The current issue and full text archive of this journal is available atwww.emeraldinsight.com/0007-070X.htm Biotechnological innovations in kefir production: a review Innovations in kefir production S. Sarkar Metro Dairy Limited, Neelgunj Bazar, West Bengal, India 283 Abstract Purpose – The purpose of the paper is to show that traditionally, kefir was obtained by fermenting milk with kefir grains. Wide variation in microflora of kefir grains makes it difficult to obtain an optimal and uniform starter culture necessary for obtaining a quality kefir. Reviewed literature on microbiological and technological innovations in kefir production would enrich the scientific knowledge resulting in production of kefir with superior physical, chemical, nutritional, therapeutic and sanitary qualities. Design/methodology/approach – An attempt is made to highlight the microbiological and technological aspects of kefir production with regard to the microflora of kefir grains, suitability of different types of milk, treatment of milk, starter inoculation and incubation, packaging, storage and post-production treatment of kefir as well as methods of preservation of kefir grains. Findings – Diverse microflora of kefir grains is the prime cause for the wide variation in kefir quality. Production of kefir is based on symbiotic relation between lactic acid bacteria and yeasts and the type of milk, their heat-treatment, size of inoculating starters and temperature of incubation influence their metabolic activities. Application of a suitable combination of lactic acid bacteria and yeasts would enable production of kefir with more uniform product with specific properties Packaging of kefir in a suitable container and storage at low temperature are suggested to retain its qualities. Originality/value – Fermentation of milk with a suitable starter combination consisting of lactic acid bacteria and yeasts rather than application of kefir grains during the production of kefir would be more scientific to yield a product with enhanced nutritional and therapeutic qualities. Keywords Heat treatment, Milk, Food products, Drinks Paper type Research paper Introduction Yeasts are commercially significant in food industries, particularly in dairy processing, because they bring about desirable fermentative changes in some of the fermented milk products (Westall and Filtenborg, 1998) and have remarkably stable association with lactic acid bacteria (LAB), especially lactobacilli (Wood, 1981). kefir is a self-carbonated, sour beverage obtained with a fermenting agent called “kefir grain”, consisting of casein and gelatinous colonies of LAB and yeasts grown together symbiotically (Webb et al., 1987). Yeasts and lactobacilli are mutually dependent and grow in balanced proportion in kefir grains (Wood and Hodge, 1985) and symbiosis between yeasts, lactobacilli and streptococci was noted during the production of kefir (Loretan et al., 2003). Kefir has gained popularity in various part of the world including Southwest Asia, Eastern and Northern Europe, North America, Japan (Otles and Cagindi, 2003), Middle East, North Africa and Russia (Koroleva, 1982, IDF, 1988) owing to its nutritional (Guzel-Seydim et al., 2003, Liut Kevicius and Sarkinas, 2004) and therapeutic properties (Marquina et al., 2002, Liu et al., 2002, Zacconi et al., 2003, Czamanski et al., 2004) and has been recommended for consumption as a dietetic beverage (Sarkar, 2007). Wide variation in microflora of kefir grains makes it difficult to obtain an optimal and British Food Journal Vol. 110 No. 3, 2008 pp. 283-295 q Emerald Group Publishing Limited 0007-070X DOI 10.1108/00070700810858691 BFJ 110,3 284 uniform starter culture necessary for obtaining a quality kefir beverage. Factors affecting kefir flora and consequently the quality of kefir include media used, grain to milk ratio, cultures used for kefir production, time and temperature of incubation, extent of agitation, type of package and storage conditions (Koroleva, 1988a, 1991). According to the Codex Standard, composition of kefir must be constituted of , 10.0 per cent (m/m) milk fat, a minimum of 2.80 per cent (w/w) milk protein, 0.6 per cent (m/m lactic acid) titratable acidity, 107cfu/g total microbial count and 104cfu/g yeast, however no specification for ethanol content has been mentioned (FAO/WHO, 2001). In the present endeavor biotechnological innovations for production of kefir with improved dietetic properties have been reviewed. Production of kefir Kefir was first made from goat milk with kefir grains in goatskin bag by hanging in the house during winter and outside during summer (Ozer and Ozer, 1999). Traditionally, kefir was produced by inoculating milk with kefir grains (Pijanowski, 1980) or by the widely adopted European method, which involved use of bulk milk culture obtained by kefir grains for milk inoculation (Puhan and Vogt, 1985). Kurmann (1984) classified kefir under the class of mixed lactic acid and ethanol fermented milk and can be further sub-classified as kefir obtained using kefir grains and artificial kefir obtained without kefir grains Various steps involved during kefir production are enunciated below. Microflora of kefir grains Kefir grains are sometimes called “Millet of the prophet” or “Mohomet grains”, dump shaped, gelatinous granules measuring about 1-2 mm to 3-6 mm, sometimes up to 2-15 mm in diameter with irregular, rough and convoluted surfaces (Koroleva, 1991). It has been reported that kefir grains are constituted of 108-109cfu/ml LAB, 105-106cfu/ ml yeasts and 105-106cfu/ml acetic acid bacteria (Koroleva, 1991; Garrote et al., 2001). Basic microflora of kefir grain consists of LAB such as lactobacilli (thermophilic and mesophilic), leuconostoc, streptococci (homofermentative and heterofermentative), lactococci and acetic acid bacteria as well as yeasts (Bottazzi et al., 1994, Rea et al., 1996), all held together by water soluble polysaccharide called kefiran (Kosikowski, 1977), which is composed of glucose and galactose (Yokoi et al., 1991). Bosch et al. (2006) identified Lactobacillus kefir, Lactobacillus parakefir and Lactobacillus brevis (heterofermentative) and Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus kefirgranum, Lactobacillus kefiranofaciens and Lactobacillus casei (homofermentative) from kefir grains. Ninane et al. (2005) reported greater variability in the population of lactic acid streptococci (443 per cent) than lactobacilli (28 per cent) and yeasts (35 per cent), isolated from kefir grains. Witthuhm et al. (2004) reported that LAB and yeasts present in kefir grains vary widely and counts ranged from 6.4 £ 104 – 8.5 £ 108 and 1.5 £ 105 – 3.7 £ 108 cfu/ml, respectively. Identified LAB and yeasts in kefir grains and kefir are shown in Tables I and II. Duitschaever et al. (1988) observed the presence of short and elongated bacilli as well as yeasts embedded amongst a densely packed fibrillar, amorphous matrix but no cocci in kefir grains. They further reported appearance of some long and curved bacteria in the surface section of the grain indicating disparity in the microflora at the edge and near the centre of kefir grains. Dominance of outer layer of kefir grain with rod shaped LAB, yeasts at the core, a balanced bacteria and yeast at the intermediate zone and a progressive change Constituents of bacterial flora Lactobacilli Lactobacillus kefir Lactobacillus kefiranofaciens Lactobacillus kefirgranum Lactobacillus parakefir Lactobacillus brevis Lactobacillus plantarum Lactobacillus paraplantarum Lactobacillus gasseri Lactobacillus helveticus Lactobacillus acidophilus Lactobacillus delbrueckii Lactobacillus rhamnosus Lactobacillus casei Lactobacillus paracasei Lactobacillus fructivorans Lactobacillus hilgardii Lactobacillus fermentum Lactobacillus viridescens Lactobacillus bulgaricus Lactococci Lactococcus lactis subsp. lactis Lactococcus lactis subsp. cremoris Streptococci Streptococcus thermophilus Enterococci Enterococcus durans Enterococcus faecium Leuconostocs Leuconostoc mesenteroides Leuconostoc mesenteroides subsp. cremoris Acetic acid bacteria Acetobacter aceti Acetobacter pasteurianus Other bacteria Escherichia coli Bacillus subtilis Micrococcus sp. References Takizawa et al. (1994) Takizawa et al. (1994) Takizawa et al. (1994) Takizawa et al. (1994) Santos et al. (2003) Santos et al. (2003) Anana et al. (2005) Anana et al. (2005) Simova et al. (2002) Santos et al. (2003) Santos et al. (2003) Santos et al. (2003) Simova et al. (2002) Santos et al. (2003) Yoshida and Toyoshima (1994) Yoshida and Toyoshima (1994) Angulo et al. (1993) Angulo et al. (1993) Wang et al. (2004) Innovations in kefir production 285 Yoshida and Toyoshima (1994) Koroleva (1991) Simova et al. (2002) Yu¨ksekdag et al. (2004) Wang et al. (2004) Koroleva (1991) Mainville et al. (2006) Koroleva (1991) Ottogalli et al. (1973) Angulo et al. (1993) Ottogalli et al. (1973) Angulo et al. (1993) according to the distance from the core has been reported (Ottogalli et al., 1973; Rea et al., 1996). Microflora of kefir may vary with the type of culturing media used as well as the method of kefir production employed. No disparity in microflora and polysaccharide composition of kefir grains were observed during culturing in milk or whey (Fil’ Chakova and Koroleva, 1997), however higher polysaccharide contents were recorded in cow milk than in soya milk (Abraham and Antoni, 1999). Microflora detected in kefir was lactobacilli, streptococci and yeasts, when made from these pure cultures or cocci and few yeast, when made by direct-set culture (Duitschaever et al., 1988). Recently, scanning electron microscopy and transmission electron microscopy of Table I. Bacterial flora of kefir grains and kefir BFJ 110,3 286 Table II. Yeast flora of kefir grains and kefir Constituents of yeast flora Klyveromyces species Klyveromyces matxianus Klyveromyces lactis Saccharomyces species Saccharomyces cerevesiae Saccharomyces unisporus Saccharomyces exiguss Saccharomyces turicensis Saccharomyces delbrueckii Torulaspora species Torulaspora delbrus Torulaspora delbrueckii Candida species Candida pseudotropicalis Candida tenuis Candida inconspicua Candida maris Candida lambica Candida tannotelerans Candida valida 6 Candida kefyr Candida holmii Other yeasts Pichia fermentans Zygosaccharomyces rouxii Debaryomyces hansenii Bretannomyces anomalus Issatchenkia occidentalis References Loretan et al. (2003) Loretan et al. (2003) Loretan et al. (2003) Loretan et al. (2003) Iwasawa et al. (1982) Wyder and Puhan (1997) Rosi (1978) Loretan et al. (2003) Loretan et al. (2003) Ottogalli et al. (1973) Ottogalli et al. (1973) Simova et al. (2002) Simova et al. (2002) Engel et al. (1986) Dousset and Caillet (1993) Dousset and Caillet (1993) Engel et al. (1986) Engel et al. (1986) Angulo et al. (1993) Loretan et al. (2003) Loretan et al. (2003) Wyder and Puhan (1997) Engel et al. (1986) kefir grains revealed most denser colonized portion at the exterior surface, consisting mainly of bacteria and yeasts, many of them autolysing but could not pass through the polysaccharide matrix (Zhang et al., 1998). Lin et al.(1999) also reported that LAB were localized mainly on the surface layer consisting of Klyveromyces marxianus and Leuconostoc mesenteroides whereas yeasts such as Pichia fermentas at the center of the grains. Type of milk Milk intended for fermented milk production should comply with the following requirements of low bacterial count, absence of pathogens or inhibitory substances such as antibiotic residues and sanitizer residues (Roginski, 1988). kefir is frequently made from whole, part skim or skim milk (Webb et al., 1987) employing milk from cow, ewe, goat, mare (Kneifel and Mayer, 1991), camel (Garg, 1989) or sheep (Wojtowski et al., 2003). Goat milk is not suitable for kefir manufacture due to lower viscosity and sensory properties in contrast to cow milk kefir and no significant improvement in the sensory scores due to softer consistency could be achieved with 2 per cent supplementation of goat milk kefir with milk powder, whey protein concentrate and inulin in comparison to un-supplemented kefir (Bozanic et al., 2003). Recently literature indicated that quality of kefir could be influenced with the acquiring period of milk. Jasinska et al. (2005) concluded that best quality kefir with good consistency, highest acidification and least perceptible goaty flavour could be obtained by utilizing goat milk collected at the end of October. Amongst milk from different breeds (cow, goat and sheep), sheep milk should be preferred for kefir production due to certain health benefits owing to its lowest contents of medium-chain saturated acids and highest linoleic and / -linolenic acid (Wojtowski et al., 2003). Recently, production of kefir from soy milk has also been reported (Halle et al., 1994; Kuo and Lin, 1999). Treatment of milk Concentration of skim milk to . 1.3 to 1.8 fold adopting ultrafiltration technique is suggested for obtaining denser kefir (Chagarovsky and Lipatov, 1990). Milk intended for cultured milk production must be heat-treated with the objective of pasteurizing the product, rendering milk more stimulatory growth medium for starter cultures due to elaboration of amino acids and other growth factors, reduction of the redox potential and elimination of inhibitory substances, improve physical properties of fermented milk and to reduce syneresis and prevent hydrolytic rancidity through inactivation of enzyme lipase (Kessler, 1981). Various temperature-time combinations have been suggested for heat-treatment of milk during kefir production. Suggested heat-treatments were 858C/30 min (Vedamuthu, 1977), 85-908C/15-20 min (Klyavinya, 1980), 90-938C/15 min (Penido et al., 2001), 928C/20 min (Simova et al., 2006), 958C/15 min (Ozer and Ozer, 1999), 90-958C/2-3 min (Koroleva, 1988b) and 958C/10-15 min (Dies, 2000). Starter inoculation and incubation Kefir can be obtained either by inoculating milk with kefir grains or by inoculating milk with bulk starter obtained by culturing with kefir grains. Beshkova et al. (2002) produced kefir involving a starter comprising of two bacteria (Lactobacillus helveticus and Lactobacillus lactis subsp. lactis), single yeast culture isolated from kefir grains (Saccharomyces cerevesiae) along with two yoghurt strains (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus). In USA, kefir is produced employing only starter cultures consisting of Streptococcus lactis, Lactobacillus plantarum, Streptococcus cremoris, Lactobacillus casei, Streptococcus diacetylactis, Leuconostoc cremoris and Saccharomyces florentinus (Hertzler and Clancy, 2003). Factors influencing extent of acidification during kefir production are size of inoculation of kefir grains, agitation and temperature of incubation (Irigoyen et al., 2003). Extent of acidification and carbondioxide production was influenced by the concentration of kefir grains, higher being noted at a level of 100 g than 10 g/l milk (Garrote et al., 1998). kefir production with 5 per cent kefir grains proved to be optimum for ethanol and volatile acid production (Korovkena et al., 1978). Rate of inoculation also influence the kefir flora. kefir made with 1 per cent inoculation had LAB as the dominant flora but those made with 5 per cent inoculation were predominated with yeasts and acetic acid bacteria (Irigoyen et al., 2005). The fermentation temperature affects distinctly both the total capacity of acidification and the acid production speed (Irigoyen et al., 2003). Chagarovsky and Zholkevskaya (2003) reported that acid production occurred within 6 h at 408C and 308C for thermophilic and mesophilic bacteria, respectively. Mesophilic microflora considerably increased during the first 24 h of fermentation and then remained stable Innovations in kefir production 287 BFJ 110,3 288 with a slight increase at the end of kefir production (Penido et al., 2001). During the same period of fermentation, the viable population of bacteria (lactobacilli and lactococci), yeasts and acetic acid bacteria attain a level of 108, 105 and 106cfu/ml, respectively (Irigoyen et al., 2005). Garcia-Fortan et al. (2006) noted predominance of Lactococcus sp. during the first 48 h of fermentation (< 8.0 log cfu/g), followed by predominance of Lactobacillus sp. (< 8.5 log cfu/g). Liu et al. (2002) recorded that immediately after inoculation of soya milk with kefir grains, counts of LAB was higher than yeasts and their activity enhanced with fortification of soya milk with 1 per cent glucose. During fermentation various flavouring compounds are produced by starter cultures. Acetaldehyde is produced by L. delbrueckii subsp. bulgaricus, diacetyl by S. thermophilus, L. helveticus and L. lactis subsp. lactis, acetone by L. delbrueckii subsp. bulgaricus and L. helveticus, ethanol by S. cerevesiae (Beshkova et al., 2002). Garg (1989) mentioned other flavouring compounds traced in kefir to be propionaldehyde, 2-butanone, isoamyl alcohol and diacetyl. Acceptability of kefir produced by pure cultures could be enhanced either by sweetening (Duitschaever et al., 1987, 1991), addition of peach flavour or modification of fermentation process with the addition of lactococci, lactobacilli or yeasts (Duitschaever et al., 1991; Muir et al., 1999). Recommended incubation temperature-time combinations are 208C/20 h 11 h (Chen et al., 2006), 208C/48 h (Abraham and Antoni, 1999), 20-238C/12-14 h (Pijanowski, 1980), 228C/11 h (Li et al., 2004), 22-238C/20 h (Bottazzi, 1985), 24-278C/20 h (Klupsch, 1985) and 22-258C/8-12 h (Koroleva, 1988b) for kefir production. Recently, Simova et al. (2006) recommended two-stage fermentation process (288C/5 h and 208C/16 h) for kefir production. Packaging Kessler (1981) mentioned that the containers to be used for packaging of fermented milk should be impermeable to water and odours, insoluble in water and free from foreign odours. Kwak et al. (1996) mentioned that growth of yeasts continue after packaging, therefore the containers intended for kefir packing must be either strong enough to withstand the buildup pressure such as glass or flexible enough to retain the amount of gas produced such as plastic with an aluminium foil top. Special containers designed for kefir had lids consisted of three layers that allows the escape of carbon dioxide generated by viable yeasts, which prevents swelling and bulging of kefir cups (Fluckiger, 1986). Storage Ozer and Ozer (1999) suggested that post-fermentation cooling of kefir to 4-68C should be done slowly within 10-12 h to ensure retention of its pronounced aroma and typical taste. A slight abatement in contents of orotic and citric acid but a significant enhancement in lactic acid to 7,739ppm, ethanol to 0.08 per cent, acetaldehyde to 11mg/g, decline in acetoin to 16ppm and presence of non-detectable levels of diacetyl during storage of kefir at 48C/21 days were encountered (Guzel-Seydim et al., 2000). Irigoyen et al. (2005) reported a decline in lactic flora (1.5 log units) but a stability for acetic acid bacteria and yeasts during storage of kefir at 5 ^ 18C/28 days. Storage of kefir for two days prior to its consumption is recommended for better sensory qualities and highest viable population of Lactobacillus gasseri (Anana et al., 2005). Post-production treatments Shelf-life of kefir is dependent on the type of packaging material and varies from eight to ten days at 3-48C (Koroleva, 1988b). Post-production treatments such as autoclaving, irradiation, ohmic heating and high pressure treatment can be done on kefir (Mainville et al., 2001) with the objective of enhancing the shelf-life. High pressure treatment of kefir at 400 MPa/30 minutes induced deactivation of bacteria and yeasts (Mainville et al., 2001) and loss of ability of LAB to inhibit test organisms (Jankowska et al., 2001) but no change in protein and lipid structure (Mainville et al., 2001). Since loss of viability and antibacterial activity of starter cultures will reduce the therapeutic value of kefir, application of high-pressure treatment for kefir intended for therapeutic application is not recommended. Freeze drying of kefir also induced a loss in viability of microorganisms but the survival can be improved with the addition of 10% galactose or 10% sucrose prior to freeze drying (Chen et al., 2006). Biotechnological innovations in kefir production Traditional kefir made from goat milk had low viscosity and sensory properties in contrast to cow milk kefir and contained 0.04-0.3 ml/100 ml ethanol (Seiler, 2003). Tratnik et al. (2006) recommended supplementation of goat milk with whey protein concentrate at a level of 60.0-60.5 g/100 g proteins for enhancement in ethanol production (0.35 ml/100 ml). To comply with the consumers demand for more healthy foods, soya milk could be a suitable substitute for kefir production owing to its low saturated fat and cholesterol (Berry, 2000), higher polyunsaturated fatty acids, lecithin, linolenic acid, magnesium, iron, folic acid and vitamin E (Hermann, 1991). Koca et al. (2002) reported that though soya milk is a satisfactory medium for the growth and activity of starter cultures, a slower rate of acidification is noted in soya milk in contrast to those in cow milk Fortification of soya milk with glucose (Liu and Lin, 2000) and low-fat milk with tryptose (Schoevers and Britz, 2003) are suggested for growth stimulation LAB and yeasts. Fermentation of milk by traditional methods employing kefir grains resulted in disparity in product quality due to diverse microflora and uncontrolled fermentation. Two methods have been suggested to overcome the drawbacks of traditional methods of kefir production kefir can be produced either by simultaneous (Tamai et al., 1996) or consecutive lactic acid and yeast fermentation (Beshkova et al., 2002) Production of kefir employing pure cultures induced production of higher concentration of carbonyl compounds (Beshkova et al., 2003) and carbondioxide (1.7 vs 0.85 to 1.05 g/L) in contrast to those obtained adopting kefir grains (Gobbetti et al., 1990, Beshkova et al., 2002; Simova et al., 2002). Klupsch (1985) recommended primarily fermentation of milk with a 2 per cent starter comprising of streptococcus and lactococci, followed by a secondary fermentation with 0.05-0.5 per cent starter containing Candida kefir and Lactobacillus brevis. Best quality kefir could be produced by incubating homogenized milk with 3 per cent (V/V) each of bacteria and yeast at 258C for 24 h (Assadi et al., 2000). Probiotic kefir capable of exhibiting antimicrobial properties could be obtained employing L. acidophilus, Lactobacillus kefiranofuctens and Lactobacillus kefiranofaciens (Santos et al., 2003) or Bifidobacterium bifidum (Murashova et al., 1997). Introduction of Candida kefyr during kefir production may be advantageous as it did not disappeared completely at pH 2.0, retains 97.2 per cent viability in presence of 1 Innovations in kefir production 289 BFJ 110,3 290 per cent bile salts and not inhibited by most antibiotics including tetracycline (You et al., 2006). During kefir production introduction of 3 per cent L. delbrueckii subsp. bulgaricus HP 1 with S. thermophilus T 15 (1:1), 1 per cent L. lactis subsp. lactis C 15, 3 per cent L. helveticus MP 12 (Simova et al., 2006) have been recommended and further inclusion of Sacch. cerevesiae AB shortened the time to reach the maximum exopolysaccharide production (824.3 mg/L) by 6 h. In order to meet the consumer’s demand for healthful foods in the current era of self-care and complementary medicine kefir with enhanced dietetic properties could be obtained by co-inoculation of soya milk with yeasts (Sacch. cerevesiae, Candida kefir), lactic acid bacteria (Streptococcus thermophilus, Lactobacillus bulgaricus) and probiotic cultures (Lactobacillus acidophilus, Bifidobacterium bifidum). Preservation of kefir grains During fermentation, kefir grains comes at the surface due to production of carbon dioxide and are strained, washed, dried and stored for longer periods between their application. Liu et al. (1999) recommended storage of lyphilized kefir grains at 2 208C and viability of LAB retained stable and resulted in kefir with viable population similar to those obtained from non-stored grains. kefir grains could be preserved by frozen storage without milk at 2 188C, refrigerated storage at 48C, air drying at room temperature for three weeks in a dessicator or freeze drying but air drying is not recommended for commercial application due to unacceptable colour and flavour of grain (Witthuhm et al., 2005a). Among various packaging materials low-density polyethylene film (LPDE), oriented polyster film (OPET) and methallized oriented polyster film (MOPET), adoption of MOPET is recommended for retaining the activity of kefir grains for extended storage period (Witthuhm et al., 2005b). Selection of proper packaging material and preservation technique is of utmost imporancy for longer period of storage of kefir grains. Conclusion Traditional method of kefir production involved unpredictable and slow souring of milk using kefir grains, resulting in disparity in quality of kefir produced. Biotechnological innovations must be considered to upgrade the dietetic characteristics of kefir There is exigency to screen out the method for enhancing the shelf life of kefir to extend its market reach. Every effort should be exercised to popularize kefir as a dietetic beverage. References Abraham, A.G. and Antoni, G.L.D. (1999), “Characterization of kefir grains in cow’s milk and in soya milk”, J. Dairy Res., Vol. 66, pp. 327-33. Anana, I., Ortigosa, M., Irigoyen, A., Ibanez, F.C. and Torre, P. (2005), “Isolation and identification of potentially probiotic lactobacillus strains from kefir”, Milchwiss., Vol. 60, pp. 292-4. Angulo, L., Lopez, E. and Lema, C. (1993), “Microflora present in kefir grains of the Galician region (North-West of Spain)”, J. Dairy Res., Vol. 60, pp. 263-7. 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