STARTER CULTURES AND FERMENTED MILK PRODUCTS

Module 16. Kefir

Lesson 30
KEFIR-MANUFACTURE, COMPOSITION, NUTRITIONAL AND THERAPEUTIC PROPERTIES

30.1 Introduction

Kefir is a viscous, slightly carbonated dairy beverage that contains small quantities of alcohol and, like yoghurt, is believed to have its origins in the Caucasian mountains of the former USSR. It is also manufactured under a variety of names including kephir, kiaphur, kefer, knapon, kepi and kippi with artisanal production of kefir occurring in countries as widespread as Argentina, Taiwan, Portugal, Turkey and France. It is not clear whether all kefirs originate from a single original starter culture, since microbial analyses of kefir samples taken from different locations indicate microflora population differences.

30.2 Definition

Although no clear definition of kefir exists, it is a viscous, acidic, and mildly alcoholic milk beverage produced by fermentation of milk with a kefir grain as the starter culture (FAO/WHO 2003). The Codex Alimentarius description of kefir state it as Starter culture prepared from kefir grains, Lactobacillus kefir, and species of the genera Leuconostoc, Lactococcus and Acetobacter growing in a strong specific relationship. Kefir grains constitute both lactose-fermenting yeasts (Kluyveromyces marxianus) and non-lactose-fermenting yeasts (Saccharomyces unisporus, Saccharomyces cerevisiae and Saccharomyces exiguus).

30.3 Composition

The composition of kefir will be essentially dependant on the type of milk that was used. The major change caused by fermentation measured in term of acid production and alcohol production may also reflect in the composition.Table-30.1 shows the composition standards prescribed by the Codex

Table 30.1 Codex standard for kefir

Milk protein (% w/w)	min. 2.8
Milk fat (% m/m)	<10
Titratable acidity, expressed as % of lactic acid	min. 0.6
Ethanol (% vol. /w)	not stated
Sum of specific microorganisms constituting the starter culture (cfu/g, in total)	107 (minimum)
Yeasts (cfu /g)	104 (minimum)

(From Codex Standard for Fermented Milks CODEX STAN 243 – 2003)

30.4 Kefir Manufacture

Although commercial kefir is traditionally manufactured from cow’s milk, it has also been made from the milk of ewes, goats and buffalos. Moreover, kefir produced using soy milk has also been recently reported. The various steps of kefir manufacture are depicted in Figure 30.1.

Traditionally, kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, mesophilic, homofermentative and heterofermentative lactic acid streptocci, thermophilic and mesophilic lactobacilli, acetic acid bacteria, and yeast) to a quantity of milk. The size of the initial kefir grain inoculum affects the pH, viscosity and microbiological profile of the final product. A grain to milk ratios of 1:30 to 1:50 were found optimum. In some manufacturing procedures, a percolate of the grains from a coarse sieve is used as the mother culture to inoculate fresh milk. Fermentation of the milk by the inoculum proceeds for approximately 24 hours, during which time homofermentative lactic acid streptococci grow rapidly, initially causing a drop in pH. This low pH favours the growth of lactobacilli, but causes the streptococci numbers to decline. The presence of yeasts in the mixture, together with fermentation temperature (21-23°C), encourages the growth of aroma producing heterofermentative streptococci. As fermentation proceeds, growth of lactic acid bacteria is favoured over growth of yeasts and acetic acid bacteria.

Fig. 30.1 Method of manufacture of kefir

Kefir grains are key to kefir production, and it has been found that the finished product has a different microbiological profile from the grains and therefore cannot be used to inoculate a new batch of milk. Grains have been shown to possess a dynamic and complex flora which is not conducive to commercial production of a uniform, stable product; this has prompted researchers to try to produce kefir from a mixture of pure cultures. Some researchers produced a starter consisting of two bacteria (Lactobacillus helveticus and Lactococcus lactis subsp lactis) and one yeast (S. cerevisiae) isolated from kefir grain and combined with two yoghurt strains (Lactobacillus delbrueckii subsp bulgaricus, and Streptococcus thermophilus). Yeast was added to the starter with sucrose either at the beginning, or after lactic acid fermentation. The two resulting kefirs produced were found to have high numbers of viable cocci and lactobacilli and had chemical and organoleptic properties that were similar to traditional kefir. A commercial kefir is being produced in the United States using a mixture of defined microorganisms rather than kefir grains. This starter culture mixture has been reported to contain Streptococcus lactis, Lb. plantarum, Streptococcus cremoris, Lb. casei, Lactococcus lactis subsp Lactis biovar diacetylactis, Leuconostoc cremoris and Saccharomyces florentinus.

30.5 Characteristics of Kefir

The flavour, viscosity and microbial/chemical composition of the final kefir product can be affected by the size of the inoculum added to the milk, the occurrence of any agitation during fermentation, and the rate, temperature and duration of the cooling and ripening stages following fermentation. Natural kefir has a refreshing, yeasty taste and a ‘sparkling’ mouth feel. Modern manufacturing procedures for kefir result in ethanol levels in the finished product of 0.01– 0.1% although kefir with ethanol concentrations as high as 0.25% have been produced from grains in the laboratory. The amounts of ethanol and CO2 produced during fermentation of kefir depend on the production conditions used. CO2 content of kefir has been said to be ‘comparatively low’ in relation to other fermented drinks. The distinctive taste of kefir results from the presence of several flavour compounds which are produced during fermentation. Kefir produced from pure cultures did not receive high sensory evaluation scores. Acetaldehyde and acetoin have received particular attention with regard to their roles during kefir manufacture because of their contribution in the taste; both have been found to increase in concentration during kefir fermentation. During storage, acetaldehyde increases in concentration and acetoin decreases.

30.6 Kefir Grains

Kefir grains (Figure-30.2) resemble small cauliflower florets: they measure 1-3 cm in length, are lobed, irregularly shaped, white to yellow-white in colour, and have a slimy but firm texture. Grains are kept viable by transferring them daily into fresh milk and allowing them to grow for approximately 20 hours; during this time, the grains will have increased their mass by 25%. Grains must be replicated in this way to retain their viability, since old and dried kefir grains have little or no ability to replicate. In addition, washing grains in water also reduced viability. It has been recommended that in a commercial operation using grains to produce kefir, grains should be kept viable through daily transfers and should only be replaced if their ability to ferment milk becomes impaired. Low temperature storage appears to be the best way to maintain kefir grains for long periods. Storage of kefir grains at 80° or 20°C for 120 days did not change their fermentation properties compared to grains that had not been stored; however, grains stored at 4°C did not produce acceptable kefir after thawing.

Fig. 30.2 Kefir grains

30.6.1 Microbiology of kefir grains

30.6.1.1 Bacteria found in kefir grains and kefir

The microbial population (Figure 30.3) found in kefir grains have been used as an example of a symbiotic community. This symbiotic nature has made identification and study of the constituent microorganisms within kefir grains difficult.

Table 30.2 Bacteria found in kefir grains and kefir

Lactobacilli Lactobacillus kefir	Lactobacillus delbrueckii
Lactobacillus kefiranofaciens	Lactobacillus rhamnosus
Lactobacillus kefirgranum	Lactobacillus casei
Lactobacillus parakefir	Lactobacillus paracasei
Lactobacillus brevis	Lactobacillus fructivorans
Lactobacillus plantarum	Lactobacillus hilgardii
Lactobacillus helveticus	Lactobacillus fermentum
Lactobacillus acidophilus	Lactobacillus viridescens
Lactococci Lactococcus lactis subsp lactis Lactococcus lactis subsp cremoris	Enterococci Enterococcus durans
Streptococci Streptococcus thermophilus	Leuconostocs Leuconostoc mesenteroides
Acetic acid bacteria Acetobacter pasteurianus Acetobacter aceti	Other Bacteria Bacillus spp, Micrococcus spp. Bacillus subtilis, Escherichia coli

30.6.1.2 Yeasts found in kefir grains and kefir

The yeasts in kefir (Table-30.3) have been less well studied than kefir bacteria, although it is obvious that the yeasts in kefir grains provide an environment for the growth of kefir bacteria, producing metabolites that contribute to the flavour and mouthfeel of kefir. To prevent excessive CO2 production (particularly after fermentation), a two stage fermentation process starting with a non-lactose fermenting yeast such as Saccharomyces cerevisiae can be done. The properties of yeasts found in kefir grains vary. For example, some of the yeast found in kefir grains are capable of fermenting lactose, while some are not. Also, it has been observed that some type of yeasts are located at the surface of the grain, while others inhabit the interior. It may be that yeasts located at different locations in the kefir grains play different roles in the fermentation process. Like kefir bacteria, the profile of yeasts is different in kefir grains when compared to the final kefir product.

Fig. 30.3 Electron Micrograph of a kefir grain
Table 30.3 Yeasts found in kefir grains and kefir

30.7 Nutritional Significance of Kefir

The composition of kefir depends greatly on the type of milk that was fermented. However, during the fermentation, changes in composition of nutrients and other ingredients have also been shown to occur. L(+) lactic acid is the organic acid in highest concentrations after fermentation and is derived from approximately 25% of the original lactose in the starter milk. The amino acids valine, leucine, lysine and serine are formed during fermentation, while the quantities of alanine and aspartic acid increase as compared to raw milk. Appreciable amounts of pyridoxine, vitamin B12, folic acid and biotin were synthesized during kefir production, depending on the source of kefir grains used, while thiamine and riboflavin levels were reduced. Some workers reported decreases in biotin, vitamin B12 and pyridoxine, and significant increases in folic acid, as compared to non-fermented milk.

30.8 Bio Active Ingredients in kefir

Kefir has a long tradition of offering heath benefits. There are several compounds in kefir that may have bioactive properties.

(i) Exopolysaccharides

Exopolysaccharides of differing structures and compositions are produced by a variety of lactic acid bacteria including Lactobacillus, Streptococcus, Lactococcus and Leuconostoc. These cell-surface carbohydrates confer protective and adaptive properties on their bacterial producers; since they are often loosely bound to the cell membrane, they are therefore, easily lost to their environment. In food products, exopolysaccharides often contribute to organoleptic and stability characteristics. A unique polysaccharide called kefiran has been found in kefir grains. Grains may also contain other exopolysaccharides. Kefiran contains D-glucose and D-galactose only in a ratio of 1:1. Kefiran dissolves slowly in cold water and quickly in hot water, and forms a viscous solution at 2% concentration.

(ii) Bioactive peptides

Many organisms possess enzymes (e.g. proteinases and peptidases) that are able to hydrolyse the protein in a medium, thereby supporting growth of the organism by liberating peptides and amino acids. The action of proteinase and peptidase enzymes on milk proteins can theoretically result in a very large number of possible peptides. An analysis of the proteinase activity of kefir grain bacterial isolates has shown that several isolates have high proteinase activities which increases the possibility that bioactive peptides may be present in kefir. Studies on the peptide content of kefir drink have shown that kefir contains a large number of peptides.

30.9 Therapeutic Significance of kefir

Kefir has had a long history of being beneficial to health in Eastern European countries, where it is associated with general wellbeing. It is easily digested and is often the first weaning food received by babies.

• It has been proposed that stimulation of the immune system may be one mechanism whereby probiotic bacteria may exert many of their beneficial effects. Peptides formed during the fermentation process or during digestion have also been shown to be bioactive, and demonstrate a variety of physiological activities, including stimulation of the immune system in animal models. Stimulation of the immune system may also occur due to the action of exopolysaccharides found in kefir grains.

• Anti tumour effects of a water-soluble polysaccharide (approximate molecular weight 10,000,00 Da) isolated from kefir grains is reported.
• A water soluble polysaccharide fraction from kefir grains was shown to inhibit pulmonary metastasis of Lewis lung carcinoma, whether the kefir-derived polysaccharide was given orally before or after tumour transplantation.

• Some kefir grains have been shown to possess b-galactosidase activity which remains active when consumed and thus can be beneficial for lactose intolerant people.

• Many lactobacilli are capable of producing a wide range of antimicrobial compounds, including organic acids (lactic and acetic acids), carbon dioxide, hydrogen peroxide, ethanol, diacetyl and peptides (bacteriocins) that may be beneficial not only in the reduction of food borne pathogens and spoilage bacteria during food production and storage, but also in the treatment and prevention of gastrointestinal disorders and vaginal infections. Fresh kefir grains were found to inhibit the growth of the pathogens Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Leuconostoc mesenteroides and Lactobacillus plantarum, isolated from kefir grains, have been shown to produce antimicrobial compounds which can inhibit Gram-positive and Gram-negative bacteria. These antimicrobial compounds are found to be heat stable. But their antimicrobial properties are reduced after exposure to proteolytic enzymes. Lactobacilli isolated from kefir grains had antimicrobial activities against E. coli, Listeria monocytogenes, Salmonella Typhimurium, S. Enteritidis, S. flexneri and Yersinia enterocolitica. Bacteriocins were thought to be responsible for the antimicrobial activities .

Kefir is a microbiologically complex product with a large number of different bacteria and yeast involved in its making . Many of these microorganisms are only now being identified by using advanced molecular biological techniques. The study of kefir is made more difficult, because it appears that many different sources of kefir grains exist that are being used to produce kefir. The production of kefir depends on the synergistic interaction of the microflora in kefir grains. During the fermentation process, the yeasts and bacteria in kefir grains produce a variety of ingredients that give kefir its unique taste and texture. After fermentation, the finished kefir product contains many ingredients that are proving to be bioactive and may be used as functional ingredients.