735 Bulgarian Journal of Agricultural Science, 12 (2006), 735-740 National Centre for Agrarian Sciences Effect of Some Plant Supplements on the Number of Lactic Acid Bacteria in Bulgarian Fermented Milk S. BOYCHEVA, T. DIMITROV and D. PAVLOV Trakia University, BG - 6000 Stara Zagora, Bulgaria Abstract BOYCHEVA, S., T. DIMITROV and D. PAVLOV, 2006. Effect of some plant supplements on the number of lactic acid bacteria in Bulgarian fermented milk. Bulg. J. Agric. Sci., 12: 735-740 Fermented milk (yogurt) with extract of linseed and sesame seed was prepared. Dynamics of acidification at coagulation of milk and after 24 h and 48 h storage at 4-6 0 C was investigated. The number of lactic acid bacteria - rods and cocci in the produced fermented milk was determined now of coagulation and after 24 and 48 h cold storage. Fermented milk, prepared from cow's milk, supplemented with extract of linseed and sesame seed, coagulated more quickly than natural milk. The extracts of linseed and sesame seed had beneficial effect on the growth of Lactobacillus delbrueckii ssp. bulgaricus. Fermented milk, produced from milk with extract of linseed and sesame seed had more compact and homogenous coagulum with no whey secretion. Key words: fermented milk, lactic acid bacteria, linseed, sesame seed, Lactobacillus delbrueckii ssp. bulgaricus Introduction Original Bulgarian sour milk, which belongs to fermented milks, has increased its world popularity and consumption. This milk completely satisfies the human needs to consume dietetic and healthy foods. Sour milk combines useful features of the fresh milk compounds and the beneficial effect of lactic acid bacteria. It is well known that lactic acid bacteria possess antimutagenic and anticarcinogenic effect (Fuller, 1997; Lukasova, 1990; Tavan et al., 2002; Saikali et al., 2004) and lead to reduction of the cholesterol level in the blood serum (Hapner et al., 1979; Liong and Shah, 2005). They also stimulate the organism immunological system activity and suppress the development of enteropathogenic microorganisms and the formation of toxic products in the intestinal tract (Perdigon et al., 1990; Reddy et al., 1988). Recently more investigations have been carried out with Bulgarian fermented milk incorporating different additives aiming at increase of its organoleptic and healthy characteristics. Fermented milk is supplemented by sugar, fruit juice, purees, etc.
736 Sour milk supplemented by sorbitol or fructose has been consumed better by diabetes mellitus patients. The objective of the present study was to establish the effect of extract of sesame seed and linseed on the amount of lactic acid bacteria in Bulgarian fermented milk and its physicochemical and organoleptic features. Material and Methods Fermented milk preparation. Fermented milk was prepared from fresh cow's milk produced in a private cow farm in the village of Bogomilovo. Fresh milk was heated on water bath at 85-90 o C for 30 min. Before heating 3 % of ground linseeds or sesame seeds were added to the milk. After heating the milk was filtrated by a sterile gauze to separate seed's larger fragments, cooled to 45 o C and 1.5 % 18 hour starter for Bulgarian fermented milk (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) from the Microbiology section collection was added. The samples were incubated at 42 o C to milk coagulation. After coagulation samples were stored at 4-6 o C. Physicochemical analysis of the milk. The contents of dry matter, milk fat, non-fatty substances and total protein in the raw milk were determined by the ultrasonic analyzer "Ecomilk". Microbiological analysis. The total bacteria count, number of lactic acid bacteria, coli bacteria and psychotrophs in the raw milk were investigated using standard methods and media - nutrient agar, China blue hydrolyzed milk agar, Endo agar and nutrient agar with yeast extract respectively. S. Boycheva, T. Dimitrov and D. Pavlov Determining the dynamics of acidification. The titratable acidity was determined at the end of the first and second hour after adding the starter and after milk coagulation. Active acidity and time for coagulation were also determined. Determining lactic acid bacteria count in the coagulated milk. The number of L. delbrueckii ssp. bulgaricus and S. thermophilus were determined by direct count on stained preparations according to the Breed method, performed at the moment of coagulation of the milk and after 24 and 48 hours of cold storage. Results and Discussion The cow's milk used in the investigation was up to the requirements for extra quality in accordance with Instruction N 30 for qualification of raw milk (Table 1). It differed from the requirements by the milk fat index only. The data about milk microflora before and after pasteurization (Table 2) suggested that the microorganisms count in the pasteurized milk was strongly reduced and met the requirements of the standard. It was evident from the data about acidification dynamics of natural and linseed and sesame seed supplemented milk (Table 3) that the acidity of both experimental samples was higher than the control sample, even on the first hour after adding the starter (P<0.05). The same tendency was observed on the second hour and it was more clearly expressed (P<0.01). The milk supplemented with extract of linseed coagulated 16 minutes earlier (P<0.05), but its acidity was lower - 62.62 o T than the control sample. Its lower acidity compared to the control sample
Effect of Some Plant Supplements on the Number of Lactic Acid Bacteria... 737 Table 1 Composition and properties of raw milk (n=7) Indices х Sх Dry matter, % 11.870 0.100 Fat, % 3.280 0.100 Non-fatty substances, % 8.590 0.040 Total protein, % 3.240 0.001 Density 28.900 0.018 Titratable, Т о 18.100 0.320 рн 6.610 0.010 Table 2 Microflora of raw and pasteurized milk (n=7) Microorganisms (CFU/сm 3 ) Total bacteria count Before pasteurization 978 800 After pasteurization 416 Lactic acid bacteria 511 900 61 Colibacteria 6 960 0 Psychotrophic bacteria 230 000 166 remained after 24 and 48 hours cold storage. That was probably due to mucin like substances liberated by linseed in the milk during pasteurization. These substances increased the buffer capacity of milk. The milk supplemented with extract of sesame seed coagulated at almost the same acidity as natural milk but 26 minutes earlier (P<0.05). Acidity of control sample increased during cold storage as well as the experimental sample. Concerning to the number of L. delbrueskii ssp. bulgaricus and S. thermophilus in the produced fermented milk, it is evident that immediately after milk coagulation the number of L. bulgaricus in the sample supplemented with linseed extract was almost 1.7 times higher than in the control sample (P<0.001), while the number of S. thermophilus increased but at a lower rate - 1.3 times (Table 4). After 24 and 48 hours of cold storage (4-6 o C) the L. bulgaricus number in the experimental sample increased by 28.7% and 36.6% and exceeded the control group - 1.64 and 1.47 times, respectively (P<0.01). It can be assumed that the higher number of rodlike bacteria in milk supplemented with linseed extract was due to the enriched composition of the milk by the functional nutrient compounds liberated by the linseed. There is, at first glance, some contradiction between the higher number of L. bulgaricus cells in the sample with linseed extract and the lower acidity at coagulation and after 24 and 48 hours cold
738 S. Boycheva, T. Dimitrov and D. Pavlov Тable 3 Dynamics of acidification of milk supplemented with linseed and sesame seed extract Indices Control Linseed Sesame seed x S x x S x x S x Initial acidity 18.2 0.33 18.48 0.38 18.48 0.38 Acidity after 1 h., Т о 21.33 0.47 24.26 1.01 a* 24 0.990 a* Acidity after 2 hours, T o 36.69 2.12 46.18 2.87 a** 47.94 1.760 a** Acidity at coagulation 64.47 4a 62.62 2.83 a 63.78 4.34 a Time for coagulation, min. 165 3.93 149 6.000 a* 139 5.790 a* рн 4.84 0.090 a 4.98 0.140 a 4.83 0.14 a A f t e r 2 4 h o u r s Titratable acidity, T o 82.96 3.221 a 75.56 2.704 a 82.24 3.249 a рн 4.36 0.08 a 4.48 0.120 a 4.34 0.081 a A f t e r 4 8 h o u r s Titratable acidity, T o 97.35 2.208 a 92.77 3.539 a 91.5 2.978 a рн 4.14 0.098 a 4.21 0.068 a 4.21 0.080 a * The differences among control and additives for respective parameters are statistically significant if not equal letters at P<0.05 (*) and P<0.01 (**). storage. Probably there is a positive effect of the nutrients from the supplemented linseed which improve the growth of lactic acid bacteria. The organoleptic test of sour milk confirmed these conclusions. The coagulum of fermented milk supplemented by linseed extract was more compact and homogeneous, without whey secretion. Considerable increase of the lactic acid bacteria count in fermented milk with sesame seed extract was observed compared to natural milk-about 1.7 times. The increase of L. bulgaricus number was more strongly expressed - more than twice (P<0.01). The tendency of higher number of lactic acid bacteria in milk supplemented with sesame seed extract was kept during the cold storage of milk for 24 and 48 hours but the difference bet-ween experimental and control sample decreased to 1.49-1.46 times, respectively (P<0.01). The increase was again at the expense of rodlike bacteria mostly, as well as in the milk supplemented with linseed extract. The extracts of linseeds and sesame seeds incorporated in fermented milk production changed the ratio at the main microflora ingredients responsible for the quality characteristics of the product. They decreased the share of S. thermophilus and increased that of L. bulgaricus. It is well known that Bulgarian fermented milk is a natural probiotic, which our nation has used since the remote past. To provide their beneficial healthy properties, the minimum level of viable bacteria is approximately 10 6 cfu/ml and the suggested therapeutic dose is 10 8-10 9 vi-
Effect of Some Plant Supplements on the Number of Lactic Acid Bacteria... 739 Table 4 Number of lactic acid bacteria in coagulated milk with extract of linseed and sesame seeds, thousand/cm 3 Samples 1.L. bulgaricus 2. S. thermophilus Total count Ratio 1/2 At coagulation Control 173 216 414 180 a 587 396 1 : 2.4 Linseed 308 865 a*** 561 310 a,b 870 175 a** 1 : 1.8 Sesame seed 356 389 ** 637 642 b** 994 033 b** 1 : 1.79 A f t e r 2 4 h o u r s Control 242 047 557 904 799 951 1 : 2.3 Linseed 397 807 a** 674 370 a* 1 072 177 a*** 1 : 1.8 Sesame seed 436 305 b** 759 772 a* 1 196 077 a*** 1 : 1.74 A f t e r 4 8 h o u r s Control 285 855 614 190 a 900 045 1 : 2.15 Linseed 422 145 a** 694 282 a 1 116 427 a** 1 : 1.64 Sesame seed 489 847 b** 827 917 b** 1 317 765 b** 1 : 1.69 * The differences among control and additives are statistically significant if not equal letters at P<0.01 (**) and P<0.001 (***). able cells per day (Kurman and Rasic, 1991). However, studies indicate that probiotic bacteria may not survive in sufficient number and retain their activity before its consumption or in the host gastrointestinal tract (Hamilton-Miller et al., 1999). That's why we think that fermented milk produced from milk supplemented with linseed and sesame seed extracts is a product beneficial for human health containing high number of viable and active cells of L. delbrueskii ssp. bulgaricus. This conclusion is supported by the study of Hou et al. (2003), according to which sesame oil emulsion may serve as a suitable biocapsule to encapsulate bacteria and especially L. bulgaricus from the unfavourable conditions in the intestinal tract. Conclusion Supplementation of extracts of linseed and sesame seeds into the raw milk during the process of production of Bulgarian fermented milk reduces the time for coagulation with 9.7% and 15.8%. The acidity of fermented milk with supplements increases more slowly at 48 hours cold storage compared to pure fermented milk, which is guarantees the good quality. Extracts of linseeds and sesame seeds had a beneficial effect in the production of Bulgarian fermented milk improving microbial activity. The total number of L. bulgaricus and S. thermophilus increased with 48.14% and 69.2% now of coagulation and with 24% and 46.4% respectively after 48 h of cold storage compared to pure fermented milk.
740 Applied linseed and sesame seed extracts had a better effect on the activity of L. delbrueskii ssp. bulgaricus increasing their number at a higher degree - 1.7 and 2.1 times compared to S. thermophilus - 1.3 and 1.5 times. The ratio of L. bulgaricus to S. thermophilus decreased from 1:2.15 to 1:1.69. Sesame seed had the highest effect on the microbial growth, increasing the number of L. delbrueskii ssp. bulgaricus 2.05 times and of S. thermophilus 1.54 times, respectively. Bulgarian fermented milk supplemented with linseed extract had more compact and homogeneous coagulum without whey secretion. It was probably due to its strongly expressed buffering. References Bogdanov, I., P. Popkhristov and L. Marinov, 1962. VII International Cancer Congress, Moscow, 364 pp. (Ru). Fuller, R., 1997. Probiotics 2: Applications and practical aspects. Chapman & Hall. Hamilton-Miller, J. M. T., S. Shah and J. T. Winkler, 1999. Public health issues arising from microbiological and labeling quality of foods and supplements containing probiotic microorganisms. Public Health Nutr., 2: 223-229. Hepner, G., R. Fried, S. StJeor, L. Fusetti and R. Morin, 1979. Hypocho lesterolemic effect of yogurt and milk. American J. of Clinical Nutrition, 32: 19-24. Hou, R. C. W., M. Y. Lin, M. M. C Wang S. Boycheva, T. Dimitrov and D. Pavlov and J. T. C. Tzen, 2003. Increase of viability of entrapped cells of Lactobacillus delbrueckii ssp. Bulgaricus in artificial sesame oil emulsions. J. Dairy Science, 86: 424-428. Kurmann, J. A. and J. L. Rasic, 1991. The health potential of products containing bifidobacteria. In Robinson R. K. ed. Therapeutic properties of fermented milks. London: Elsevier Science Publishers, Ltd., pp. 117-158. Liong, M. T. and N. P. Shah, 2005. Acid and bile tolerance and cholesterol removal ability of lactobacillus strain. J. of Dairy Science, 88: 55-66. Lucasova, J., 1990. Antibacterial activity of lactic fermentation bacteria. Vet. Medicina, 35 (3): 187-192 (Ru). Perdigon, G., S. Alvares, M. Nader de Macias, M. Roux, Pesce de Ruiz and A. Holgado, 1990. The oral administrations of lactic acid bacteria increase the mucosal intestinal immunity in response to enteropathogens. J. of Food Protection, 53 (5): 404-410. Reddy, N. R., S. M. Roth, W. N. Eigel and M. D. Pierson, 1988. Foods and food ingredients for prevention of diarrheal disease in children in developing countries. J. of Food Protection, 51 (1): 66-75. Saikali, J., C. Picard, M. Freitas and P. Holt, 2004. Fermented milks. Probiotic cultures and colon cancer. Nutrition and Cancer, 49: 14-24. Tavan, E., C. Cayuela, J. M. Antoine and P. Cassand, 2002. Antimutagenic activities of various lactic acid bacteria against food mutagens. J. of Dairy Res., 69: 335-341. Received July, 12, 2006; accepted September, 12, 2006.