Kefir production in Iran

World Journal of Microbiology & Biotechnology 13, 579±581 Short Communication: Ke®r production in Iran M. Motaghi, M. Mazaheri,* N. Moazami, A. Farkhondeh, M.H. Fooladi and E.M. Goltapeh Ke®r grains were prepared in a goat-hide bag using pasteurized milk inoculated with sheep intestinal ¯ora, followed by culture of the surface layer in milk. From the grain, 11 strains of lactic acid bacteria, non-lactic acid bacteria and yeasts were isolated and identi®ed. Six samples of ke®r were prepared by fermenting pasteurized milk for different lengths of time. Sensory evaluation identi®ed the sample prepared by 24 h fermentation as the best product. Key words: Ke®r grain, microorganisms. Ke®r, a traditional popular middle eastern drink originates from the Caucasus in Central Asia. It is produced by adding activated ke®r grain to cow or goat milk under mesophilic conditions. The resulting fermentation produces an acidic alcoholic beverage that has become increasingly popular in many countries during the last decade. Ke®r has frequently been claimed to be effective against a variety of complaints and diseases (Honsono et al. 1990). There is evidence to support the antitumour activity of polysaccharides from ke®r grain (Shiomi et al. 1982). The microbial ¯ora of the ke®r grains seems to differ according to the place of the origin. The microbiological and product quality of isolated ke®r grain in Iran has been investigated. Materials and Methods A goat-hide bag (4-l capacity) obtained from Pariz and Babak villages in Kerman (Southwest Iran) was washed several times with sterile water, ®lled with pasteurized milk and intestinal ¯ora from sheep. It was kept at 24 to 26 °C for 48 h and shaken hourly. When the milk was coagulated, 75% was replaced with fresh milk. This procedure was repeated for 12 weeks. Gradually a polysaccharide layer (spongy form) appeared on the surface of the hide. The layer was removed aseptically from the hides and propagated in pasteurized cow's milk. Ke®r grains of variable size (0.5±3.2 cm in diameter) were added several times to the fresh cow's milk. shed with sterile water and ground in 1% (w/v) sterile saline (10 ml). Portions (0.1 ml) of a known dilution were plated in duplicate on different culture media similar to the method of Angulo et al. (1993). The isolation of yeasts was carried out by surface spreading on plates of malt extract agar and yeast extract±malt extract agar. After incubation at 28 °C for 3±6 days colonies of different morphology were obtained. The methods of Barnett et al. (1990) were used for their identi®cation. Isolation and Identi®cation of Bacteria For isolation of lactic acid bacteria (LAB), 0.1 ml portions were plated on MRS agar and Rogosa agar (Difco). Cycloheximide (0.05% w/v) was added to inhibit yeast growth. The plates were incubated at 30 °C for 7±12 days in aerobic and anaerobic (10% CO2) atmospheres. For the isolation of lactic streptococci, azide agar medium (Difco) was used aerobically. The isolation medium for Acetobacter contained 0.5% Bacto yeast extract (Difco), 1.5% ethanol and 2.5% agar. Leuconostoc mesenteroides was isolated on whey agar and tomato juice agar with an initial pH below 4.5. The colonies were provisionally considered LAB on the basis of their cellular morphology, Gram-positive and catalase-negative reactions. The criteria applied in the taxonomic characterization were those described by Garvie (1986), Hardie (1986) and Kandler & Weiss (1986). All the cultures were veri®ed and deposited in the Persian Type Culture Collection (Tehran MIRCEN). Isolation and Identi®cation of Yeasts Each of the grain samples was cut so that a portion of the grains' interior could be obtained. These pieces were thoroughly wa- Ke®r Manufacturing Process Ke®r grain (5%) was added to pasteurized (85 °C for 25 min) homogenized milk and incubated at 25 °C for 12, 24, 36, 48, 60 and 72 h. Each tested ke®r was mixed with 1% sucrose. After through mixing, the ke®r was diluted with sterile water and distributed in 200-ml glass bottles (196 g per bottle) capable of withstanding a pressure of atmosphere closed with a crown cap and incubated at room temperature. M. Motaghi, M. Mazaheri, N. Moazami, A. Farkhondeh and M.H. Fooladi are with the Biotechnology Centre, Iranian Research Organization for Science and Technology, No. 71 Forsat Street, Enghelab Ave, Tehran, Iran; fax: (+98) 21 6413417. E.M. Goltapeh is with the School of Agriculture, Tarbiat Modarres University Tehran, Iran. *Corresponding author. Titrable Acidity, pH, Alcohol Titrable acidity was determined using a potentiometer: 10 ml of ke®r was transferred to a beaker containing 100 ml of distilled water, a magnetic stirrer used to expel the carbon dioxide when the contents were heated to 45 °C, cooled to 20 °C, and titrated ã 1997 Rapid Science Publishers World Journal of Microbiology & Biotechnology, Vol 13, 1997 579 M. Motaghi et al. with 0.1 N NaOH to pH 8.2. The acidity was expressed as percent lactic acid. For other products heating was omitted. A Radiometer model 26 pH meter (Copenhagen) was used for the determination of pH and titrable acidity. Alcohol was determined using the enzymatic method of Boehringer Mannheim. Viscosity, Carbon dioxide and Fat Content of Ke®r Viscosity was determined according to Duitschaever et al. (1987). Carbon dioxide was measured manometricaly according to the method of Sandine et al. (1957). The fat content was estimated by Plummer's method (1989). Sensory Evaluation The ke®rs were evaluated by a trained panel of judges, for the characteristics acidity, viscosity, ¯avour, effervescence and degree of liking, using standard evaluation forms. The ke®rs were stored at 4±5 °C for 10 days before being evaluated. Results and Discussion Ke®r differs from other fermented milk products in that it is not produced by the metabolic activity of an evenly distributed micro¯ora. It is made by fermentation with a mixed micro¯ora which is con®ned to discrete ke®r grains which contain several types of LAB, non-lactic acid bacteria and yeasts and which are recoverable after fermentation (Table 1). Among the yeasts, Saccharomyces cerevisiae, S. fragilis, S. lactis and Candida ke®r were isolated. The predominace of isolated yeasts in the grains was consistent with the studies carried out by Rossi & Govvetti (1991) and Angulo et al. (1993). A total of 11 lactic acid and non-lactic acid bacteria and yeast species were isolated and identi®ed. Among the isolated bacteria L. brevis, L. Ke®r, Leuconostoc mesenteroides, and Acetobacter aceti were predominant over the rest of the bacteria. Duitschaever et al. (1988), Hon- Table 1. The Micro¯ora of ke®r grain. Micro¯ora Yeast Candida ke®r Saccharomyces lactis S. cerevisiae S. fragilis Bacteria Lactobacillus ke®r L. brevis L. casei L. Plantarum Streptococus lactis Leuconostoc mesenteroides Acetobacter aceti 580 Table 2. The characteristics of ke®r produced in different time intervals. Ke®r A B C D E F time (h) pH 12 24 36 48 60 72 4.0 3.89 3.01 3.0 2.98 2.98 Acidity Fat (g/100g) 1.18 1.47 2.35 2.40 2.41 2.45 2.1 1.95 1.93 1.90 1.87 1.90 Viscosity CO2 Alcohol (cm/10s) (%) (%w/w) 7.1 7.0 6.83 8.0 6.75 6.75 3.0 3.0 3.5 3.5 3.0 3.0 0.10 0.15 0.15 0.18 0.20 0.20 sono et al. (1990), Rossi & Govvetti (1991) and Angulo et al. (1993) also noted the presence of most of these isolated bacteria and yeast from ke®r grain of different countries. Candida ke®r and Saccharomyces cerevisiae are the most commonly isolated species as compared to the rest or ke®r isolated yeast. As a result of fermentation of milk with Iranian ke®r grain a foamy, effervescent drink has been achieved by varying the ®rst step fermentation incubation time (12, 24, 36, 48, 60 and 72 h). There was no signi®cant difference in pH, acidity, fat, sugar, CO2 and alcohol content between the ke®r samples A and B whereas in ke®rs C, D and F the acidity increased (Table 2). Reduction in sugar content was observed as a function of incubation time. There was no signi®cant difference in alcohol contents of all the tested ke®r samples. Duitschever et al. (1987) found similar results in pH, total acidity and viscosity between the three tested ke®rs. The trained panel of judges accepted ke®r B although there was no signi®cant effect as compared to the rest of the tested ke®rs. Ke®r B was kept for one month in order to check the shelf-life at room temperature. Fortunately there was a negligible increase in acidity and CO2 in the beverages with this duration of storage. The sensory qualities was still judged to be excellent after 30 days. Acknowledgement Number per g ke®r grain 5 1 1 1 1 ´ ´ ´ ´ 10 104 106 105 1 1 1 1 1 1 1 ´ ´ ´ ´ ´ ´ ´ 109 106 106 106 104 105 106 World Journal of Microbiology & Biotechnology, Vol 13, 1997 This work was supported by Iranian Research Organization for Science and Technology. Research Grant No. 26234/IROST. References Angulo, L., Lopez, E. & Lema Cesar, E. 1993 Micro¯ora present in ke®r grains of the Galician Region (north-west of Spain). Journal of Dairy Research 60, 263±267. Barnett, J.A., Payne, R.W. & Yarrow, D. 1990 Yeast characteristics and identi®cation, 2nd edn. Cambridge: Cambridge University press. Ke®r production in Iran Duitschaever, C.L., Kemp, N. & Emmons, D. 1987 Pure culture formulation and procedure for the production of ke®r Milchwissenschaft 42, 80±82. Garvie, E. I. 1986 Genus Leuconostoc. In Bergey's manual of Systematic Bacteriology, Vol. 2, eds Sneath. P.H.A., Mair N.S. & Sharpe, M.E. pp. 1075±1070. Baltimore, MD: Wiliams and Wilkins. Hardie, J.M. 1986 Genus Streptococcus. In Bergey's manual of Systematic Bacteriology, Vol. 2, eds Sneath, P.H.A, Mair, N.S. & Sharpe, M.E. pp. 1043±1071. Baltimore, MD: Wiliams and Wilkins. Honsono, A., Tanare, T. & Otani, H. 1990 Binding properties of lactic acid bacteria isolated from Ke®r milk mutagenic amino acid pyrolyzates. Milchwissenschaft 45, 647±657. Kandler, O. & Weiss, N. 1986 Genus Lactobacillus. In Bergey's Manual of Systematic Bacteriology, Vol. 1, eds Sneath, P.H.A., Mair, N.S. & Sharpe M.E. pp. 1209±1234, Baltimore, MD: Wiliams and Wilkins. Plummer, D.T. 1989 An introduction to practical biochemistry, 3rd edn. London: McGraw-Hill. Rossi, J. & Govvetti, M. 1991 Multistarter for making Ke®r by continuous process. Annali di Microbiologia and Enzimologia 4, 223±226. Sandine, W.E., Ellider, P.R. & Anderson, A.W. 1957 A simple apparatus for measurement of gas production and activity of lactic starter cultures. Milk product Journal, 48, 12±15. Shiomi, M., Sasaki, K., Murofushi, M. & Aibara, K. 1982 Antitumor activity in mice orally administered polysaccharide from Ke®r grains. Japanese Journal of Medical Science and Biology 35, 75±80. (Received in revised form 26 August 1996; accepted 30 August 1996) World Journal of Microbiology & Biotechnology, Vol 13, 1997 581