SYNOPSIS

PH.D IN FOOD SCIENCE AND NUTRITION

ASSESSMENT OF NUTRITIONAL AND FUNCTIONAL PROPERTIES OF

PROBIOTIC COMPLEMENTARY FOOD MIXES FROM LOCALLY
AVAILABLE CEREALS AND LEGUMES

BY
MANISHA SHARMA
(18PHFNF010)

Supervisor

Dr. S.KOWSALYA
Registrar
Professor, Food Science and Nutrition

SUBMITTED TO

AVINASHILINGAM INSTITUTE FOR HOME SCIENCE AND HIGHER

EDUCATION FOR WOMEN, COIMBATORE- 641043

MARCH 2022

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A. Title of the Thesis
Assessment of nutritional and functional properties of probiotic complementary
food mixes from locally available cereals and legumes.

B. Brief Description on the state of the art of the research topic

The process by which infant progresses from a diet composed of only breast milk to a
family diet consisting of wide varieties of food which is necessary to ensure that nutrient
intakes continue to be adequate for healthy growth and development throughout childhood
(WHO, 2003). Fermentation is mainly by a complex microflora, which develops
spontaneously with lactic acid bacteria generally overcoming the other flora as it is difficult
to control, sometimes not safe since they are liable to contamination by pathogen. Therefore
the need to replace the prevailing uncontrolled fermentation with a pure culture probiotic so
as to obtain a consistent product quality. The probiotic complementary food mixes was
designed in the present study to help restore and maintain the natural flora in the gut and
support the immune system of the 6-23 months along with it reduces the diarrheal frequency
as it is one of the root causes of childhood disability that contributes towards third major
cause of under age five mortalities (Semwal et al., 2015). Since there is no such data on
probiotification of complementary foods, an attempt was made to assess the acceptability of
the developed probiotic complementary food mixes based on locally available cereals and
legumes and to study its efficacy on the Experimental rats.
C. Definition of the problem
Diarrhoea contributes towards 3rd major cause of malnutrition, mortality and
morbidity among under five children especially in the developing countries like India.
According to the latest report of World Health Organisation (2020), nearly eight percentage
of under age five children death in developing countries is due to diarrhoeal disease. It is,
thus, clear that this disease is a major public health issue in India (Omona et al., 2020).
United Nations International Children’s Emergency Fund in 2021 revealed that deaths among
under five children are mostly are caused by malnourishment (45%), Pneumonia (15%)
diarrhoea (8%) and malaria (5%). Five countries namely India, Nigeria, Congo, Pakistan and
China together contribute for half of the diarrhoea death among children (4.249 Million) in
2018. The National Family Health Survey (NFHS-5) shows that the prevalence of childhood
diarrhoea in urban areas are 6.2% and rural areas are 7.7% in the year 2019-21.

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D. Objectives
The present study entitled “Assessment of nutritional and functional properties of
probiotic complementary food mixes from locally available cereals and legumes” has
been undertaken with the following objectives:
• Development of probiotic complementary food mixes from locally available cereals and
legumes
• Assessment of the physico-chemical and functional properties of the developed
complementary food mixes
• In-vivo efficacy of developed probiotic complementary food mixes
• Study the shelf life properties of the developed complementary food mixes

E. Methodology:

Phase I:

a. Sample selection
Based on the guidelines given by Indian Council of Medical Research (ICMR)
for complementary foods, different food ingredients for complementary food formulations
under the present study were selected like rice (Oryza sativa), pearl millet (Pennisetum
glaucum), finger millet (Eleusine coracana), malted wheat flour (Triticum), mung bean
(Vigna radiata), soyabean (Glycine max), sesame seeds (Sesamum indicum) and pumpkin
seeds (Cucurbita sp.).
b. Procurement of raw materials
The selected samples were procured from the local market of Coimbatore and
Chennai, Tamil Nadu, India. The samples were selected for their easy accessibility,
availability and high therapeutic and nutraceutical properties. The samples were stored in
high density polyethylene virgin containers for future use.
c. Processing of raw materials
The raw samples (rice, pearl millet, finger millet, mung bean,pumpkin seeds, sesame
seeds) were sorted and cleaned, hot air oven was used for drying the raw ingredients. The
dried samples were then roasted and grinded into flour using electric grinder. The grinded
flour was then sieved through different mesh size and stored in airtight containers. Soyabeans
were sorted, cleaned and soaked overnight for 12 hours. Boiling of the seeds along with water
was done for 30 minutes. Removal of seed coat was done manually. The seeds were then
oven dried for six hours and then grinded into flour using electric grinder. The grinded flour
was then sieved through different mesh size and stored in airtight containers.

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d. Development of the ARF
Wheat grains were soaked in water for 12 hour and then kept in muslin cloth for
germination for 72 hours. Hot air oven dryer was used for drying for 12 hours, then grinded
into flour using electric grinder, sieved and stored in airtight container.

Phase II:

Determination of nutritional characteristics of raw ingredients:

The proximate composition i.e moisture, crude fat, crude protein, crude fibre and total
minerals were determined by A.O.A.C methods. The available carbohydrate content was
estimated by the method of Hedge and Hofreiter (1962). Mineral elements like iron(Fe) was
determined according by Ranganna (1997) by using spectrophotometer, zinc (Zn) content
was estimated in the sample by using Atomic Absorption Spectrophotometer, calcium (Ca)
was determined by using flame photometer according to the method A.O.A.C, Phosphorus
(P) was determined by Photometric method by Wheeler and Ferrel (1971) and tannins were
determined by modified Vanillin-HCL in methanol method by Broadhurst and Jones(1978).

Phase III:

a. Formulation and standardization of complementary food mixes using ARF:

Based on the guidelines given by Bureau of Indian Standards (2006) for
complementary foods, six different compositions (T1 to T6) for complementary food mixes
under the present study were formulated. The formulation with the highest energy density
(ED) was subjected to further Probiotification. The total energy content of the formulations
were determined by adding the estimated value of carbohydrate, protein and fat of all the
ingredients for each test samples and then multiplying with a constant factor of 4 for
carbohydrate and protein and 9 for fat. A total of six test samples were formulated using
different level of incorporation. The energy density value of the formulations should be
between 300– 550 kcal per 100g of sample.
Total energy (kcal/100 g) = [(% available carbohydrates × 4) + (% protein × 4) + (% fat × 9)].
Standard principles outlined by ICMR (2010).Formulation T4 with rice and mung bean had
the highest ED value 1830KJ (437.50Kcal). Formulation T6 with finger millet and mung
bean had second highest ED value 1855 KJ (443.49 Kcal).

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Phase IV:
a. Probiotification of selected complementary food mixes:

The probiotic cultures were obtained from National Collection of Dairy Cultures
(NCDC), ICAR – National Dairy Research Institute Karnal, Haryana. The probiotics selected
were Lactobacillus fermentum (NDRI-141), Lactobacillus casei (NDRI 297), Lactobacillus
rhamnosus (NDRI-353) and Lactobacillus plantarum (NDRI-375). Nutrient Agar and MRS
(de Man Rogosa Sharpe) broth were selected as the suitable medium for the growth and
culturing of probiotic bacteria. Culture was incubated at 37oC temperature for 24 hours under
appropriate gaseous conditions. Four conical flasks of 50 ml were used for the preparation.
Mixing of pure culture from agar plates in 50 ml conical flask with 25 ml of MRS broth and
incubating in a BOD incubator for 24 hours at 37o C. Centrifuging bacterial cultures grown in
broth at 5000rpm for 15 minutes to obtain pure palate. The bacterial cultures grown in broth
were then centrifuged once at 5000 rpm for 15 minutes to obtain pure palate. The supernatant
was thrown and pure palate were taken and added with 10 ml of normal saline. The density of
bacterial cell suspension/inoculum (stored pure palate) was compared to the freshly prepared
0.5 McFarland turbidity standard scale by holding them both in front of a light against a
contrast background. The density of inoculum was adjusted by diluting it with more sterile
normal saline if needed. Submerged fermentation (1:3) was carried out by adding 297 ml of
sterile distilled water aseptically in 500 ml of conical flask and mixed thoroughly with 100 g
of mixes separately (T4 and T6) in a homogenizer and sterilized at a temperature of 121oC for
20 minutes. The fermentation jars were cooled and inoculated at 1% of 10 8 CFU m/L, tightly
covered with aluminium foil to keep it away from contamination and kept undisturbed in a
BOD incubator for 24 hours at 37o C and then freeze dried for 72 hours and packed in airtight
containers. The mixes after Probiotification were named as CFM I (T4) and CFM II (T6).

b. Acceptability trials of developed probiotic complementary food mixes.

For evaluating the acceptability of the developed formulations, score card method was
selected. In the present study, a score card consisting a table utilizing the Hedonic ratings of
nine point scale (Peryam and Pilgrim, 1957) from like extremely to dislike extremely. The
acceptability of the formulated complementary mixes was assessed by score card for sensory
characteristics. Semi trained panel i.e a total of 30 staff members and students from the
Department of Food Science and Nutrition, Avinashilingam Institute for Home Science and
Higher Education for Women, Coimbatore evaluated the prepared samples for acceptability
trials. The IHEC Approval number is AUW/IHEC/FSN-21-22/XPD-33.The product has been

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patented under the name “A process of preparation of probiotic complementary food mix
and product thereof” with application number 202241007163”.

Phase V:
I. Analysis of physical and nutritional composition of complementary food mixes
a. The morphological and biochemical characterization of bacterial cultures were done
by the methods described by Giuliano et al., (2019).
b. Resistance to gastric acidity of food mixes at different ph values at different exposure
time were done by standard protocol given by Awan and Rahman (2005).
c. Bile acid resistance was done by Awan and Rahman (2005)
d. Antimicrobial activity was carried out by disc diffusion method (Daoud et al., 2015).

II. Analysis of physical composition of complementary food mixes

Physical characteristics of the developed complementary food mixes: Bulk density
was measured by the method suggested by Lewis, (1987), Swelling capacity were also
determined with the method suggested by Appiah et al., (2011), Viscosity was measured by
the modified method of Hallic and Kelly (1959) by using Visco basic plus viscometer, Water
and fat absorption capacity of the flours was determined by the method of Sosulski et al.
(1976). The sedimentation volume was described according to AACC (2002). Dispersibility
is calculated by Kulkarni et al., (1991). Texture was analysed by the modified method
given by Mantihal et al., (2019) by using a TA.XT-Plus Texture analyser.

a. Proximate composition of the probiotic complementary food mixes were done according
to the methods stated in Phase II.
b. Phytochemicals like flavonoids, alkaloids, terpenoids, glycosides, tannins, phenolic, and
saponins were analyzed by qualitative chemical method and quantitative method
Gierschner, et al and Latif et al., (2020).
c. Mineral composition of Probiotic complementary food mixes were done by A.O.A.C
methods.
d. Free radical scavenging activity was measured by using DPPH method according to Vani
et al., (1997).
e. In vitro starch digestibility was assessed with modification of the methods given by Som
et al. (1992). In vitro protein digestibility was determined according to the modified
method described by Walter et al., (1983).

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III. In vivo efficacy for quality of the developed complementary food mixes

The experiments were conducted in the Animal house, Department of Biochemistry,

Avinashilingam Institute for Home Science and Higher Education for Women. Coimbatore.
The IAEC Approval number is AIW:IAEC.2020:FSN:01.

a. The impact of feeding probioticated CFM I and CFM II on the gut microflora of rats
through fecal microbial count were done through the standard protocol described by Barai
et al., 2018.
b. Net protein ratio (Bender and Miller, 1953) is the simplest procedure to evaluate the
protein quality. It focuses on weight loss of protein free group to the weight gain of group
fed with protein diet.
c. The food conversion efficiency will be calculated as per method of Shingari and
Sapra(1991).
d. The blood glucose level will be measured using SIEMENS Glucose in vitro diagnostic
kit.
e. The total blood protein levels will be measured by using SIEMENS Total Protein in vitro
diagnostic kit.

Phase VI:

I. Shelf life properties of the developed complementary food mixes

a. Total bacterial count was analyzed by the Standard operating procedure for the
enumeration of micro-organisms Colony Count Technique at 35°C.
b. Peroxide value of the samples was determined by following AOAC (1975) method.
c. Free fatty acid content of the samples were determined by following AOAC (1970)

II. Data collection & Statistical analysis

All data were analyzed using SPSS v.17.0 for Windows (SPSS Inc., Chicago, IL).
Statistical significance was set at P< 0.05 and P<0.01

F. The salient findings of the study are as follows:

 The moisture content was found to be highest in malted wheat flour 4.01±0.10 g among
cereals, soyabean 5.02±0.10 g among pulses and sesame seeds 3.30±0.10 g among oil
seeds.
 The available carbohydrate was found to be highest in finger millet 88.50±0.015 g among
cereals, mung bean 73.00±.005 g among pulses and sesame seeds 25.40±.005 g among oil
seeds.
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 The crude protein was found to be highest in 30±.005 g in malted wheat flour among
cereals, soyabean 39.28±.005 g among pulses and pumpkin seeds 30.10±.001 g among oil
seeds.
 The crude fat content was found to be highest in malted wheat flour 9.8±0.002 g among
cereals, soyabean 10.48±0.13 g among pulses and pumpkin seeds 51.90±0.15 g among oil
seeds.
 Finger millet had the highest calcium 330.01±0.010 mg, iron 6.71±0.010 mg and zinc
3.00±0.005 mg content among cereals, Phosphorus was found to be highest in malted
wheat flour 811.00±0.005 mg content. Among pulses, soybean had the highest calcium
220.01±0.010 mg, iron 11.01±0.005 mg, zinc 3.31±0.010 mg and phosphorus
675.01±0.015 mg content. Among oil seeds, sesame seeds had the highest calcium
1330.01±0.010 mg, iron 8.91±0.010 mg, zinc 12.11±0.010 mg and phosphorus
551.69±0.010 mg content.
 The phytate content was found to be highest in finger millet 699±0.010 mg/100g, oxalates
39±0.005 mg/100g and tannins 280±0.010 mg/100 g was found to be highest in pearl
millet.
 The highest energy density values i.e T4 with rice and mung bean with 1830KJ (437.50
Kcal) and T6 with finger millet and mung bean with 1855 KJ (443.49 Kcal) were selected
for further analysis.
 The protein content 12.24±0.010 percentage, Tannins 10.91±0.005 percentage, water
absorption capacity 0.356±0.005 ml/g and bulk density 2.58±0.005 g/ml was found to be
highest in T6.
 The viable number of probiotic bacteria was found to be highest in T4 with Lactobacillus
plantarum (CFM I) i.e 6×10 8 and T6 with Lactobacillus casei (CFM II)i.e 7.2×108 .
 Resistance to gastric acidity of food mixes at different ph values at different exposure
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time was found to be highest in CFM I at P.H 3 at 3 hours i.e 4.5×10 and at 6 hours
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8 and at and at 6 hours 5.5×108 .
3.9×10 and CFM II at P.H 3 at 3 hours i.e 5.6×10
 Tolerance to bile salts was found to be highest in CFM I, 0.5 oxgall i.e 5.5×108
percentage and CFM II, oxgall 6.9×108 percentage.
 Out of the four formulations prepared, the probiotic complementary food mix developed
from Finger Millet (CFM II) was best scored in colour, flavour, appearance and overall
acceptability. CFM I developed from rice scored highest in consistency.

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 The bulk density (g/ml) 0.54±0.005, Sedimentation volume (ml) 5.80±0.015, Water
holding capacity (%) 220±1.00, Fat holding capacity (%) 237±1.00 ,Dispersibility (%)
64.66±1.15 of CFM I was found to be highest among the four formulations. The
Swelling capacity (%) 19.00±0.10 of CFM II was found to be highest among the four
formulations.
 The viscosity (cP) was found to be lowest in CFM II 951.00±1.00 at 12 rpm,
566.16±1.04 at 30 rpm and 401.20±1.08 at 60 rpm. A low viscosity value with high
nutrient content is a desirable characteristic of complementary foods (Ariahu et al.,
1999).
 The Adhesiveness (J) 4.16±.03, stickiness -5.63±0.06 and Stringiness (mm) 3.30
±0.04 was found to be highest in CFM II. Resilience 0.35±.010 was found to be highest
in standard commercial sample.
 The moisture (g) 3.85±.010, available carbohydrate (g) 82.69±.0.10, crude protein (g)
19.90±.0.09, crude fat (g) 6.60±.05, crude fibre (g) 6.12±.07, total minerals (g)
1.81±..05, total energy 490 kcal of CFM II was significantly higher than CFM I.
 The nutraceuticals content of CFM II was significantly higher than CFMI. The
nutraceuticals content of CFM II are given as follows, flavonoids content (mg/100g)
130±0.95, Alkaloids (mg/100g) 41.06±.51, Cyanogenic glycosides (mg/100g)
26.53±0.45, tannins(mg/100g) 6.29±.06, oxalates (mg/100g) 0.55±.01, trypsin inhibitor
(mg/100g) 4.20±0.25, saponins (mg/100g) 13.41±.07, phytate (mg/100g) 1.6±.03.
 The mineral composition of CFM II was significantly higher than CFM I. The mineral
content of CFM II (in PPM) values are given as sodium 5.72±0.24, potassium
432.38±0.33, calcium 259.58±0.38, magnesium 6.85±0.30, manganese 15.48±0.42, iron
13.65±0.522, zinc 4.84±0.14, copper 14.15±0.217, phosphorus 55.11±0.99.
 The free radical scavenging activity of CFM II was significantly higher than CFM I. The
free radical scavenging activity of CFM II was 74.3 percentage and CFM I was 72.2
percentage.
 The protein digestibility of the formulated complementary food mixes ranged from
70.26±0.37 percentage in CFM I to 73.46±0.50 percentage in CFM II and were
statistically different at (p≤0.05) from each other.
 The reduction of diarrhea was found to be highest in positive control fed with
Loperamide 3mg/kg 70.68 percentage. Among the test sampes fed it was found to be
significantly higher in CFM II i.e 68.88 percentage and CFM I i.e 64.84 percentage.

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 Impact of feeding probioticated on the gut microflora of experimental rats was found to
be increasing till the 4th day in both the test samples ie CFM I 7.8 and CFM II 9.2. The
probiotic count were statistically different at (p≤0.05) from each other.
 The net protein ratio was found to be highest in reference diet fed with casein diet i.e
31.37±0.28. Among the test diet the CFM I had net protein ratio of 26.11±0.67 and CFM
II had net protein ration 30.17±0.48 which was significantly higher than CFM I.
 The food conversion efficiency was found to be lowest in CFM II i.e 0.18±.015, CFM I
0.22±0.20. A low FCE is an indication of a high quality feed.
 Blood glucose levels in experimental animals was found to be statistically different at
(p≤0.05) from each other till 21st day. The blood glucose levels was found to be
102.9433±0.91 for group I, 88.4433±1.38 for Group II and 91.1667±1.25 for Group III.
 Total blood protein levels in experimental animals was found to be statistically different
at (p≤0.05) from each other till 21st day. Total blood protein levels was 7.35±0.12 for
Group I, 12.51±0.48 for Group II, 13.29±0.611 for Group III.
 The total bacterial count for CFM I was 5.18 ×108 and CFM II was 6.18×108 till 60th day
of storage. The total bacterial count were statistically different at (p≤0.05) from each
other.
 Free fatty acid for CFM I was 4.29±0.55 percentage and CFM II was 4.05±.05
percentage for 70th day and was not fit for consumption. No remarkable change was
observed up to 2 months of storage which may be due to the packing of the flours in
polyethylene bags, sealed and stored in cool temperature (4 degree).
 The peroxide values for 70th day for CFM I was 6.95±0.05 m moles/kg fat and CFM II
was 7.05±0.04 m moles/kg fat. The peroxide value of the developed products was below
the standard values prescribed by Prevention of Food Adulteration Rule, 1991.

G. Conclusion

The present work focused on nutritional enhancement of complementary food mixes from
locally available cereals and legumes. Developed complementary food mixes are
nutritionally rich baby food developed using locally available food materials that are low cost
and from all the food groups i.e. cereals, pulses, oilseeds. Six different (T1 to T6)
compositions were formulated out of which T4 and T6 had the highest energy density values
adding ARF. The addition of ARF to existing complementary food for malnourished and
growth-faltering young children is a simple and effective means to change the consistency of

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the food to facilitate its ingestion by young children and thus optimize their dietary (and
energy) intake. Fermentation of both the mixes (T4 and T6) was carried out to enhance the
bioaccessibility and bioavailability of nutrients and improves organoleptic properties as well
as in extending the shelf life. The fermentation process was carried out in aseptic
environment in Laminar Air flow using safety measures such as gloves. Freeze drying was
done at a temperature of -530C was done for the samples to avoid contamination and dried
samples were then stored in polyethylene bags. The mixes after Probiotification were named
as CFM I (T4) and CFM II (T6). The mixes after probiotification were further accessed for
nutritional and functional characteristics. Out of the two formulations CFM II was found to
be highly acceptable and possessed high nutritional and functional contents, free radical
scavenging activity and nutraceutical potentials. The in-vivo studies stated that both the
product were effective in reducing the episodes of diarrhea along with it increasing the
beneficial bacteria in the gut microflora, increasing the total protein and total weight and
maintaining the blood glucose. The shelf life studies stated that both the products are good for
consumption for a period of 60 days when kept under refrigerated temperature (4OC).
Dissemination of this technology in to the rural areas on large scale will be helpful to the
rural farm women to feed their children a nutritionally balanced feed. The probiotic
fermentation resulted in favourable changes in nutritional profile of complementary food
mixes resulting in increase in nutritional quality and decrease in antinutrient contents. Hence,
it can be recommended that the probiotic fermentation technology possess a good potential to
develop nutritionally enriched complementary mixes.

H. Paper Published:
1. Manisha Sharma and Kowsalya, S., Comparative Study on Different Drying Techniques
for determining the Moisture Content of Cereals /Millets and Pulses, The Indian Journal
of Nutrition and Dietetics, Supplement - 2, January - March 2021. UGC CARE list-
Sciences S. No. 392.

2. Manisha Sharma and Kowsalya, S. A review on: functional foods and its growing trend
in India, Journal of Xi'an University of Architecture & Technology, Volume XIII, Issue 8,
574-584. Scopus Indexed. Issn No. : 1006-7930
I. Paper Presented
1. Presented in Home science association meet on “Functional Foods: The Need Of The
Hour” on 22nd - 23rd January 2020
2. Presented in International web conference on food technology and nutrition-prospects for
health on “A comparative study on different drying techniques for determining the
moisture content of cereals/millets and pulses” on 28th – 29th January 2021

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J. Recommendations
 The product as a published patent technology of product development should be
transferred to industries so that this will reach the community.
 To create awareness on homemade complementary food mixes prepared using locally
available cereals and legumes, popularization can be done through social media aids.
 The research work has used only in vitro models to assess the protein digestibility of the
developed complementary food mixes. In future work, new studies can be done at
molecular and cellular level to understand the protein digestibility of weaning foods
which helps to improve infant health.

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