The word ‘probiotic’ is derived from the Greek language ‘pro bios’ which means ‘for life’. Probiotics are defined as “live microorganisms which on ingestion in certain numbers, exert health benefits beyond basic nutrition” (Guarner and Schaafsma, 1998). When the milk or milk products are added with these probiotics micro-organisms, they contributes in improvement of health and provide numerous health benefits. The lactic acid bacteria (LAB) are the most common probiotic micro-organisms that is mostly used as human probiotic micro-organisms. In LAB, Lactobacillus and Bifidobacterium constitutes the most frequently used genera (Holzapfel and Schillinger, 2002), either singly or in combination, constitutes a significant proportion of probiotic cultures in nutritional supplements, pharmaceuticals and functional foods (Piano et al., 2006). The applications of probiotic bacteria in food products are increasing due to their potential health benefits mainly through maintenance of normal intestinal microflora, protection against gastrointestinal pathogens (D’Aimmo et al., 2007), enhancement of the immune system (Gilliland, 1990), reduction of serum cholesterol level, blood pressure and anti-carcinogenic activity (Rasic, 2003), improved utilization of nutrients and improved nutritional value of food (Lourens-Hattingh and Viljoen, 2001). Therapeutic applications of probiotics include prevention of infantile diarrhea, urogenital diseases, osteoporosis, food allergy and atopic diseases; reduction of antibody-induced diarrhea; alleviation of constipation and hypercholesterolemia; control of inflammatory bowel diseases; and protection against colon and bladder cancer (Mattila-Sandholm et al., 2002; Salminen, 1996; Venturi et al., 1999).

From the past few decades, a significant success has been achieved in the development of dairy based probiotic products such as fermented milks, ice cream, various types of cheeses, baby food, milk powder, frozen dairy desserts, whey based beverages, sour cream, buttermilk, normal and flavored milk (Mortazavian et al., 2012). It is also be consumed as live probiotic cells in many nutraceutical products. Probiotic food products only provide significant health benefits when consumed in adequate amounts of 10⁶-10⁷cfu/ml of viable microorganisms count. The approximate quantity of probiotic products consumed regularly for desired health benefits should be 100 g/day in order to deliver required amount of probiotic microorganisms (Karimi et al., 2011). The efficacy of added probiotic bacteria in food systems depends largely on dose level and their viability must be maintained throughout storage period, and they must survive and colonize easily in the gut environment (Gustaw et al., 2011). For improving these features of probiotic bacteria, probiotic products should be supplemented with non-digestible food ingredients i.e. prebiotics that beneficially affect the host by selectively stimulating the growth of the bacteria in the colon. The product obtained after probiotic microorganisms and prebiotic substrates is termed as synbiotic product. This combination of probiotics and prebiotics can improve the survival of probiotic organism.

Cereals are one of the most suitable components for the production of food products containing a probiotic microorganisms and may also have prebiotic properties due to the presence of non-digestible components (Gupta et al., 2010). Oats, unlike other cereals have received considerable interest in recent years as a delivery vehicles for probiotics due to their high content of soluble and insoluble fibres. Oats are an excellent source of soluble fibre β–glucan and the content of β–glucan in oats is in the range of 3-7 % (Skendi et al., 2003). These oats due to the presence of nutrients shows the high ferment ability upon applying probiotic lactic acid bacteria.

Materials and Methods

Bacterial Strains

Two probiotic cultures i.e. Lactobacillus casei 19 and Lactobacillus acidophilus 14 have been utilized in the preparation of the milk drink. The cultures were obtained in freeze dried form from the National Collection of Dairy Cultures (NCDC), National Dairy Research Institute (NDRI), Karnal, Haryana, India. The cultures were maintained by sub-culturing into sterilised skim milk and stored at 4^oC.

Oat Substrate

Oats ground into fine oat flour were used as a substrate in different concentrations (0.5, 1.0 and 1.5%). The slurry of oat flour was prepared in small quantity of milk then added to prevent formation of lumps in the milk.

Synbiotic Milk Drink Production

The synbiotic milk drink was prepared from fully pasteurized and homogenized milk with 1.5 % fat and 9 % SNF. The milk was pre-heated at 35-45⁰C. Sugar was added @ 7% w/v followed by addition of oat flour in different concentrations of 0.5 % (T₁), 1.0 % (T₂) and 1.5 % (T₃) and control (T₀) was prepared by without addition of oat flour. Sterilization was carried out in the conical flask by putting cotton plug as a capping material to maintain hygienic condition of the product and for attaining sterility, temperature is maintained at 121⁰C for about 15 min. After sterilization, milk is allowed to cool in atmospheric conditions to 37⁰C for inoculation of probiotic cultures (L. acidophilus and L. casei in 1:1 ratio). The inoculated milk was transferred to clean, pre-sanitized plastic bottles and then incubated for 2 h at 37⁰C. The synbiotic milk drink was stored at refrigerated temperature.

Microbial Analysis

The microbial analysis of synbiotic milk drink was done for 21 days in the interval of every 7 days to determine to viability of probiotic microorganisms (APHA, 1992) during storage. The total viable count (TVC), coliform count and Yeast and mold count were also determined during storage period (APHA, 1992).

Statistical Analysis

Data were subjected to analysis of variance for a one factor completely randomized design (CRD) and for two factor factorial completely randomized design (FCRD) procedure at a significance level of 0.05. (Gupta and Kapoor, 2007).

Results and Discussion

Effect on Microbial Quality in Synbiotic Milk Drink Samples during Refrigerated Storage for 21 Days

The influence of the refrigerated storage on microbial quality of control and experimental samples of synbiotic milk drink were analysed for probiotic count, yeast and mold count, coliform count and total viable count (TVC) during storage period of 21 days in the interval of every 7 days and is expressed in log₁₀cfu/ml. Microbial population in the food products is of utmost importance when it is related to the safety of the product. The study of microbial population in the samples of symbiotic milk drink supplemented with oats was of primary importance to produce safe and good quality products. Each microorganisms can be either beneficial or harmful to the consumer. Hence, the microorganisms involved in the preparation of the synbiotic milk drink and their microbial load must be known so as to decide the safety and quality of the product. In the control and experimental samples of synbiotic milk drink, its probiotic count, coliform count, yeast and mold count and total viable count (TVC) were determined and shown in Table 1.

Table 1: Effect of addition of oat flour on the microbial population (in log cfu/ml) of synbiotic milk drink samples during refrigerated storage (Mean ±SE)

Changes in the Probiotic Count during Storage of 21 Days

The probiotic count in the synbiotic milk drink represents the microbial load of Lactobacillus casei 19 and Lactobacillus acidophilus 14 in the product. The effect of addition of oat flour on the probiotic count of synbiotic milk drink during storage period of 21 days is presented in the Table 1 and graphically in Fig.1.

Fig. 1: Effect of addition of oat flour on probiotic count of synbiotic milk drink samples during storage at refrigerated temperature

The probiotic count of all the samples in fresh synbiotic milk drink were determined and found to have the lowest probiotic count of 8.79 log cfu/ml in T₀, whereas in the experimental samples the probiotic count increased from 8.82 log cfu/ml in T₁, 8.88 log cfu/ml in T₂ and found highest in T₃ with the probiotic count of 8.92 log cfu/ml. For providing health benefits, the minimum probiotic count in the products when ingested must contain a minimum of 6-7 log cfu/ml in the final product (Shah, 2007). The results of probiotic count in synbiotic milk drink was fulfilling the minimum required dosage in the probiotic products. Based on existing standards and from a health point of view, it is very important that probiotic microorganisms retain their viability and functional activity throughout the shelf life of product. The L. casei 19 and L. acidophilus 14 were added as a probiotic culture in the symbiotic milk drink. The probiotic count of synbiotic milk drink in the samples T₀, T₁, T₂ and T₃ at 0^th day were 8.79, 8.82, 8.88 and 8.92 log₁₀ cfu/ml respectively. Whereas the corresponding values of probiotic count for the samples T₀, T₁, T₂ and T₃ at the end of storage period (21 days) were found to be 5.78, 5.95, 7.04 and 7.08 log₁₀ cfu/ml respectively. The experimental samples T₂ and T₃ of synbiotic milk drink had maintained the minimum number of probiotic microorganisms that must be present in the product to exert health benefits, whereas the probiotic count in samples T₀ and T₁ were below the minimum probiotic count in the product at the end of storage period. The statistical analysis showed that the probiotic count in the synbiotic milk drink samples were decreased significantly (p<0.05) during storage period. The above results revealed that the addition of oat flour above 1.0% level maintained the minimum probiotic count in the product during storage. The probiotic count in the product remained ≥7.0 log₁₀ cfu/ml even after 21 days of storage period in T₂ and T₃ which are considered sufficient to exert health beneficial effects. The shelf life of the synbiotic milk drink samples T₂ and T₃ were estimated to be 21 days under refrigerated storage.

The probiotic count in the synbiotic milk drink was decreased during storage period. The decrease in the count of probiotic microorganisms during storage may be due to the decrease in the viability of probiotic microorganisms in the synbiotic milk drink samples. The decrease in the viability of probiotic microorganisms in the milk drink is due to a number of factors including H₂O₂ produced by starter bacteria, oxygen content, pH, storage environment and concentration of metabolites such as lactic and acetic acids (Akalin et al., 2007). Yadav et al. (2007) reported for decrease in the probiotic count of dahi during storage period. Bolin et al. (1998) analyzed the viability of L. acidophilus strains in milk cultures in refrigeration conditions (7⁰C) and showed that the number of bacteria significantly decreased during storage (35 days). The mean difference between initial count (7.97 log cfu/ml) and end count (6.21 log cfu/ml) was 1.76 log cfu/ml (Bolin et al., 1998). Dave and Shah, (1997) studied the viability of bacteria from commercial starter cultures during yoghurt manufacture and storage. The count of L. acidophilus was in the range of 1.8-3.8×10⁷cfu/g after incubation but decreased during storage and recommended level of 10⁶ cells was maintained only for 20-25 days. Nighswonger et al. (1996) reported for decrease in the viability of L. casei and L. acidophilus in buttermilk and yoghurt during refrigerated storage.

Changes in the Total Viable Count (TVC) of Synbiotic Milk Drink during Storage

The total viable count (TVC) of the synbiotic milk drink represents the total microbial load in the product and is presented in the Table 1 and graphically in Fig. 2. The TVC in the fresh product was found to have the lowest microbial count of 8.80 log₁₀ cfu/ml in control (T₀) sample, whereas in the experimental samples the microbial count increased from 8.84 log₁₀ cfu/ml in T₁, 8.90 log₁₀ cfu/ml in T₂ and found highest in T₃ with the total viable count of 8.94 log₁₀ cfu/ml. During storage period, the TVC was significantly (p<0.05) decreased in the samples T₀, T₁, T₂ and T₃ and were found to be 5.78, 5.96, 7.05 and 7.11 log₁₀ cfu/ml respectively.  There was no major difference found between the probiotic count and TVC of the synbiotic milk drink samples, as the probiotic products contains only probiotic micro-organisms.

Fig. 2: Effect of addition of oat flour on total viable count of synbiotic milk drink samples during storage at refrigerated temperature

The TVC count in the synbiotic milk drink samples were decreased as the storage period proceeds. The total viable count in the fresh synbiotic milk drink was higher in experimental samples T₂ and T₃ containing 1.0 % and 1.5 % oat flour. The oat flour addition to synbiotic milk drink protects the microbial population due to its prebiotic effect. Tesfaye, (2013) prepared bio-yoghurt by adding oat flour and reported that the microbial population were decreased at the end of storage period of 21 days.

Coliform Count of Synbiotic Milk Drink during Storage

The Table 1 shows that the no coliform was observed in the fresh samples of symbiotic milk drink supplemented with oats after 2 h of incubation. The absence of coliforms indicated that the synbiotic milk drink was prepared under hygienic conditions. The absence of coliform in the synbiotic milk drink samples revealed that all samples have undergone the better sterilization treatment and also the probiotic culture that was added in the product does not contain any coliforms which ultimately resulted in the absence of coliform in the final product. During storage of 21 days, no coliform growth was found in any of the milk drink samples. Hence, the synbiotic milk drink samples were safe for consumption until 21 days of storage period. Kale et al. (2011) prepared value added dahi with the addition of oat flour and reported for the absence of coliform in the control as well as in the experimental samples during storage period.

Yeast and Mold Count of Synbiotic Milk Drink during Storage

The Table 1 shows the yeast and molds count in the fresh samples of synbiotic milk drinks. The yeasts and molds growth were not detected in all the samples of fresh synbiotic milk drinks. Yeasts and molds may get entry into the product from atmosphere, utensils and human hands under natural conditions. Yeasts and molds in the milk based products produces gassiness and flavour defects associated with lipolysis of milk fat. The experimental samples of synbiotic milk drink showed the absence of yeast and mold growth even after 21 days of storage period at refrigeration temperature. Hence, the samples of synbiotic milk drink added with oat flour were safe for consumption until 21 days as it is preserved at refrigeration temperature.

Conclusion

In the synbiotic milk drink, the addition of oat flour had maintained the minimum level of probiotic microorganisms that must be present in the product for providing health benefits. During storage, the samples T₂ and T₃ had maintained the recommended level of probiotic microorganisms of 7.0 log cfu/ml in T₂ and T₃ samples during storage. Coliform count and yeast and mold count were neither found in the fresh nor in the stored samples of synbiotic milk drinks. It is concluded that the synbiotic milk drink prepared by adding oat flour above 1.0 % have the ability to maintain the viability of probiotic microorganisms in the fresh as well as in stored product.

References

1. Aimmo, D. M. R., Modesto, M. and Biavati, B. (2007). Antibiotic resistance of lactic acid bacteria and Bifidobacterium isolated from dairy and pharmaceutical products. International Journal of Food Microbiology, 115: 35-42.
2. Akalin, A.S., Tokusoglu, O., Gonc, S. and Aycan, S. (2007). Occurrence of conjugated linoleic acid in probiotic yoghurts supplemented with fructo-oligosaccharide. International Dairy Journal, 17: 1089–1095.
3. (1992). Microbiological methods for dairy products. In: Standard methods for examination of dairy products.16th edition. Marshall, R.T. Ed. American public health association, Washington, DC, 287- 307.
4. Bolin, P.C., Hutchison, W.D., Andow, D.A. and Ostlie, K.R. (1998). Monitoring for European corn borer (Lepidoptera: Crambidae) resistance to Bacillus thuringiensis: logistical considerations when sampling larvae. Journal of Agricultural Entomology, 15: 231-238.
5. Dave, R.I. and Shah, N.P. (1997). Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter culture. International Dairy Journal, 7: 31–41.
6. Gilliland, S.E. (1990). Health and nutritional benefits from lactic acid bacteria. FEMS Microbiology Letters, 87: 175-188.
7. Guarner, F. and Schaafsma, G.J. (1998). Probiotics. International Journal of Food Microbiology, 39: 237-238.
8. Gupta, S., Cox, S. and Abu-Ghannam, N. (2010). Process optimization for the development of a functional beverage based on lactic acid fermentation of oats. Biochemical Engineering Journal, 52: 199-204.
9. Gupta, S.C. and Kapoor, V.K. (2007). Fundamentals of Mathematical Statistics. Sultan Chand and Sons.
10. Gustaw, W., Kordowska-Wiater, M. and Koziol, J. (2011). The influence of selected prebiotics on the growth of lactic acid bacteria for bio-yoghurt production. Acta Scientiarum Poloorum Technologia Alimentaria, 10: 455-466.
11. Holzapfel, W.H. and Schillinger, U. (2002). Introduction to pre-and probiotics. Food Research International, 35: 109-116.
12. Kale, A.K., Dhanalakshmi, B. and Kumar, U. (2011). Development of value added dahi by incorporating cereal and fruits. Journal of Food Science and Engineering, 1: 379-385.
13. Karimi, R., Mortazavian, A. and Cruz, A. (2011). Viability of probiotic microorganisms in cheese during production and storage: a review. Dairy Science and Technology, 91: 283–308.
14. Lourens-Hattingh, A. and Viljoen, B.C. 2001. Yoghurt as probiotic carrier food. International Dairy Journal, 11: 1-17.
15. Mattila-Sandholm, T., Myllarinen, P.M., Crittenden, R., Mogensen, G., Fonden, R., and Saarela, M. (2002). Technological challenges for future probiotic foods. International Dairy Journal, 12: 173-182.
16. Mortazavian, A.M., Mohammadi, R. and Sohrabvandi, S. (2012). Delivery of probiotic microorganisms into gastrointestinal tract by food products. In: Prof. Tomasz Brzozowski (Eds) New Advances in the Basic and Clinical Gastroenterology, 122-146.
17. Nighswonger, B.D., Brashears, M.M. and Gilliland, S.E. (1996). Viability of Lactobacillus acidophilusand Lactobacillus casei in fermented milk products during refrigerated storage. Journal of Dairy Science, 79: 212-219.
18. Piano, D.M., Morelli, L., Strozzi, G.P., Allesina, S., Barba, M., Deidda, F., Lorenzini, P., Ballare, M., Montino, F., Orsello, M., Sartori, M., Garello, E., Carmagnola, S., Pagliarulo, M. and Capurso, L. (2006). Probiotics: from research to consumer. Digestive Liver Disease, 38: 248-255.
19. Rasic, J.L. (2003). Microflora of the intestine probiotics. In: Caballero, B., Trugo, L. and Finglas, P. (Eds),Encyclopedia of food sciences and nutrition, Academic Press, Oxford, 3911-3916.
20. Salminen, S. (1996). Uniqueness of probiotic strains. IDF Nutrition Newsletter, 5: 16-18.
21. Shah NP. 2007. Functional cultures and health benefits. Int Dairy J 17:1262–77.
22. Skendi, A., Biliaderis, C. G., Lazaridou, A. and Izydorczyk, M. S. (2003). Structure and rheological properties of water soluble β-glucans from oat cultivars of Avena sativa and Avena bysantina. Journal of Cereal Science, 38: 15-31.
23. Tesfaye, Y. (2013). Effect of oat flour concentration on the physico-chemical and microbiological quality of probiotic bio-yoghurt. MSc. Thesis. Addis Ababa Institute of Technology, Ethopia.
24. Venturi, A., Gionchetti, P., Rizzello, F., Johansson, R., Zucconi, E. and Brigidi, P. (1999). Impact on the composition of the faecal flora by a new probiotic preparation: by a new probiotic preparation: Preliminary data on maintenance treatment of patients with ulcerative colitis. Alimentary Pharmacology and Therapeutics, 13: 1103-1108.
25. Yadav, H. Jain, S. and Sinha, P.R. (2007). Production of free fatty acids and conjugated linoleic acid in probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei during fermentation and storage. International Dairy Journal, 17:1006-1010.