www.questreach.com www.academicpublishingplatforms.com PRADEC Conference Proceedings Volume 1 Issue 1 December 2012 pp.113-117 Selection of lactic acid bacteria and yeast for koumiss starter and its impact on quality of koumiss Zubaira Kozhahmetova, Gulmira Kasenova Department Biological safety, Kazakh National Agrarian University Almaty, Kazakhstan The objective of current research is to determine the optimal composition of the yeast cultures consisting of Lactobacillus acidophilus, strain 74/17 + Streptococcus lactis, strain 31/11 + Torulopsis sphaerica, strain 117k, which facilitates intensive fermentation of horse milk and improves the quality of the product. Keywords: Koumiss, lactic acid bacteria, yeasts, sourdough, strain Introduction The purpose of this study was the selection of new strains of lactic acid bacteria and yeast separated from local koumiss for the improvement of koumiss ferment and its impact on quality of koumiss. The introduction of the leaven koumiss acidophilus was intended to accelerate the formation of the finished product, which meant it had a higher energy of acid than Bulgarian bacteria. In addition, the introduction of Lactobacillus acidophilus in the starter improved nutritional properties of natural koumiss because of it being the only lactic acid bacilli existing in human intestines and because of it having a long positive therapeutic effect on the body (Shigaeva and Ospanova, 1983; Zhilin, 1960; Duysembaev, Seytov, Khasenova et al., 1979) While studying koumiss prepared using natural starters in different regions of Kazakhstan, acidophilus were selected. These samples of koumiss were recognized as the best due to their taste. Based on production-valuable characteristics of koumiss best strains were selected from the identified cultures of lactic acid bacteria and Lb. acidophilus (74/17, 75/20, 87/75), Lb. bulgaricus (78/30, 79/31), Str. lactis (31/11), Torulopsis sphaerica (105k, 109k, 117k), Torulopsis kefir. var. kumis (114z), Candida mycoderma (125k). With the purpose of determining the compatibility of testing organisms in joint cultivation, coagulation of milk, acid production, and microscopic examination after 16-18 hour growth were assessed. A total of 14 combinations of lactic streptococci and bacilli were tested. Strains of Str. lactis monoculture were inferior to acid production strains of Lactobacillus acidophilus and Bulgaria, but the joint cultivation with the latter proved the acidity to be much improved. In previous experiments on the combination of acid strains 31/11 (Str. lactis) 74/17 (Lb. acidophilus) and 31/11 (Str. lactis) 78/30 (Lb. bulgaricus) surpassed all other combinations of lactic acid bacteria. It is a well-known fact that yeast plays a key role in preparing koumiss and therefore, the further work concerned the selection of yeast for making sourdough with lactic acid bacteria. In the laboratory the cultivation of microbial fermentation of koumiss was conducted in an incubator (FHC-12-250) at the Department of Microbiology, Virology, and Immunology (current "Biological Safety") of KazNAU. Five - 113 -
different strains of yeast were tested and they are the following: Torulopsis sphaerica, sht.105k, 109k, 117k Torulopsis kefir. var. kumis, pcs. 114z and Candida mycoderma, 125k units isolated from koumiss starters, horse milk in combination with lactic acid bacteria Lb. acidophilus, Lb. bulgaricus, Str. lactis, selected from previous experiments. During cultivation the formation features of starters under consideration were studied. The energy of acid, the growth rate of yeast, and lactic acid bacteria were the points of focus in the experiment. The master batch koumiss sourdough prepared on sterile milk. Cultures of lactic acid bacteria Lb. acidophilus, Lb. bulgaricus, Str. lactis and yeast Torulopsis sphaerica were grown separately and mixed in 1:1:0.5 ratios just before the fermentation of koumiss on raw horse milk. The sequence of the experiment is shown in the following combinations, where 20 combinations of koumiss starters were used. Koumiss was prepared with fermentation of the following composition (Table 1): TABLE 1. COMBINATIONS OF KOUMISS STARTER No. Composition of sourdough 1 Str.lactis,31/11(10%) + Lb.acidophilus, 74/17(10%)+T.sphаerica, 105к (5%) 2 Str.lactis,31/11(10%)+ Lb.acidophilus, 74/17(10%)+T.sphаerica, 109к (5%) 3 Str.lactis, 31/11(10%)+ Lb.acidophilus, 74/17(10%)+T.kefir var.kumis, 114з (5%) 4 Str.lactis, 31/11(10%)+ Lb.acidophilus, 74/17(10%)+T.sphаerica, 117к (5%) 5 Str.lactis, 31/11(10%)+ Lb.acidophilus, 74/17(10%)+C.mycoderma, 125к (5%) 6 Str.lactis, 31/11(10%)+ Lb.acidophilus, 75/20(10%)+T.sphаerica, 117к (5%) 7 Str.lactis, 31/11(10%)+ Lb.acidophilus, 75/20(10%)+T.sphаerica, 105к (5%) 8 Str.lactis, 31/11(10%)+ Lb.acidophilus,.75/20(10%)+T.sphаerica, 109к (5%) 9 Str.lactis, 31/11(10%)+ Lb.acidophilus, 75/20(10%)+T.kefir var.kumis, 114з (5%) 10 Str.lactis, 31/11(10%)+ Lb.acidophilus, 75/20(10%)+C.mycoderma, 125к (5%) 11 Str.lactis, 31/11(10%)+ Lb.acidophilus, 87/75(10%)+T.sphаerica, 117к (5%) 12 Str.lactis, 31/11(10%)+ Lb.acidophilus, 87/75(10%)+T.sphаerica, 105к (5%) 13 Str.lactis, 31/11(10%)+ Lb.acidophilus,87/75(10%)+T.sphаerica, 109к (5%) 14 Str.lactis,31/11(10%)+ Lb.acidophilus, 87/75(10%)+T. kefir var.kumis, 114з (5%) 15 Str.lactis, 31/11(10%)+ Lb.acidophilus,.87/75(10%)+C.mycoderma, 125к (5%) 16 Str.lactis,31/11(10%)+ Lb.bulgaricus,78/30(10%)+T.sphаerica, 117к (5%) 17 Str.lactis, 31/11(10%)+ Lb.bulgaricus, 78/30(10%)+T.sphаerica, 105к (5%) 18 Str.lactis, 31/11(10%)+ Lb.bulgaricus, 78/30(10%)+T.sphаerica, 109к (5%) 19 Str.lactis, 31/11(10%)+ Lb.bulgaricus, 78/30(10%)+T. kefir var.kumis, 114з (5%) 20 Str.lactis, 31/11(10%)+ Lb.bulgaricus, 78/30(10%)+C.mycoderma, 125к (5%) Natural koumiss produced in the farm "Seysebek" in Talgar district of Almaty region served as the controller. Biochemical parameters were determined after koumiss fermentation. The results of these studies are presented in Table 2. Results were checked hourly during fermentation based on taste. Koumiss produced with yeast has a sour taste and is free from foreign smell that gives the drink a smooth consistency and a better flavor. Koumiss prepared with these starters organoleptically complies with the standard. Along with this antagonist activity of koumiss with the most optimal combination of pathogens most favorable to make koumiss fermentation starters was also determined. The results of these experiments are shown in Table 3. - 114 -
TABLE 2. IMPACT OF DIFFERENT COMBINATIONS OF LACTIC ACID BACTERIA AND YEASTS ON QUALITYOF KOUMISS Option, Fermentation time, h Acidity, 0 Т Biochemical parameters рн Carbohydrates, % Alcohol,% Diacetyl, mg% Acetoin, mg% Organoleptic parameters 1 20 75.0 4.0 4.80 1.20 0.05 scent Koumiss non-carbonated, sweet, fermented, uniform 2 20 90.0 4.0 3.20 2.20 0.12 scent Koumiss pleasant, fermented, carbonated, uniform 3 20 75.0 4.0 4.31 1.60 0.1 scent Koumiss pleasant, sour milk, fermented, lightly carbonated 4 20 110.0 4.0 3.00 2.60 0.14 0.09 Koumiss pleasant, refreshing, sparkling, homogeneous, fermented 5 22 60.0 4.5 5.30 0.70 0.08 scent Koumiss lightly carbonated, watery taste 6 22 75.0 4.0 4.25 1.63 0.14 0.08 Koumiss fermented, fermented, uniform 7 21 80.0 4.5 4.00 1.90 0.08 scent Koumiss pleasant, cultured milk, fermented, refreshing 8 22 85.0 4.0 4.10 1.70 0.12 scent Koumiss pleasant, watery taste 9 20 90.0 4.0 3.20 2.40 0.09 scent Koumiss pleasant, cultured milk, fermented, highly carbonated 10 22 60.0 4.5 5.30 0.80 0.08 scent Koumiss sour, lightly carbonated, uniform 11 22 60.0 4.5 5.00 1.00 0.1 scent Koumiss fermented, lightly carbonated, sweet, uniform 12 22 81.0 4.5 3.50 2.00 0.05 scent Koumiss fermented, little fermented, uniform 13 22 85.0 4.0 3.20 2.40 0.1 scent Koumiss pleasant, sparkling, fermented, refreshing, uniform 14 22 67.0 4.5 5.00 1.00 0.08 scent Koumiss pleasant, lightly carbonated, fermented 15 22 69.0 4.5 4.40 1.51 0.04 scent Koumiss fermented, sparkling, sweet 16 22 76.0 4.0 4.10 1.70 0.09 0.08 Koumiss fermented, sparkling, sweet 17 22 62.0 4.5 5.10 0.70 0.04 scent Koumiss fermented, fermented, lightly carbonated 18 22 65.0 4.5 5.00 1.00 0.09 0.05 Koumiss pleasant, sparkling, fermented, refreshing, uniform 19 22 67.0 4.5 5.00 1.00 0.05 scent Koumiss pleasant lightly carbonated, fermented, uniform 20 22 60.0 4.6 5.50 0.60 0.05 scent Koumiss fermented, lightly carbonated, blank taste The results of the studies in this field show that the finished product obtained through a new method shows high antagonistic activity against the saprophytic, conditionally pathogenic, and pathogenic bacteria (zone of suppression of 12-28 mm). Thus, among the tested sourdough starters, sourdough No.4 was found to as the best one, which included: Lb. acidophilus, st.74/17 + Str. lactis, st.31/11 + T. sphaerica, st.117k The effect of yeast No.4 on the nature and intensity of fermented mare's milk, the quality of koumiss as well as acid and aroma formation in koumiss were studied as well. The results of repeated experiment also confirm the high quality of this product. Microflora resulting from natural koumiss starter No.4 is shown in Figure 1. - 115 -
TABLE 3. ANTAGONISTIC ACTIVITY OF KOUMISS STARTERS TO THE SAPROPHYTIC, CONDITIONALLY PATHOGENIC AND PATHOGENIC BACTERIA composition sourdough Sarcina flava Bac. mycoides S. albus, st.165 E. coli, st. О.Б. E.coli,st. f D. septicus Proteus vulgaris S.choleraesuis, st.177 S.abortus equi,st 841 1 24 12 23 22 22 22 12 18 18 16 12 12 14 25 15 15 16 12 2 24 12 23 22 22 22 12 18 18 16 12 12 14 25 15 15 16 12 3 20 10 20 20 20 23 10 18 20 10 12 10 14 19 12 12 12 10 4 24 14 24 28 26 24 12 22 22 22 18 12 20 23 18 18 16 16 5 16 10 10 20 20 22 8 18 18 12 10 11 10 20 13 13 13 10 6 24 12 23 22 22 22 12 18 18 16 12 12 14 25 15 15 16 12 7 16 12 14 17 15 17 10 10 10 11 10 10 10 15 16 16 12 12 8 14 12 16 17 15 20 10 10 10 11 10 10 10 15 13 13 12 12 9 16 10 20 20 20 22 10 16 16 16 10 10 10 20 13 13 12 10 10 16 12 14 17 15 17 10 10 10 11 10 10 10 15 16 16 12 12 11 17 10 18 16 16 16 10 10 10 14 10 10 10 17 12 12 13 10 12 16 10 20 20 20 22 10 16 16 16 10 10 10 20 13 13 12 10 13 16 10 10 20 20 22 8 18 18 12 10 11 10 20 13 13 13 10 14 13 10 14 17 15 15 8 14 14 15 10 10 10 12 13 13 12 10 15 16 10 10 20 20 22 8 18 18 12 10 11 10 20 13 13 13 10 16 24 12 23 22 22 22 12 18 18 16 12 12 14 25 15 15 16 12 17 16 10 10 20 20 22 8 18 18 12 10 11 10 20 13 13 13 10 18 20 10 20 20 20 23 10 18 20 10 12 10 14 19 12 12 12 10 19 16 10 20 20 20 22 10 16 16 16 10 10 10 20 13 13 12 10 20 14 12 16 17 15 20 10 10 10 11 10 10 10 15 13 13 12 12 S. abortus ovis S. typhimurium S. dublin S. gallinarum P. multocida L. monocytogenes E. rhusipathiae C. faetus Bac. anthracis FIGURE 1. NEW NATURAL MICROFLORA OF KOUMISS Having identified the optimal combination of koumiss starter, improving the way of obtaining koumiss, which is to speed up the cooking time of koumiss was the next priority. For this purpose, the experiment, where seven different versions - 116 -
with different mixing rates (in revolutions per minute) under changing temperature conditions on the basis of koumiss was conducted: 1- option 185 rev / min 27-28 С 2- option 185 rev / min 30-31 С 3- option 185 rev / min 33-34 С 4- option 220 rev / min 27-28 С 5- option 220 rev / min 30-31 С 6- option 220 rev / min 33-34 С 7- option 330 rev / min 30-31 С In this experiment the best results were obtained while conducting the test in the version No.2. The time necessary for koumiss to be ready decreased from 51 hours to 20 hours or by 2.5 times. The volume of masterbatch starter remained at level of 25-30%, which increased the organoleptic qualities of the product. References Duysembaev, K., Seytov, Z., Khasenova, Z., Cherepanov, A., Belokobylenko, V., 1979. Koumiss and Shubat [Koumiss i shubat], in Russian, Alma-Ata Shigaeva, M., Ospanova, M., 1983. Microflora National Milk Beverages [Mikroflora nacionalnyh kislomolochnyh napitkov], in Russian, Alma-Ata: Nauka Zhilin, M., 1960 Antibiotic Properties of Yeast Koumiss, In.: Proceedings of the conference on one horse breeding and dairy koumissodeliyu [Trudy konferencii po molochnomu konevodstvu i koumissodeliyu], in Russian, Moscow, pp.162-74 - 117 -