This is a refereed journal and all articles are professionally screened and reviewed

Advances in Environmental Biology, 6(9): 2471-2475, 2012 ISSN 1995-0756 2471 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Some Physicochemical And Sensorial Properties And Culture Viability In Milk Cultured With Different Content Of Mesophilic Starter Culture 1 Sara Nasiri Boosjin, 2 Vajiheh Fadaei and 3 Mahnaz Hashemiravan 1 M.Sc. student of Food Science and Technology, Varamin Pishva Branch, Islamic Azad University, Varamin, Iran 2,3 Department of Food Science and Technology, Varamin Pishva Branch, Islamic Azad University, Varamin, Iran. Sara Nasiri Boosjin, Vajiheh Fadaei and Mahnaz Hashemiravan: Some Physicochemical And Sensorial Properties And Culture Viability In Milk Cultured With Different Content Of Mesophilic Starter Culture ABSTRACT This research is based on the production of Långfil, that it is a new product in Iran. The milk standardized, homogenized, heat treated and cooled was inoculated with 1%(T1), 2%(T2) and 3%(T3) of mesophilic starter culture, DL-type. On the 5 th, 10 th and 15 th days of storage at 4 C, fermented milk samples were taken for determination of some physicochemical and sensorial properties and culture viability. Therefore, the aim of the present investigation was to assess the impact of different content of mesophilic starter culture inoculated on some physicochemical and sensorial properties and culture viability in fermented milk obtained,and on basis of Iranian acceptance, to select the preferred treatment. It was demonstrated that T2 could be chosen as the preferred treatment. However, this product had a low viscosity; hence, it should be improved and adjusted in taste and texture to meet Iranian consumer demand. Key words: Långfil; mesophilic starter culture; sensory properties; culture viability; physicochemical properties Introduction Fermented milks result from the selective growth of specific bacteria in milk [11]. These products have evolved around the world over thousands of years and are believed to have originated in the area that is now the Middle East [11,17]. Many health benefits have been attributed to fermented milk products [16]. A considerable amount of evidence has been accumulated for some benefits such as improved lactose tolerance. Physiological benefits include antimicrobial activity and gastrointestinal infections, anticancer effects, reduction in serum cholesterol, and immune system stimulation [16,5]. Traditional fermented milks in the Nordic countries are fermented at a low temperature (17 22 C), and mesophilic lactic acid bacteria mostly complete the fermentation [6]. These products are made from encapsulated EPS producing lactococci, primarily Lactococcus lactis ssp. cremoris. They are suggested to play immunomodulating role. This is primarily due to antigenic structures of the surface of lactococci [16]. The fermented milks of Scandinavia are distinctive in flavor and texture. They are generally characterized by a ropy and viscous body, and include viili, ymer, skyr, långfil, keldermilk, and several local products [20,11,19,3,13]. Långfil is the modern variant of the traditional tätmjölk and it is produced in Norway and Sweden [6,21]. It has a very mild and slightly acidic taste, high viscosity and ropy consistency [7,10,9,6]; this product is poured or pressed out of the package onto a plate and eaten as it is with a spoon. Due to the production of EPS, the product is very stable and has a low tendency to wheying off [6]. Långfil is made with the addition of lactococci strains which are able to produce ropiness [12]. In the absence of starter culture, leaves of the butterwort plant (pinguicula vulgaris) is added to the milk and incubated at 17-20 C until the milk has coagulated [7,20,6]. In some instance the leaves of Dorsera spp. are used; both plants are known locally as täätegras or tätgräs,meaning thickening grass [20]. Modern science and technology offer many opportunities to improve and develop present products. By taking advantage of these opportunities, it will become possible to market Nordic fermented milks not only in the Scandinavian countries but in other markets as well. Iranians have a high per capita consumption of fermented milks, especially yogurt. Hence, we decided to produce a product the same as Corresponding Author Vajiheh Fadaei,Department of Food Science and Technology,Varamin-Pishva Branch,Islamic Azad University,Varamin,Iran. E-mail:vn.fadaei@gmail.com and v.fadaie@shahryariau.ac.ir

yogurt texture but with different flavor and aroma on basis of Iranian acceptance. For this, the aim of the present investigation was to assess the impact of 1%(T1), 2%(T2) and 3%(T3) of mesophilic starter culture inoculated on some physicochemical and sensorial properties and culture viability in fermented milk obtained,and on basis of Iranian acceptance, to select the preferred treatment. Material and Methods Starter Culture: Starter culture was purchased from Chr. Hansen, Denmark. Treatments are fermented milks made with different content of DL-type starter culture; characteristics of starter culture and treatments used in the research are described in Table 1. Sample Preparation: At first, the milk was standardized to a fat content of 2.5 g 100 g 1, homogenized and heat treated at 95 C for 5 minutes. The milk is cooled to 20 C and then inoculated with 1%, 2% and 3% of mesophilic starter culture, DL-type, which were defined as treatments T 1, T 2, and T 3, respectively. Then incubated at 20 C for 20-24 hours in 200-g plastic cups and when acidity value of samples reached 86 D, they were Cooled to the storage temperature, 4 C. On the 5 th, 10 th and 15 th days of storage at 4 C, samples were taken for determination of ph, titratable acidity, viscosity, viability of starter culture and sensory properties. Chemical Analysis: All ph measurements were made with a phmeter (Metrohm 827,Swiss). Titratable acidity (TA), as percent lactic acid, was measured for all treatments on d 5,10 and 15 using 0.1 N NaOH and 1% phenolphthalein (Sigma chemical Co.) solution in 95% ethanol as an endpoint indicator. Viscosity Measurement: Viscosity of fermented milks was measured at 10 ± 1 C using a Brookfield DV-II+Pro viscometer (Brookfield Engineering Laboratories, USA). Viscometer was operated at 50 rpm with spindle number 3 after 15s (Cinbas & Yazici, 2008). Microbiological Analysis: Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were counted on M17 agar incubated aerobically at 25 C for 72 h. Viable cell numbers of Leuconostoc bacteria in the samples were counted on Tomato juice agar aerobically at 30 C for 2472 72 h using pour plate method according to the standard. Serial dilutions of samples were made in strength Ringer solution and spread plated in duplicate on their special media [2,1]. Sensory Evaluation: Samples were evaluated using nine-point Hedonic scale. A panel of trained judges evaluated fermented milks for flavor, odor, texture, color characteristics and overall acceptability. Statistical Analysis: All measurements were performed in three replicates. All data were submitted to ANOVA procedure using SAS 9.1 software and GLM procedure. Duncan s multiple range test was used for comparison of means. Results and Discussion Chemical Analysis: Table 2 shows the chemical characteristics of samples during storage at 4 C. As it shows, on the 5 th day the highest TA value was observed in the product containing 2% mesophilic starter culture, T 2. This factor in all treatments decreased during storage time. On the 5 th day the highest ph value was measured in T 3 and the lowest ph value was obtained in T 2. So, the increase in ph and concurrent decrease in TA observed during storage for all the treatments Viscosity Measurement: The results showed that there was significant difference in viscosity between different treatments (p<0.05). As shown in Table 3, the highest viscosity value of 8.07 cp was observed on the 5 th day for treatment 2, and the value increased to 8.45 cp on the 10th day, and finally decreased to 8.08 cp at the end of the storage period. Treatment 3 showed a similar trend but treatment 1 had decreased trend during 15 days storage. Viscosity, and the structure of the gel, is influenced by several factors, including incubation temperature, casein concentration, heat treatment of the milk, acidity and type of starter culture [14]. As mentioned elsewhere långfil has a very mild and slightly acidic taste, high viscosity and ropy consistency. Due to the production of EPS, the product is very stable and has a low tendency to wheying off [6]. In this research, we used medium- EPS producing starter culture, so that the texture of fermented milks was not as ropy as it should be. In general, the viscosity of all treatments was low. Microbiological Analysis:

The result of mesophilic bacteria count are presented in Table 4. Leuconostoc bacteria count of samples in different treatments showed significant difference (p<0.05) and also there was significant difference between the Leuconostoc bacteria count of samples during storage at 4 C (p<0.05). Lactococcus bacteria count of fermented milks during cold storage showed significant difference (p<0.0001) but there was no significant difference between the Lactococcus bacteria count in different treatments. In general, the highest mesophilic bacteria count was observed on the 5 th day and then decreased at the end of the storage period in all samples. Sensory Evaluation: Table 5 shows the sensory evaluation scores of samples during storage at 4 C. Flavor scores of samples in different treatments showed significant difference (p<0.0001) but there was no significant difference in the flavor between samples during storage. Odor of the samples showed significant difference between different treatments (p<0.05) and also there was significant difference in the odor between the samples during storage at 4 C (p<0.0001). Scores attributed by panelists to flavor and odor of samples was low. However, the product containing 2% DL-type starter culture,t 2, had the highest score during cold storage. Even though the level of citrate metabolism in lactic acid bacteria is very low, it is an important property of some mesophilic strains [18]. The starter culture used in the research contains a blend of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Lactococcus lactis subsp. lactis and Lactococcus 2473 lactis subsp. cremoris are capable of rapid acicification of milk. The latter two organisms are the main aroma-forming bacteria in the product [8]; They are used in products such as cultured buttermilk to produce diacetyl via citrate fermentation [11]. Certain carbonyl/flavoring compounds, such as acetate, diacetyl, acetoin and 2,3 butanediol, and CO 2 are produced in milk through the metabolism of citrate. While acetoin and butanediol are tasteless and not involved in flavor, diacetyl is an important flavor component [18]. It was noted that Iranians do not preferred high diacetyl flavor. Color of the treatments showed significant difference (p<0.05) but there was no significant difference between the color of fermented milks during storage. Texture scores of fermented milks showed significant difference (p<0.0001) and also there was significant difference in the texture between the samples during storage at 4 C (p<0.0001). It is evident that EPSs, which are produced by the starter cultures during the fermentation stage, modify or significantly affect the texture properties of fermented milks. Such products have been reported to obtain a higher viscosity and a lower degree of syneresis (whey separation) compared with products produced with non-eps producing cultures [15,18]. As mentioned elsewhere, in this research we used medium-eps producing culture, so that the texture of fermented milks was not as ropy as it must be. Finally, overall acceptability scores of fermented milks showed significant difference (p<0.0001) and also there was significant difference in that between samples during storage at 4 C (p<0.05). As it was shown in Table 5, the product containing 2% mesophilic starter culture, T 2, had the highest score during cold storage. This indicated that the texture and odor of the fermented milks were important in determining liking of them. Table 1: Treatments used in the research Treatment % Inoculation of DL-type starter culture (CHN-22) * T 1 1 T 2 2 T 3 3 *CHN-22: high flavour/ medium texture. Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc mesenteroides subsp. cremoris. Table 2: Chemical properties of fermented milks (T 1, T 2 and T 3) during cold storage* Treatment Storage(day) Acidity( D) ph T 1 5 87.33 1.53 ab 4.41 0.09 a 10 86.33 3.21 ab 4.65 0.39 a 15 85.00 2.65 ab 4.50 0.02 a T 2 5 89.67 1.53 a 4.38 0.07 a 10 87.67 2.08 ab 4.55 0.06 a 15 88.00 2.65 ab 4.50 0.05 a T 3 5 88.00 1.00 ab 4.50 0.05 a 10 85.00 1.00 ab 4.38 0.33 a 15 83.00 1.00 b 4.53 0.04 a Table 3: Viscosity (cp) of fermented milks (T 1, T 2 and T 3) during cold storage*

2474 Treatment storage (day) 5 10 15 T 1 8.02 0.55 ab 7.54 0.23 ab 6.74 0.52 b T 2 8.07 0.30 ab 8.45 0.48 a 8.08 0.80 ab T 3 4.99 0.86 c 7.19 0.42 ab 7.17 0.25 ab Table 4: Mesophilic bacteria count (log cfu/ml) of fermented milks (T 1, T 2 and T 3) during cold storage* Treatme nt Storage(day) Lactococcus Leuconostoc T 1 5 8.10 0.09 a 8.04 0.11 a 10 7.70 0.16 abcde 7.81 0.29 abc 15 7.39 0.21 def 7.56 0.25 abc T 2 5 8.03 0.18 abc 8.04 0.01 ab 10 7.81 0.03 abcd 7.59 0.07 abc 15 7.47 0.11 def 7.64 0.18 abc T 3 5 8.05 0.26 ab 7.93 0.11 abc 10 7.54 0.18 def 7.38 0.17 c 15 7.21 0.05 f 7.35 0.37 c Table 5: sensory properties of fermented milks (T 1, T 2 and T 3) during cold storage* Treatment Storage(day) Sensory properties Flavor Odor Color Texture Overall acceptability T 1 5 4.58 1.16 d 4.92 0.79 abc 5.83 0.72 ab 3.75 1.06 cdef 4.25 0.75 de 10 5.42 1.08 abcd 5.00 0.95 abc 6.00 0.60 ab 4.17 0.83 bcdef 4.17 1.03 de 15 5.25 0.75 abcd 6.00 0.95 c 5.92 1.00 ab 4.75 0.87 abc 4.83 0.72 abcd T 2 5 6.25 1.66 a 5.25 0.75 abc 6.17 1.11 ab 4.58 1.16 abcd 5.67 0.49 ab 10 5.29 1.38 abc 5.58 1.00 bc 6.17 0.58 ab 5.00 0.74 ab 5.50 0.80 abc 15 6.25 0.97 ab 6.00 0.13 c 6.50 0.80 a 5.42 0.51 a 5.75 0.45 a T 3 5 4.75 1.36 d 4.33 0.89 a 6.00 1.21 ab 3.33 0.98 f 3.92 0.79 de 10 4.42 0.90 d 4.67 1.07 ab 5.58 0.90 ab 4.00 0.85 bcdef 3.67 0.78 e 15 4.67 0.89 d 5.58 0.79 bc 5.25 0.87 b 4.50 0.52 abcde 4.42 1.00 de Conclusion: Understanding consumer needs and preferences are critical to successful marketing and enhancing marketing value of a product. This study showed that långfil made by 1(T1), 2(T2) and 3%(T3) of mesophilic starter culture, DL-type, with medium- EPS producing had a low viscosity. However, T2 was selected as preferential treatment; it had a higher viscosity and a lower tendency to wheying off. We found that viscosity and flavor of the fermented milk should be improved. Acknowledgements The authors gratefully thank Tehran milk industry Co-shahdad (Varna) for providing laboratory and processing facilities. References 1. Atlas, R.M., 2006. Microbiologycal media for the examination of food, CRC Press, Boca Raton, pp: 211-286. 2. Barrette, J., C.P. Champagne, D. Roy and N. Rodrigue, 2000. The production of mixed cultures containing strains of Lactococcus lactis, Leuconostoc cremoris and Lactobacillus rhamnosus, on commercial starter media. Journal of Industrial Microbiology & Biotechnology, 25: 288-297 3. Chandan, R.C., 2006. History and consumption trends. in Manufacturing yogurt and fermented milks. Chandan, R. C.Blackwell Publishing Ltd, Oxford, UK, pp: 3-15. 4. Cinbas, A. and F. Yazici. 2008. Effect of the addition of blueberries on selected physicochemical and sensory properties of yoghurts, blueberry addition to yoghurts. Food Technol. Biotechnol, 46(4): 434-441. 5. Ebringer, L., M. Ferencik and J. Krajcovic, 2008. Beneficial health effects of milk and fermented dairy products-review. Folia Microbiol, 53(5): 378-394 6. Fondén, R., K. Leporanta and U. Svensson, 2006. Nordic/Scandinavian fermented milk products. in Fermented milks. Tamime, A.Y. Blackwell Science Ltd,Oxford, UK, pp: 156-173.

7. Kurmann, J.A., J.L.Rasic and M. Kroger, 1992. Encyclopedia of fermented fresh milk products: an international inventory of fermented milk, cream, buttermilk, whey, and related products. Van Nostrand Reinhold, New York, pp: 202-255. 8. Lyck, S., L.E. Nilsson and A.Y. Tamime, 2006. Miscellaneous fermented milk products. in Fermented milks. Tamime, A.Y.Blackwell Science Ltd,Oxford, UK, pp: 217-236. 9. Mistry,V.V., 2006. Fermented liquid milk production. in Handbook of food science, technology and engineering. Hui,Y.H. Taylor & Francis Group, New York, pp: (66) 1-8. 10. Mistry, V.V., 2004. Fermented liquid milk products. in Handbook of food and beverage fermentation technology. Hui,Y.H. Marcel Dekker Inc, New York, pp: 833-847. 11. Mistry, V.V, 2001. Fermented Milks and Cream. in Applied Dairy Microbiology. Marth, E. H. and J.L.Steele.Marcel Dekker, Inc. US, pp: 301-325. 12. Oberman, H. and Z. Libudzisz, 1998. Fermented milks. in Microbiology Of Fermented Foods. Wood, B. J. B. 2ed, Blackie Academic and Professional, London, pp: 308-349. 13. Ray, B. and A.K. Bhunia, 2007. Fundamental Food Microbiology.4th edition, CRC, Boca Raton, FL, pp: 188-190 14. Robinson, R.K. and P. Itsaranuwat, 2006. Properties of yoghurt and their appraisal. in Fermented Milks. Tamime, A.Y. Blackwell Science Ltd, Oxford, UK, pp: 76-94. 15. Ruas-Madiedo, P.R., Tuinier, M. Kanning and P. Zoon, 2002. Role of exopolysaccharide 2475 produced by Lactococcus lactis subsp. Cremoris on the viscosity of fermented milks. International Dairy Journal, 12: 689-695. 16. Shah, N.P., 2006. Health benefits of yogurt and fermented milks. in Manufacturing yogurt and fermented milks. Chandan, R. C. Blackwell Publishing Ltd, Oxford, UK, pp: 327-340. 17. Tamime, A.Y., 2002. Fermented milks: a historical food with modern applications-a review. European Journal of Clinical Nutrition, 56: 2-15. 18. Tamime, A.Y., A. Skriver and L.E. Nilsson, 2006. Starter cultures. in Fermented milks. Tamime, A.Y. Blackwell Science Ltd,Oxford, UK, pp: 11-52. 19. Tamime, A.Y., M. Saarela, A. Korslund Søndergaard, V.V. Mistry and N.P. Shah, 2005. Production and maintenance of viability of probiotic micro-organisms in dairy products. in Probiotic dairy products. Tamime, A.Y.Blackwell Publishing Ltd, Oxford, pp: 39-72. 20. Tamime, A.Y. and V.M.E. Marshall, 1997. Microbiology and technology of fermented milks. in Microbiology and biochemistry of cheese and fermented milk. Law, B. A. 2nd edition, Blackie Academic & Professional, London, pp: 57-152. 21. Vedamuthu, E.R., 2006. Other Fermented and Culture-Containing Milks. in Manufacturing yogurt and fermented milks. Chandan, R. C. Blackwell Publishing Ltd, Oxford, UK, pp: 295-308.