratory scale production
In the present study, the sourdough propagation was continued once in a 24

h during 7 days. However, the well-known wheat sourdough Lb. sanfranciscensis
was not detected in any of the refreshments. The results of the present study
conﬁrm the results obtained from some other studies related to laboratory
sourdough fermentations (Van Der Meulen et al., 2007; Vrancken et al., 2011).
Vrancken et al. (2011) reported the absence of Lb. sanfranciscensis in laboratory
sourdough fermentations performed under semi-sterile conditions and hypothesized
the non-flour origin of this species. However, wheat LAB was monitored from ear
harvest until the first step of fermentation and Lb. sanfranciscensis wasidentified as
the only from durum wheat semolina (Alfonzo et al., 2013) but not from the ear,
kernel, dough and also semolina in another monitoring study (Alfonzo et al., 2017).
Minervini et al. (2012a) investigated the laboratory and artisan propagated
sourdoughs and reported the LAB flora difference between artisan bakery and
laboratory levels. In their study, strains of Lb. sanfranciscensis were found in some
129
sourdoughs. Results of the study showed the undoubted influence of the

refreshment environment on the composition of sourdough yeast and LAB.
Figure 4.18. Distribution of the LAB strains at the family level in the sourdough
fermentations
LAB detected in the sourdough samples at the family level are shown in
Figure 4.18. The isolated strains from sourdough samples belonged to four
families, i.e., Lactobacillaceae, Enterococcaceae, Leuconostocaceae and
Streptococcaceae. As it can be seen, many of the isolated strains belonged to the
Lactobacillaceae family. Among 178 isolates, the number of strains in the
Lactobacillaceae, Enterococcaceae, Leuconostocaceae and Streptococcaceae
families were 145, 17, 15 and 1, respectively. Distribution as a percentage is given
in Figure 4.19.
130
Figure 4 19. Frequency of the strains at the family level
LAB detected in the sourdough samples at the genus level are shown in
Figure 4.20. The isolated strains from sourdough samples belonged to 6 genera,
i.e., Lactobacillus, Pediococcus, Enterococcus, Leuconostoc, Weissella and
Lactococcus. As it can be seen, many of the isolated strains were Lactobacillus
spp. Totally 166 strains were identified at the genus level and number of the
Lactobacillus, Enterococcus, Weissella, Leuconostoc, Pediococcus and
Lactococcus spp. were 135, 11, 8, 7, 4 and 1, respectively. Distribution as a
percentage is given in Figure 4.21.
131
Figure 4. 20. Distribution of the LAB strains at the genus level in the sourdough
fermentations
Figure 4.21. Frequency of the strains at the genus level

132
In the present study, 81% of the identified strains belonged to the genus
Lactobacillus spp. Microbial patterns of the sourdoughs collected from different
bakeries differed between each other. The predominant LAB species Lb.
sanfranciscensis mainly dominated the sourdough ecosystem. As reported
previously, this species is well known in natural sourdough habitats of the artisan
and industrial bakeries (Kline and Sugihara, 1971; Corsetti et al., 2001; Meroth et
al., 2003; Siragusa et al., 2009; Vrancken et al., 2011; Venturi et al., 2012;
Lhomme et al., 2016). In both rye and wheat sourdoughs produced with continuous
propagation by back-slopping procedures, Lb. sanfranciscensis was reported as
probably the most adapted species in the sourdough microbiota (Gobbetti and
Corsetti, 1997; Vogel et al., 2002; Vogel et al., 2011). As reported previously, its
good adaptation to the sourdough environment can be related to the utilization of
sourdough carbohydrates, activated proteolytic enzymes and synthesis of
antimicrobial compounds (Gobbetti and Corsetti, 1997; Corsetti et al., 2001;
Lattanzi et al., 2013). Lb. sanfranciscensis was followed by Lb. plantarum and Lb.
paralimentarius. Minervini et al. (2012a) investigated the microbiota of 19 Italian
sourdoughs and the most frequent LAB isolates belonged to Lb. sanfranciscensis,
Lb. plantarum and Lb. paralimentarius accounting for 28, 16 and 14% of the total
LAB isolates (Minervini et al., 2012a). As reported previously, Lb. plantarum is
associated with Lb. sanfranciscensis in sourdoughs (Gobbetti, 1998). Lb.
paralimentarius has been frequently reported in many sourdoughs (Cai et al., 1999;
Minervini et al., 2012a; Taccari et al., 2016). Many sourdoughs contain
associations of different hetero- and homofermentative LAB strains. It was
reported that homofermentative LAB dominate in spontaneous fermentation
processes and heterofermentative species drive sourdough fermentation processes
produced via back-slopping (De Vuyst and Neysens, 2005). Most
heterofermentative LAB, especially Lactobacillus spp., occur in stable sourdough
ecosystems and the facultatively heterofermentative Lb. paralimentarius also
seems to be optimally adapted to the sourdough ecosystem (Huys et al., 2013).
133
Other less predominant LAB species, including members of the genera Weissella,
Pediococcus, Leuconostoc, Lactococcus, Enterococcus and Streptococcus can be
found in sourdoughs but at lower levels than Lactobacillus spp. (Corsetti and
Settanni, 2007). According to Corsetti et al. (2007b), different non-Lactobacillus
species are mainly found at the initial stages of sourdough production to prepare
the environment for the growth of typical species of mature sourdoughs. In this
study, besides the dominant Lb. sanfranciscensis, Lb. plantarum, Lb.
paralimentarius species, also Lb. paracasei, Leu. mesenteroides, W. confusa, Lb.
curvatus and Lb. brevis were found as minor species. Other less frequently isolated
species were Lb. pentosus, Leu. citreum, Lb. paraplantarum, Lb. acidophilus, E.
faecium, Pd. inopinatus, Lb. parabrevis, Lc. lactis subsp. cremoris, W. cibaria and
Pd. pentosaceus in our study. The presence of isolated species in sourdoughs has
been reported in other studies (Vogel et al., 1994; Corsetti et al., 2004; Gül et al.,
2005; Iacumin et al., 2009; Minervini et al., 2012a; Rossi et al., 2012; Amari et al.,
2013; Settanni et al., 2013; Rizzello et al., 2014; Lhomme et al., 2015; Yagmur et
al., 2016; Alfonzo et al., 2017; Bartkiene et al., 2017). However, the species
distribution and the dominant flora vary in the collected sourdough samples and
showed the importance of the environment on the sourdough ecosystem. According
to De Vuyst et al. (2017), sourdough ecosystem can contain a simple microflora
characterized by Lb. plantarum or Lb. sanfranciscensis or a restricted LAB species
diversity or with a complex microbial consortium including different LAB species
generally less than three species. Besides geographical origin, sampling, isolation,
and identification techniques are also important in the estimation of the sourdough
ecosystem. However, the flour type, quality and the process parameters such as
fermentation temperature, pH and pH evolution, dough yield, water
activity, oxygen tension, back-slopping procedure and fermentation duration
directly determine the dynamics and outcome of backslopped sourdough
fermentation processes
134
Other species belonging to the genera Enterococcus, Lactococcus,

Leuconostoc, Pediococcus, Streptococcus and Weissella are generally determined
at lower levels compared with Lactobacillus spp. as shown in the present study
(Corsetti and Settanni, 2007). Corsetti et al. (2007b) reported the occurence of
different non-Lactobacillus species during the first stages of sourdough
fermentations, which prepare the environment for the establishment of typical
species.
Figure 4. 22. Distribution of species in the laboratory sourdough production
In the present study, identified LAB in the sourdough produced at

laboratory scale belonged to 6 genera including Lactobacillus, Enterococcus,
Lactococcus, Leuconostoc, Pediococcus and Weissella. The distribution of the
genera in the laboratory sourdough production is shown in Figure 4.22. Van Der
Meulen et al. (2007) reported the occurence of LAB species that were not speciﬁc
for sourdough from the beginning until the 2nd day of the sourdough fermentation.
4.1.4.2. Phylogenetic Relation of the LAB Strains

Phylogenetic trees were constructed based on the 16S rRNA gene
sequences (1400 bp≤) of the identified strains at the species level using two
135
possible tree reconstruction methods, minimum evolution and UPMGA, in MEGA

7 (Kumar et al., 2016).
100 Lb. paralimentarius RL833

99
Lb. paralimentarius RL1628
82
Lb. plantarum RL1624
67 Lb. paralimentarius BL734
Lb. paralimentarius BL740
8 99 Lb. brevis RL826
100 Lb. curvatus BL1578
2 67 Lb. plantarum RL1046
32 Lb. sanfranciscensis RL976

Leu. mesenteroides XL1558
43 Lb. curvatus RL1617

3
Lb. sanfranciscensis RL658
0
23 Lb. sanfranciscensis BL1023
100 Lb. plantarum BL635

100 Lb. plantarum RL749
28 Lb. brevis RL177

Lb. paraplantarum BL45

E. faecium XL959
12
Lc. lactis subsp. cremoris XL1542
12
Lb. pentosus XL640
15
57 Lb. acidophilus RL214
100 W. confusa BL741

Lb. sanfranciscensis BL631
0 58 Leu. citreum RL975
W. cibaria RL1546
4 Pd. inopinatus RL670

Lb. curvatus RL1551
24
93
100 Lb. paracasei XL958
100 Lb. plantarum XL963

100 Pd. pentosaceus BL1649
48
14 Lb. paracasei RL227
Lb. plantarum XL24
100 Leu. mesenteroides RL1545
94 Lb. parabrevis RL1042

0 34 Leu. mesenteroides BL1577
Leu. citreum BL969
99 Leu. mesenteroides BL1579
5 42 Lb. plantarum XL170
Leu. mesenteroides RL1633

Lb. plantarum BL735
100
Figure 4.23. Evolutionary relationships of taxa by using the Minimum Evolution

method
136
The evolutionary relationships of taxa that was constructed using the

Minimum Evolution method is shown in Figure 4.23 (Rzhetsky and Nei, 1992).
The bootstrap consensus tree inferred from 500 replicates is taken to represent the
evolutionary history of the taxa analyzed. Branches corresponding to partitions
reproduced in less than 50% bootstrap replicates are collapsed. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test
(500 replicates) are shown next to the branches (Felsenstein, 1985). The
evolutionary distances were computed using the number of differences method
(Nei and Kumar, 2000) and are in the units of the number of base differences per
sequence. The analysis involved 52 nucleotide sequences. All ambiguous positions
were removed for each sequence pair.
The second evolutionary history was inferred using the UPGMA
(Unweighted Pair Group Method with Arithmetic Mean) method as shown in
Figure 4.24 (Sneath and Sokal, 1973). The bootstrap consensus tree inferred from
500 replicates is taken to represent the evolutionary history of the taxa analyzed
(Felsenstein, 1985). Branches corresponding to partitions reproduced in less than
50% bootstrap replicates are collapsed. The evolutionary distances were computed
using the number of differences method (Nei and Kumar, 2000) and are in the units
of the number of base differences per sequence. The analysis involved 52
nucleotide sequences. All ambiguous positions were removed for each sequence
pair.
137

100
94
Lb. plantarum RL1624
93 Lb. paralimentarius BL734
Lb. paralimentarius BL740

100 Lb. curvatus BL1578
Lb. curvatus RL1617
25 90
Lb. plantarum XL170
13
96
100
4
1
Lb. brevis RL177
0
Lb. paraplantarum BL45
0 9 Lb. plantarum RL164

26 E. faecium XL959
11
Lc. lactis subsp. cremoris XL1542
16 Lb. pentosus XL640
0 Lb. acidophilus RL214
44
100 W. confusa BL741

100 Pd. pentosaceus BL1649
48
Lb. paracasei RL227
2 98 Leu. citreum BL969

0
Leu. mesenteroides BL1579
91 Lb. parabrevis RL1042

7
15

49 Leu. mesenteroides XL1558
Leu. citreum RL975
45 W. cibaria RL1546
Lb. curvatus RL1551
48
98
100 Lb. paracasei XL958
Pd. inopinatus RL670
Figure 4.24. Evolutionary relationships of taxa by using the UPMGA method
138
4.1.4.3. Yeast Identification

A total of 181 presumptive yeast colonies were picked from YPD and L-
lysine media of 8 sourdough samples collected from bakeries. In addition, 54
colonies were collected from laboratory scale sourdough production. All of the 235
presumptive yeast cultures were grown in YPD medium for 24-36 hours and
subjected to DNA extraction using Instagene matrix kit. Before extraction, all of
the isolated yeasts were treated with lyticase enzyme to degrade the cell walls. In
total, 205 genomic DNA was extracted and subjected to 5.8S ITS rRNA region
amplification using primers ITS1 and ITS4. A gel image of the 5.8S ITS rRNA
region amplification is attached to Appendix 9. PCR products showing visible
bands on the agarose gel were subsequently digested using the restriction
endonucleases Hae III, Hha I and Hinf I. A gel image of the RFLP with restriction
endonucleases Hae III, Hha I and Hinf I is attached to Appendix 10. A total of 7
profiles were determined according to the restriction fragments as shown in Table
4.13. Strains exhibited a unique restriction pattern for each species with the three
endonucleases used.
Figure 4. 25. Distribution of the genera of the identified yeast isolates
139

gene sequencing as shown in Table 4.14. For a species-level identification,
identity more than 99% with the sequence length at least 400 bp was selected
(Romanelli et al., 2010). Seven species were identified that belonged to 4 genera,
i.e., Saccharomyces, Hanseniaspora (H’spora), Pichia and Kazachstania, as shown
in Figure 4.22. One species was identified in the genera Saccharomyces and
Hanseniaspora. On the other hand, two and three species belonged to the genera
Pichia and Kazachstania, respectively.
140
Table 4.13. Restriction fragments of the identified yeast species from sourdoughs
PCR Restriction fragments (bp)
RFLP products
Profile Species (bp) Hae III Hha I Hinf I
I S. cerevisiae 880 315+240+180+145 385+365+130 390+130
II P. membranifaciens 500 320+90+50 175+110+90 275+200
III K. servazzii 750 320+240+190 320+200+150 360
IV P. fermentans 450 340+80+30 170+100+80 250+200
V H'spora valbyensis 750 750 630+120 260+215+175+100
VI K. bulderi 700 500+200 350+280 300+250+130
VII K. unispora 750 300+220+180 310+200+160 385+360
141
Table 4.14. Accession numbers of the identified yeast species with their closest relatives and type strains
Closest relative Type strain
RFLP Accession Accession number / Accession Divergent
N1 bp3
Profile Species Strain2 number Identity(%)4 number/ bases6
Identity(%)5
S. cerevisiae S. cerevisiae
I S. cerevisiae 111 PM 1 MH704179 598 SFM35 NRRL Y-12632 7
MG017576.1/99 NG_042623.1/99
P. membranifaciens P.
NM CBS:598 membranifaciens
II P. membranifaciens 8 MH704180 600 4
1004 KY108894.1/99 NRRL Y-2026
NG_042444.1/99
K. servazzii NRRL K. servazzii NRRL

PM 603 MH704181 614 5
III K. servazzii 7 Y-12661 Y-12661
PM 604 MH704182 624 3
NG_055029.1/99 NG_055029.1/99
P. fermentans
lhWW149 P. fermentans
PM 801 MH704183 594
IV P. fermentans 9 MF462777.1/100 NRRL Y-1619 5
NM 816 MH704184 603
P. fermentans A16 NG_055109.1/99 4
KM589463.1/99
142
Table 4.14 (Continued)

H'spora
H'spora valbyensis
NM 625 MH704185 586 valbyensis NRRL 3
V H'spora valbyensis 3 CBS:479
NM 626 MH704186 621 Y-1626 8
KY107857.1/99
NG_042630.1/99
K. bulderi PD-1 K. bulderi NRRL
VI K. bulderi 11 PM 190 MH704187 588 MG983971.1/99 Y-27203 2
NG_055022.1/99
K. unispora CBS
K. unispora
VII K. unispora 4 PM 617 MH704188 562 398 3
MG525064.1/99
NG_055027.1/99
1
Number of species 226S rRNA gene sequenced strain representing each RFLP profile, 3sequence lengh, 4Sequence identity in
the D1 ⁄ D2 region of isolates with species in the GenBank, 5Sequence identity in the D1 ⁄ D2 region of isolates with type
strain of the same species in the GenBank, 6Number of the divergent bases from type strain
143
In present study, 153 yeast strains belonging to 7 species were identified as

shown in Table 4.15. S. cerevisiae (72.5%) was the dominant yeast species. Other
isolated yeast species were K. bulderi (7.2%), P. fermentans (5.9%), P.
membranifaciens (5.2%), K. servazzii (4.6%), K. unispora (2.6%) and H'spora
valbyensis (2%).
Table 4.15. Percentage of the isolated yeast species in sourdoughs

Species Number of the species %
S. cerevisiae 111 72.5
K. bulderi 11 7.2
P. fermentans 9 5.9
P. membranifaciens 8 5.2
K. servazzii 7 4.6
K. unispora 4 2.6
H'spora valbyensis 3 2.0
Total 153 100%
The distribution of yeast species among sourdough samples is shown in

Table 4.16. S. cerevisiae was the only species identified in SD-M sourdoughs at
both sampling times. S. cerevisiae was isolated from thr SD-T2 sample as the
dominant species. In addition, only one strain was determined as K. bulderi in the
SD-T2 sample. S. cerevisiae and K. bulderi co-dominated the first sampling of the
same bakery, SD-T1. In the SD-K1 sample, S. cerevisiae, H'spora valbyensis, K.
servazzii and K. unispora were co-dominant. On the other hand, S. cerevisiae and
P. membranifaciens were co-dominant in the SD-K2 sample. In rye sourdough, S.
cerevisiae was dominant at the first sampling. S. cerevisiae and P. fermentans co-
dominate the second sampling. In laboratory scale productions, none of the isolated
strains were identified as yeast at the first 2 days. On the other, S. cerevisiae was
isolated from 4 and 7 day refreshment.
144
Table 4.16. Distribution of the yeast species among sourdough samples

Yeast species M1 M2 T1 T2 K1 K2 R1 R2 L4 L7
S. cerevisiae 13/13 18/18 13/23 17/18 7/21 9/17 16/16 13/22 1/1 4/4
P. fermentans 9/22
K. bulderi 10/23 1/18
K. servazzii 7/21
P. membranifaciens 8/17
K. unispora 4/21
H'spora valbyensis 3/21
Total LAB 13 18 23 18 21 17 16 22 1 4
145
In the present study, yeast diversity was less than that of the LAB
microbiota, since only seven yeast species were identified in the collected
sourdoughs. This finding is in consistent with the literature (Minervini et al., 2015).
In the present study, S. cerevisiae was the most frequently isolated yeast species.
Collected sourdoughs were produced without using baker’s yeast. However S.
cerevisiae was isolated from all of the samples including laboratory produced
sourdough. As reported previously the presence of S. cerevisiae in the bakery
sourdoughs can be related to contamination of the bakery environment with
commercial baker’s yeast (Vrancken et al., 2010; Minervini et al., 2015). On the
other hand, baker’s yeast was also not used for laboratory production. However, S.
cerevisiae was detected at the 4th and 7th day of the refreshment steps. Flour
could have been a source of S. cerevisiae in the laboratory scale production
(Vrancken et al., 2010). Previous studies showed that S. cerevisiae is the most
reported yeast species in both wheat and rye sourdoughs (Vernocchi et al., 2004;
Vrancken et al., 2010; De Vuyst et al., 2016).
The SD-K sourdough sample showed a rich yeast biodiversity compared
with other sourdough samples, as illustrated in Figure 4.26. Isolates of this bakery
belonged to 4 different genera and 5 different species. Yeast species, determined at
two different sampling times, exhibited differences. S. cerevisiae, H'spora
valbyensis, K. servazzii and K. unispora were co-dominant in the SD-K1 sample
and S. cerevisiae and P. membranifaciens were co-dominant in the SD-K2 sample.
K. bulderi was identified in the SD-T sourdough samples. P. fermentans was only
determined in the second sampling of the rye sourdough. The dominant yeast
species in the collected sourdough samples was S. cerevisiae. However, the
presence of other yeast species differed between bakeries. There were 7 yeast
species over all 8 bakeries and 3 species representing 14.4% of all the identified
strains belonged to Kazachstania clade including K. servazzii, K. bulderi and K.
unispora. Kazachstania spp. have been previously isolated from sourdough and the
bakery environment (Vrancken et al., 2010; Minervini et al., 2012a; De Vuyst et
146
al., 2014; Lhomme et al., 2015; Minervini et al., 2015; De Vuyst et al., 2016;
Lhomme et al., 2016; Sarilar et al., 2017). As previously it was reported, S.
cerevisiae may not compete with other sourdough and bread dough yeast species
and Kazachstania spp. could be better adapted to these environments (Lhomme et
al., 2015). K. bulderi (formerly known as S. bulderi), detected from maize silage as
a novel species, was closely related to S. barnettii and S. exiguus (Middelhoven et
al., 2000). In 2016, it was isolated from French sourdough for the first time
(Lhomme et al., 2016). K.unispora (formerly known as S. unisporus) was
previously determined in sourdoughs (Vrancken et al., 2010; De Vuyst et al.,
2016). In the study of Vrancken et al. (2010), only one artisan sourdough isolate
was identified as K. unispora. K. unispora and K. servazzii (formerly S. servazzii)
have been reported in Italian sourdoughs (Di Cagno et al., 2014).
Figure 4.26. Distribution of yeast species in sourdough samples
Other yeast species P. membranifaciens, P. fermentans and H'spora

valbyensis were isolated to a lesser extent from sourdough samples. In the present
study, P. membranifaciens and P. fermentans were isolated from wheat and rye
sourdough samples, respectively. Pichia spp. is rarely isolated from sourdough
147
fermentations (Paramithiotis et al., 2000; Vernocchi et al., 2004; Vogelmann et al.,

2009; Yagmur et al., 2016). The occurence of P. guiliermondii in Turkish and
Italian sourdoughs has been previously reported (Yagmur et al., 2016; Gaglio et al.,
2017). Paramithiotis et al. (2000) isolated P. membranfaciens from Greek
sourdoughs. Also presence of P. membranifaciens and P. fermentans species was
reported in southern Italian sourdoughs (Succi et al., 2003). Recently, P.
membranifaciens was also isolated from Chinese sourdoughs (Liu et al., 2018).
In the present study, H'spora valbyensis was isolated from sourdoughs for
the first time. To my knowledge, the presence of this yeast species in sourdough
fermentations has not been documented previously. H'spora valbyensis was
isolated from industrial-scale Kombucha and cider fermentations (Coton et al.,
2015; Coton et al., 2017).
The association of yeasts with LAB is necessary in order to protect the
variety of regional specialities as previously reported (Corsetti and Settanni, 2007).
De Vuyst et al. (2016) investigated the yeast diversity of sourdoughs and reported
the adaptation of sourdough yeasts to the harsh conditions including nutrient
starvation, acidic, oxidative, thermal, and osmotic stresses. Yeasts in sourdough
fermentations primarily contribute to the leavening and flavor of sourdough
products. Besides ethanol and carbon dioxide, some metabolites that affect flavour
can be produced by yeast species. In addition, some functional properties such as
vitamin production, improvement of the bioavailability of phenolic compounds, the
dephosphorylation of phytic acid, the presence of probiotic potential and the
inhibition of fungi and their mycotoxin production lead to nutritional and safety
advantages.
4.1.4.4. Phylogenetic Relation of the Yeast Strains

sequences (400 bp≤) of the identified yeast strains using a possible tree
reconstruction method, UPMGA, in MEGA 7 (Kumar et al., 2016).
148
The evolutionary relation of the isolated yeast strans from sourdough

samples was inferred using the UPGMA method as shown in Figure 4.27 (Sneath
and Sokal, 1973). The bootstrap consensus tree inferred from 200 replicates is
taken to represent the evolutionary history of the taxa analyzed. Branches
corresponding to partitions reproduced in less than 50% bootstrap replicates are
collapsed. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (200 replicates) are shown next to the branches
(Felsenstein, 1985). The evolutionary distances were computed using the
Maximum Composite Likelihood method and are in the units of the number of base
substitutions per site (Tamura et al., 2004). The analysis involved 10 nucleotide
sequences. All positions with less than 95% site coverage were eliminated. That is,
fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at
any position.
100 K. servazzii PM603

100 K. unispora PM617
65
H spora valbyensis NM626
86 H spora valbyensis NM625
K. bulderi PM190
53
100 K. servazzii PM604
P. membranifaciens NM1004
100 S. cerevisiae PM1
P. fermentans PM801
P. fermentans NM816
Figure 4. 27. Evolutionary relationships of yeast with UPMGA method
4.2. Chickpea Liquid Starter and Dough Samples

4.2.1. Chemical Characteristics of Chickpea Liquid Starter and Dough
Samples
Chickpea liquid starter and dough samples were collected from three
different bakeries located in different cities at two different times. The bakeries are
149
well-known in their regions and have been producing chickpea bread traditionally
for many years. Chickpea liquid starter samples were obtained by seperating
chickpeas from the fermentation liquid at the end of the fermentation. Chickpea
dough samples were collected by taking a piece of leavened dough. Codes were
assigned to each collected sample as letters and numbers without expressing the
bakery names due to the special request by the bakeries. Chickpea liquid starter
samples were coded as CLS-A1, CLS-A2, CLS-B1, CLS-B2, CLS-N1 and CLS-
N2 and the chickpea doughs as CD-A1, CD-A2, CD-B1, CD-B2, CD-N1 and CD-
N2. Codes were given with a randomly chosen letter for the bakery and number 1
or 2 indicating the first or second sampling. First samples (coded as 1) were
collected from bakeries in the spring or at the beginning of summer and the second
samples (coded as 2) were collected at the end of the autumn or in winter, resulting
in chickpea fermentations with different characteristics.
4.2.1.1. pH
Results of the pH measurements of the 6 chickpea liquid starter samples,
including both sampling are shown in Table 4.17.
Table 4.17. pH levels of the chickpea liquid starter samples

CLS-A1 5.13b 0.00
CLS-A2 4.82a 0.00
CLS-B1 5.28c 0.01
CLS-B2 5.13b 0.02
CLS-N1 5.50d 0.01
CLS-N2 5.67e 0.02
150
The pH levels of the chickpea liquid starter samples ranged from 4.82 to
5.67. and the mean and median pH values were 5.25 and 5.21, respectively. The
lowest pH level was measured in the CL-A2 sample; whereas the highest pH value
was measured as 5.67 in the CL-N2 chickpea liquid sample. The pH value of the
first sampling of this bakery was the second highest pH as 5.50. There was a wide
variation among pH levels of the chickpea liquid samples and sampling times.
Testing of homogenity of variances showed that variances ccould be treated as
equal (p>0.05) and a parametric ANOVA test was conducted. According to the
statistical results, the differences between the samples collected from different
bakeries were significant (p<0.05). The pH differences between samples could be
due to the different microflora in the dough samples, different production methods
and incubation conditions as the samples were collected from different cities. In
addition, every bakery has its own traditional production parameters and these
parameters directly affect the pH of the end product, therefore, chickpea liquids
produced in different places exhibit different biochemical patterns. On the other
hand, differences were observed between some samples collected from the same
bakery at two different times.
Results of the pH measurements of the 6 chickpea dough samples,
including both samplings are shown in Table 4.18.
Table 4.18. pH levels of the chickpea dough samples

CD-A1 5.53d 0.14
CD-A2 5.20ab 0.01
CD-B1 5.12a 0.02
CD-B2 5.32bc 0.12
CD-N1 5.52d 0.07
CD-N2 5.43cd 0.01
a-dDifferent superscript letters within a column indicate
a significant difference (Duncan p<0.05)
151
The pH levels of the chickpea dough samples ranged from 5.12 to 5.53 and
the mean and median pH values were 5.35 and 5.41, respectively. The lowest and
highest pH levels were measured in the CD-B1 and CD-A1 samples, respectively.
Testing of homogenity of variances showed that variances could be treated as equal
(p>0.05) and a parametric ANOVA test was conducted. According to the statistical
results, the differences between the samples collected from different bakeries were
significant (p<0.05). The pH differences between samples could be due to the same
reasons stated above, i.e., different microflora in the dough samples, different
production methods and incubation conditions as the samples were collected from
different cities. In addition, every bakery has its own traditional production
parameters and these parameters directly affect the pH of the end product,
therefore, chickpea liquids produced in different places exhibited different
biochemical patterns. On the other hand, differences were observed between some
samples collected from the same bakery at two different times. Among the
chickpea dough samples, only CD-N samples collected at two different times did
not show any significant difference between two samplings (p>0.05).
Figure 4.28. pH variation among chickpea liquid starter and dough samples
152
As it can be seen from Figure 4.25, the pH of chickpea dough starter

samples were higher than the chickpea liquid in the A1, A2, B2 and N1 samples.
Different production methods and incubation conditions directly affect the pH,
therefore, chickpea dough starters produced in differing places exhibited different
biochemical patterns. Similar results have been obtained in other studies.
Erginkaya et al. (2016) reported higher pH levels in dough than the chickpea liquid
starter. In the study of Hatzikamari et al. (2007a), the pH of the fermenting liquid
was reported to be 5.35 after 18 h. Çebi (2009) also determined higher pH values in
chickpea dough than the chickpea liquid starter. Katsaboxakis and Mallidis (1996),
determined pH values at 32, 37 and 42°C to be 4.89, 4.66 and 4.60 at the end of a
30 hour fermentation of the coarsely ground chickpeas in the soak water,
respectively. However, 12 and 24 hour fermentations of coarsely ground chickpeas
in water resulted in pH values of 6.22 and 5.17, 5.61 and 4.61 and 5. 26 and 4.69 at
32, 37, and 42°C, respectively. As expected, the pH decreased with increasing time
and temperature. The samples in this study were fermented for 16-18 hours at
higher temperatures inside the bakery and reached lower pH values than reported at
12 hours; however, close pH values determined for 24 hour fermentation.
4.2.1.2. Total Titratable Acidity

Total titratable acidity was given as mL of 0.1 N NaOH consumed and
results of the 6 chickpea liquid samples including both sampling are shown in
Table 4.19.
153
Table 4.19. TTA levels of the chickpea liquid samples

Sample TTA (mL 0.1 N NaOH Std.
/10 g sample) deviation
CLS-A1 2.24c 0.03

CLS-A2 3.20e 0.00
CLS-B1 1.70a 0.00
CLS-B2 1.65a 0.10
CLS-N1 2.43d 0.06
CLS-N2 2.02b 0.03
Acidity levels of the collected chickpea liquid samples ranged from 1.65 to
3.20 mL 0.1 N NaOH/10 g sample. In terms of per cent lactic acid, TTA values
ranged from 0.15 to 0.29 %. The acidity content was significantly (p<0.05)
different between chickpea liquid samples. The values also showed differences
among samples at different sampling times. Testing of homogenity of variances
showed that variances were unequal and the differences between the samples were
significant (p<0.05). On the other hand, among the chickpea liquid samples, only
CLS-B samples collected at two different times did not show any significant
difference between two sampling (p>0.05).
TTA was given as mL of 0.1 N NaOH consumed and results of the 6
chickpea dough samples including both sampling are shown in Table 4.20.
154
Table 4.20. TTA levels of the chickpea dough samples

Sample TTA (mL 0.1 N Std.
NaOH /10 g sample) deviation
CD-A1 3.03a 0.15
CD-A2 3.90b 0.00
CD-B1 5.40d 0.40
CD-B2 4.80c 0.00
CD-N1 3.17a 0.47
CD-N2 3.58ab 0.42
a-d
Different superscript letters within a column indicate a
significant difference (Duncan p<0.05)
The acidity levels of the collected chickpea dough samples ranged from
3.03 to 5.40 mL 0.1 N NaOH/10 g sample. The median acidity level was
determined to be 3.73 mL 0.1 N NaOH/10 g sample. In terms of per cent lactic
acid, TTA values ranged from 0.29 to 0.49%. The acidity content was significantly
(p<0.05) different among chickpea dough samples. Testing of homogenity of
variances showed that variances were unequal and the differences between the
samples were significant (p<0.05). The values also showed a significant difference
among samples collected on two different sampling times, except CD-N samples,
i.e., the CD-N sample did not show any significant difference between the two
sampling (p>0.05).
155
Figure 4.29. TTA variation among chickpea liquid starter and dough samples
In the A and N samples, the acidity values increased at the second

sampling. The highest acidity levels of the dough samples were determined in the
B sample (Fig 4.29) at both sampling times. Conversely, the lowest acidity among
liquid samples was observed in the chickpea liquid samples taken from Bakery B at
both sampling times. The production and incubation conditions at that bakery
resulted in a chickpea dough with a higher acidity level than the fermented
chickpea liquid starter. Hence, it is very difficult to find a correlation between the
characteristics of liquid and dough samples collected from different bakeries.
However, the samples collected from the same bakeries exhibited the same pattern
at both sampling times in terms of pH and acidity. As it is known, acid production
is related to the compounds and microbial flora in the sample. The flour used in the
production contains varying levels of carbohydrate sources. Therefore, the raw
material used in the production and microflora results in dough samples with
different acidity level. The bakeries are located in different places and therefore
different flours are used depending on the regions. In addition, production methods
and incubation conditions can affect the pH and acidity levels in the samples.
156
Total acidity values of the chickpea liquid starter and dough samples
displayed a wide variation. Hatzikamari et al. (2007a) reported the changes in total
titratable acidity during a submerged chickpea fermentation liquid, at 37 °C for 18
h, with the final value as 0.34% lactic acid, which concurs with the present study.
Another study reported the acidity value of the soak water, including coarsely
ground chickpea seeds, as 0.04% at the beginning of the fermentation. The study
reported the acidity values at the end of the 30 h fermentation as 0.22, 0.33 and
0.29% at 32, 37 and 42°C, respectively. On the other hand, 12 and 24 h
fermentation of coarsely ground chickpeas in water resulted in acidity values of
0.07 and 0.20% at 32 °C, 0.10 and 0.35% at 37°C, and 0.20 and 0.31% at 42°C
(Katsaboxakis and Mallidis, 1996). Total acidity values of this study was
determined to be in the range of the values found at 37 °C and almost at 42°C, as
expected.
The control chickpea liquid starter and dough samples were produced in
duplicate under laboratory conditions. Fermentations were conducted at 32 and
37°C. Samples were taken at the beginning (CLS-0h) and end of the fermentation
of the chickpea liquid starter (CLS-32 and CLS-37) and dough samples at both
temperatures (CD-32-0h, CD-32, CD-37-0h and CD-37). The first samples were
taken from the unfermented chickpea liquid immediately after water addition. As it
can be seen in Figure 4.30, in the fermented chickpea liquid starters, the pH
decreased and TTA increased during 18 hours of fermentation. At the end of the
fermentation, the pH level at 32 and 37°C decreased to 4.91 and 4.75, respectively.
The total titratable acidity value was higher in the chickpea liquid starter fermented
at 37°C compared with 32°C. TTA values were 1.95 and 2.95 mL 0.1 N NaOH/10
g sample fermented at 32 and 37°C, respectively. Following chickpea liquid
fermentations, the fermented liquid starter was used in chickpea dough production.
Due to the addition of flour, the pH increased and acidity decreased. At the end of
4 hours of fermentation, the final pH values of both fermentations were close to
each other as 4.84 at 32°C and 4.81 at 37°C. TTA values were 4.80 and 5.00 mL
157
0.1 N NaOH/10 g dough sample fermented at 32 and 37°C, respectively.

Differences in the pH and TTA values of the chickpea liquid starter and dough
samples fermented at two different temperatures were not statistically significant
(p˃0.05).
Figure 4. 30. pH and TTA levels of the chickpea liquid starter and dough samples
produced under laboratory conditions
(CLS0h: unfermented chickpea liquid, CLS32: chickpea liquid starter fermented at
32°C, CLS37:chickpea liquid starter fermented at 37°C, CD-32-0h:unfermented
dough produced with chickpea liquid starter fermented at 32°C, CD-37-
0h:unfermented dough produced with chickpea liquid starter fermented at 37°C,
CD-32: chickpea dough fermented at 32°C, CD-37: chickpea dough fermented at
37°C).
The pH and TTA values of the collected chickpea liquid starter samples
were in the range of 4.82-5.67 and 1.65-3.20 mL 0.1 N NaOH/10 g liquid starter.
Acidity values of the laboratory produced samples were in agreement with the
collected samples; however, chickpea liquid starters produced under laboratory
conditions were characterized by lower pH values. The pH and TTA values of the
158
collected chickpea dough samples were in the range of 5.12-5.53 and 3.03-4.80 mL
0.1 N NaOH/10 g dough. Under laboratory conditions, chickpea doughs exhibited
higher acidity values compared with fermented chickpea liquid starters, which
were in agreement with some of the collected samples. Acidity values of chickpea
liquid starter and dough samples at 32-37°C were 0.18-0.27 % and 0.43-0.45 %,
respectively. Hatzikamari et al. (2007a) reported the TTA for a submerged
chickpea fermentation liquid fermented at 37°C for 18 h as 0.34 % lactic acid,
which was higher than found in the present study. Katsaboxakis and Mallidis
(1996) reported acidity values of the coarsely ground chickpeas after fermentation
for 12-24 hours as 0.07-0.20% at 32°C and 0.10-0.35% at 37°C. The chickpea
liquid acidity values of this our study were in agreement with that study at both
temperatures. The pH values after 12-24 hours fermentation were 6.22-5.17 at
32°C and 5.61-4.61 at 37°C (Katsaboxakis and Mallidis, 1996). In this study,
fermentations were conducted for 16–18 hours and the resulting pH values were in
consistent with the pH values of the fermentations conducted at 37°C. In another
study, the pH of the liquid was reported to be 5.35 after 18 h, which is higher than
obtained in this study (Hatzikamari et al., 2007a). On the other hand, Erginkaya et
al. (2016) reported the pH value of the unfermented and fermented chickpea liquids
as almost 7 and below 5, respectively. The results of the chickpea liquid samples
are in agreement with this study, both at the beginning and end of fermentation.
However, the final pH value of the chickpea dough after 2 hours fermentation at
37°C was reported to be above 5 in their study In this study, the pH value of the
dough was below 5 following 4 hours of fermentation at both temperatures.
Increasing the time and temperature leads to a pH decrease and acidity increase, as
differences in production methods, fermentation conditions, contents of the raw
materials and also temperature of the water used in the production result in
different final products with different biochemical patterns.
159
4.2.1.3. Carbohydrate, Organic Acid and Ethanol Contents of the Chickpea

Liquid Starter and Dough Samples
The content of maltose, sucrose, glucose, fructose and ethanol were
determined by HPLC with a RID detector and lactic and acetic acid levels were
assessed with an UV detector. As reported in the previous section, discrimination
of the maltose and sucrose peaks was difficult as their retention times were close to
each other. Therefore, the results of these compounds are given in combination as
maltose+sucrose. Chromatogram images of chickpea liquid starter and dough
samples are shown in Appendix 6 and 7, respectively.
Mean values of the carbohydrate and ethanol content of the collected
samples are given in Table 4.21. The mean-median values of maltose+sucrose,
glucose, fructose and ethanol contents in chickpea liquid starter samples were 2.71-
2.89, 4.60-4.58, 4.09-4.19 and 2.54-2.54 g/kg, respectively. The mean-median
values of maltose+sucrose, glucose, fructose and ethanol content in chickpea dough
samples were 25.22-24.69, 6.96-6.54, 6.95-7.04 and 2.66-2.67 g/kg, respectively.
Table 4.21. Mean carbohydrate and ethanol content (g/kg) of the chickpea liquid
starter and dough samples
Samples Maltose+sucrose Glucose Fructose Ethanol
CLS-A1 1.66a±0.08 3.90b±0.49 3.26b±0.31 2.49ab±0.06
CD-A1 20.38c±1.76 5.70c±1.10 4.35c±0.65 2.81e±0.07
CLS-A2 1.25 ±0.17
a 2.59 ±0.02
a 2.18a±0.07 2.52ab±0.08
CD-A2 29.26f±0.76 7.38d±0.60 6.56e±0.33 2.80e±0.08
CLS-B1 4.50 ±0.98
b 5.78 ±0.32
c 5.11d±0.01 2.58bcd±0.07
CD-B1 23.89de±0.55 7.66d±0.13 7.50f±0.16 2.45a±0.03
CLS-B2 3.06ab±0.50 3.12ab±0.53 2.42a±0.24 2.55abc±0.04
CD-B2 25.49 ±1.54
e 9.80 ±1.10
e 6.58e±0.29 2.59 bcd ±0.09
CLS-N1 3.10ab±0.40 6.94d±0.50 6.44e±0.72 2.53ab±0.02
CD-N1 29.38 ±1.18
f 5.54 ±0.27
c 8.44g±0.20 2.65cd±0.05
CLS-N2 2.71ab±0.63 5.26c±0.75 5.15d±0.25 2.59 bcd ±0.06
CD-N2 22.93 ±1.58
d 5.68 ±0.22
c 8.24g±0.32 2.70de±0.05
Results indicate mean values± SD, Different superscript letters within a column
160
The carbohydrate content in the chickpea dough samples was generally

determined higher than the chickpea liquid starter samples. In the chickpea liquid
starter samples, the content of maltose+sucrose, glucose and fructose ranged from
1.25-4.50, 2.59-6.94 and 2.18-6.44 g/kg, respectively. In chickpea dough samples,
the maltose+sucrose, glucose and fructose contents ranged from 20.38-29.38, 5.54-
9.80 and 4.35-8.44 g/kg, respectively. Among the chickpea liquid starter samples,
the highest maltose+sucrose content was measured in CLS-B1, and glucose and
fructose contents were determined in the CLS-N1 sample. The CLS-A2 sample
exhibited the lowest carbohydrate content. Among the chickpea dough samples, the
highest maltose+sucrose and fructose contents were determined in the CD-N1
sample; however, the lowest glucose content was detected in that sample. The
ethanol content of the samples was in the range of 2.45–2.81 g/kg.
The organic acid content of the chickpea liquid starter and dough samples
are given in Table 4.22. The mean-median contents in the chickpea liquid starter
samples were 0.39-0.25 for lactic acid and 0.99-0.94 g/kg for acetic acid,
respectively. The mean-median values of lactic acid content in chickpea dough
samples were 0.52-0.68 g/kg. The mean-median acetic acid levels were determined
as 3.38-3.55 g/kg. In the samples collected from Bakery N, the lactic acid content
was below the LOQ, except for the CD-N2 sample. Furthermore, CLS-A1 and CD-
A2 samples contained lactic acid below the LOQ. Acetic acid was determined in all
of the liquid samples in the range of 0.86-1.23 g/kg. With the exception of the
CLS-B2, acetic acid content was higher than lactic acid in chickpea liquid samples.
The levels of the acetic acid in the dough samples were below the LOQ.
161
Table 4.22. Mean organic acid content (g/kg) of the chickpea liquid starter and
dough samples
Samples Lactic acid Acetic acid
CLS-A1 <LOQ 1.23c±0.16

CD-A1 0.68b±0.02 <LOQ
CLS-A2 0.90cd±0.00 0.97ab±0.12
CD-A2 <LOQ <LOQ
CLS-B1 0.50a±0.04 0.86a±0.00
CD-B1 0.68 ±0.03
b <LOQ
CLS-B2 0.93d±0.01 0.90a±0.01
CD-B2 0.85 ±0.15
c <LOQ
CLS-N1 <LOQ 1.10bc±0.10
CD-N1 <LOQ <LOQ
CLS-N2 <LOQ 0.90a±0.02
CD-N2 0.94d±0.94 <LOQ
Results indicate mean values± SD, Different superscript
letters within a column indicate a significant difference
(Duncan p<0.05)
Control chickpea liquid starter and dough samples were produced at 32 and
37°C under laboratory conditions. Samples were taken at the beginning and end of
fermentation of the chickpea liquid starter and dough samples at both temperatures.
The maltose+sucrose was not detected in the chickpea liquid starter samples
(<LOQ). The concentration of maltose+sucrose were 21.80 and 19.18 g/kg in the
dough samples fermented at 32 and 37°C, respectively. The glucose and fructose
content was higher in the chickpea liquid starter fermented at 32°C than at 37°C.
On the other hand, the organic acid content was lower in the chickpea liquid starter
fermented at 32°C than at 37°C. Similarly, the lactic acid content was higher in the
doughs fermented at 37°C than at 32°C. Acetic acid was not determined in the
dough samples. Between chickpea liquid starter samples fermented at two different
temperatures, fructose and lactic acid content differences were statistically
significant (p˃0.05). Between chickpea dough samples, only the difference in the
lactic acid content was statistically significant (p˃0.05). Figure 4.31 shows the
carbohydrate, ethanol and organic acid contents of the chickpea liquid starter and
dough samples produced under laboratory conditions.
162
Figure 4.31. Carbohydrate, ethanol and organic acid contents of the chickpea liquid
starter and dough samples produced under laboratory conditions
32°C, CLS37:chickpea liquid starter fermented at 37°C, CD-32-0h:unfermented
37°C).
In the present study, the total fructose and glucose content was 1.79 g/kg at
the end of the 18 h fermentation at 37°C. Hatzikamari et al. (2007a) investigated
changes in chemical characteristics during a submerged chickpea fermentation at
37°C for 18 h and reported the reducing sugar content of 1.46 mg glucose/mL at
the end of the chickpea liquid fermentation. The results of that study are almost in
agreement with the results of the present study. In their study, the amount of
reducing sugars gradually increased and then decreased at the end of the
fermentation. They also reported the occurence of soluble starch. According to the
Hatzikamari et al. (2007a), the increase in reducing sugars could be related to the
enzyme activities and then decrease of them can be due to utilization as carbon
source (Hatzikamari et al., 2007a). Another study investigated traditional chickpea
fermentation in the Aegean region of Turkey and produced chickpea liquids
fermenting at 42 °C for 16 hours. Final reducing sugar contents were in the range
163
of 1.78-2.32 mg/mL and changed according to the chickpea variety and (Kasım,
2014).
In the present study, variations among the carbohydrate concentrations in
the collected samples were observed. The carbohydrate content of the chickpea
liquids can vary according to the chickpea variety and fermentation time (Kasım,
2014). Samples were collected from different bakeries. The production method is
usually the same in a particular bakery, however, differences in the chickpea
fermentations conducted in the same bakery at two different times were observed.
This could be related to the temperature change of the environment, as chickpea
fermentations are conducted inside the bakery at the environmental temperature.
Differences among different bakeries could be related to the using a different
chickpea variety, the ratio of chickpeas to water, temperature of water, incubation
time and temperature. In almost all of the chickpea dough samples, the
carbohydrate contents were determined higher than determined in the liquid
starters, which is related to the addition of flour during dough production as flour
contains carbohydrates. However, the chickpea fermentation liquid also contained
starch and carbohydrates, which originated from chickpeas (Kasım, 2014). The
level of solubilized starch decreased and reducing sugars increased with the
progression of the chickpea fermentation. However, compared with the dough
fermentation, the liquid fermentation is conducted over longer periods of time;
therefore, carbohydrates can be consumed via enzymatic and microbial activity. It
was reported that an 18 h period results in considerable substrate modification in
the chickpea liquid (Hatzikamari et al., 2007a).
4.2.2. Microbiological Characteristics of Chickpea Fermentations

Cell densities of LAB, yeasts, total aerobic bacteria and molds in the 12
collected chickpea liquid starter and dough samples are shown in Table 4.23.
164
Table 4.23. Mean values of cell counts (log CFU/g) of chickpea liquid starter and
dough samples on different media
LAB Yeasts Total aerobic Molds
Sample mMRS gM1 YPD L-lysine NA bacteria
PCA MXA
sCLS-A1 1.60 7
4.40 5.18 5.00 5.30 5.30 0
CLS-A2 3.15 3.42 0 0 4.78 5.48 0
CLS-B1 5.69 5.58 2.55 2.30 2.20 3.45 1.60
CLS-B2 7.18 7.23 2.76 2.74 7.70 7.11 0
CLS-N1 5.45 5.65 5.85 4.34 5.99 5.45 3.58
CLS-N2 3.00 0 0 0 5.68 5.41 0
CD-A1 5.90 5.76 6.83 3.97 4.04 6.09 <1
CD-A2 4.30 4.60 3.86 3.45 4.78 4.20 2.30
CD-B1 6.86 6.68 4.00 2.62 3.53 5.49 1.78
CD-B2 6.41 7.85 3.73 3.53 7.39 8.08 2.15
CD-N1 5.32 5.51 5.58 3.88 5.30 5.49 4.40
CD-N2 6.89 7.16 2.20 <1 6.64 6.96 2.60
4.2.2.1. Presumptive LAB Cell Counts

Cell counts of presumptive LAB in collected chickpea liquid starters were
found to be in the range of 1.60-7.18 log CFU/g on mMRS medium. On the other
hand, cell counts of presumptive LAB cell counts on gM17 medium ranged from 0
to 7.23 log CFU/g for chickpea liquid starter samples. The mean, 25th, median, and
75th percentiles of the cell counts were determined to be 4.5, 4.3, 2.65 and 6.81 log
CFU/g on mMRS and 4.38, 4.99, 2.56 and 6.04 log CFU/g on gM17. The highest
cell densities on mMRS and gM17 agar media were counted in the CLS-B2
sample. In addition, the cell counts of the CLS-B1 sample was high compared with
the other samples. The lowest cell density on mMRS agar was 1.60 for the CLS-A1
sample. Bacterial growth was not detected in CLS-N2 sample on gM17 agar.
The mean cell counts of presumptive LAB in collected chickpea dough
samples were determined to be in the range of 4.30-6.89 and 4.60-7.85 log CFU/g
on mMRS and gM17 media, respectively. The mean, 25th, median, and 75th
percentiles of the cell counts were determined as 5.94, 6.15, 5.06 and 6.86 log
CFU/g on mMRS and 6.26, 6.22, 5.28 and 7.33 log CFU/g on gM17. The highest
165
cell densities on mMRS and gM17 agar media were found in the CD-N2 and CD-
B2 samples, respectively. Among the chickpea dough samples, the lowest cell
densities on mMRS and gM17 media were counted in the CD-A samples at both
sampling times. The growth of presumptive LAB from chickpea liquid and dough
samples is shown in Figure 4.32.
Figure 4.32. Presumptive LAB count of chickpea liquid starter and dough samples
on MRS and M17 agar
Çebi (2009) reported the LAB count of chickpea liquids at the end of 16
hours fermentation conducted at 40°C as 6.43 and 6.31 log CFU/g on MRS and
M17 agar media, respectively. In the same study, LAB count of chickpea dough
samples were reported as 6.76 on MRS and 6.72 log CFU/g on M17 agar media. In
their study, a slightly higher bacteria count was observed in the chickpea dough
samples compared with chickpea liquid samples. Conversely, another study
reported higher LAB counts in chickpea liquid starter fermented at 37°C on MRS
agar, compared with chickpea dough fermented at the same temperature. The cell
counts of LAB on MRS agar were reported as 8.07 and 5.60 log CFU/g for
chickpea liquid starter and dough, respectively (Erginkaya et al., 2016). Hancıoğlu-
166
Hancıoglu-Sıkılı (2003) reported the LAB counts in the range of 6.85-9.45 log
CFU/g for chickpea liquid and 5.32-7.49 log CFU/g for chickpea dough samples
collected from the Aegen region in Turkey. Another study reported LAB counts
around 7-7.5 log CFU/mL in chickpea liquid starters fermented at 42°C for 16 h
(Kasım, 2014).
4.2.2.2. Presumptive Yeast Cell Counts

Presumptive yeast cells were counted on YPD and L-lysine agar media.
Cell counts of chickpea liquid samples were in the range of 0-5.85 log CFU/g on
two different media. The mean, 25th, median, and 75th percentiles of the cell
counts were determined to be 2.72, 2.65, 0 and 5.34 log CFU/g on YPD and 2.40,
2.52, 0 and 4.50 log CFU/g on L-lysine media. The highest cell densities on YPD
and L-lysine agar media were detected in the CLS-N1 and CLS-A1 samples,
respectively. On the other hand, according to the enumeration results on YPD and
L-lysine media, no cells grew on the plates of the second sampling of A and N
chickpea liquid samples. The samples of the second sampling were collected in
winter and it was observed that bakeries use very hot or boiling water in colder
weather. This result is in consistent with the maximum growth temperatures of
yeasts. Growth temperatures of yeasts, which varies with species, but in general,
many species are unable to grow at a temperature above 35°C (Madan and Thind,
2000).
Presumptive yeast cell counts of chickpea dough samples were in the range
of 2.20-6.83 and <1-3.97 log CFU/g on YPD and L-lysine media, respectively. The
mean, 25th, median, and 75th percentiles of the cell counts were determined as
4.36, 3.93, 3.35 and 5.90 log CFU/g on YPD and 2.90, 3.50, 1.96 and 3.90 log
CFU/g on L-lysine. The highest cell densities on both media were counted in CD-
A1 and CD-N2 samples, respectively. The growth of presumptive yeasts in
chickpea liquid and dough samples on two different media is shown in Figure 4.33.
167
Figure 4.33. Presumptive yeast counts of chickpea liquid starter and dough samples
on YPD and L-lysine agar
Erginkaya et al. (2016) reported yeast counts of approximately 4 log

CFU/g for chickpea liquid and dough starter samples, which is inconsistent with
the mean yeast count of the present study. Another study reported the yeast counts
in the range of <1-4.90 and <1-4.23 log CFU/g for chickpea liquid and chickpea
dough samples collected from the Aegean region of Turkey, respectively
(Hancıoglu-Sıkılı, 2003).
4.2.2.3. Enumeration of other Microorganisms

According to the microbiological analysis results, the total bacteria counted
on NA medium was in the range of 2.20-7.70 and 3.53-7.39 log CFU/g for
chickpea liquid starter and dough samples, respectively. The mean, median, 25th
and 75th percentiles of the cell counts were determined to be 5.28, 5.49, 4.13 and
6.41 log CFU/g for chickpea liquid starter and 5.28, 5.04, 3.91 and 6.82 log CFU/g
for chickpea dough. The highest counts of chickpea liquid starter and chickpea
168
dough samples on NA medium were observed in the B2 sample, reaching values of

7.70 and 7.39 log CFU/g for the chickpea liquid starter and dough, respectively.
Conversely, the lowest counts were observed in the first sampling of the B sample
in both products, which could be related to the sampling time as Bacillus spp. have
been reported to increase during chickpea liquid fermentation (Hatzikamari et al.,
2007b). In that study, Bacillus spp. counts on NA agar increased during the
fermentation of a chickpea liquid starter from 1.37 log CFU/mL at the beginning of
the fermentation to 7.72 log CFU/mL at the end of fermentaton. During the first 8
hours, the count increased rapidly to 6.46 log CFU/mL and at the end of the
fermentation it reached 7.72 log CFU/mL. The count of an adapted dough at the
end of fermentation was reported to be 7.18 log CFU/mL (Hatzikamari et al.,
2007b). Another study determined Bacillus spp. based on the aerobic growth and
spore-forming properties. According to the results, aerobic spore-forming bacteria
in a chickpea liquid starter and dough were less than 3 log CFU/mL and more than
2 log CFU/mL, respectively (Erginkaya et al., 2016).
In the present study, total mesophilic aerobic bacteria was enumerated on
PCA and was in the range of 3.45-7.11 and 4.20-8.08 log CFU/g for chickpea
liquid starter and dough samples, respectively. The mean, median, 25th and 75th
percentiles of the cell counts were determined to be 5.36, 5.43, 4.83 and 5.89 log
CFU/g for chickpea liquid starter and 6.05, 5.79, 5.17 and 7.24 log CFU/g for
chickpea dough samples. The highest and lowest cell counts on NA medium were
in the CLS-B2 and CLS-B1 samples, respectively. Among the dough samples, CD-
B2 showed the highest cell counts on NA medium, whereas the lowest count was
found in the CD-A2 sample. Çebi (2009) reported the mesophilic aerobic bacteria
count of chickpea liquid starters and doughs on PCA media as 6.14 and 6.81 log
CFU/g, respectively (Çebi, 2009). Hatzikamari et al. (2007b) determined the
mesophilic aerobic bacteria count on PCA medium to be 7.94 and 7.63 log
CFU/mL at the end of the chickpea liquid primary starter and adapted dough
starter, respectively. According to Katsaboxakis and Mallidis (1996), regardless of
169
the incubation temperature of 32, 37 and 42 °C, soak water of ground chickpeas
resulted in almost 8 log CFU/mL viable counts of bacteria on PCA medium at the
end of the 30 hours. As expected, incubation at higher temperatures caused a
significant increase. Between 16-18 hours, the counts on PCA was almost 6 log
CFU/ml at 32 °C and more than 8 log cfu/mL at 37 and 42 °C (Katsaboxakis and
Mallidis, 1996).
With the exception of the CLS-B1 and CLS-N1 samples, molds were not
observed in chickpea liquid starter samples. Conversely, mold count in CLS-B1
and CLS-N1 samples were 1.60 and 3.58 log CFU/g, respectively. Among the
chickpea dough samples, the CD-A1 sample count was <1 log CFU/g and the
highest mold count was enumerated in the CD-N1 sample (4.40 log CFU/g).
Presumptive coliforms were assessed by growth in LST broth and gas
production in the Durham tube. Presumptive total coliform bacteria were detected
at <0.3 MPN/g in chickpea liquid samples with the exception of CLS-B2, CLS-N1
and CLS-N2 samples. Among the chickpea doughs, the first sampling of A and B
samples were found to be <3 MPN/g, as shown in Table 3. The highest counts were
determined in CD-N1 and CD-N2 samples as 1100 and 460 MPN/g, respectively.
Development of a red color in upper the layer of the gas positive tubes after the
addition of Kovacs' indole reagent was recorded as presumptive Escherichia coli
(Halkman, 2005). As Escherichia coli can break down tryptophan into indole by
tryptophanase enzyme resulting in red color due to reaction with p-
dimethylaminobenzaldehyde contained in the Kovacs reagent (Macfaddin, 2000).
Escherichia coli is indole-positive culture but for complete identification of the
Escherichia coli isolates additional biochemical confirmation is needed (Yousef
and Carlstrom, 2003). Therefore results are shown as probable Escherichia coli in
Table 4.24.
170
Table 4.24. Presumptive total coliform bacteria and indol test in chickpea
fermentations
Sample Presumptive total coliform Indol test/Presumptive
bacteria (MPN/g) Escherichia coli (MPN/g)
CLS-A1 <0.3 -
CD-A1 <3 -
CLS-A2 <0.3 -
CD-A2 35 -
CLS-B1 <0.3 -
CD-B1 <3 -
CLS-B2 3 -
CD-B2 9.20 +/3.60
CLS-N1 0.36 -
CD-N1 1100 +/150
CLS-N2 0.36 -
CD-N2 460 +/240
Microbial cell densities were investigated for each group of

microorganisms in the laboratory produced chickpea liquid starter and dough
samples. Microbiological analysis was conducted at the beginning and end of the
liquid and dough fermentations at 32 and 37°C and the cell counts on the mMRS,
gM17, YPD, NA and PCA media are shown in Figure 4.34.
171
Figure 4.34. Cell counts in the laboratory scale chickpea fermentations

32°C, CLS37: chickpea liquid starter fermented at 37°C, CD-32-0h:unfermented
37°C).
At the beginning of the chickpea liquid fermentation, presumptive LAB

counts on mMRS and gM17 were determined as 0.30 and 0.85 log CFU/g,
respectively. At the end of the 18 hour fermentation, mMRS and gM17 counts
were 5.70 and 6.42 log CFU/g at 32°C and 5.35 and 7.00 log CFU/g at 37°C. In the
chickpea doughs, counts on mMRS and gM17 were determined to be 8.95 and
8.20 log CFU/g at 32°C and 9.07 and 8.44 log CFU/g at 37°C. The total bacteria
counts on PCA and NA agar media were very high, both counts were above 9 log
CFU/g in the chickpea liquid starter samples. In the chickpea doughs, PCA counts
were 9.40 and 9.95 log CFU/g at 32 and 37°C, respectively. Total bacteria count on
172
NA medium at at 32 and 37°C was determined as 8.30 and 8.40 log CFU/g,
respectively. Yeast counts were less compared with the bacteria counts. In
chickpea doughs, the final yeast counts were 3.10 and 3.00 log CFU/g at 32 and
37°C, respectively.
Growth was not observed in L-lysine and MXA agar media prepared for
non-Saccharomyces yeasts and molds, respectively. In the chickpea liquid starter,
presumptive total coliform counts were ˂0.3 MPN/g as no gas bubbles were
detected in Durham tubes. In the chickpea doughs, presumptive total coliform
bacteria were 15 MPN/g in unfermented dough and 11 and 9.2 MPN/g in the
doughs fermented at 32 and 37°C, respectively.
4.2.3. Multivariate Statistical Analysis of the Chickpea Liquid Starter and

Dough Samples
The microbiological and chemical parameters of chickpea liquid starter and
dough samples were subjected to the multivariate analysis to evaluate the
differences/variabilities among the samples. AHC classified the samples in
accordance to their mutual dissimilarity and relationship (Figure 4.35). This
analysis basically generated two main mega-clusters. As expected, all of the
chickpea doughs were gathered together with the control trial. Similarly, all of the
chickpea liquid starters were included in another cluster. On the other hand, the
CLS-N samples and first sampling of the A and B liquid starters were included in a
different class from the second sampling of the A and B liquid starters. Laboratory
produced liquid starters were in the same class with them. The chickpea dough
group was more homogeneous than the other groups.
173
Figure 4.35. Dendrogram resulting from hierarchical cluster analysis on chickpea

liquid starter and dough samples
Data of the microbiological and chemical inputs of chcikpea liquid starter

and dough samples were also subjected to PCa to express the important
information as principal components. Three eigen-values were higher than 1
and correspond to 79.45% of the variance. The eigenvalues and the corresponding
factors by descending order with the variability they represent is shown in Figure
4.36.
Factor 1 and Factor 2 explained 36.34 and 33.76 % of variability,
respectively. A large part of the variability was taken into account by the first two
axes since the percentage of variability represented by these two factors was
70.10% of total variability as shown in Figure 4.36. Correlation matrix (Pearson
(n)) of the variables is shown in Table 4.25.
174
Figure 4.36. The Eigenvalues and the corresponding factors by descending order
withl the variability they represent
Correlation circle (Figure 4.37A) composed of two distinct groups with

regards to F1. The variables including acetic acid, ethanol, PCA and NA were in
the negative correlation compared to others with regards to F1. LAB counts on
MRS, M17 and TTA and lactic acid were positively correlated as expected. The pH
was negatively correlated with TTA and positively correlated with YPD, fructose
and glucose. The score plot (Figure 4.37B) clearly shows the far distance among
the samples collected from different bakeries. Chickpea dough samples were
seperated from liquid starter samples along with F1. As it can be seen, close
relation was found between laboratory produed samples at two different
temperatures. Chickpea liquid starter samples were characterized with acetic acid.
Laboratory produced doughs were characterized with MRS, M17 counts and also
lactic acid contents.
175
component analysis of chickpea fermentations
176
Table 4.25. Correlation matrix (Pearson (n)) of the variables

maltose+ lactic acetic
Variables MRS NA YPD M17 PCA pH TTA glucose fructose ethanol
sucrose acid acid
MRS 1 0.343 0.121 0.829 0.535 -0.238 0.545 0.449 0.202 0.360 0.667 -0.568 -0.054
NA 0.343 1 -0.332 0.461 0.917 -0.501 0.082 -0.176 -0.458 -0.358 0.557 0.147 0.154
YPD 0.121 -0.332 1 0.247 -0.270 0.416 0.160 0.467 0.503 0.445 -0.282 -0.378 0.034
M17 0.829 0.461 0.247 1 0.627 -0.457 0.503 0.352 0.071 0.142 0.679 -0.393 0.004
PCA 0.535 0.917 -0.270 0.627 1 -0.590 0.300 -0.045 -0.394 -0.295 0.757 -0.046 0.114
pH -0.238 -0.501 0.416 -0.457 -0.590 1 -0.268 0.239 0.553 0.512 -0.668 -0.196 0.120
TTA 0.545 0.082 0.160 0.503 0.300 -0.268 1 0.737 0.505 0.548 0.449 -0.744 -0.121
maltose+
0.449 -0.176 0.467 0.352 -0.045 0.239 0.737 1 0.682 0.796 0.041 -0.954 0.193
sucrose
glucose 0.202 -0.458 0.503 0.071 -0.394 0.553 0.505 0.682 1 0.808 -0.261 -0.632 -0.139
fructose 0.360 -0.358 0.445 0.142 -0.295 0.512 0.548 0.796 0.808 1 -0.146 -0.756 -0.148
lactic acid 0.667 0.557 -0.282 0.679 0.757 -0.668 0.449 0.041 -0.261 -0.146 1 -0.172 0.029
acetic acid -0.568 0.147 -0.378 -0.393 -0.046 -0.196 -0.744 -0.954 -0.632 -0.756 -0.172 1 -0.115
ethanol -0.054 0.154 0.034 0.004 0.114 0.120 -0.121 0.193 -0.139 -0.148 0.029 -0.115 1
177
4.2.4. Biodiversity of the LAB and Yeasts in Chickpea Fermentations

A total of 395 LAB and 238 yeast isolates were collected from chickpea
liquid starter and dough samples, including laboratory scale production. The
distribution of all the isolates is shown in Figure 4.38. No yeast was isolated from
CLS-N2 and CLS-A2 samples; moreover LAB were not isolated from the CLS-N2
sample.
Figure 4.38. Distribution of presumptive LAB and yeasts in the chickpea

fermentations
4.2.4.1. LAB Identification

A total of 366 colonies were isolated from the petri dishes of the 12
chickpea liquid starter and chickpea dough samples collected from bakeries. In
addition, 29 colonies were collected from the fermentation of the chickpea liquid
starter and dough samples produced under laboratory conditions. All of the 395
LAB cultures were subjected to microscopic inspection and Gram-stain and
catalase tests. After Gram-stain characterization and catalase testing, 360 strains
were still considered putative LAB cultures (Gram-positive and catalase-negative).
All of the LAB cultures were grown in the MRS or M17 broth 12-24 hours and
178
subjected to DNA extraction by using Instagene matrix kit. Then genomic DNA of
the isolates were subjected to RAPD analysis using M13 primer. Some strains
showed weak band profile and were eliminated for further analysis. Bands were
evaluated according to the DNA marker by using the Infinity gel documentation
imaging system software. Band patterns of RAPD-PCR profiles of 269 strains were
scored as band absent (0) or present (1) and data were entered into a binary matrix.
The dissimilarity index was calculated on the basis of the Jaccard coefficient
generated with the DARwin (6.0.15) software package. A dendrogram was also
constructed based on the genetic distances with the UPGMA method as shown in
Figure 4.12. According to the calculated genetic distance matrix, a total of 74
strains were chosen for sequence analysis that had a genetic distance at the level
0.4≤.
Figure 4.39. Dendrogram obtained from RAPD-PCR(M13) band profiles of LAB

isolates in chickpea fermentations
Selected strains were subjected to 16s rRNA gene sequencing analysis.

Obtained sequences and their ABI chromatograms were examined with Bioedit
Sequence Alignment Editor 7.2.6. (Hall, 1999). The sequences more than 1400 bp
were compared by BLAST with nucleotide sequences deposited at the database
National Center for Biotechnology Information (NCBI) (Altschul et al., 1990).
179
Sequences with at least 98% identity to the sequences of the closest relative
available within the NCBI database showed strains belonging to the same species.
Strains with less than 98 % identity were identified at the genus (94%<) and family
(86%<) level (Yarza et al., 2014).
A total of 54 strains representing 149 isolates were confirmed to be
members of the LAB group with a sequence length of more than 1250 bp. Based on
the 16s rRNA sequence analysis, a total of 35 strains (1400 bp≤) representing 121
isolates were identified at the species level (98%≤). The identified strains along
with their accession numbers are given in Table 4.26.
180
Table 4.26. Identified LAB isolates (sequence length 1400 bp≤) at the species level
(98%≤) in chickpea fermentations
Similarity %
Strain of of closest relative by Sequence Accession
isolates Species GenBank) length (bp) number
RL419 1 W. confusa 98 (NR_113258.1) 1526 MH704144
RL425 26 W. confusa 98 (NR_113258.1) 1461 MH704145
RL453 1 Pd. acidilactici 98 (NR_042057.1) 1510 MH704146
RL458 12 W. cibaria 98 (NR_036924.1) 1446 MH704147
XL484 5 E. faecium 98 (NR_114742.1) 1412 MH704148
BL512 1 Pd. pentosaceus 98 (NR_042058.1) 1467 MH704150
RL898 1 W. confusa 98 (NR_113258.1) 1476 MH704152
RL899 1 W. cibaria 98 (NR_036924.1) 1512 MH704153
RL900 2 W.confusa 99 (NR_113258.1) 1523 MH704154
RL902 2 W.confusa 98 (NR_113258.1) 1552 MH704155
RL910 8 W.confusa 98 (NR_113258.1) 1455 MH704156
RL1139 12 W.confusa 98 (NR_113258.1) 1481 MH704157
XL1150 9 E. faecium 98 (NR_114742.1) 1413 MH704158
RL1165 1 Lb. brevis 98 (NR_116238.1) 1407 MH704159
RL1169 1 Lb. brevis 98 (NR_116238.1) 1482 MH704160
BL1171 3 E. faecium 98 (NR_114742.1) 1424 MH704161
RL1184 2 E. faecium 98 (NR_114742.1) 1477 MH704162
RL1220 1 Pd. acidilactici 98 (NR_042057.1) 1538 MH704164
RL1223 1 E. faecium 98 (NR_114742.1) 1522 MH704165
RL1227 5 E. faecium 98 (NR_114742.1) 1580 MH704166
BL1229 1 E. lactis 98 (NR_117562.1) 1559 MH704167
BL1233 2 Lb. brevis 98 (NR_116238.1) 1489 MH704168
RL1252 1 W.confusa 98 (NR_113258.1) 1487 MH704169
Leu. mesenteroides
RL1253 1 98 (NR_040817.1) 1508 MH704170
subsp. dextranium
RL1346 1 St. lutetiensis 99 (NR_115719.1) 1496 MH704171
BL1361 1 W. cibaria 98 (NR_036924.1) 1540 MH704172
BL1362 1 St.lutetiensis 98 (NR_042051.1) 1494 MH704173
RL1386 1 St.lutetiensis 98 (NR_042051.1) 1591 MH704174
BL1406 1 W.confusa 98 (NR_113258.1) 1533 MH704175
RL1734 4 E. faecium 98 (NR_114742.1) 1500 MH704176
XL1742 2 St. salivarius 98 (NR_042776.1) 1416 MH704177
XL1747 2 E. faecium 98 (NR_114742.1) 1485 MH704178
19 strains representing 28 isolates were identified only at the genus (94%≤)

or family (86%≤) level as shown in Table 4.27. Strains sequence length less than
1400 bp also identified as species level despite of the similarity of 98%.
181
Table 4.27. Identified LAB isolates at the genus (94%≤) or family level (86%≤) in
chickpea fermentations
Similarity %
of of closest relative by Sequence Accession
Strain isolates Family/Genus GenBank) length (bp) number
RL498 1 Weissella spp. 95 (NR_113258.1) 1525 MH704229
BL504 1 Weissella spp. 96 (NR_113258.1) 1465 MH704230
BL514 1 Enterococcus spp. 94 (NR_114453.1) 1458 MH704231
XL890 3 Streptococcaceae 94 (NR_040956.1) 1424 MH704233
RL1137 1 Enterococcus spp. 97 (NR_114742.1) 1477 MH704235
RL1189 2 Enterococcaceae 86 (NR_114742.1) 1369 MH704237
BL1367 3 Streptococcus spp. 95 (NR_115719.1) 1507 MH704240
XL1368 1 Weissella spp. 98 (NR_113258.1) 1335 MH704241
XL1377 1 Streptococcus spp. 95 (NR_115719.1) 1527 MH704242
RL1387 4 Streptococcus spp. 98 (NR_115719.1) 1368 MH704243
XL1400 1 Streptococcus spp. 95 (NR_042051.1) 1555 MH704244
In the present study, 121 strains belonging to 12 LAB species were

identified at the species level as shown in Table 4.28. W. confusa (44.6%) was the
dominant species, followed by E. faecium (25.6%) and W. cibaria (11.6%).
Furthermore, Leu. mesenteroides (5%), Lb. brevis (3.3%) and Streptococcus
lutetiensis (2.5%) were found as minor species. Conversely, Lb. plantarum, Pd.
acidilactici, St. salivarius, E. lactis, Pd. pentosaceus and Leu. mesenteroides subsp.
dextranium were only isolated from 1 or 2 samples.
182
Table 4.28. Percentage of the isolated species in chickpea fermentations

Species Number of species %
W. confusa 54 44.6
E. faecium 31 25.6
W. cibaria 14 11.6
Leu. mesenteroides 6 5.0
Lb. brevis 4 3.3
St. lutetiensis 3 2.5
Lb. plantarum 2 1.7
Pd. acidilactici 2 1.7
St. salivarius 2 1.7
E. lactis 1 0.8
Pd. pentosaceus 1 0.8
Leu. mesenteroides subsp. 1 0.8
dextranium
Total 121 100 %
The number of W. confusa was 54 in a total of 121 strains and comprised

almost half of the isolates. W. confusa was isolated from all collected chickpea
liquid starter and dough samples at both sampling times, except the A2 sample. No
W. confusa strains were isolated from the chickpea liquid starter and dough
samples of Bakery A at the second sampling. E. faecium was commonly isolated
from collected samples, except the B1 and N1 chickpea liquid starter and dough
samples. This species was also identified in laboratory-scale chickpea
fermentations conducted at 37°C. W. cibaria was identified in A and N chickpea
dough samples at both sampling times. It was also identified in the CLS-B1, CD-
B2 and CLS-B2 samples. Of the other isolated strains, Leu. mesenteroides was
only identified in the CD-A1 sample. Other LAB species Lb. brevis, St. lutetiensis,
Lb. plantarum, Pd. acidilactici, St. salivarius, E. lactis and Leu. mesenteroides
subsp. dextranium were isolated from different bakeries. None of the strains were
identified as LAB in the CLS-A1 and CLS-N2 samples. The number of LAB
identified at the species level in chickpea liquid starter and dough samples is shown
in Table 4.29.
183
The identified species in the fermentations of Bakery A are detailed in

Figure 4.40. No LAB species were detected in the chickpea liquid starters at the
first sampling. On the other hand, 5 species were identified in the A1 dough
adapted from that liquid starter, and the flour and fermentation environment could
be the source of these species. In the CD-A1 sample, Leu. mesenteroides and E.
faecium co-dominated the fermentation. Other identified minor species were W.
cibaria, Pd. pentosaceus and W. confusa. In the second sampling, E. faecium
dominated the chickpea fermentation. E. faecium and Lb. plantarum were
identified in both chickpea liquid starter and the dough that produced that liquid
starter. On the other hand, Lb. brevis, Pd. acidilactici, E. lactis and W. cibaria were
only identified in the chickpea liquid starter and dough at the second sampling of
Bakery A, respectively. The chickpea liquid starter contained a more complex LAB
flora than dough since only certain species dominated the chickpea dough
fermentations. On the other hand, W. cibaria was identified in the dough sample
despite not being isolated in the chickpea liquid starter; the flour and production
equipment could be the source of this species.
184
Table 4.29. Number of LAB identified at the species level in chickpea liquid starter and dough samples
CD- CLS- CD- CLS- CD- CLS- CD- CLS CD- CD- CLS CD- CLS- CD-
Species A1 A2 A2 B1 B1 B2 B2 -N1 N1 N2 -32 32 37 37
W. confusa 1/15 11/15 15/15 2/13 6/14 1/2 5/8 13/14
E. faecium 5/15 7/12 3/5 5/13 5/14 3/3 3/3
W. cibaria 2/15 1/5 3/15 3/13 3/14 1/8 1/14
Leu. mesenteroides 6/15
Lb. brevis 2/12 2/13
St.lutetiensis 1/2 2/8
Lb. plantarum 1/12 1/5
Pd. acidilactici 1/12 1/15
St. salivarius 1/1 1/1
E. lactis 1/12
Pd. pentosaceus 1/15
Leu. mesenteroides 1/13
subsp.
dextranium
Total LAB 15 12 5 15 15 13 14 2 8 14 1 1 3 3
185
Figure 4. 40. LAB species identified in Bakery A chickpea fermentations
The identified species in the fermentations of the Bakery B are shown in

Figure 4.41. W. confusa was isolated from all of the samples collected from Bakery
B at both sampling times. W. confusa, W. cibaria and Pd. acidilactici were
identified in the CLS-B1 sample. However, W. confusa alone dominated the
chickpea dough fermentation adapted from that starter. The second sampling
exhibited a richer diversity than the first sampling, with E. faecium and W. cibaria
dominating the liquid starter fermentation. Furthermore, W. confusa, Lb. brevis and
Leu. mesenteroides subsp. dextranium were identified as minor species. Certain
species continue to dominate the dough fermentation. From the most dominant to
186
the least, W. confusa, E. faecium and W. cibaria were identified in the chickpea
dough of Bakery B at the second sampling.
Figure 4.41. LAB species identified in Bakery B chickpea fermentations
The identified species in the fermentations of the Bakery N are given in

Figure 4.42. The number of isolated species was very low in the first sampling of
the Bakery N to discuss the dominant species. Only two strains belonging to W.
confusa and St. lutetiensis species were isolated from the CLS-N1 sample. The
187
chickpea dough adapted from that liquid starter contained W. confusa as the
dominant species. On the other hand, St. lutetiensis and W. cibaria were identified
in the CD-N1 sample. No LAB species were detected in the chickpea liquid starter
at the second sampling. Conversely, two species were identified in the N2 dough
produced with that liquid starter. The flour and fermentation environment could be
the source of these species. W. confusa dominated the dough fermentation at the
first sampling.
Figure 4.42. LAB species identified in Bakery N chickpea fermentations
188
Laboratory-scale chickpea fermentations were conducted at 32 and 37°C.

A total of 8 LAB strains were isolated from the laboratory-scale chickpea
fermentations. St. salivarius species was isolated from samples fermented at 32°C
and E. faecium species was isolated from samples fermented at 37°C. Both species
were isolated from the chickpea liquid starter and dough as shown in Figure 4.43.
As production was performed under semi-sterile conditions, the flora observed in
the liquid starter was seen in the dough producing that starter. However, the
number of the isolated strains was too low to discuss the dominant flora.
Figure 4.43. LAB species identified in the laboratory scale chickpea fermentations
189
Figure 4. 44. Distribution of the LAB strains in the chickpea fermentations at the
family level
LAB detected in the chickpea fermentations at the family level is shown in

Figure 4.44. The isolated strains from the chickpea liquid starter and dough
samples belonged to four families, i.e., Lactobacillaceae, Enterococcaceae,
Leuconostocaceae and Streptococcaceae. The distribution as a percentage is shown
in Figure 4.45.
190
Figure 4.45. Frequency of the LAB strains in the chickpea fermentations at the
family level
As it can be seen, many of the isolated strains belonged to the

Leuconostocaceae family, followed by Enterococcaceae. Among 149 isolates, the
number of strains in the Leuconostocaceae, Enterococcaceae, Lactobacillaceae,
and Streptococcaceae families were 78, 40, 14 and 17, respectively. The
Leuconostocaceae family was dominant in the CLS-B1, CD-B1, CD-N1 and CD-
N2 samples. On the other hand, it was co-dominant with Enterococcaceae in the A
and B2 chickpea liquid starter and dough samples.
The distribution of LAB strains detected in the chickpea fermentations at
the genus level is shown in Figure 4.46. The isolated strains from sourdough
samples belonged to 6 genera, i.e., Lactobacillus, Pediococcus, Enterococcus,
Leuconostoc, Weissella and Streptococcus. As it can be seen, many of the isolated
strains belonged to Weisella spp. 143 strains were identified at the genus level and
191
the number of Weissella, Enterococcus, Streptococcus, Lactobacillus, Leuconostoc

and Pediococcus were 71, 38, 14, 10, 7 and 3, respectively.
Figure 4.46. Frequency of LAB strains in the chickpea fermentations at the genus
level
192
Figure 4.47. Distribution of LAB strains in the chickpea fermentations at the genus
level
As it can be seen from Figure 4.47, the chickpea fermentation in Bakery B

was dominated by Weissella spp. at the first sampling; whereas, Weissella spp. and
Enterococcus spp. were co-dominant in the second sampling. Enterococcus spp.
was frequently isolated from the chickpea fermentations of Bakery A. In the N
Bakery fermentations, Streptococcus spp. and Weissella spp. were co-dominant;
however, Weissella spp. was dominant in the second sampling.
In the present study, half of the identified strains belonged to the genus
Weissella spp. and the most frequenly isolated species was W. confusa. The second
most frequenly isolated species was E. faecium. The microbial patterns of the
chickpea liquid starter and dough samples collected from different bakeries were
different from each other. The processing parameters and raw materials used
during production differed in the different bakeries. The production environment,
fermentation conditions, type of chickpea seeds, flour and even temperature of the
193
water used in the production can affect the microflora of the fermentations; hence,
the dominant species differed among different bakeries.
In a study conducted on the LAB flora of chickpea fermentations, Lc.
lactis, Lb. brevis and Lb. plantarum were identified, via phenotypic methods, in
the chickpea liquid starter. In the chickpea dough, the same species and also Lb.
pentosus and W.confusa were detected (Çebi, 2009). In another study, E.
mundtii/E. gallinarum, E. casseliflavus, Lb. plantarum/pentosus, Lb. sanfrancisco,
Lb. viridescens, Lb. bifermantans, Pd. urinea-equi, St. thermophiles and Lc. lactis
subsp. cremoris were isolated from chickpea fermentations (Hancıoglu-Sıkılı,
2003). The species Leuconostoc, Lactobacillus, Streptococcus and Pediococcus
spp. were previously reported in chickpea-containing fermented foods made in
India (Reddy et al., 1982)
In the present study, non-Lactobacillus spp. dominated the chickpea
fermentations. The chickpea dough is characterized by a higher pH compared with
the sourdough. Final pH values of the chickpea liquid starter and dough samples
were in the range of 4.82–5.67. Lactobacillus spp. are more resistant to acidic
conditions than other LAB and are able to grow well at pH values as low as 3-4
(Hammes and Hertel, 2009). Therefore, Lactobacillus spp. dominate in an acidic
sourdough environment. However, other species that grow at higher pH values are
commonly identified in chickpea fermentations. In addition, chickpea
fermentations are conducted in a very hot environment compared with sourdough
fermentations. The range of pH conditions for Weissella spp. growth is 5–7 and
they can grow up to 42–45°C (Fusco et al., 2015). Enterococcus species can
survive temperatures above 60°C for short periods (around 30 min), whereas the
optimum temperature is 37°C for Enterococcus and Streptococcus (Švec and Franz
2014). Leuconostoc species are non-acidophilic and the optimal temperature for
their growth is in the range of 10–37°C (Pikuta and Hoover, 2014).
194
4.2.4.2. Phylogenetic Relation of the LAB Strains

sequences (1400 bp≤) of the identified strains at the species level using two
possible tree reconstruction methods, minimum evolution and UPMGA, in MEGA
7 (Kumar et al., 2016).
The evolutionary history inferred using the Minimum Evolution method is
shown in Figure 4.48 (Rzhetsky and Nei, 1992). The bootstrap consensus tree
inferred from 500 replicates is taken to represent the evolutionary history of the
taxa analyzed. Branches corresponding to partitions reproduced in less than 50%
bootstrap replicates are collapsed. The percentage of replicate trees in which the
associated taxa clustered together in the bootstrap test (500 replicates) are shown
next to the branches (Felsenstein, 1985). The evolutionary distances were
computed using the number of differences method and are in the units of the
number of base differences per sequence. The ME tree was searched using the
Close-Neighbor-Interchange (CNI) algorithm at a search level of 1 (Nei and
Kumar, 2000). The Neighbor-joining algorithm was used to generate the initial tree
(Saitou and Nei, 1987). The analysis involved 35 nucleotide sequences.
195
100 Lb. brevis RL1169

85 Pd. acidilactici RL453
52
W. confusa BL1406
7 Lb. brevis RL1165
E. faecium XL1747
1 95 E. faecium BL1171
100 E. faecium RL1223
W. cibaria RL458
0 E. faecium RL1227
19
34 S. lutetiensis RL1346
Lb. brevis BL1233
0
58 E. lactis BL1229
61 E. faecium XL484
20 W. confusa RL900
7 W. confusa RL910

100 W. confusa RL425

Leu. mesenteroides subsp. dextranium RL1253

5
W. confusa RL1139
86 S. lutetiensis BL1362

100 S. salivarius XL1742

29 W. cibaria RL899
S. lutetiensis RL1386
5 E. faecium XL1150
Pd. pentosaceus BL512
18
36
W. confusa RL902
100 W. cibaria BL1361
Figure 4.48. The evolutionary history inferred using the Minimum Evolution
method
196
The evolutionary history inferred using the UPGMA method is shown in

Figure 4.49 (Sneath and Sokal, 1973). The bootstrap consensus tree inferred from
500 replicates is taken to represent the evolutionary history of the taxa analyzed.
Branches corresponding to partitions reproduced in less than 50% bootstrap
replicates are collapsed. The percentage of replicate trees in which the associated
taxa clustered together in the bootstrap test (500 replicates) are shown next to the
branches (Felsenstein, 1985). The evolutionary distances were computed using the
number of differences method and are in the units of the number of base
differences per sequence (Nei and Kumar, 2000).
197

56 S. lutetiensis BL1362
E. faecium RL1184
0 100 W. confusa RL1139

Leu. mesenteroides subsp. dextranium RL1253
31 E. faecium XL484
0
68 W. confusa RL900

33 W. confusa RL425
W. confusa RL910
21 E. faecium XL1150
0
0
Pd. pentosaceus BL512
15 S. salivarius XL1742
100 W. cibaria RL899
100 E. faecium BL1171

E. faecium XL1747
W. confusa BL1406
38
57 Lb. brevis RL1169
14 E. lactis BL1229
Lb. brevis BL1233

11
W. confusa RL902
100 W. cibaria BL1361
W. cibaria RL458
E. faecium RL1227
Figure 4.49. The evolutionary history inferred using the UPGMA method
198
4.2.4.3. Yeast Identification

A total of 216 presumptive yeast colonies were picked from YPD and L-
lysine media of the 12 chickpea liquid starter and dough samples collected from
bakeries. In addition, 22 colonies were collected from the laboratory-scale chickpea
fermentations. All of the 238 presumptive yeast cultures were grown in YPD
medium for 24-36 hours and subjected to DNA extraction using Instagene matrix
kit. Before extraction, all of the yeasts isolated were treated with lyticase enzyme
to degrade the cell walls. Totally 126 genomic DNA were extracted and subjected
to 5.8S ITS rRNA region amplification using primers ITS1 and ITS4. PCR
products showing visible bands on the agarose gel were subsequently digested
using the restriction endonucleases Hae III, Hha I and Hinf I. For a total of 6
profiles were determined according to the restriction fragments as shown in Table
4.30. Strains showed a unique restriction pattern for each species with the three
endonucleases used.
199
Table 4.30. Restriction fragments of the identified yeast species from chickpea fermentations
Restriction fragments (bp)
RFLP PCR products
Profile Species (bp) Hae III Hha I Hinf I
I S. cerevisiae 880 315+240+180+145 385+365+130 390+130

II P. fermentans 450 340+80+30 170+100+80 250+200
III C. parapsilosis 550 420+115 300+240 270+240
IV M. guilliermondii 625 400+120+50 300+250 320+300
V Cr. albidosimilis 630 500 330+300 350+280
VI Wickerhamiella spp. 420 420 280+200 180
200

gene sequencing as shown in Table 4.31. Only one isolate was identified at the
genus level as Wickerhamiella spp.For a species-level identification identity more
than 99% with the sequence length at least 400 bp was selected (Romanelli et al.,
2010).
The strains belonged to the 6 genera Saccharomyces, Candida,
Meyerozyma, Pichia, Cryptococcus (Cr.) and Wickerhamiella as shown in Figure
4.50.
Figure 4.50. Distribution of the 6 genera in the identified yeast isolates
201
Table 4.31. Accession numbers of the identified yeast species with their closest relatives and type strains
Closest relative
Accession Type strain
RFLP Accession Divergent
N1 2 bp3 number / Accession number/
Profile Species Strain number bases6
Identity(%)4 Identity(%)5
I S. cerevisiae 24 PM 343 MH704189 579 S. cerevisiae S. cerevisiae 5

SFM45 NRRL Y-12632
MG017586.1/99 NG_042623.1/99
II P. fermantans 2 NM 1088 MH704190 537 P. fermentans P.fermentans 4

A16 NRRL Y-1619
KM589463.1/99 NG_055109.1/99
III C. parapsilosis 20 PM 1076 MH704191 601 C. parapsilosis C. parapsilosis 0

PM 1124 MH704192 593 M66 ATCC 22019 4
GU080053.1/99 NG_054833.1/100
C. parapsilosis
ATCC 22019
NG_054833.1/99
202
Table 4.31. (Continued)

IV M. guilliermondii 12 NM 1119 MH704193 569 M. guilliermondii M. guilliermondii 3
NM 322 MH704194 599 Y2M NRRL Y-2075 5
MG478478.1/99 NG_042640.1/99
M. guilliermondii M. guilliermondii
SSA1523 NRRL Y-2075
KX791409.1/99 NG_042640.1/98
V Cr. albidosimilis 1 NM 1115 MH704195 622 Cr. albidosimilis N. albidosimilis 12

OF-17 CBS 77117
JQ916060.1 /99 NG_057653.1 /98
VI Wickerhamiella 1 PM 331 MH704196 613 Wi. pararugosa Wi. pararugosa 18
spp. QWD NRRL Y-17089
KF268260.1 /97 NG_055327.1/96
1
Number of species 226S rRNA gene sequenced strain representing each RFLP profile, 3sequence lengh, 4Sequence identity in
the D1 ⁄ D2 region of isolates with species in the GenBank, 5Sequence identity in the D1 ⁄ D2 region of isolates with type
strain of the same species in the GenBank, 6Number of the divergent bases from type strain 7Naganishia albidosimilis,
Synonymy ≡Cryptococcus albidosimilis -Vishniac & Kurtzman, International Journal of Systematic Bacteriology 42: 550
(1992).
203
In present study, 59 yeast strains belonging to 5 species were identified as

shown in Table 4.32. S. cerevisiae (40.7 %) was the dominant yeast species among
the identified strains. Other identified yeast species were P. fermentans (3.4 %), C.
parapsilosis (33.9 %), M. guilliermondii (20.3 %) and Cr. albidosimilis (1.7 %).
Table 4.32. Percentage of the isolated yeast species in chickpea fermentations

Species Number of the species %
S. cerevisiae 24 40.7
C. parapsilosis 20 33.9
M. guilliermondii 12 20.3
P. fermentans 2 3.4
Cr. albidosimilis 1 1.7
Total 59 100%
204
Table 4.33. Number of yeasts identified at the species level in chickpea fermentations
Yeast species CLS- CD- CD- CLS- CD- CLS- CD- CD-
A1 A1 A2 B1 B1 B2 B2 N2
S. cerevisiae 4/4 9/9 4/4 1/3 3/21 2/15 1/1
C. parapsilosis 16/21 4/15
M. guilliermondii 2/2 2/3 1/21 7/15
P. fermentans 2/15
Cr. albidosimilis 1/21
Total LAB 4 9 4 2 3 21 15 1
205
In the present study, yeast diversity was less than that of the LAB
microbiota, as 5 yeast species were identified in the collected chickpea liquid
starter and dough samples. S. cerevisiae was the most frequently isolated yeast
species. Collected chickpea doughs were produced without using baker’s yeast;
however, S. cerevisiae was isolated from all of the samples including the
laboratory-produced sourdough (Table 4.33). The presence of S. cerevisiae in the
bakery sourdoughs could be related to contamination of the bakery environment
and flour. No yeasts were identified in the CLS-A2, CLS-N1, CD-N1 and CLS-N2
samples. In the N Bakery, only one S. cerevisiae strain was isolated from the
chickpea dough at the second sampling. S. cerevisiae was the only identified
species from the A Bakery at both sampling times. On the other hand, chickpea
fermentations in Bakery B showed a rich biodiversity especially at the second
sampling (Figure 4.51). At the first sampling, M. guilliermondii was identified
from the CLS-B1 sample. Together with M. guilliermondii, S. cerevisiae was also
identified in the chickpea dough sample produced from that liquid. In addition, one
strain in the CLS-B1 sample was identified at the genus level and belonged to
Wickerhamiella spp. Conversely, B2 chickpea liquid starter and dough samples
showed a rich biodiversity. C. parapsilosis was the most identified yeast strain in
the CLS-B2 sample. Minor species identified in that chickpea liquid starter were S.
cerevisiae, M. guilliermondii and Cr. albidosimilis. In the chickpea dough
produced from that liquid starter, M. guilliermondii was dominant. Furthermore, C.
parapsilosis, S. cerevisiae and P. fermentans were also isolated; whereas, P.
fermentans was only isolated from the dough sample and the source of this species
could be the flour.
206
Figure 4.51. Distribution of the yeast species in chickpea fermentations
The collected samples showed different yeast diversity. The differences in

bakeries could be related to the location, as samples were collected from different
cities. Furthermore, the production environment, fermentation conditions, variety
of the chickpeas, flour and even temperature of the water used in the production
can affect the microflora of the fermentations, hence the dominant species differed
among different bakeries. Since chickpea doughs were produced in different
regions, the chickpeas and flour used in the production of these liquid starters and
doughs were region specific. In Bakery B, wholemeal flour was used in the
production of chickpea dough. Since nutrients in chickpea flour are different to that
of white wheat flour, different yeast species can grow. On the other hand, A and N
bakeries use boiling water, however, Bakery B uses hot water in the production of
the chickpea liquid starter and high water temperatures can prevent the
development of the microflora. In addition, no yeasts were identified in the
laboratory-scale chickpea fermentations. The temperature of water was 50°C for
the production of chickpea liquid; in addition, the fermentation was conducted
under semi-sterile conditions. After hot water is added to the seeds, the lid of the
207
jar was closed; therefore, if the microorganisms were killed by the hot water, no
microorganisms from the external environment could reach the liquid starter.
Katssboxakis and Mallidis (1996) reported that yeasts were unable to grow during
the fermentation of chickpea seeds (Katsaboxakis and Mallidis, 1996). However, to
evaluate the relationship of processing parameters with the microflora, every
processing parameter should be investigated indivdually both under bakery and
laboratory conditions.
There is limited research focusing on yeasts in chickpea fermentations,
with only one study reporting S. cerevisiae in chickpea fermentations (Hancıoglu-
Sıkılı, 2003).
C. parapsilosis was previously isolated from food fermentations of pozol
(a Mexican fermented maize dough), chinese steamed wheat buns, sourdough in
China and also Turkish sourdoughs (Ulloa et al., 1987; Luangsakul et al., 2009;
Zhang et al., 2011; Yagmur et al., 2016) However, this yeast species was
recognized as potantially pathogenic fungi (Trofa et al., 2008). M. guilliermondii
(formerly P. guilliermondii) was isolated from Turkish sourdough and Spanish
laboratory-made wheat sourdough (Barber and Baguena, 1988; Yagmur et al.,
2016; Gordún et al., 2018). Another study reported the presence of P. fermentans
species in southern Italian sourdoughs (Succi et al., 2003). Cr. albidosimilis
(synonym Naganishia albidosimilis) was only isolated from one chickpea liquid
starter. Interestingly, this yeast species was first isolated from soil in Antarctica
(Vishniac and Kurtzman, 1992). On the other hand, Cr. albidosimilis was identified
during the initial stages of the processing of barley (steeping and germination) in
an industrial malting facility in Finland (Laitila et al., 2006).
208
4.2.4.4. Phylogenetic Relation of the Yeast Strains

sequences (400 bp≤) of the identified yeast strains using a possible tree
reconstruction method UPMGA in MEGA 7 (Kumar et al., 2016).
The evolutionary relation of the isolated yeast strans from sourdough
samples was inferred using the UPGMA method as shown in Figure 4.52 (Sneath
and Sokal, 1973). The bootstrap consensus tree inferred from 200 replicates is
taken to represent the evolutionary history of the taxa analyzed. Branches
corresponding to partitions reproduced in less than 50% bootstrap replicates are
collapsed. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (200 replicates) are shown next to the branches
(Felsenstein, 1985). The evolutionary distances were computed using the
Maximum Composite Likelihood method and are in the units of the number of base
substitutions per site (Tamura et al., 2004). The analysis involved 8 nucleotide
sequences. All positions with less than 95% site coverage were eliminated. That is,
fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at
any position.
Figure 4.52. Evolutionary relationships of yeast with UPMGA method
209
4.3. Evaluation of the Technological Attributes of Selected LAB

4.3.1. LAB Strain Selection as Starter Culture in Sourdough and Chickpea
Fermentations
The most frequently isolated species were selected for further analysis in
sourdough and chickpea fermentations. For that purpose, some strains of Lb.
sanfranciscensis, Lb. plantarum and Lb. paralimentarius were investigated for
technological potential to be used as starter culture in sourdough fermentations.
Experimental production with starter culture was planned with mono- and dual-
culture. Lb. sanfranciscensis was the most frequent species in sourdoughs due to
the good adaptation of this strain to sourdough environmental conditions.
Therefore, strains of Lb. sanfranciscensis were chosen and investigated for their
technological potential for use as a starter culture in sourdough fermentations. For
strain 2, the properties of Lb. plantarum and Lb. paralimentarious species were
compared. Then, depending on their technological properties, strains belong to Lb.
sanfranciscensis and Lb. plantarum or Lb. paralimentarious were chosen for
experimental sourdough production. In chickpea fermentations, W. confusa was the
most frequent species and strains of W. confusa were investigated for technological
potential to be used as starter culture in chickpea fermentations. Strains
investigated for technological evaluation in sourdough and chickpea fermentations
are shown in Table 4.34.
210
Table 4.34. Strains investigated for technological evaluation in sourdough and

chickpea fermetations
Isolation
Strain Species/Family/Genus source
RL658 Lb. sanfranciscensis SD-K1
RL976 Lb. sanfranciscensis SD-T2
BL631 Lb. sanfranciscensis SD-K1
RL986 Lb. sanfranciscensis SD-T2
BL1023 Lb. sanfranciscensis SD-K2
RL1046 Lb. plantarum SD-K2
XL24 Lb. plantarum SD-M1
XL23 Lb. plantarum SD-M1
RL749 Lb. plantarum SD-W2
RL233 Lb. paralimentarius SD-T1
RL17 Lb. paralimentarius SD-M1
RL982 Lb. paralimentarius SD-T2
BL740 Lb. paralimentarius SD-W2
RL1639 Lb. paralimentarius SD-L7
RL1628 Lb. paralimentarius SD-L4
RL425 W.confusa CD-B1
RL1139 W.confusa CD-B2
RL898 W.confusa CD-N2
RL910 W.confusa CD-N2
RL1252 W.confusa CLS-B2
BL1406 W.confusa CLS-N1
211
Figure 4.53. Acidification kinetics of SFE by the 21 strains
To investigate acidification activity, LAB cultures were inoculated into the

SFE. The results of the acidification kinetics of SFE by the 21 strains are shown in
212
Figure 4.53 and Table 4.35. 10 LAB strains (W. confusa RL898, RL1252, RL1139,
RL425, RL910; Lb. plantarum XL23, RL749, XL24; Lb. paralimentarius BL740,
RL982) were able to decrease the pH below 5.0 after 8 h. At 24 h, almost all of the
strains acidified the medium to below pH 4.0. After 3 days, the lowest pH values
were measured in the SFEs inoculated with Lb. plantarum species. Lb. plantarum
XL23 showed the lowest pH value at the 7th day. Among the Lb. sanfranciscensis
strains, RL976 exhibited the lowest acidity values.
As reported previously, the 11 LAB strains belonging to different species
were able to decrease the SFE pH below 5.0 after 6 h and almost all of the strains
acidified the medium to below pH 4.0 after 24 hours (Alfonzo et al., 2013).
213
Table 4.35. Acidification kinetics of SFE by LAB strains

Lactic Acetic acid
0h 2h 4h 6h 8h 24 h 48 h 72 h 7d FQ
acid (mM) (mM)
Lb. paralimentarius BL740 6.0 5.83 5.54 5.13 4.74 3.77 3.70 3.65 3.40 8.00 11.13 0.72
Lb. paralimentarius RL1628 6.1
1 6.15 6.07 5.87 5.57 3.82 3.48 3.42 3.39 5.21 12.06 0.43
5 6.12 6.02 5.77 5.50 3.78 3.63 3.53 3.26 5.42 13.25 0.41
4 5.87 5.59 5.28 5.15 3.74 3.60 3.57 3.25 7.26 11.63 0.62
1 5.96 5.78 5.42 5.30 3.76 3.64 3.58 3.37 7.35 11.38 0.65
7 5.70 5.49 5.17 5.00 3.78 3.53 3.44 3.39 7.16 11.36 0.63
Lb. plantarum RL1046 6.0
0 6.05 5.96 5.73 5.25 3.80 3.54 3.38 3.28 8.44 12.75 0.66
Lb. plantarum RL749 6.0
9 5.92 5.46 4.87 4.44 3.61 3.38 3.29 3.28 8.44 12.75 0.66
Lb. plantarum XL23 6.0
6 5.79 5.32 4.64 4.34 3.62 3.41 3.30 3.18 12.81 18.57 0.69
Lb. plantarum XL24 6.2
9 6.14 5.56 4.93 4.48 3.68 3.46 3.35 3.21 8.85 10.29 0.86
Lb. sanfranciscensis BL1023 6.1
8 6.08 5.96 5.81 5.50 3.77 3.62 3.55 3.38 7.53 17.68 0.43
Lb. sanfranciscensis BL631 6.0
5 5.99 5.88 5.66 5.18 3.85 3.69 3.53 3.39 6.38 11.07 0.58
Lb. sanfranciscensis RL658 6.1
8 6.06 5.95 5.83 5.42 4.87 4.75 4.38 3.93 11.33 12.80 0.88
0 6.00 5.85 5.66 5.30 3.84 3.67 3.51 3.33 7.36 10.75 0.68
0 6.09 6.02 5.73 5.25 3.76 3.63 3.49 3.40 8.68 17.64 0.49
W. confusa BL1406 6.1
4 6.06 5.87 5.58 5.29 3.80 3.71 3.62 3.36 7.26 17.39 0.42
W. confusa RL1139 6.0
3 5.82 5.28 4.71 4.49 3.94 3.65 3.51 3.34 10.14 26.45 0.38
2 5.67 5.09 4.56 4.30 3.98 3.81 3.63 3,30 9.04 18.26 0.50
W. confusa RL425 6,1
7 6,00 5,42 4.79 4.48 3.99 3.86 3.65 3,26 7.79 17.63 0.44
7 5.55 4.72 4.32 4.13 3.66 3.52 3.47 3.36 8.78 14.03 0.63
9 5.95 5.55 5.16 4.48 3.68 3.49 3.40 3.33 7.23 12.20 0.59
8 214
After 8 hours of fermentation, acidified SFE samples were analysed for

their lactic and acetic acid content. The highest lactic acid was detected in the
acidified SFE inoculated with Lb plantarum XL23. The lactic acid content of
acidified SFE was determined to be in the range of 5.21-12.81 mM, with the lowest
amount corresponding to Lb. paralimentarius RL1628 and the highest to Lb
plantarum XL23. Acetic acid production was very high among strains. In terms of
mass per mass, the content of lactic and acetic acids were in the range of 0.47–1.15
and 0.62–1.58 mg/g, respectively. In the present study, all of the investigated
strains were heterofermentative. The acetic acid levels were also higher than those
of previously reported studies, which could be related to the composition of the
flour extract. In the present study, the supernantant of the flour extract was in the
semi-solid form, therefore the dry matter composition could be higher than the SFE
taken as liquid supernatant. Alfonzo et al. (2013) reported the highest acetic acid
content as 0.11 mg/g in the SFE inocukated with a Weisella spp. Settanni et al.
(2013) reported the lactic and acetic acid contents produced by different LAB
strains in sourdoughs processed with non sterile flour in the range of 1.36-6.47 and
0.15-1.08 mg/g after 8 h of fermentation, respectively. In another study,
experimental sourdoughs were produced by inoculating Lb. plantarum and Lb.
sanfranciscensis and lactic and acetic acid contents of the inoculated sourdoughs
were reported in the range of 1.48-4.19 and 0.33-1.05 mg/g after 8 hours
fermentation, respectively (Ventimiglia et al., 2015).
In the present study, according to the acidification activity results, Lb.
paralimentarious exhibited less acidification compared with the Lb. plantarum
species. Therefore, Lb. plantarum strains were further investigated for
technological potential and Lb. paralimentarious species were eliminated.
Furthermore, it has been reported previously that, Lb. plantarum could be an ideal
starter culture for Type I sourdoughs (Minervini et al., 2010).
215
Total EPS yields were determined gravimetrically. In addition, the colonies

were investigated for EPS formation on agar media as mucoid colonies. Among the
strains, only three W. confusa strains, RL1139, RL425 and RL1252, showed EPS
production. Figure 4.54 shows the growth of colonies that produced EPS on agar
media supplemented with sucrose (50 g/L). The EPS yield was determined as
0.00236, 0.00204 and 0.00173 g/mL for W. confusa RL1139, RL1252 and RL425,
respectively. Dextran production from sucrose by some W. confusa strains has been
reported previously (Collins et al., 1993; Katina et al., 2009; Björkroth et al.,
2014). A study reported the significant production (11–16 g/kg DW) of polymeric
dextran in wheat sourdoughs by W. confusa strains (Katina et al., 2009). Another
study determined produced EPS in wheat broth media by W. confusa as 0.43 g/100
ml (Lim et al., 2018).
Figure 4.54. EPS production on agar media
The proteolytic activity of the strains was tested using MRS agar media
supplemented with skimmed milk powder. With the exception of Lb.
sanfranciscensis RL658, zone formation was observed by all the strains. Different
strains of Lb. sanfranciscensis and Lb. plantarum have previously been reported to
exhibit proteolytic activity during sourdough fermentation (Gobbetti et al., 1994;
216
Gobbetti et al., 1996a; Rollán et al., 2005). However, proteolytic activity depends
on the strain and should be characterized at the strain level. In a study, the
proteolytic system of Lb. sanfranciscensis strain DSM 20451 was characterized
based on a genome-sampling approach (Vermeulen et al., 2005). In addition, by
adaptation to the protein environment, proteolytic activity can be increased. A
higher capacity of the Lb. plantarum strain, isolated from pickles, previously
showed better adaptation to protein-enriched medium than other LAB species
(Güler and Özcelik, 2017).
4.3.2. Investigaton of the Some Properties of the Selected LAB Strains

In the present study, sourdough strains were selected based on the
acidification capability. Acid production capacity of Lb. plantarum XL23 was very
high among Lb. plantarum species. After 8 hours, pH of the acidified flour extract
was the lowest in the Lb. plantarum XL23. Also according to the final pH values at
the 7th day, the lowest pH was determined in the same strain among Lb. plantarum
strains. After 8 hours, pH values were close to each other among Lb.
sanfranciscensis strains. On the other hand, final pH values at the 7th day was
determined in the acidified flour extract inoculated with Lb. sanfranciscensis
RL976 among other Lb. sanfranciscensis species. On the other hand, the FQ values
were investigated for strain selection for Lb. sanfranciscensis and RL976 and
RL658 were close to optimum value. Also, lactic acid production by Lb.
sanfranciscensis RL658 was the highest. However, this strain exhibited a very slow
acidification process and proteolytic activity was not detected. Therefore, Lb.
sanfranciscensis RL976 was chosen for experimental sourdough production. W.
confusa strains were selected based on the EPS production and less acidification
activity during 8 hours. Because chickpea doughs exhibited higher pH values
compared to sourdough fermentations. After 8 hours, W. confusa RL1139 and
217
BL1406 showed the highest pH determined in the fermented flour extract.

Therefore W. confusa RL1139 was used as the starter culture in the experimental
chickpea fermentations as the strain both producing EPS and showing less
acidification.
Antimicrobial activities, growth under different conditions and enzyme
profiles of the three selected strains for sourdough and chickpea fermentations
were investigated.
In the present study, antimicrobial activities of three strains selected as
starters were determined against B. subtilis, B. lincheniformis, Escherichia coli,
Penicillium expansum and Penicillium digitatum using the dual culture overlay
technique. The LAB strain Lb. plantarum XL23 showed inhibitory activity against
B. subtilis, B. lincheniformis, Escherichia coli and Penicillium expansum. Lb.
sanfranciscensis RL976 showed inhibitory activity only against B. subtilis. On the
other hand, W. confusa RL1139 did not exhibit any antimicrobial activity.
It was reported that Lb. plantarum 21B showed a very broad spectrum of
activity and inhibited many fungus species including Penicillium spp.
(Lavermicocca et al., 2000). Corsetti et al. (1996) investigated the Lactobacillus
spp. isolated from sourdoughs and reported all the strains were inhibitory to B.
subtilis and among the strains Lb. sanfranciscensis and Lb. plantarum strains had
the largest inhibitory spectrum. However, the inhibitory spectrum among strains of
the same species varied.
218
Figure 4.55. Acid production and color change with different carbohydrate sources
Growth of the selected strains at 15, 28, 37 and 45°C was investigated. Lb.
plantarum XL23 and Lb. sanfranciscensis RL976 strains did not grow at 45°C,
whereas W. confusa RL1139 strain grown at all temperatures. Growth at 45°C
generally varies among strains but many of the Lactobacillus spp. do not grow at
that temperature (Pot et al., 2014). Among W. confusa strains, growth at 45°C is
strain dependent with some strains showing good growth at this temperature
(Collins et al., 1993). Lb. plantarum XL23 tolerated all the conditions. On the other
hand, Lb. sanfranciscensis RL976 did not grow in the presence of 8% NaCl and at
pH 3.5. As reported previously, Lb. sanfranciscensis growth was inhibited at pH
4.0 (Brandt et al., 2004). W. confusa RL1139 did not grow in the presence of 6 and
8% NaCl and at pH 3.5. Acid was produced from glucose, fructose, sucrose,
maltose and mannose in all strains. The color of the tubes changed from red to
yellow as a result of low pH caused by acid production (Figure 4.55). Consumption
of other sugars changed according to the strain. Raffinose and xylose were only
used by Lb. plantarum XL23 and W. confusa RL1139, respectively. Acid
production from xylose, but not from arabinose, lactose, and raffinose was reported
for W. confusa strains previously (Fusco et al., 2015). None of the investigated
strains used ramnose and arabinose as carbohydrate sources. Growth of the selected
strains under different conditions are shown in Table 4.36.
219
Table 4.36. Growth of the selected strains under different conditions

Growth Lb. Lb. W.
conditions plantarum sanfranciscensis confusa
XL23 RL976 RL1139
15°C + + +
28°C + + +
37°C + + +
45°C - - +
%4 NaCl + + +
%6 NaCl + + -
%8 NaCl + - -
pH 3.5 + - -
pH 4.5 + + +
pH 6.5 + + +
Glucose + + +
Fructose + + +
Sucrose + + +
Maltose + + +
Galactose + + -
Lactose + + -
Mannose + + +
Mannitol + + -
Raffinose + - -
Xylose - - +
Ramnose - - -
Arabinose - - -
The enzyme profile of the selected strains was investigated using the API
ZYM enzyme testing system. An image of the color changes in the wells resulting
from enzyme activity is shown in Figure 4.56. Enzyme pattern results are given in
Table 4.37. Lb. plantarum XL23 produces enzymes as follows: leucine
arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-Bi-
phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase and N-acetyl-β-
glucosaminidase. Similar enzyme profiles of other Lb. plantarum strains have been
previously reported (Park and Lim, 2015; Mikelsaar et al., 2016). Lb.
sanfranciscensis RL976 produces enzymes as follows: leucine arylamidase, valine
arylamidase, acid phosphatase, naphthol-AS-Bi-phosphohydrolase, α-glucosidase,
220
β-glucosidase and N-acetyl-β- glucosaminidase. In a study, positive enzyme

activities were reported as only leucine arylamidase, valine arylamidase, cystine
arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase in API ZYM
kit (Hoang et al., 2015). W. confusa RL1139 produces alkaline phosphatase, acid
phosphatase and naphthol-AS-Bi-phosphohydrolase.
Figure 4.56. Investigation of the enzyme activities by API ZYM kit
221
Table 4.37. Enzyme activities of the strains

Lb. Lb. W.
Enzyme Code plantarum sanfranciscensis confusa
XL23 RL976 RL1139
Control 1 - - -
Alkaline phosphatase 2 - - +
Esterase 3 - - -
Esterase Lipase 4 - - -
Lipase 5 - - -
Leucine arylamidase 6 + + -
Valine arylamidase 7 + + -
Cystine arylamidase 8 - - -
Trypsin 9 - - -
α-chymotrypsin 10 - - -
Acid phosphatase 11 + + +
Naphthol-AS-Bi-
12 + + +
phosphohydrolase
α -galactosidase 13 - - -
β -galactosidase 14 + - -
β -glucuronidase 15 - - -
α -glucosidase 16 + + -
β - glucosidase 17 + + -
N-acetyl- β-
18 + + -
glucosaminidase
α -mannosidase 19 - - -
α -fucosidase 20 - - -
4.3.3. Production of Experimental Sourdoughs and Evaluation of Chemical

and Microbiological Properties
Based on the technological screening, Lb. plantarum XL23 and Lb.
sanfranciscensis RL976 were selected to act as starter for experimental sourdough
production. Experimental sourdoughs were produced using selected strains.
Overnight LAB cultures with an optical density (OD) of ca.1.00, corresponds to an
approximate concentration of 109 CFU/g, was used as inocula at the concentration
1 % (Settanni et al., 2013). The final concentration of the inoculum for each strain
was approximately 106 CFU/g in dough. Lb. sanfranciscensis 976 and Lb.
222
plantarum XL23 strains were used indivually and as dual-culture in the production
of experimental sourdoughs as shown below:
-Sourdough C (SD-C)-Control
-Sourdough 1 (SD-1)-Lb. plantarum XL23
-Sourdough 2 (SD-2)-Lb. sanfranciscensis RL976
-Sourdough 3 (SD-3)-Lb. plantarum XL23+Lb. sanfranciscensis RL976
Sourdough fermentations were conducted at 28 °C for 3 days with daily

refreshment. Some microbiological and chemical properties of the produced
sourdoughs were investigated and compared with the control sourdough. The pH
and TTA values registered for the experimental sourdoughs are shown in Figure
4.57.
Figure 4.57. pH and TTA values of the experimental sourdoughs
223
The initial pH and TTA of the dough were 5.98 and 3.2 mL, respectively.
Doughs inoculated with mono- or dual-culture of Lb. plantarum XL23 reached the
pH values less than 4.0 in 12 hours. Dough inoculated with Lb. sanfranciscensis
RL976 reached pH values less than 4.0 after 24 hours. At 24 hours, all of the
inoculated sourdoughs reached pH values around 3.75 and were stable until the last
refreshment. Conversely, in the control sourdough the pH decreased very slowly
and reached similar pH values with the inoculated sourdoughs after 48 h. pH of the
control sourdough was 3.85 at the last refreshment.
After 12 hours, the TTA of the inoculated sourdoughs was determined to
be in the range of 8.25-11.75 mL 0.1 N NaOH/10 g dough. The dough inoculated
with mono-culture of Lb. plantarum XL23 reached the highest acidity value as
11.75 mL 0.1 N NaOH/10 g dough. The dough inoculated with dual-culture of Lb.
plantarum XL23 and with Lb. sanfranciscensis RL976 reached an acidity value of
10.05 mL 0.1 N NaOH/10 g dough. Among the inoculated strains, the lowest
acidity at 12 hours was determined in the SD-2 dough inoculated with Lb.
sanfranciscensis RL976 as 8.25 mL 0.1 N NaOH/10 g dough. After 24 hours, the
acidity of the inoculated sourdoughs were in the range of 15.35-16.03 mL 0.1 N
NaOH/10 g dough. On the other hand, acidity values of the control dough
confirmed the trend showed by pH. TTA increased after 48 hours to 13.05 mL 0.1
N NaOH/10 g dough. At the last refreshment, the highest acidity was determined in
the SD-3 sourdough produced with the dual-culture inoculum. The highest acidity
was registered in the presence of Lb. plantarum XL23, alone and in combination
with Lb. sanfranciscensis RL-976. Acidifying capacity varies among strains and
Lb. plantarum XL23 showed good acidification in the present study. In a study,
experimental sourdoughs were produced by using mono- and dual-starter culture
combinations of Lb. plantarum and Lb. sanfranciscensis. At the end of the
224
fermentation, pH and TTA values were in the range of 3.44-4.09 and 10.10-12.60
mL (Ventimiglia et al., 2015).
Presumptive LAB, yeast, total mesophilic aerobic, mold and coliform
bacteria counts of the sourdoughs were investigated. The results of the cell counts
on mMRS agar are shown in Figure 4.58. Sourdoughs were inoculated with LAB
cultures at 6 log CFU/g; hence, LAB counts of the inoculated strains were around 6
log CFU/g. Inoculated strains dominated the fermentations as observed on the
morphological investigation of the petri dishes. Sourdoughs produced with mono-
culture inoculums contained colonies with the same appearence, whereas in the
multi-culture there were 2 colonies of different appearance. Figure 4.59 shows the
mMRS petri dishes of the mono- and dual-culture inoculums at the beginning of
the fermentations. On the first day, the lowest count was determined in the control
sourdough (8 log CFU/g). On the other hand, the counts of the sourdoughs
inoculated with starter cultures were close to each other. After 2 days, LAB counts
reached more than 11 log CFU/g in all sourdoughs.
Figure 4.58. Cell counts of LAB on the mMRS agar

(SD-C: control, SD-1: Lb. plantarum XL23, SD-2: Lb. sanfranciscensis, SD-3:Lb.
plantarum+Lb. sanfranciscensis)
225
SD-1 SD-2 SD-3
Figure 4.59. mMRS petri dishes of the mono- and dual-culture inoculums at the
beginning of the fermentations
Figure 4.60. Cell counts of yeasts and molds

In the unfermented doughs, yeast and mold counts were 3 and 2.47 log
CFU/g, respectively (Figure 4.60). Mold counts were <1 log CFU/g in the
sourdoughs produced with Lb. plantarum XL23 on the first day. At the 3rd day of
refreshment, no colonies were not detected on agar media. Yeast counts exhibited
variations. On the first day, yeast growth was not observed on agar media;
however, some of the sourdoughs showed different patterns every refreshment.
Presumptive yeast counts were 1.30 and 4.46 log CFU/g in the control and
226
SD-1 sourdough samples at the last back-slopping stage, respectively. As it can be

seen, every flour addition changed the flora in the doughs.
Figure 4.61. Cell counts of total mesophilic aerobic counts

In the unfermented doughs, total mesophilic aerobic bacteria counts were

determined as 3.7 log CFU/g. All of the TMAB counts were increased on the first
day. At the last refreshment, TMAB counts were in the range of 11.17-11.69 log
CFU/g in the sourdoughs as shown in Figure 4.61.
Presumptive total coliform bacteria counts of the sourdoughs were 120
MPN/g at the begining of the fermentation. At the 1st day of refreshment, no gas
bubbles were observed in any of the LST broth tubes of the sourdoughs inoculated
with starter culture, hence the presumptive coliform group bacteria was <3 MPN/g.
In the control sourdough, presumptive total coliform bacteria counts were 75
MPN/g on the first day of refreshment and then decreased after 2 days. As it can be
seen, control sourdough reached the characteristics of the inoculated sourdough
227
after 48 hours. Disappearance of the presumptive coliform bacteria can be related

to the pH decrease. In the inoculated sourdoughs, acidification was faster than in
control dough. Inoculated sourdoughs were characterized with high LAB counts,
fast acidification and low pH values. At the first refreshment, pH values of the
inoculated doughs were decreased below 4.0. On the other hand, sourdough sample
produced without starter culture addition reached this pH at the 2nd refreshment.
Acidity values and LAB counts of the samples confirmed the trend showed by pH.
After two days the control sourdough exhibited the same patterns with the
inoculated sourdoughs.
Carbohydrate, ethanol and organic acid content of the experimental
sourdoughs were also investigated and the results are given in Table 4.38. At the
final refreshment, the maltose+sucrose content was in the range of 9.19-13.34 g/kg
and was less in inoculated sourdoughs than the control sourdough. Differences
among the inoculated sourdough samples were not significant (p>0.05). At the
final refreshment, the glucose content was in the range of 6.17-10.51 g/kg. The
highest glucose content was detected in the control sourdough. Differences
between the glucose content of sourdoughs inoculated with mono and dual-culture
of Lb. plantarum XL23 were not significant (p>0.05). The final fructose contents
were in the range of 2.17-3.80 g/kg. The fructose content was less in inoculated
sourdoughs than the control sourdough. Differences in fructose content among the
inoculated sourdough samples were not significant (p>0.05). Lactic acid
production at the last refreshment was determined to be in the range of 8.87-11.53
g/kg. Among the inoculated sourdoughs, the lowest lactic acid production was
determined in the SD-1 sourdough, however, differences among the inoculated
sourdoughs were not significant (p>0.05). The acetic acid content was the highest
in the control dough, followed by the SD-S sourdough inoculated with Lb.
228
sanfranciscensis RL976 as a mono-culture. Differences in ethanol content among

the sourdough samples were not significant (p>0.05).
Table 4.38. Carbohydrate, ethanol and organic acid contents of the experimental
sourdoughs at the last refreshment
Compounds (g/kg) Experimental sourdoughs
Refreshment day SD-C SD-1 SD-2 SD-3
Maltose+sucrose
0 12.03 a ±0.098 12.03 a ±0.098 12.03 a ±0.098 12.03 a ±0.098
1 13.25 a ±0.09 11.11 b ±0.38 13.15 a ±1.15 13.92 a ±0.41
2 14.48 a ±0.68 9.84 b ±0.042 9.87b±0.29 10.07b±0.44
3 13.34a±0.47 9.19b±0.89 9.76b±0.14 9.87b±0.90
Glucose
0 7.41a±0.27 7.41a±0.27 7.41a±0.27 7.41a±0.27
1 7.08 ±1.09
ab
9.64 ±0.49
b
8.18 ±0.04
ab
6.15a±0.41
2 10.03 ±1.03
a
9.36 ±0.12
ab
8.89 ±0.50
a
7.81b±0.46
3 10.51 ±0.90
c
8.45 ±0.28
a
10.03 ±0.50
bc
6.17a±0.74
Fructose
0 6.21a±0.66 6.21a±0.66 6.21a±0.66 6.21a±0.66
1 4.08 b±0.60 2.36 a±0.17 4.50 b±0.11 2.76 a±0.06
2 3.17b±0.24 2.31 a±0.04 3.01bc±0.02 2.56 ab±0.31
3 3.80 ±0.89
b
2.17 ±0.25
a
2.52 ±0.44
ab
2.47 ab±0.19
Ethanol
0 <LOQ <LOQ <LOQ <LOQ
1 2.70b±0.00 2.44 a±0.12 2.52a±0.02 <LOQ
2 3.01b±0.04 2.42a±0.00 2.64a±0.06 <LOQ
3 2.54 a±0.02 2.40 a±0.00 2.62 a±0.06 <LOQ
Lactic acid
1 1.41a±0.40 11.34b±1.02 12.38b±0.09 10.80b±0.36
2 6.15a±0.79 10.74b±0.11 11.77b±0.97 11.38b±0.17
3 8.87 ±0.62
a
10.85 ±0.37
b
11.05 ±0.85
b
11.53 b±0.95
Acetic acid
1 1.70a±0.57 1.07a±0.20 2.10a±0.44 1.85a±0.17
2 2.48b±0.13 1.66ab±0.18 1.63ab±0.38 1.47a±0.54
3 1.76 ±0.12
b
1.20 ±0.62
ab
1.56 ±0.28
b
0.75a±0.26
a-c
Different superscript letters within same line indicate significant difference (Duncan
p<0.05) Results are given mean±SD (SD-C: control, SD-1: Lb. plantarum XL23, SD-2: Lb.
sanfranciscensis, SD-3:Lb. plantarum+Lb. sanfranciscensis)
229
As a result of the activities in sourdoughs, some VOC compounds are

generated. The SPME-GC-MS chromatographic analysis of the experimental
sourdoughs revealed the presence of 37 VOC compounds belonged to different
chemical groups as shown Table 4.39. VOC compounds were determined based on
the relative peak area. A GC-MS chromatogram image of VOCs is shown in
Appendix 11. In control dough at 0h, lower number of chemicals was detected
(n=13). At the end of the third refreshment, number of the VOC compounds
detected in the SD-C, SD-1, SD-2 and SD-3 sourdoughs were 22, 20, 19 and 21,
respectively. According to the relative peak area, formamide was the most detected
in the unfermented dough. Ethyl acetate and D-limonene were the most found in
SD-C and SD-1 sample. Besides these VOCs, heptenal and acetic acid in SD-C and
pentane and formamide in SD-1 was found. In SD-2, formamide, D-limonene,
ethenyl acetate, hexanal, heptenal and pentane and in SD-3 D-limonene, acetic
acid, ethenyl acetate, formamide, (1-methylbutyl)-oxirane, 1-hexanol, 3-methyl-1-
butanol and pentane were the most determined VOC compounds. In the present
study, D-limonene was detected in all sourdoughs. On the other hand, 3-methyl-1-
butanol and 1-hexanol was only determined in the SD-3 sourdough. In another
study, these alcohol comounds, 3-methyl-1-butanol and 1-hexanol, were detected
in the control, Lb. plantarum and Lb. sanfranciscensis inoculated sourdoughs
(Ventimiglia et al., 2015). Some VOC compounds as the metabolite of Lb.
plantarum were reviewd and among them ethyl acetate, acetaldehyde, 3-methyl-1-
butanal and heptenal were also detected in our study (Salim Ur et al., 2006).
230
Table 4.39. VOCs in the experimental sourdough samples as relative peak area (%)
VOC compoundsa D-0b SD-Cc SD-1c SD-2c SD-3c
2-Octen-1-ol (E) 5.42 n.d. 0.14 n.d. n.d.
(1-methylbutyl)-Oxirane n.d. 1.78 1.2 n.d. 9.12
(E-E)-2, 4-nonadienal 0.28 0.88 n.d. 0.59 n.d.
1-Hexanol n.d. n.d. n.d. n.d. 7.96
1-Pentanol n.d. n.d. n.d. n.d. 4.44
2-Penten-1-ol n.d. 0.78 n.d. 0.28 n.d.
2-pentyl-furan n.d. 0.47 1.29 0.57 0.84
3-methyl-1-Butanol n.d. n.d. n.d. n.d. 6.42
3-methyl-butanal 3.87 n.d. 2.135 n.d. n.d.
4-amino-1-Pentanol 1.25 0.43 n.d. n.d. n.d.
4-methyl- trans-Cyclohexanol n.d. n.d. 0.295 n.d. n.d.
5-(pentyloxy)-1-Pentene n.d. n.d. 0.59 0.75 n.d.
5-ethyl-4-methyl-3-Heptanone n.d. n.d. 0.28 n.d. n.d.
Acetaldehyde 2.84 0.29 1.27 5.14 4.31
Acetic acid n.d. 6.50 n.d. n.d. 13.69
Butyl acetate n.d. 1.00 n.d. n.d. n.d.
Ethenyl acetate n.d. n.d. n.d. 14.41 11.18
Hexyl acetate n.d. n.d. 0.25 n.d. n.d.
Pentyl acetate n.d. n.d. 0.52 n.d. n.d.
Benzene, 1,3-dichloro- 1.02 n.d. n.d. 0.26 0.22
Cyclobutanol 1.42 0.44 n.d. 0.39 0.21
Cyclopentanol 1.88 0.19 0.41 1.45 0.95
D-Limonene n.d. 12.32 11.03 17.92 13.96
Ethyl Acetate n.d. 52.59 59.70 n.d. n.d.
Formamide 40.87 3.88 6.14 25.11 12.62
Ethenyl formate 2.58 n.d. n.d. 0.89 0.50
Ɣ-Terpinene n.d. 0.19 0.50 0.33 0.35
Heptanal 1.14 6.75 2.67 8.41 n.d.
Hexanal 29.20 2.01 1.62 14.41 0.04
Humulene n.d. 0.15 0.32 0.48 1.41
l-Menthone n.d. n.d. n.d. n.d. 0.23
o-Cymene n.d. 0.33 0.66 1.08 0.99
Pentanal 2.55 n.d. n.d. n.d. n.d.
Pentane n.d. 3.93 8.82 6.22 7.91
propyl-Propanedioic acid n.d. 0.69 n.d. n.d. n.d.
tert-butyl-benzene n.d. 0.59 n.d. n.d. n.d.
trans-1,2-Cyclopentanediol n.d. 2.1 n.d. 0.9 1.74
Results indicate mean values of two measurements and are expressed as relative peak areas (peak area of each
compound/total area) × l00 ±SD.n.d., not detected. a The chemicals are shown alphabetically. bD-0:unfermented
doughcSourdoughs at the final refreshment (3rd day) SD-C: control sourdough, SD-1: fermented dough with Lb.
plantarum XL23, SD-2: inoculated dough with Lb. sanfranciscensis RL976, SD-1: inoculated dough with Lb.
plantarum XL23+ Lb. sanfranciscensis RL976
231
4.3.4. Multivariate Statistical Analysis of Experimental Sourdoughs

The microbiological and chemical parameters of sourdough samples were
subjected to the multivariate analysis to evaluate the differences/variabilities
among the samples. Data of the sourdough samples were subjected to PCa and a
total of 47 variables were investigated. They were grouped as microbiological,
chemical and VOC compounds and coded as M, C and V letters, respectively. The
loading and score plots of PCa analysis in Figure 4.62 shows that an overall
77.04% of variance was explained by the first component (F1 of 43.20%) and
second component (F2 of 33.84%).
As it can be seen, control sourdough, SD-C, differed from the inoculated
sourdoughs along Factor 1. SD-C sourdough was explained by the higher pH,
maltose+sucrose, glucose and acetic acid contents than other sourdoughs. Acidity
was the lowest in that sample as it can be seen in the negative correlation of pH and
TTA. Also control sourdough was characterized by the VOC compounds including
4-amino-1-Pentanol, (E-E)-2, 4-nonadienal, 2-Penten-1-ol, butyl acetate, tert-butyl-
benzene and propyl-Propanedioic acid. Among the inoculated sourdoughs, SD-1
differed from SD-2 and SD-3 sourdoughs along Factor 2. Especially, YPD and
PCA counts were the highest in that sample and relation was observed in the bi-
plot. SD-1 sourdough was characterized the VOC compounds especially 2-Octen-
1-ol (E), 5-ethyl-4-methyl-3-Heptanone, hexyl acetate, pentyl acetate, 3-methyl-
butanal, 4-methyl- trans-cyclohexanol and also Ɣ-Terpinene, 2-pentyl-furan,
pentane. SD-2 and SD-3 sourdoughs, inoculated with mono- and dual-culture of
Lb. sanfranciscensis RL976, were characterized with many VOC compounds and
acidity. SD-3 was mostly characterized with the high MRS counts, TTA, lactic acid
and VOC compounds.
232
component analysis of variables determined on sourdoughs
(M1:MRS, M2:YPD, M3:PCA, C4:pH, C5:TTA, C6:maltose+sucrose, C7: glucose,

C8:fructose, C9:lactic acid, C10:acetic acid, C11:ethanol,V12:Ɣ-Terpinene, V13:trans-1,2-
Cyclopentanediol, V14:3-methyl-1-Butanol, V15:1-Hexanol, V16:1-Pentanol, V17:4-
amino-1-Pentanol, V18:5-(pentyloxy)-1-Pentene, V19:(E-E)-2, 4-nonadienal, V20:2-Octen-
1-ol (E), V21:2-Penten-1-ol, V22:5-ethyl-4-methyl-3-Heptanone, V23:Acetaldehyde,
V24:Acetic acid, V25:Ethenyl acetate, V26:Butyl acetate, V27:Hexyl acetate, V28:Pentyl
acetate, V29: 1,3-dichloro Benzene, V30: tert-butyl-benzene, V31:3-methyl-butanal,
V32:Cyclobutanol, V33:4-methyl- trans-Cyclohexanol, V34:Cyclopentanol, V35:D-
Limonene, V36:Ethyl Acetate, V37:Formamide, V38:Ethenyl formate, V39:2-pentyl-furan,
V40:Heptanal, V41:Hexanal, V42:Humulene, V43:l-Menthone, V44:o-Cymene, V45:(1-
methylbutyl)-Oxirane, V46:Pentane, V47:propyl Propanedioic acid)
233
4.3.5. Production of Experimental Chickpea Liquid Starter and Dough

Samples and Evaluation of Chemical and Microbiological Properties
Experimental chickpea liquid starter and dough samples were produced
using W. confusa RL1139. Overnight LAB cultures with an optical density (OD) of
ca.1.00, corresponds to an approximate concentration of 109 CFU/g, was used as
inocula at the concentration 1 % (Settanni et al., 2013). The final concentration of
the inoculum was approximately 106 CFU/mL in chickpea liquid. W. confusa
RL1139 strain was used as mono-culture in the production of experimental
chickpea liquid starters as shown below:
- Chickpea liquid without starter addition (Control): CLS-C and CD-C

- Chickpea liquid with W. confusa RL1139: CLS-W and CD-W
Chickpea liquid was prepared by mixing ground chickpeas with boiled and
cooled water (37 °C). W. confusa RL1139 strain was inoculated to the chickpea
liquid at the beginning. Fermentations were conducted at 37 °C for 18 hours. Then
chickpea liquid starters were used in the production of adapted chickpea dough.
Some microbiological and chemical properties of the produced liquid starter and
dough samples were investigated and compared with control samples.
The pH and TTA values registered for the chickpea liquid starter and doughs are
shown in Figure 4.63.
234
Figure 4.63. pH and TTA values of the experimental chickpea liquid starter and
dough samples
The initial pH of the chickpea liquid was 6.93. The pH values of the
control and inoculated chickpea liquid starters were 4.92 and 4.82 at the end of the
fermentation, respectively. The final pH values of the control and inoculated
chickpea doughs were 4.82 and 4.79, respectively.
The pH of chickpea liquid fermentation was monitored in the first 10 hours of the
fermentation. According to the results, pH started to decrease after 6 hours in the control
liquid. Conversely, the pH of the inoculated liquid decresased after 2 hours, as shown in
Figure 4.64.
235
Figure 4.64. pH values monitored during 10 hours
After 18 hours, the TTA of the control and inoculated liquid starter
samples were 4.4 and 4.1 mL 0.1 N NaOH/10 g sample, respectively. Final TTA
values of the control and inoculated doughs produced were 5.26 and 5.97 mL 0.1 N
NaOH/10 g sample, respectively. TTA was determined as 0.47% in the control
dough. On the other hand, acidity in the chickpea dough produced with inoculated
chickpea liquid starter was 0.54%. Lower acidity values were determined in the
chikpea fermentations compared with sourdough fermentations. Chickpea dough is
generally referred as “sweet dough” in many regions. In order to reach the desired
level of acidification in chickpea dough, strains showing strong acidification
should not be used as starter culture.
Hancıoglu-Sıkılı (2003) used three different starter cultures, Lc. lactis
subsp. cremoris, Lb. bifermantas and Lb. viridescens, as mono-cultures for the
production of chickpea liquid starter. Higher acidification was detected in the
dough samples produced by Lactabacillus spp. than produced Lactoccocus spp.
Final pH and TTA values were in the range of 4.91-5.25 and 0.407-0.740%,
respectively (Hancıoglu-Sıkılı, 2003). In another study, chickpea fermentations
236
were conducted with three different LAB cultures as Lb. brevis FK2, Lc. lactis FK5
and Lb. plantarum FK25 and the pH values of the chickpea doughs were
determined in the range of 4.83-4.92. It was reported that differences in the pH
values of the chickpea doughs were not significant and spontanous flora in the
chickpea fermentations could affect the final pH values (Çebi, 2014). Reported
acidity values were in accordance with the present study.
Figure 4.65. Cell counts on mMRS and YPD media
Presumptive LAB, yeast, total mesophilic aerobic, mold and coliform

bacteria counts of the chickpea liquid starter and dough samples were investigated.
The results of the cell counts on the mMRS and YPD agar are shown in Figure
4.65. Chickpea liquid starter was inoculated with LAB culture at 6 log CFU/g;
hence, LAB counts of the inoculated sample was around 6-7 log CFU/g. On the
other hand, presumptive LAB counts of the control liquid was very low. The cells
counts of coth liquid starters on the mMRS were increased at the end of the 18 hour
237
fermentation, however, increase in the inoculated liquid starter was higher than the
control liquid starter. Cell counts on mMRS agar were higher in the chickpea
dough produced with the inoculated chickpea liquid starter compared with control
dough. Final LAB counts were 9.56 and 11.01 log CFU/g in the CD-C and CD-W,
respectively. Presumptive yeast counts varied during fermentations. Counts were 1
log CFU/g at the begining of the fermentation and then increased in the inoculated
chickpea liquid starter to 5.5 log CFU/g at the end of the fermentation. Yeast
counts were 3.02 and 4.86 log CFU/g in the dough samples at the end of the
fermentation, respectively. In the present study, yeast counts were determined less
than LAB counts. In a study, used three different starter cultures, Lc. lactis subsp.
cremoris, Lb. bifermantas and Lb. viridescens, as mono-cultures for the production
of chickpea liquid starter and reported the final LAB and yeast counts to be in the
range of 7.33-8.99 and 4.06-5.61 log CFU/g in the adapted chickpea dough
samples, respectively (Hancıoglu-Sıkılı, 2003).
In the present study, total bacteria counts during chickpea fermentations
are given in Figure 4.66. Total bacteria were enumerated on PCA and NA agar
media incubated at 30 and 37 °C, respectively. Also spore-forming bacteria were
investigated during the experimental chickpea fermentations. Spore-forming
bacteria counts were given as presumptive Bacillus spp. Total bacteria counts on
NA and PCA agar media showed similar patterns, but, number of the colonies
enumerated on PCA agar were higher than the colonies counted on NA agar.
Bacillus spp. were less than the total bacteria counts. At the end of the
fermentations, Bacillus spp. were 4.60, 3.65, 5.0 and 4.7 log CFU/g in the CLS-C,
CLS-W, CD-C and CD-W, respectively.
238
Figure 4.66. Total bacteria counts on PCA and NA media
Bacillus spp. are reported as the dominant microflora in chickpea liquids

previously (Hatzikamari et al., 2007b). In the present study, total bacteria and
Bacillus spp. counts on NA agar of chickpea liquid samples were monitored during
10 hours. Figure 4.67 shows the total and spore forming bacteria enumerated on
NA agar incubated at 37 °C for 10 hours. In the control liquid starters, Bacillus
spp. was counted as 4.69, 4.00 and 1.30 log CFU/g at the 2, 4 and 6 hours,
respectively. Any colony was not observed in the inoculated liquid starter plates.
Total bacteria counts were increased in both liquid samples during 10 hours.
239
Figure 4.67. Total and spore forming bacteria during the first 10 hours of chickpea
liquid fermentation
Table 4.40 shows the mold, coliform counts and indole test. Mold was
observed only in the unfermented dough samples. Presumptive total coliform
bacteria were <0.3 MPN/g in the liquid starter samples.
Table 4. 40. Mold and coliform counts during chickpea fermentations

Indole test /
Total coliform
Mold Presumptive
Samples bacteria
(log CFU/g) Escherichia
(MPN/g)
coli (MPN/g)
CLS-C-0 0 <0.3 -
CLS-C-18 0 <0.3 -
CLS-W-0 0 <0.3 -
CLS-W-18 0 <0.3 -
CD-C-0 2 11 3.6
CD-C-4 <1 3.6 -
CD-W-0 0.3 11 3.6
CD-W-4 <1 3.6 -
240
Table 4.41. Carbohydrate, ethanol and organic acid contents (g/kg) of the
experimental chickpea liquid starter and dough samples
Samples Maltose+ Glucose Fructose Lactic acid Acetic acid
sucrose
CLS- 0 <LOQ <LOQ <LOQ <LOQ <LOQ
CLS-C-18 0.30a±0.07 0.75ab±0.08 0.34a±0.00 1.05bc±0.14 1.29a±0.01
CLS-W-18 0.32a±0.07 0.81ab±0.01 <LOQ 0.70a±0.02 1.57a±0.33
CD-C-0 11.83ab±0.29 2.31bc±0.19 4.71b±0.99 0.99ab±0.29 2.23b±0.07
CD-C-4 13.29c±0.25 3.69c±0.98 5.42b±0.49 0.74a±0.03 2.74c±0.13
CD-W-0 14.76d±0.95 3.11c±0.33 4.79b±0.65 1.72d±0.63 1.96b±0.09
CD-W-4 14.60d±0.58 3.51c±0.02 5.33b±0.27 1.84d±0.05 3.43d±0.09
a-c
Different superscript letters within same column indicate significant difference (Duncan
p<0.05) Results are given mean±SD
The contents of carbohydrate, ethanol and organic acid in the experimental

chickpea liquid starter and dough samples are given in Table 4.41. Carbohydrate
contents of the chickpea liquid starters were below the quantification limit at the
beginning of the fermentation. Sample representing the beginning of the
fermentation is taken directly from water after mixed with chickpeas; hence,
compounds in the chickpeas could not passed into water yet. Therefore, all of the
detected compounds were <LOQ in the liquid at the beginning of the fermentation.
At the end of the chickpea liquid fermentation, differences in the maltose+sucrose
and glucose contents were not significant between the liquid starters produced with
and without starter culture (p<0.05). Lactic acid production was higher in the
control liquid starter than inoculated liquid. This can be related to the spontanous
flora present in the chickpea liquid since fermentations were conducted under
semi-sterile conditions. Differences in the maltose+sucrose, lactic and acetic acid
contents were significant but glucose and fructose contents were not signigicant
between the liquid starters produced with and without starter culture (p<0.05).
Acetic acid was produced more than lactic acid in the doughs. Lactic acid content
in the chickpea dough produced with inoculated liquid starter was higher than the
control dough. Ethanol contents of the all samples determined <LOQ. The possible
241
explanation can be low yeast counts in the samples or evaporation of ethanol

during the sampling.
The SPME-GC-MS chromatographic analysis revealed the presence of 32
VOC compounds in experimental chickpea fermentations (Table 4.42). VOC
compounds were determined based on the relative peak area. In control liquid
starter at 0 h, lower number of chemicals was detected (n=8). In the control and
inoculated chickpea liquid starters, number of the VOC compounds were 14 and
15, respectively. The number of VOC compounds in the chickpea doughs were in
the range of 16-19. According to the relative peak area, formamid, hexanal and
acetaldehyde were the most detected VOC compounds in the unfermented chickpea
liquid. Butanoic acid (synonym butyric acid) was found in all of the fermented
chickpea liquid starter and dough samples. In the fermented chickpea liquid
starters, butanoic acid had the biggest relative area and followed with 2,3,4-
trimethyloxetane, butyl butanoate and formamide. Relative peak area of butanoic
acid were 59.30 and 72.81%, in the control and adapted dough produced with
inoculated chickpea liquid starter, respectively. In chickpea fermentations,
production of butyric acid can be related to the presence of Clostridium species as
reported previously (Katsaboxakis and Mallidis, 1996). Because some strains of
Clostridium spp. produce butyric acid (He et al., 2005; Yang et al., 2011).
Hancıoglu-Sıkılı (2003) reported the occurence of butanoic and acetic acid acid in
the chickpea liquid starter and dough samples produced with various starter
cultures. Çebi (2014) investigated the volatile profile of the chickpea dough and
bread samples produced with different starter cultures and 1% baker's yeast and
determined alcohols including ethanol, 1-butanol, 1-hexanol, 1-octan-3-ol,
aldehydes including hexanal and acetaldehyde, esters including ethyl acetate and
hexyl butanoate more than other compounds in chickpea dough samples.
242
Table 4.42. VOCs in the experimental chickpea fermentations as relative peak area
(%)
CLS- CLS- CLS- CD-C- CD-C- CD-W-
VOC compoundsa
0b C-18c W-18c 0c CD-W-0c 4c 4c
1-fluoro-butane n.d. n.d. 0.19 n.d. n.d. n.d. n.d.
3-hydroxy-butanal n.d. n.d. n.d. n.d. n.d. n.d. 0.09
3-methyl-butanal n.d. n.d. 0.14 n.d. n.d. n.d. n.d.
Propyl-propanedioic acid n.d. n.d. 0.04 5.27 0.31 0.14 0.07
1,3-dichloro-benzene 0.55 n.d. n.d. 0.05 0.09 n.d. n.d.
Methoxyacetone n.d. 1.1 n.d. n.d. 3.14 n.d. 0.22
2,3,4-Trimethyloxetane n.d. 25.61 41.36 31.72 42.24 20.87 7.32
2,3-Butanedione n.d. n.d. n.d. 6.24 n.d. n.d. n.d.
2-amino-, (S)-1-propanol n.d. 0.05 n.d. n.d. n.d. 0.05 n.d.
2-Nonynoic acid n.d. n.d. n.d. n.d. n.d. 0.06 n.d.
2-Octynoic acid n.d. n.d. n.d. n.d. n.d. n.d. 0.04
3-methyl-pentanal n.d. 0.35 1.17 0.29 n.d. n.d. n.d.
5-Methyloxazolidine n.d. n.d. n.d. n.d. 0.51 0.48 n.d.
Acetaldehyde 11.23 n.d. 0.09 0.04 n.d. n.d. n.d.
Acetic acid n.d. n.d. n.d. n.d. 1.65 3.34 4.01
Acetone n.d. 5.08 4.9 4.58 3.86 3.6 n.d.
Butanoic acid n.d. 41.04 30.45 16.43 21.14 59.3 72.81
Butyl acetate n.d. 1.82 n.d. 1.26 n.d. 2.29 2.05
Butyl butanoate n.d. 13.5 5.7 2.99 0.37 3.49 0.78
Cyclobutanol n.d. 0.28 0.4 0.14 0.43 0.18 0.22
Ethenyl acetate n.d. n.d. n.d. 4.32 1.57 0.97 n.d.
Ethenyl formate 1.17 0.14 1.73 n.d. n.d. n.d. 0.02
Ethyl acetate n.d. 0.44 0.62 7.47 7.18 n.d. 7.26
Ethyl butanoate n.d. 4.28 5.31 1.32 0.61 0.64 1.9
Ethylene oxide 1.8 n.d. n.d. 0.76 0.02 0.28 0.01
Formamide 43.59 5.15 7.78 11.94 6.05 3.37 3.1
Formic acid n.d. n.d. n.d. n.d. 6.3 n.d. n.d.
Formyl acetate n.d. 1.15 n.d. 2.41 2.41 n.d. n.d.
Heptanal n.d. n.d. n.d. 0.61 n.d. 0.05 n.d.
Hexanal 39.36 n.d. 0.12 2.09 2.17 0.94 0.15
Pentanal 0.9 n.d. n.d. n.d. n.d. n.d. n.d.
β-Terpinyl acetate 0.96 n.d. n.d. n.d. n.d. n.d. n.d.
Results indicate mean values of two measurements and are expressed as relative peak areas (peak area of each
compound/total area) × l00 ±SD., n.d., not detected., a The chemicals are shown alphabetically., b CLS-
0:unfermented chickpea liquid, cCLS-C-18: Fermented chickpea liquid starter without inoculation, CLS-W-18:
Fermented chickpea liquid starter inoculated with W.confusa RL1139, CD-C-0:Unfermented control chickpea
dough, CD-W-0:Unfermented chickpea dough produced with inoculated chickpea liquid starter, CD-C-
4:Fermented control chickpea dough, CD-W-4:Fermented chickpea dough produced with inoculated chickpea
4.3.6. Multivariate Statistical Analysis of Experimental Chickpea

Fermentations
The microbiological and chemical parameters of chickpea liquid starter and
dough samples were subjected to the multivariate analysis to evaluate the
differences/variabilities among the samples. Data of the chickepea fermentations
243
were subjected to PCa and a total of 44 variables were investigated. They were
grouped as microbiological, chemical and VOC compounds and coded as M, C and
V letters, respectively. The loading and score plots of PCa analysis in Figure 4.68
shows that an overall 60.27% of variance was explained by the first component (F1
of 40.48%) and second component (F2 of 19.79%).
As it can be seen, unfermented chickpea liquid differed from the other
samples with regards to F1. Along with F2, chickpea liquid starters and dough
samples were seperated from each other. Control and inoculated samples were
close to each other but characterized with different VOC compounds. CLS-W-18
sample was characterized by VOC compounds including 3-methyl-pentanal, 3-
methyl-butanal and also butane1-fluoro. Chickpea dough samples were
characterized with many VOC compounds. Among the VOCs, butanoic acid and
also acetic acid are positively correlated with fermented chickpea dough samples.
244
component analysis of variables determined on chickpea
fermentations
(M1:MRS, M2:YPD, M3:NA, M4:NA-spore forming bacteria, M5:PCA, C6:pH, C7:TTA,

C8:maltose+sucrose, C9:glucose, C10:fructose, C11:lactic acid, C12: acetic acid, V13:3-
hydroxy-butanal, V14: 3-methyl-butanal, V15: Propyl-propanedioic acid, V16:1,3dichloro-
benzene, V17: Methoxyacetone, V18:2,3-Butanedione, V19:2-amino-,(S)-1-propanol, V20:2-
Nonynoic acid, V21:2-Octynoic acid, V22:3-methyl-pentanal, V23:5-Methyloxazolidine,
V24:Acetaldehyde, V25:Acetic acid, V26:Ethenyl acetate, V27: Formyl acetate, V28:Butyl
acetate, V29:Acetone, V30: 1-fluoro-Butane, V31:Butanoic acid, V32:Butyl butanoate, V33:
Ethyl butanoate, V34:Cyclobutanol, V35: β-Terpinyl acetate, V36:Ethyl Acetate, V37:Ethylene
oxide, V38:Formamide, V39:Formic acid, V40: Ethenyl formate, V41:Heptanal, V42:Hexanal,
V43: 2,3,4-Trimethyloxetane, V44:Pentanal)
245
246
5. CONCLUSION Cennet Pelin BOYACI GÜNDÜZ
5. CONCLUSION
In this study, microbiological and chemical characteristics and LAB and

yeasts biodiversity of the sourdough and chickpea fermentations were investigated
in the collected samples and laboratory scale productions. Technological potential
of some LAB strains were investigated and used in the sourdough and chickpea
liquid starter productions.
Results of the sourdough fermentations:
 Sourdough samples, produced by traditional method without baker's

yeast addition, were collected from the bakeries in three different
cities of Turkey at two different times.
 The pH levels of the collected sourdough samples ranged from 3.71 to
3.96 and the pH exhibited a mean value of 3.87. The highest pH value
was measured in the rye sourdough sample. pH values showed
differences among sourdoughs and sampling times.
 TTA levels of the collected sourdough samples ranged from 6.78 to
23.93 mL 0.1 N NaOH /10 g dough. TTA values exhibited differences
among sourdoughs and sampling times and the differences between
the samples were significant (p<0.05).
 According to the statistical results, the differences in sugar content
between the samples collected from the different bakeries were
significant (p<0.05).
 In the collected sourdoughs, lactic and acetic acids varied from 57 to
156 and 9 to 39 mM, respectively. The differences between the lactic
and acetic acid contents in the collected samples from different
bakeries were significant (p<0.05).
247
 The highest cell density on mMRS agar was in the rye sourdough.
 The highest cell density on YPD agar was in the rye sourdough.
 For presumptive total coliform bacteria, all of the tubes gave negative
results (<3MPN/g).
 Under laboratory conditions, sourdough was produced at 28°C by
propagating over a period of 7 days using the daily back-slopping
(refreshment) procedure.
 The pH of the prepared sourdough did not change during the first 12 h
of fermentation, but decreased to 4.58 after 24 hours. TTA was stable
during the first 12 hours, but then increased. During the following
days, the acidity continued to increase, but not greatly, reaching at
final value of 17.56 mL 0.1 N NaOH.
 In the present study, FQ levels were determined in the range of 2.48-
5.90. The FQ of the laboratory produced sourdough was high and
determined as 10.84. Favored conditions such as propagation ratio,
fermentation time and temperature provided the growth of LAB and
metabolite production increased.
 According to the multivariate statistical analysis of the sourdoughs,
acetic acid, TTA, lactic acid and SDB were positively correlated.
Samples collected from the same bakery at two different times were
included in a group except SD-T sample.
 Properties of the SD-T sample was different in the second sampling
since processing conditions changed. Changes in the processing
conditions at two different times, result in sourdoughs with different
characteristics.
248
 Lb. sanfranciscensis was the dominant species in sourdough

fermentations and Lb. plantarum and Lb. paralimentarious were other
frequently isolated species.
 Lb. paracasei, Leu. mesenteroides, W. confusa, Lb. curvatus and Lb.
brevis were found to be minor species. Furthermore, Lb. pentosus,
Leu. citreum, Lb. paraplantarum, Lb. acidophilus, E. faecium, Pd.
inopinatus, Lb. parabrevis, Lc. lactis subsp. cremoris, W. cibaria and
Pd. pentosaceus were only isolated from 1 or 2 samples.
 In the SD-M sample, Lb. plantarum was detected as the predominant
species at the first sampling and Lb. paralimentarious and W. confusa
were co-dominant species at the second sampling.
 In the SD-T1 sample, Lb. paralimentarious and Lb. paracasei were
detected as the predominant species and Lb. sanfranciscensis was the
dominant species at the second sampling.
 The SD-K sample contained Lb. sanfranciscensis as the dominant
species at the first sampling and at the second sampling Lb. plantarum
was the dominant species.
 Rye sourdough contained Lb. paralimentarious as the dominant
species at both sampling.
 In the laboratory produced sourdough, Lb. sanfranciscensis was not
isolated during 7 days of fermentation.
 S. cerevisiae (72.5%) was the dominant yeast species in sourdoughs.
Collected sourdoughs were produced without using baker’s yeast,
however, the presence of S. cerevisiae in the bakery sourdoughs can
be related to contamination of the bakery environment with baker’s
yeast.
249
 Other isolated yeast species were K. bulderi (7.2%), P. fermentans

(5.9%), P. membranifaciens (5.2%), K. servazzii (4.6%), K. unispora
(2.6%) and H'spora valbyensis (2%).
 In the present study, H'spora valbyensis was isolated from sourdoughs
for the first time. To my knowledge, the presence of this yeast species
in sourdough fermentations has not been documented previously.
Results of the chickpea fermentations:
 Chickpea liquid starter and dough samples were collected from the
bakeries that are well-known in their regions in Turkey and have been
producing chickpea bread for many years.
 The pH levels of the chickpea liquid starter samples ranged from 4.82
to 5.67. There was a wide variation among pH levels of the chickpea
liquid samples and sampling times. According to the statistical results,
the differences between the samples collected from different bakeries
were significant (p<0.05).
 The pH levels of the chickpea dough samples ranged from 5.12 to
5.53. According to the statistical results, the differences between the
samples collected from different bakeries were significant (p<0.05).
 Acidity levels of the collected chickpea liquid samples ranged from
1.65 to 3.20 ml 0.1 N NaOH/10 g sample. The acidity content was
significantly (p<0.05) different between chickpea liquid samples.
 The acidity levels of the collected chickpea dough samples ranged
from 3.03 to 5.40 mL 0.1 N NaOH/10 g sample. The acidity content
was significantly (p<0.05) different among chickpea dough samples.
The values also showed a significant difference among some samples
collected on two different sampling times.
250
 The levels of the acetic acid in the dough samples were below the
LOQ.
 The highest cell densities on mMRS and gM17 agar media were
counted in the CLS-B2 sample.
 The highest cell densities on YPD and L-lysine agar media were
detected in the CLS-N1 and CLS-A1 samples, respectively.
 The control chickpea liquid starter and dough samples were produced
in duplicate under laboratory conditions at 32 and 37°C. The total
titratable acidity value was higher in the chickpea liquid starter
fermented at 37°C (2.95 mL 0.1 N NaOH/10 g sample) compared with
32°C (1.95 mL 0.1 N NaOH/10 g sample).
 According to the multivariate statistical analysis of the chickpea
fermentations, all of the chickpea doughs were gathered together with
the control trial. Similarly, all of the chickpea liquid starters were
included in another cluster.
 LAB counts on MRS, M17 and TTA and lactic acid contents were
positively correlated as expected. The pH was negatively correlated
with TTA and positively correlated with YPD, fructose and glucose.
 Chickpea dough samples were seperated from liquid starter samples in
the dendogram constructed according to the PCa analysis.
 W. confusa was the dominant species, followed by E. faecium and W.
cibaria.
 Leu. mesenteroides, Lb. brevis and St. lutetiensis were found as minor
species. Conversely, Lb. plantarum, Pd. acidilactici, St. salivarius, E.
lactis and Leu. mesenteroides subsp. dextranium were only isolated
from 1 or 2 samples.
251
 In the CD-A1 sample, Leu. mesenteroides and E. faecium co-

dominated the fermentation. In the second sampling, E. faecium
dominated the chickpea fermentation.
 W. confusa was isolated from all of the samples collected from Bakery
B at both sampling times. The second sampling exhibited a richer
diversity than the first sampling, with E. faecium and W. cibaria
dominating the liquid starter fermentation. From the most dominant to
the least, W. confusa, E. faecium and W. cibaria were identified in the
chickpea dough of Bakery B at the second sampling.
 W. confusa dominated the fermentations in N Bakery.
 Non-Lactobacillus spp. dominated the chickpea fermentations.
Lactobacillus spp. are more resistant to acidic conditions than other
LAB and therefore, Lactobacillus spp. dominate in an acidic
sourdough environment. However, other species that grow at higher
pH values were commonly identified in chickpea fermentations.
 S. cerevisiae was the dominant yeast species.
 Other isolated yeast species were P. fermentans, C. parapsilosis, M.
guilliermondii and Cr. albidosimilis. All of them were reported for the
first time in chickpea fermentations.
 Also Bacillus spp. are found in chickpea fermentations. Some of the
sequences showed high identity with Bacillus spp. (data not shown)
Results of the experimental sourdough and chickpea fermentations:
 Strains of Lb. sanfranciscensis, Lb. plantarum and Lb.
paralimentarius were investigated for technological potential to be
used as starter culture in sourdough fermentations.
252
 In chickpea fermentations, W. confusa was the most frequent species

and strains of W. confusa were investigated for technological potential
to be used as starter culture in chickpea fermentations.
 The lowest pH values were measured in the sterile flour extract
inoculated with Lb. plantarum species.
 Among the strains, only three W. confusa strains, RL1139, RL425 and
RL1252, showed EPS production.
 Based on the technological screening, Lb. plantarum XL23 and Lb.
sanfranciscensis RL976 were selected to act as starter culture for
experimental sourdough production.
 Doughs inoculated with mono- or dual-culture of Lb. plantarum XL23
reached the pH values less than 4.0 in 12 hours. However, dough
inoculated with Lb. sanfranciscensis RL976 reached pH values less
than 4.0 after 24 hours.
 In the control sourdough the pH decreased very slowly and reached
similar pH values with the inoculated sourdoughs after 48 h.
 Inoculated sourdoughs were characterized with high LAB counts, fast
acidification and low pH values. At the first refreshment, pH of the
inoculated doughs were decreased below 4.0. On the other hand,
sourdough sample produced without starter culture addition reached
this pH at the 2nd refreshment. Acidity values and LAB counts of the
samples confirmed the trend showed by pH. After two days the
control sourdough exhibited the same patterns with the inoculated
sourdoughs.
 Ethyl acetate and D-limonene were the most found in SD-C and SD-1
sample. In SD-2, formamide, D-limonene, ethenyl acetate, hexanal,
heptenal and pentane and in SD-3, D-limonene, acetic acid, ethenyl
253
acetate, formamide, (1-methylbutyl)-oxirane, 1-hexanol, 3-methyl-1-

butanol and pentane were the most determined VOC compounds.
 D-limonene was detected in all sourdoughs.
 SD-2 and SD-3 sourdoughs, inoculated with mono- and dual-culture
of Lb. sanfranciscensis RL976, were characterized with many VOC
compounds and acidity. SD-3 was mostly characterized with the high
MRS counts, TTA, lactic acid content and VOC compounds.
 Experimental chickpea liquid starter and dough samples were
produced using W. confusa RL1139.
 Lower acidity values were determined in the chikpea fermentations
compared with sourdough fermentations. Chickpea dough is generally
referred as “sweet dough” in many regions. In order to reach the
desired level of acidification in chickpea dough, strains showing
strong acidification should not be used as starter culture.
 Total bacteria and Bacillus spp. counts on NA agar of chickpea liquid
samples were monitored during 10 hours. Total bacteria counts were
increased in both liquid samples during 10 hours.
 Butanoic acid was found in all of the fermented chickpea liquid starter
and dough samples.
 Chickpea fermentations were characterized with butanoic acid.
The following suggestions can be given for further studies based

on the results of this thesis:
 Traditional bread production has gained importance due to increasing

demand by consumers for more organic and healthy foods. Sourdough
and chickpea breads are produced with fermentation without baker's
254
yeast addition. Therefore, potentiality of the industrial production of

sourdough and chickpea breads should be investigated.
 For the industrial production, starter fermentations should be
examined. In future studies, starter culture characteristics of the
identified microorganisms can be investigated to develop starter
culture combinations for sourdough and chickpea fermentations. To
obtain a final product with same characteristics, same conditions
should be applied during fermentations. Starter culture addition and
providing same conditions will result in doughs with same
characteristics. Therefore, by starter culture addition, industrial
production of standard sourdough and chickpea breads will be
possible everytime at the same quality.
 The quality parameters of the breads should be examined by using
combinations of LAB strains to improve the quality parameters.
 In chickpea fermentations, besides LAB, some Bacillus spp. can be
investigated to be used as starter cultures.
255
256
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302
CURRICULUM VITAE
The author was born in 1988 in Isparta. She completed primary, secondary
and high school education in Antalya. In 2010, she graduated from Ege University,
Faculty of Engineering, Department of Food Engineering, İzmir, Turkey. In 2008,
she participated in the Erasmus Student Exchange Program for a six-month period
in the Food Engineering Department of Helsinki University, Helsinki, Finland. In
2010, she was selected as fellow for the 2211-E National Scholarship Programme
of the The Scientific and Technological Research Council of Turkey (TÜBİTAK)
and was supported during Msc and PhD education. In 2012, she successfully
completed her MSc degree in Akdeniz University, Department of Food
Engineering, Antalya, Turkey. In 2013, she was admitted into the PhD programme
in the Institute of Natural and Applied Science, Faculty of Agriculture, Department
of Food Engineering, Çukurova University, Adana, Turkey. She has been working
as a Research Assisstant in the Food Engineering Department of Adana Science
and Technology University, Adana, Turkey since 2013. She has been continuing
her PhD education in the Department of Food Engineering, Çukurova University.
303
304
APPENDICES
305
306
Appendix 1. Chromatogram image of the maltose+sucrose, glucose and fructose standards
307
Appendix 2. Chromatogram image of the ethanol standard
308
Appendix 3. Chromatogram image of the lactic acid and acetic acid standards
309
Appendix 4. Calibration curves for standards
310
Appendix 5. Chromatogram images of a sourdough sample
311
Appendix 6. Chromatogram images of a chickpea liquid starter
312
Appendix 7. Chromatogram images of a chickpea dough
313
Appendix 8. A gel image of the RAPD-PCR analysis with M13 primer
314
Appendix 9. A gel image of the 5.8S ITS rRNA region amplification
315
Appendix 10. A gel image of the RFLP with restriction endonucleases Hae III,
Hha I and Hinf I
316
Appendix 11. GC-MS chromatogram image of VOCs
317

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ÇUKUROVA UNIVERSITY

INSTITUTE OF NATURAL AND APPLIED SCIENCES

Cennet Pelin BOYACI GÜNDÜZ

MOLECULAR CHARACTERIZATION OF THE

DEPARTMENT OF FOOD ENGINEERING

MOLECULAR CHARACTERIZATION OF THE PREDOMINANT

Cennet Pelin BOYACI GÜNDÜZ

DEPARTMENT OF FOOD ENGINEERING

………………………….. ……………………........ …………………………………

This PhD Thesis is written at the Food Engineering Department of Institute of

Prof. Dr. Mustafa GÖK

A part of this study was financially supported by Çukurova University Academic

MOLECULAR CHARACTERIZATION OF THE PREDOMINANT

Cennet Pelin BOYACI GÜNDÜZ

Supervisor : Prof. Dr. Hüseyin ERTEN

Key Words: Sourdough, chickpea liquid starter, LAB, yeasts, PCR

NOHUT MAYASI VE EKŞİ HAMUR FERMANTASYONLARINDAKİ

Cennet Pelin BOYACI GÜNDÜZ

Danışman : Prof. Dr. Hüseyin ERTEN

Bu çalışmada, ekşi hamur ve nohut mayası fermantasyonlarında etkili

I would like to sincerely thank to my supervisor, Prof. Dr. Hüseyin

Table 2.1. Fermentation metabolism of some LAB .............................................. 13

Figure 2.1. Fermentation of glucose in homofermentatives ................................. 15

Fermentation is one of the oldest known methods of food preservation and

and Macedonia (Hatzikamari et al., 2007a). In the literature, research on chickpea

 Isolation of LAB and yeasts from sourdough samples produced using

1. Foods are preserved by fermentation as a result of the formation of

A diverse range of fermented foods are found worldwide and a number of

2.1.1. Biochemistry of the Fermentation Process

produces a microbial product by the mass culture of microorganisms (Stanbury,

Fermentation and respiration are alternative metabolic choices available to

2.1.2. Carbohydrate Catabolism

The phosphoketolase (PK) pathway is characteristically observed in

2.1.3. Lactic Acid Bacteria (LAB)

lactate dehydrogenase under anaerobic conditions, as shown in Equation 2 on a

Table 2.1. Fermentation metabolism of some LAB

2.1.3.1. Homofermentative LAB

Homofermentative LAB include Lactococcus, Streptococcus, Pediococcus,

Figure 2.1. Fermentation of glucose in homofermentatives adapted from Endo and

2.1.3.2. Heterofermentative LAB

heterofermentative LAB produce more flavor and aroma compounds, such as

pyruvic acid produced via glycolysis is converted to acetaldehyde and CO2 by

Yeasts are an important group of eukaryotic microorganisms (Tamang and

highly aerobic conditions and at a low concentrations of sugar, yeast consume

2.2.1. History of Sourdough

2.2.2. Sourdough Production

refreshments, also known as re-buildings, replenishments, back-slopping etc.

2.2.2.1. Flour in Sourdough Production

2.2.2.1.(1). Wheat Flour

2.2.2.1.(2). Rye Flour

solubility and swelling behaviour of pentosans increase at the low pH values

2.2.2.1.(3). Other Flour Types

2.2.2.2. Classification of Sourdough Production Methods

-Type I traditional sourdoughs

Type I-traditional sourdoughs are produced by using a sourdough that was

2.2.3. Sourdough Microflora

Lactococcus, Pediococcus, Leuconostoc and Weissella species. Sourdough is rich

by conventional physiological and biochemical tests and also confirmed with

plantarum, Lb. alimentarius, Lb. paralimentarius, Lb. sanfranciscensis, Lb. brevis,

guiliermondii and T. delbrueckii were detected as the predominant yeast species in