Production of probiotic milk drink containing

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1 Oktay Yerlikaya Production of probiotic milk drink containing Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus casei Oktay Yerlikaya*, Gulsah Ender, F. Artemis Torunoglu, Necati Akbulut *Corresponding author Ege University, Faculty of Agriculture, Department of Dairy Technology, 35100, Bornova-İzmir, TURKEY KEYWORDS: Lactobacillus acidophilus; Bifidobacterium animalis subsp. lactis; Lactobacillus casei; probiotic drinks; fermented milks ABSTRACT: In this study, new fermented milk drinks containing combined three probiotic cultures (Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus casei) were developed and characterized. Fermented milk drinks were evaluated for their physical, chemical, rheological, microbiological and sensory properties. Determinations of these parameters were investigated at 1, 10, 20 and 30 days of storage. Addition of fruit pulp did not significantly affect the chemical properties, but contributed to an increase in apparent viscosity of fermented milk drinks. However, they were accepted in terms of sensory properties by panellists. All the drinks contained the recommended levels of (7 Log cfu/ml) probiotic bacteria at the end of 30 day shelf life. Milk fermented with these combined three probiotic bacteria could be manufactured as an alternative probiotic dairy product. INTRODUCTION Nowadays, consumers are increasingly demanding foods with special properties, such as pleasant flavour, lowcalorie value or low fat content, and beneficial health effects. Within this context, the food industry has been developing products with improved flavour and appearance. Functional dairy products meet various nutritional requirements, along with health benefits that are strengthened by the addition of probiotics (1). The most important and frequently used functional food compounds are probiotics and prebiotics, or they are collectively known as synbiotics. The word probiotic means for life and the world health organization defines probiotics as live microorganisms which when administered in adequate amounts confer a health benefit on the host. In the last two decades, research in the probiotic area has achieved major progresses in the selection and characterization of specific probiotic cultures and confirmed the health benefits associated with them. Traditionally, fermented foods are the main source of probiotics and hence one of the major dietary supplements of modern world. This microorganisms defend against pathogenic microorganisms. Other benefits attributed to probiotics include prevention of cancer, traveller s diarrhoea and lactose intolerance, stimulation of the immune system, lowering of serum cholesterol levels, and improvement of vitamin synthesis. The species which are the most frequently used as probiotics belong to the genera Lactobacillus and Bifidobacterium. However, due to the short lifespan of probiotics, studies have been carried out to evaluate the contribution of prebiotics to the viability of these microorganisms (2, 3, 4). Some specific strains of lactic acid bacteria such as L. acidophilus, L. casei, B. longum, L. fermentum, L. rhamnosus, L. reuteri, L. crispatus, L. plantarum, B. animalis, and B. lactis are in the market and provide good profit for several companies (5). Fermented milk drinks containing Lactobacillus acidophilus, Bifidobacterium, and Lactobacillus casei are well known probiotic beverages produced in many countries as functional products (6). Dairy products containing probiotic cultures such as Bifidobacterium sp. and Lactobacillus sp. have been produced around the world for many years and it is estimated that currently more than 80 products containing these bacteria are being produced worldwide (7). Besides their desired health properties, probiotics should meet several basic requirements for the development of marketable probiotic products including their survival and activity in the product, and stability during storage of the product. In addition, probiotics should not adversely affect the taste or aroma of the product or acidification during the shelf life of the product (8). Flavour is a crucial characteristic of foods as the sensory properties play an important role in product acceptance by consumers. In fermented dairy products, flavour perception is strongly based on the volatile components (9). Although some fermented milk drinks are consumed in their natural state, in Europe the most of these products contain sugar, fruit syrup or aroma. Fruit syrup, which may also contain small fruit particles, could be added to the fermentate. The fruit aroma, which is normally obtained in a concentrated form, will have a larger aromatic effect in less acidic products; and it is added to long shelf-life products (10). Although the fruit-added fermented milks are responsible for more than a half of the yoghurt-like products market, only a few publications on these dairy products enriched with fruit bases are available, and data regarding the possible impact of these fruits on the viability of the probiotic microorganism in the food product (11). The aim of this study is to develop a new fermented milk drink containing combined three probiotic cultures (Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus casei), sugar, some aroma agent and fruit syrup. 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2 Agro FOOD Industry Hi Tech - March/April Vol. 24(2) MATERIALS AND METHODS Materials In the production of fermented milk drinks Lactobacillus acidophilus LAFTI B10 (DSM Food Specialties BV), Bifidobacterium lactis Bb-12 (Chr. Hansens s, A/S, Hǿrsholm, Denmark), Lactobacillus casei LAFT I B26 (DSM F ood Specialties BV) cultures were used. 1g of lyophilized starter culture was added to 100 ml of sterilized reconstituted skimmed milk containing 12 % dry matter (w/v) separately. After milk coagulated at ph 4.6, it was kept at 4 ºC for 24 h. Aroma agent were obtained from Aromsa A.S. (Kocaeli, Turkey) and fruit syrup were provided from Altınkilic Ltd. (İstanbul, Turkey). Methods Production of fermented milk drinks Activated cultures were added to UHT milk containing 1.5 % fat and 8.5 % non-fat solids, 0.75 % (L. acidophilus) 1 % (B. lactis), 1 % (L. casei) rate, respectively. The fermentation lasted nearly eight hours. At the end of the fermentation ph reached 4.8 and incubation was ended. The curd was stirred and divided into five batches. One batch was the control (A) (without any aroma agent or fruit pulp); strawberry aroma (B), forest fruit aroma (C), strawberry syrup (D), and forest fruit syrup (E) were added separately to the other four batches to give average. While 6 % sucrose and 1 % aroma were added to drinks with aroma agent, syrup rate was determined as 6 % in drinks that are included syrup. After all samples were mixed well, they were put into sterilized glass cups and stored at 4 o C, until the physical-chemical, rheological, and microbiological analysis were carried out on 1 st, 10 th, 20 th and 30 th days. The experiment was repeated three times and the analyses were carried out in duplicate. Physical-chemical analyses Proteins were determined by the Kjeldahl method, ash by incineration at 550 C and total solids by drying at 105 C until constant mass according to the National Standard (12), titratable acidity of the samples was measured by titrating 10mL of sample with 0.1 N NaOH using phenolphthalein as indicator (13) and ph values were measured with Hanna Microprocessor ph 211 digital ph-meter (14). Rheological analysis A Brookfield Rotational Rheometer with LV-4 cylindrical spindle was used for determination of rheological properties of fermented milk beverages on 12 rpm at 8 C (Model DV-II Pro LV, Brookfield Engineering Laboratories Inc., Middleboro, MA, USA). Viscosity values of samples were recorded as mpa.s using RHEOCALC 32 Application Software (Brookfield Engineering Laboratories Inc.). enumeration of L. acidophilus, L. casei, and B. animalis subsp. lactis respectively (15). Sensory analysis Fermented milk samples were analysed for odour, taste, and overall acceptability after 1 day of storage at 4 C. The acceptability test was carried out with 7 trained panellists familiar with the product. A 9-point hedonic scale was used to evaluate the acceptability of the products including both the number and verbal scores were provided to the panellists. The scores were; like extremely (9), like very much (8), like moderately (5), neither like nor dislike (4), dislike moderately (3), dislike very much (2), dislike extremely (1). A section for the panellist s comments was present in the evaluation sheet (16, 17). Statistical analysis Statistical analysis of the data was done using the analysis of variance in SPSS v (SPSS Inc., Chicago, USA). Means with a significant difference were compared by Duncan s multiple range tests. P-values < 0.05 were considered statistically significant. All analyses were performed in triplicate. RESULTS AND DISCUSSION Physical-chemical properties Table 1 presents the results for composition, ph, titratable acidity and dry agent of the fermented milk beverage samples. ph values of the samples were ranged between 4.19 and 4.35 at the first day of storage (P<0.05). ph values of all samples slightly decreased on 20th day (P<0.05). At the end of the storage period (30th day), ph values were close to each other (P>0.05). Tamime and Robinson (18) related that the obtainment of fermented milks in this ph range would results in a better rearrangement and aggregation of casein particles, according to Gallardo-Escamilla et al. (19) to the formation of a more stable gel, avoiding the separation of phases, i.e., syneresis. The results of a decrease in ph with or without aroma agent or fruit syrups were the same during fermentation and storage in all samples. Therefore addition of aroma agent or fruit syrups had a negligible influence on the dynamics of ph decrease. Similar ph decreases were obtained by Shah et al. (20), Marshall and Tamime (21). Titratable acidity of samples changed between 0.66 and 0.76 on the first day, and these values increased during storage. Titratable acidity (SH) increased in all fermented milk 50 Microbiological analyses The colony counts of Lactobacillus acidophilus, B. animalis subsp. lactis, and Lactobacillus casei were enumerated in each sample (10 g) following serial dilution in Ringers solution. Appropriate dilutions were plated using the following media: MRS-sorbitol agar (MRS-S), MRSvancomycine agar (MRS-V) and MRS - nalidixic acid, neomycin sulphate, lithium chloride and paromomycin sulphate (MRS-NNLP) agar incubated anaerobically at 37 C for 72h were used for the Table 1. Chemical composition of fermented milk drink samples during 30 days storage (n=3, ± s)

3 Table 2. Rheological properties of fermented milk drink samples during 30 days storage (n=3, ± s) A: control drink (without any aroma agent or fruit pulp); B: drink with strawberry aroma, C: drink with forest fruit aroma, D: drink with strawberry syrup, and E: drink with forest fruit syrup beverage samples regardless of only sugar, aroma agent and fruit pulp addition. These results are in harmony with Akpinar (22) and Tonguc (23) studying various fermented milk drinks. Ash and protein solid contents ranged from to g/100g, and 2.71 to 2.89 g/100g respectively. No significant differences were observed in ash and protein contents; however total solids values varied significantly. These differences were significantly affected by the addition of sucrose or fruit pulp. As expected, addition of fruit pulp caused an increase in total solids (P<0.05) because of high dry matter content of fruit pulp. Rheological properties Rheological properties of fermented milk beverages have been studied to determine the flow behaviour and their effects on the textural properties. A high consistency index and high pseudo-plasticity correlated positively with the sensory acceptability of lactic beverages (24). As with the addition of fruit pulp, the cold storage time contributed to an increase in the apparent viscosity of the fermented milk drinks. Details of viscosity properties of fermented milk drink samples were shown in Table 2. The higher average values of samples with fruit pulp (D - E) are most probably due to the higher total solid. Kocak and Aydemir (25) reported that texture characteristics of fermented milk drinks were primarily related to their moisture content and protein level. D sample had the highest apparent viscosity with mpa.s on 1 th day. C samples had the lowest apparent viscosity value and was significantly different from especially D and E (P<0.05). The apparent viscosity value of the A, B, and C samples were significantly different from D and E samples (P<0.05). Our findings were higher than the results found in previous studies by Moayednia et al. (6), Tratnik et al. (26), Debon et al. (27), Pescuma et al. (28), and Demir et al. (29). lowest viable cell numbers of each probiotic strain in all tested milks remained above the legal requirement of 6 Log / ml. Storage period did not affect the viability of L. acidophilus and B. lactis, L. casei (P<0.05). Due to the fac t that ph values of samples did not fall under 3.9, viability of probiotic bacteria were not affected much. However, addition of fruit pulp to fermented milks increased the number of B. lactis at the beginning of storage (P<0.05). The viable cell counts of L. casei in A and B samples showed significant (P<0.05) decreases in all assessment times over the storage period. The largest reduction was on the last day of refrigerated storage. The maximum decrease of viability of all probiotic bacteria was after 20 days of refrigerated storage. Similar results in fermented milk products containing probiotic bacteria were obtained by De Souza Oliveira (1), Moayednia et al. (6), Donkor et al. (30). Sensory properties It is important to emphasize that all the beverages produced possessed excellent stability during 30 day of storage, and spoilage was not noticed in any sample. The sensory scores of the beverages are high, except samples fermented with selected probiotic bacteria. Sensory evaluation during cold storage was performed for all samples. The aim was to determine the acceptability of products and stability. The evaluations were performed on the 1 st, 10 th, 20 th, and 30 th days of storage. The results show that samples had satisfactory sensory scores, while samples that had sucrose, aroma agent, or fruit pulp addition were acceptable during cool storage. It could be noticed that samples with strawberry aroma or pulp addition, had the best sensory scores during storage (Table 4). Growth of probiotic bacteria had obtained satisfactory results in taste and consistence. All samples had excellent scores for appearance regardless of storage ti me. Addition of aroma agent or fruit pulp did not affect any sensory property of any samples during storage time, negatively. In the development of new products, the dairy product industry has given emphasis to the healthy and sensory properties. In this study, producing drinks that were flavoured with sugar, some aroma agent and fruit syrups had taken acceptance scores during storage period. These findings are similar to researcher s results such as Akpinar (22) and Tonguc (31) using aroma agent and fruit pulp in fermented milks. Agro FOOD Industry Hi Tech - March/April Vol. 24(2) Microbiological properties In probiotic dairy products, viability of probiotic bacteria is one of the most important subjects for product quality. The results of the research show that the usage of combined probiotics did not significantly increase the microbial flora during 30 days storage, but counts of probiotics survived at 4 C storage conditions for 30 days in a number greater than 7 Log cfu/ml (Table 3). The Table 3. Viability of probiotic bacteria in fermented milk drink samples during 30 days storage (Log cfu/ml, n=3, ± s) 51

4 Agro FOOD Industry Hi Tech - March/April Vol. 24(2) CONCLUSION In conclusion, fermented milk drinks containing three probiotic cultures were produced. The physical, chemical, rheological, microbiological and sensory properties of fermented milk drinks were found to be dependent on which flavour compounds or fruit pulp was added in its preparation. Especially, this compounds effect on sensory and rheological properties of fermented milk drinks. Aroma agent and fruit syrups did not significantly affect the chemical or microbial properties, but have affected the rheological and sensory properties ACKNOWLEDGEMENTS The authors thank the Ege University Scientific Research Fund Research Fund (2007-ZRF-009) Council for financial support to this study. REFERENCES 1. R.P. De Souza Oliveira, P. Perego et al. Int J Dairy Tech, 62, pp (2009). 2. R. Nagpal, A. Kumar et al. FEMS Microbiol Lett, 334, pp (2012). 3. J.B. Divya, K.H. Varsha et al. Eng Life Sci, 12, pp (2012). 4. F.P. Castro, T.M. Cunha et al. LWT-Food Sci Tech, 42, pp (2009). 5. D. Granato, G.F. Branco et al. Comp Rev Food Sci Food Safety, 9, pp (2010). 6. N. Moayednia, M.R. Ehsani et al. Int J Dairy Tech, 62, pp (2009). 7. H. Kesenkas, Mljekarstvo, 60, pp (2010). 8. K.J. Heller, Am J Clin Nutr, 73, pp (2001). 9. N. Kalviainen, K. Roininen et al. Food Quality and Preference, 14, pp (2003). 10. L.E. Nilsson et al. Production of Drinking Products. In: Fermented Milks (Ed: Adnan Tamime) pp (2006). 11. A.P. Espírito Santo, P. Perego et al. Trends in Food Science & Technology, 22, pp (2011). 12. TSE, TS 3810 Standard of Ayran. Ankara, Turkey: Turkish Institute of Standards (1982). 13. G. Oysun, Analysis Methods of Milk Products. Faculty of Agriculture, Ege University, Publication No: 504. Izmir, Turkey (1999). 14. Association of Official Analytical Chemists Official Methods of Analysis, 15 th edition. AOAC, Washington, DC (1992). 15. N. Tharmaraj, N.P. Shah, J Dairy Sci, 86, pp (2003). 16. F.W. Bodyfelt, M.A. Drake et al. Int Dairy J, 18, pp (1998). 17. A. Koksoy, M. Kilic, Food Hydrocolloids, 18, pp (2004). 18. A.Y. Tamime, R.K. Robinson, Tamime and Robinson s Yoghurt Science and Technology, Woodhead Publishing Limited, Cambridge (2007). 19. F.J. Gallardo-Escamilla, A.L. Kelly et al. Int Dairy J, 17, pp (2007). 20. N.P. Shah, E.V. Lankaputhra et al. Int Dairy J, 5, pp (1995). 21. V.M.E. Marshall, A.Y. Tamime, Int J Dairy Tech, 50, pp (1997) 22. A. Akpinar, Acidophilus milk properties with to produce added different flavour components. MSc Thesis. Ege Graduate School of Natural and Applied Sciences İzmir, Turkey, (2008). 23. I.E. Tonguc, A Study on Ayran production using probiotic bacteria, (MSc Thesis), Ege Graduate School of Natural and Applied Sciences İzmir, Turkey (2006). 24. A.L.B. Penna, K. Sivieri et al. J Food Engineering 49, pp (2001). 25. C. Kocak, S. Aydemir, Functional properties of milk proteins, Food Technology Association Press, 46p (1994). 26. L. Tratnik, R. Božanić et al. Int J Dairy Tech, 59, pp (2006). 27. J. Debon, E. Schwinden Prudêncio et al. E., J Food Eng, 99, pp , (2010). 28. M. Pescuma, E.M. Hébert et al. Int J Food Microbiol, 141, pp , (2010). 29. C. Demir, O. Kinik et al. Milchwissenschaft, 67, pp , (2012). 30. O.N. Donkor, S.L.I. Nilmini et al. Int Dairy J, 17, pp , (2007). 31. I.E. Tonguc, Developing fermented dairy products with low galactose content for patientswith galactosemia & lactose intolerance and determination of quality characteristics, (Ph.D. Thesis), Ege University, Department of Dairy Technology, 163 pages, (2012). 52

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