Culture craft

Introduction

Chemical composition

Contents
Microbial composition

Health Benefits

Case study
 Kombucha

 Kombucha is a beverage of manchurian origins

obtained from fermented tea by a microbial
consortium composed of several bacteria and
yeasts. (Often referred to as a SCOBY).
 The fermentation process results in a tangy,
slightly fizzy drink that can be flavored with
fruits, herbs, or spices.

de Miranda,et,al - 2021
3
It originated in northeast China (Manchuria) and later spread to Russia
and the rest of the world.

Kombucha is also frequently called “tea fungus”.

Kombucha is a traditional fermented tea whose consumption has

increased in the recent years due to its multiple functional properties
such as anti-inflammatory potential and antioxidant activity.

Villarreal-Soto S A et,.al 2018 4

Present status & Recent Trends

 nowadays there are high numbers of kombucha companies producing

commercialized kombucha.

 These kombucha products are mainly sold at local farmers’ markets. The
industrialization of kombucha in the U.S. first started in the California area
around the 1990s.

 As the kombucha industry grows, in 2014 a non-profit organization called

Kombucha Brewers International (KBI) was established to help with regulations
and legislations related to kombucha.

 Currently, as of November of 2019, there are 235 companies worldwide that are
enrolled as a member of this organization

5
• North America (U.S. and Canada), has the largest number of registered
kombucha companies in the organization. In the U.S., the West leads the
pack with 58 companies.

6
7
The value of the global kombucha market is estimated to be USD 1.5 billion in
2018. In the period of 2014–2018, the market grew at a compound annual
growth rate (CAGR) of 23%

From the late 2010s, multinational beverage companies started to show their
interest in the kombucha market.

Starting with Pepsi Co. acquiring one California-based kombucha company in

November 2016 , Molson Coors Brewing Co . has also acquired a California-
based kombucha company in June 2018 . Coca-Cola Co. has invested $20
million in a California-based kombucha company since 2014 and has acquired
an Australian-based kombucha company in 2018 .

8
• When looking at kombucha products available on the market, statements
such as ‘USDA organic’, ‘natural’ ‘healthy for your gut’, ‘live culture’,
‘raw’, ‘non-dairy probiotics’, ‘non-Genetically Modified
Organism(GMO)’, and ‘Kosher’ are commonly used.

• The common size of the kombucha products was 335–355 mL , 414–415

mL , 449–450 mL , and 473–480 mL .

9
10
 Inoculum source

Temperature
Initial sugar used
concentration Chemical
composition
Depend on

Fermentation Sugar and tea

time concentration

11
Villarreal-Soto S A et,.al 2018
 12
Villarreal-Soto S A et,.al 2018
Villarreal-Soto S A et,.al 2018 13
Microbiological composition

1. Yeasts
 commonly being Saccharomyces cerevisiae due to its high efficiency. non-
Saccharomyces yeasts are becoming increasingly used in the industry in mixed
fermentations in order to enrich the aromatic profile, and to enhance the
complexity and the kinetics of the final product .

 Microbial interactions between Saccharomyces and non-Saccharomyces yeasts

seems to be an advantageous option in mixed fermentation processing, having
several benefits like avoiding the risks of stuck fermentation, the addition of
aromas and flavors .

Villarreal-Soto S A et,.al 2018 14

 There are many yeasts genus and species in Kombucha culture, a broad
spectrum has been reported including species of Zygosac charomyces ,
Candida, Kloeckera/Hanseniaspora , Torulaspora , Pichia ,
Brettanomyces/Dekkera , Saccharomyces, Lachancea, Saccharomycoides,
Schizosaccharomyces , and Kluyveromyces .

 Reported Zygosaccharomyces as the predominant yeast with 84.1% of

relative percentage of abundance and Dekkera and Pichia species with
6% and 5%, respectively.

Villarreal-Soto S A et,.al 2018

15
 16
Villarreal-Soto S A et,.al 2018
Villarreal-Soto S A et,.al 2018 17
2. Bacteria
 The dominant bacteria of Kombucha tea culture are AAB, which are aerobic
bacteria able to use alcohol as a substrate to form acetic acid.

 The metabolic process is based on the conversion of acetaldehyde into

ethanol and acetaldehyde hydrate into acetic acid by the enzyme
acetaldehyde dehydrogenase.

 Several AAB are present in the tea fungus, including: Acetobacter

xylinoides, Bacterium gluconicum, Acetobacter aceti, Acetobacter
pasteurianus, and Gluconobacter oxydans

Villarreal-Soto S A et,.al 2018 18

 Acetobacter
Acetobacteria
pasteurianus

Villarreal-Soto S A et,.al 2018

Image Credit :Google 19
Lactobacillus Gluconobacter oxydans

Villarreal-Soto S A et,.al 2018

Image Credit :Google 20
Microorganisms Interactions

 Diverse microbial community

 Commensalism and amensalism
 Sequential and parallel actions
 Specific roles of microorganisms
 Nutrient release
 Fermentation process
 Formation of biofilm
 Inhibitory effects:

Villarreal-Soto S A et,.al 2018

21
Factors Influencing Kombucha Fermentation
 Temperature.
 PH.
 Amount of oxygen.
 CO2 dissolved.
 Operating system.
 Supply of precursors.
 Shear rate in the fermenter.
 Nature and composition of the medium .

Villarreal-Soto S A et,.al 2018

22
Villarreal-Soto S A et,.al 2018 23
Health Benefits
 Antioxidant Properties
 Detoxification
 Potential Anti-inflammatory Effects
 Anti hyperglycemic Effects
 Microbial Balance
 Organ Protection

Villarreal-Soto S A et,.al 2018

24
Potential Toxicity
 Kombucha fermentation is commonly homemade, and there fore it is
important to be cautious because pathogenic micro organisms can
contaminate the tea throughout the preparation.

 Some cases of health disorders have been reported by some

individuals with suspected dizziness and nausea, severe illness,
allergic reactions, and head pain, thus leading to its contraindication in
pregnant and lactating women

Villarreal-Soto S A et,.al 2018

25
 Case Study -1
Kombucha Fermentation and Its Antimicrobial Activity

Sreeramulu et al.,2000

Objective : To investigate the antimicrobial properties and

fermentation of Kombucha.

E. Coli, sh.Sonnei , sal.Typhimurium , sal.Enteritidis , and cm.

Jejuni,

26
MATERIALS

1. Starter Cultures

2. Kombucha

Preparation :
 The preparation involved dissolving sucrose (10% w/v) and glucose (2.5% w/v) in
demineralized water that had been boiled for 15 minutes.
 Black tea (0.5% w/v) was added, steeped for 15 minutes, and then filtered through a
sterile sieve. The tea was cooled to 25 °C before being inoculated with 5 g of freshly
grown tea fungus cultured in the same medium for 14 days.
 The mixture was then placed in a sterilized glass bottle and covered with sterile tissue
paper for aeration, allowing fermentation to occur in a dark incubator at 25 °C .

27
3. Sampling:

• Samples were taken from the incubator at two-day intervals to

measure microbial and chemical changes throughout the
fermentation process

28
METHODS

 Determination of PH
 Monitoring microbial growth:
 The wet weight of the tea fungus and the optical density (OD) of the fermentation
broth at 600 nm were measured to assess microbial growth.
 The wet weight was determined by draining the tea fungus on filter paper under
vacuum conditions until no free water remained.
 Protein estimation : The protein content in the fermentation broth was estimated using
a Cobas Mira Plus auto analyzer .

29
 Microbiological analysis:
 To monitor acetic acid-producing bacteria, liquid tea samples were
plated on WL nutrient agar containing cycloheximide to inhibit yeast
growth.
 For total yeast counting, samples were plated on OGYA medium
containing oxytetra cycline. Plates were incubated at 30 °C for bacteria
and at 25 °C for yeasts.

 Antimicrobial activity testing :

 The antimicrobial activity was assessed using an agar diffusion assay

30
 Kombucha samples were centrifuged to remove cell debris, and the
sterile supernatant was placed in wells on agar plates inoculated with
target strains.
 The plates were incubated at 37 °C, and the diameter of the inhibition
zones was measured after 12-15 hours.

 Control samples : For comparison, acetic acid samples were prepared

at the same concentration as that of fermented tea after 14 days, and
various pH samples of unfermented and fermented tea were also tested
for antimicrobial activity

31
32
-,no inhibition; +,10-15mm; ++,15-20mm; +++,20-25mm; ++++,25-30mm;
+++++,30-35mm

33
Results

 Microbial and Chemical Changes.

 PH Changes.
 Microbial Growth Patterns.

34
References :
 Guttapadu Sreeramulu, Yang Zhu,* and Wieger Knol -2000 Kombucha Fermentation
and Its Antimicrobial Activity J. Agric. Food Chem. 2000, 48, 2589−25 .

 Silvia Alejandra Villarreal-Soto , Sandra Beaufort , Jalloul Bouajila , Jean-Pierre

Souchard, and Patricia Taillandier -2018 Understanding Kombucha Tea
Fermentation: A Review Journal of Food Science Vol.83, Nr.3 , 2018.

 Jeniffer Ferreira de Miranda1 Cíntia Borges Silva1 LarissaFernandesRuiz1

ThaisMatsueUekane1 KellyAlencarSilva1 Alice Gonçalves Martins Gonzalez1
Adriene Ribeiro Lima -2021 kombucha:A review of substrates, regulations,
composition, and biological properties J. Food Sci. 2022;87:503–527.

35
Conclusion..

36
Thank you