ICAR NICHE AREA OF EXCELLENCE ON FUNCTIONAL FERMENTED DAIRY PRODUCTS WITH SYNBIOTICS

ANNUAL REPORT (2009-10)

SMC COLLEGE OF DAIRY SCIENCE ANAND AGRICULTURAL UNIVERSITY

ANAND 388110 (GUJARAT)

ICAR NICHE AREA OF EXCELLENCE
ON

FUNCTIONAL FERMENTED DAIRY PRODUCTS WITH SYNBIOTICS

Preamble Goal Capacity building in area of functional fermented dairy products with synbiotics in terms of technology as well as human resource development. Objectives To modify the existing synbiotic products and/or to formulate new synbiotic functional fermented dairy foods with fruits and vegetables. To add value to functional dairy products with synbiotics by incorporating fruits/vegetables. To study therapeutic and nutritional aspects of functional dairy products with synbiotics. To organize HRD/ training programs/ seminars on Probiotics/ Dairy Starter Cultures and Formulation of New Functional Fermented Dairy Products with Synbiotics for capacity building. Considering the significance of functional fermented foods in diet and health, the center started on September 13, 2006.

Staff Principal Investigator : Dr JB Prajapati, Professor & Head, DM

Co-Principal Investigator: Dr. Vijendra Mishra, Asso. Prof, DM Ms Suja Senan, Asst. Prof., DM Research Workers: Mr. Nihir Shah, Senior Research Fellow Mr CH.V.K. Sudheendra, Junior Research Fellow Mrs Rinku Makwana, Junior Research Fellow

Location 2

SMC College of Dairy Science, Anand 388 110, Gujarat, India

Activities During 2009-10

A: Research

Study 1: Development of oat based functional fermented product containing probiotic culture and pineapple fruit.

Research Methodology: The present investigation was conducted to formulate and develop new oat based functional fermented dairy product with probiotic Lb. rhamnosus MTCC 5462 with comparison to plain acidophilus milk. To formulate product, preliminary trials in three variants i.e. A: [milk + oat flour@4 % ( w/v)], B: [A + sugar @ 10% (w/v)] and C: [B + pineapple pieces @ 20% (w/v)] were developed. Milk fermented with probiotic organisms was taken as control (D). All blends developed were subjected to microbiological, chemical and sensory analysis for fresh (0 day) and up to 28 days of refrigerated storage at 51C. Salient findings:

Fig: Oat based functional fermented product with probiotics and prebiotics 4

1. The proximate composition of various blends is indicated below: Blends A B C D Moisture (%) 83.72 74.03 76.20 87.22 T.S. (%) 16.28 25.97 23.80 12.78 Fat (%) 4.51 4.30 4.20 4.35 Protein (%) 3.78 3.70 3.60 3.30 CHO (by diff%) 7.34 17.33 14.38 4.53 Ash (%) 0.65 0.64 0.62 0.60

2. Microbiological analysis of the products during refrigerated storage revealed that Lactobacilli count remained well above 108 cfu/ml in all blends. Blend C showed highest Lactobacillus count during complete storage period. No deleterious effect of sugar on probiotic culture was observed. 3. The absence of coliforms, yeasts and molds in all blends through out study span indicated good hygienic quality of the prodcuts. 4. The titratable acidity of all blends showed an increase from 0.63% to 1.20% lactic acid during storage for 28 days. For blend B, the rise in lactic acid value was comparatively slower during entire storage period. 5. In Sensory analysis, superior flavor scores were obtained for B & C. The body and texture score for fresh blend was highest for C (8.18) followed by B (8.08) and A (7.95). For fresh and stored blends (28 days) values for B & C were at par. 6. High color and appearance scores were obtained for all three tested blends indicating no negative effect of addition of sugar and pineapple pieces. While B & C got high overall acceptability scores but these score declined marginally during storage period although all blends were acceptable up to 28 days at 51C.

Study 2: Effects of Medicinal Herbs on Lactic Acid Bacteria and Their Use in Preparation of Probiotic Lassi
Research Methodology: The work was carried out to check effect of traditional herbs on lactic acid bacteria and for formulation of functional probiotic lassi containing such herbs. Four herbs namely, Zingiber officinale (Ginger), Asparagus racemosus (Shatavari), Chlorophytum borivillianum (Safed mushli), Withania somnifera (Ashwagantha) were selected. Effect of herbs against lactic acid bacteria was checked by cup well assay method. Optimization of incorporation of herbs was carried for three aspects viz., level of addition, stage of addition and form of addition before optimizing the procedure for preparation of lassi. The storage study was carried out at refrigerated temperature (5 1 C) to decide the shelf-life of the product. Samples were evaluated on the basis of sensory (i.e. flavour, colour and appearance, body and texture and overall acceptability score), chemical (i.e. titratable acidity, pH) and microbiological [i.e. Lactobacillus count (LAC), Streptococcus count (STC), yeast and mold count (YMC) and coliform count (CC)] parameters.

Salient findings:

The ashwagantha powder had highest inhibitory effect followed by satavari powder while Ginger and Safed Mushli powder did not have any inhibitory activity on the four test cultures. Ginger, satavari and safed mushli powder were selected for optimization of herb powder addition in preparation of herbal probiotic lassi using the best performance giving strains, one strain of Lb. acidophilus V3 and one strain of Str. thermophilus MD8. Addition of herb in the form of extract powder was found to be better than oleoresin or liquid extract. Additionally, the powder may provide added advantage of other components of plant, like protein and carbohydrates, which may have prebiotic like effect. Incorporation of herb at the level of 1% was found optimum for ginger and safed mushli while 0.5% for satavari. Incorporation of herb powder during heating of milk was better than addition at the stage of breaking of the curd. All the formulations were organoleptically acceptable, but the product with safed musli was most liked and was at par with control.
9 Fresh p rodu ct (0h) fter 48h of refrigerated storage A

Score out of 9 point hedonic scale

8 7 6 5 4 T1 T2 T3 T4 T5 T reatment T6 T7

T1- Without herb (Control), T2-Ginger added during heat treatment, T3 - Ginger added during breaking of curd, T4- Safed Musli added during heat treatment, T5- Safed Musli added during breaking of curd, T6- Satavari added during heat treatment, T7- Satavari added during breaking of curd

Fig.: Overall acceptability score of herbal probiotic lassi prepared with different herbs added at two stages. Safed mushli added product gave better organoleptic score in comparison to other herbs, showed higher acidity and higher viable count of lactobacilli and Streptococci. Thus, the final product formulation was made only with the incorporation of safed mushli at the rate of 1% during heating of milk. 6

The storage study for 21 days was carried out for safed mushli added herbal probiotic lassi as well as control (without herb) probiotic lassi stored at 5+2 C. They were assessed on the basis of sensory, chemical and microbiological changes taking place during the storage. There were no significant differences in titratable acidity or pH values between control and herbal probiotic lassi during the entire storage period. The acidity for fresh products was 0.61- 0.62% L.A.., which increased to 1.03 % LA in both cases after 21 days of refrigerated storage. The pH declined gradually during the storage from 4.84 to 3.96 in herbal probiotic lassi and from 4.78 to 3.95 in control lassi. Whey separation was negligible in both the products during 21 days storage. However, comparatively it was higher in case of safed mushli added herbal probiotic lassi compared to control. Total viable streptococci counts were 1.29 x 109 and 1.70 x 109 cfu/ml in fresh herbal probiotic lassi and control lassi, respectively. At the end of storage, control product had 2.19 x 108 and herbal product had 1.55 x 108 cfu/ml live streptococci. While total viable lactobacilli count changes from 6.60 x 107 to 1.02 x 107 cfu/ml in herbal probiotic lassi during storage. The mean yeast and mold count was non-significant in both the products during storage. Coliforms were absent in 0.1g of control and herbal probiotic lassi through out storage period. During storage, total sensory score of control and herbal probiotic lassi decreased from 8.00 to 7.00 and 7.16 to 6.83 respectively. Difference in overall acceptability score was mainly attributed to changes in flavour score. However, both the products were acceptable throughout the storage period of 21 days..

Study 3: Nutritional Profiling of Synbiotic Products

Methodology:
1.

The present investigation was carried out for nutritional analysis of new functional dairy products and comparing its effect with plain synbiotic dahi and unfermented milk.

2. For conducting the nutritional analysis of the above mentioned products freeze dried samples as listed below were sent to the Shriram Institute For Industrial Research, Delhi (Test certificate no. 000154227).
3.

Types of Sample analysed A B C D E Boiled cow milk Acidophilus milk Plain Synbiotic dahi Synbiotic dahi with tomato Synbiotic dahi with cucumber 7

F G H I 4.

Synbiotic dahi with banana Synbiotic dahi with sapota Synbiotic dahi with mango pulp Synbiotic dahi with onion

Parameters analysed i. Amino acid profiling ii. Fat Soluble vitamins (A,D,E,K) iii. Water Soluble vitamins(B1,B2,Niacinamide, B12,B6, & C) Salient findings:
1.

Folic

acid,

On comparing with plain acidophilus milk the synbiotic products show a decline in the amount of water soluble vitamin A; which could be the result of processing parameters Comparing Vit E , it does not show a marked increase or decrease

2.

3. The dahi with added banana showed to be a rich source of Vit D amounting to 1037 (IU/100g) 4. Vit B1, B2, B6, C, Folic acid and niacinamide showed levels similar to a acidophilus dahi and milk 5. Vit B12 was not detected in any of the above mentioned sample as the detection limit was 50 ppm by HPLC
6.

The amino acid profiling showed the synbiotic products to be a good source of amino acids including essential amino acids as indicated in following table:

Table: Amino Acid profiling of different synbiotic products

PRODUCTS Aspartic acid Glutamic acid Asparagine Serine Glycine Histidine Arginine Threonine Alanine Proline Cystine Tyrosine Valine Methionine Isoleucine Leucine Phenylalanine Tryptophan Lysine A 1.90 4.80 0.04 1.30 0.50 1.20 0.90 1.00 0.80 2.30 0.40 0.90 1.40 0.50 1.20 2.20 1.30 0.40 2.00 B 1.50 4.30 0.05 1.00 0.40 0.90 0.80 0.80 0.70 2.00 0.30 0.60 1.20 0.40 0.90 1.60 0.90 0.10 1.90 C 2.60 5.90 0.10 1.50 0.60 1.30 1.00 1.30 1.00 2.60 0.40 1.40 0.20 0.60 1.50 2.70 1.40 0.20 2.20 D 3.20 4.90 0.03 1.00 0.40 1.00 0.80 0.90 0.70 1.80 0.30 0.10 1.20 0.50 1.10 1.90 1.00 0.10 1.40 E 1.50 3.80 0.00 0.90 0.50 0.80 0.80 0.80 0.60 1.90 0.20 0.10 0.60 0.20 0.60 1.60 0.80 0.10 0.90 F 1.30 3.10 0.01 0.60 0.30 0.70 0.50 0.70 0.50 1.20 0.20 0.60 0.80 0.30 0.70 1.20 0.60 0.10 0.50 G 1.40 3.30 0.00 0.70 0.30 0.70 0.60 0.60 0.50 1.20 0.20 0.70 0.80 0.40 0.70 1.20 0.60 0.10 1.00 H 1.30 3.10 0.02 0.70 0.30 0.70 0.50 0.60 0.50 1.20 0.30 0.50 0.70 0.30 0.60 1.20 0.60 0.10 0.60 I 1.80 4.40 0.02 1.00 0.40 0.90 0.80 0.90 0.70 1.80 0.30 0.10 1.10 0.50 1.00 1.80 1.00 0.30 1.70

(Values in blue color indicate significant change) 8

Study 4: The effect of consumption of milk based synbiotic product on the intestinal well being and humoral immune response in healthy human subjects.
Objectives: 1. To assess the impact of synbiotic fermented milk on the general wellness in healthy subjects. 2. Examining the probiotic count in sample and human subjects stool as an indication of colonization of the strains. 3. Biochemical (Haemotology, serum lipids) and Immunological (immunoglobulins, leukocytes, monocytes) measurements as indicators of the functionality of the synbiotic fermented milk on human subjects. Methodology: The strains used in this study were the indigenous probiotic strain Lb rhamnosus V3, (MTCC 5462) and Streptococcus thermophilus MD2 (MTCC 5460) strain, a dairy starter was used for the preparation of the synbiotic product. Preparation of the product The test product was a fermented probiotic drink (lassi) with double toned milk elaborated with culture containing Str. thermophilus MD2 and Lb. rhamnosus V3and incubated aerobically till an acidity of 0.8-0.9% la was obtained. In case of the test product the sweetener added was sugar and prebiotic honey in a standardized ratio. The curd was then broken, homogenized and packed in 200 ml pouches be finally stored at 5C. The control product was a similar product but probiotic strain was not added and the addition of honey was too eliminated. Design of the trial Healthy volunteers (n=30, 15 males and 15 females) with ages ranging from 16-25 years. The participants were students from the Polytechnic school of food science and home economics and Polytechnic school of agriculture at Anand agricultural University, Anand. Before the participation in the study, all subjects were examined by physician to check their health status including gastrointestinal symptoms. The study was approved by the Ethical Committee of Anand Agricultural University and signed informed consent was obtained from every subject. The study was carried out as a double blind randomized controlled study with two weeks of baseline study, 4 weeks of consumption and two weeks of follow up. The subjects were randomized into two groups given during the consumption period either the lassi supplemented with L. rhamnosus V3 (test group) or the unsupplemented lassi (control group). The volunteers consumed 200 ml of the lassi once a day throughout the study. The daily dose of the bacteria during the consumption period was 1012 cfu/day. 9

Collection and analysis of the blood samples Blood samples were taken from each volunteer periodically using EDTA-containing vacutainers. Total cholesterol, HDL-cholesterol and triglycerides plasma concentrations were measured using enzyme-spectrophotometry kits (Siemens Medical Solutions Diagnostics Ltd. India). For the enumeration of immunoglobulin. E, A, G and M secreting cells (ISCs) in the peripheral blood the test was outsourced to the Central Diagnostic Laboratory, Karamsad, an NABL accredited and ISO 15189:2003 laboratory. Collection of faecal samples and enumeration of ingested probiotic strain Faecal samples (two half filled 50 ml plastic tubes) were collected for microbiological analysis various intervals. The sample tubes for microbial analysis were sealed in bags and transported to the laboratory. Faecal samples were serially diluted in peptone-saline and plated on MRS agar for detection of the ingested lactobacilli. The plates were incubated in aerobic incubators. The media used for the enumeration of coliforms and Enterococci counts were VRBA and Enterococcal agar respectively. Total numbers of bacteria were recorded. Bowel habit The participants were asked to annotate the frequency of stools and to compare their bowel function with that existing before the treatment by using a scale ranging from 1 to 5; where 1=bad effect, 2= moderate effect, 3= no effect, 4= positive effect, 5= drastic improvement. They were also asked to report any symptom of gastrointestinal discomfort. The final score values resulted from the addition of the scores. Statistical analysis The data were transferred to MetLab software for statistical analysis. Bacterial numbers were transformed into log10-values and presented as separately of each subjects. Salient findings: 1. No statistical differences were found in the lipid profile and hemoglobin content of test and control groups, either from baseline to consumption period or further to follow-up period. Although a significant increase in the ratio of HDL/LDL was observed between the two groups. No statistical differences were found in the Blood albumin, protein and serum triglyceride profile of volunteers. A non significant decrease in the Triglyceride level among the two groups shows a better condition. No statistical differences were found in the Level of Immunoglobulin in volunteers. There were no significant differences in the faecal flora between the two groups. But the retaining of the strain in the faecal matter of test showed that the probiotic strains showed great stability.

2.

3. 4.

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5.

The test products were well tolerated and no adverse effects were observed in healthy adults during consumption of test products thus added bacteria were not disturbing the normal health balance in the intestine. The intervention study could not confirm a beneficial effect of probiotics on health adults but confirms its acceptance to a healthy bodys microbiota.

6.

Table: Lipid profile and hemoglobin values of volunteers

Period 15th DAY 114.21 116.00 17.74 17.07 95.12 96.32 1.42 1.55 9.30 8.63 15.22 18.17

Parameter CHOLEST EROL HDL LDL HDL/LDL

HEMOGLO BULIN

Treatment C T C T C T C T C T C T

VLDL

-15th DAY 86.91 87.63 11.82 12.73 74.52 76.00 1.32 1.30 8.56 8.43 15.40 17.21

0th DAY 102.85 111.85 16.68 16.51 84.02 92.00 1.33 1.41 8.85 9.05 16.90 17.00

30th DAY 93.21 98.92 16.48 15.30 75.75 83.22 1.38 1.53 9.07 8.60 15.33 15.87

+15th DAY 96.71 92.92 14.82 13.00 81.02 76.91 1.33 1.40 9.14 8.57 15.15 15.29

Average 98.7 101.4 15.51 14.92 82.09 84.89 1.36 1.44 8.98 8.65 15.60 16.71

Significance

NS NS NS * NS NS

* significant at 5% level.

Human Resource Development

During 2008-09 a short term training programme of five days for industry personnel and a three week training programme for academic staff of universities and ICAR institutions were proposed to be organised but these could not be organised due to certain unavoidable circumstances. These programmes are now proposed to be organised during 2010-11.

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Financial Report
The expenditure incurred during period is indicated in following table: Budget Head Works Equipment (RT PCR) Recurring contingencies Total Percent utilization of grants Amount sanctioned (Rs.) 10,00,000 10,00,000 10,00,000 30,00,000 Actual expenditure (Rs.) 9,99,285 9,84,086 9,81,182 29,64,553 98.82%

Publications
Following Postgraduate theses have been approved by the university during the period:
1.

Hingu MN (2009) Physico-Chemical, Microbiological and Sensory Profile of Synbiotic Dahi added with Mango Pulp and Onion, M. Sc. Thesis submitted to AAU, Anand. Jain Shivali (2009) Development of Oat Based Functional Fermented Product Containing Probiotic Culture & Pineapple Fruit, M. Tech. Thesis submitted to AAU, Anand. Madhavi TV (2009) Development of Synbiotic Whey Drink, M. Sc. Thesis submitted to AAU, Anand. Sudheendra CH VK (2009) Development of Synbiotic Lassi, M. Sc. Thesis submitted to AAU, Anand. Momin J K (2009) Effects of medicinal herbs on lactic acid bacteria and their use in preparation of probiotic lassi, M.Sc. thesis submitted to AAU, Anand

2.

3.

4.

5.

Conference/Seminar Presentations 1. Sudheendra, Ch.V.K., Shah, R.K. (2009) Development of Synbiotic Lassi. Poster paper presented at the fourth international conference on fermented foods, health status and social well-being organized by SASNET-FF, AAU, NDDB, and LU during December 11-12, 2009 at AAU, Anand, pp. 116. 2. Akruti Joshi, Ami Patel, Nihir Shah and Prajapati JB (2009). Study of plasmid profile of lactic acid bacteria and curing thereof. Poster paper presented at National Conference on Frontiers in Biological Sciences organized under the auspices of 12

UGC-DSA Program at BRD School of Biosciences, Sardar Patel University, Vallabh Vidya Nagar, 27-28 February 2009 (Abstract No. MBBS-7). 3. Momin Jafar K. and Prajapati JB (2009). Development of herbal probiotic lassi- an ideal nutraceutical with advantage of herbs, probiotics and goodness of milk. Poster paper presented at 5th Nutraceutical summit organized by CSIR, CFTRI and MM Active at The Lalit, New Delhi during 28-30 October 2009, pp 87. 4. Shah Nihir and Prajapati JB (2009). Standardization of artificially carbonated probiotic fermented milk drink. Poster paper presented at the fourth international conference on fermented foods, health status and social well-being organized by SASNET-FF, AAU, NDDB, and LU during December 11-12, 2009 at AAU, Anand, pp. 115. 5. Suja Senan and Prajapati JB (2009). Molecular systematics- the key to precise taxonomic identification of probiotic strain Lactobacillus rhamnosus V3 (MTCC 5462) poster paper presented at XX Indian Convention of Food Scientists and Technologists, at NIMHANS, Bangalore, 21-23 December 2009, pp. P-113.

Patents
Two cultures of Probiotic Lactobacilli have been deposited under Indian Patent Deposit category at MTCC, Institute of Microbial technology, Chandigarh. possibilities of patenting some of the synbiotic products are being explored and process is in pipeline.

The

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Technical Programme for 2010-11

A: RESEARCH

Isolation and molecular characterization of lactic acid bacteria from indigenous sources. Complete genome sequencing of currently used probiotic strains namely Lactobacillu rhamnosus MTCC 5462 and L. helveticus MTCC 5463. Human Clinical Trial for assessment of hyperlipidemic effect of synbiotic lassi in patients in collaboration with PS Medical College and Sri Krishna Hospital, Karamsad, (Anand) Gujarat. Study of prebiotic effect of Spirulina and development of protein enriched synbiotic fermented milk product. Development of Iron fortified synbiotic product. Development of carbonated probiotic drink.

B: HRD

One training program (5 days) for food industrys technical staff to be organised in April 2010. One training program (3 weeks) for academic staff of universities and ICAR institutions proposed to be organised in August/September, 2010.

C: BUDGET
The tentative grants requirements for the centre are indicated in following table: Budget Head Amount Work to be done/ Justification required (Rs. lakhs) 05.00 Repair/renovation of molecular biology laboratory 10.00 Online UPS-2, Deep freezer, Bench top homogenizer, High speed blender and other routine laboratory equipments 15.00 Salaries to SRF/JRF. Contractual services, travel expenses, operational expenses, glassware, chemicals, reagents, etc 30.00 14

Works Equipment

Recurring contingencies Total

D: STAFF POSITION Project In-charge : Co-PI : Dr JB Prajapati, Professor & Head Dr Vijendra Mishra, Associate Professor Mrs Suja Senan, Assistant Professor

STAFF NEEDED FOR 2010-11 1. Research workers SRF- 1 JRF - 2 2. Administrative officer 1 3. Lab. Attendant 1 4. Sweeper -1

Dr J. B. Prajapati Project In-charge, Professor & Head, Dairy Microbiology Department, SMC College of Dairy Science, Anand Agricultural University, Anand 388 110 (Gujarat) Ph: 02692 264170, 225851, Fax: 02692 261314 (M) 09879105948, Email: prajapatijashbhai@yahoo.com

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Synbiotic whey drink with and without orange juice

Synbiotic Lassi with and without carrot juice

Synbiotic carbonated drink

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A Glimpse of Products developed by Centre