Interesting kefir research

 http://www.fibromyalgiarecovery.com/Kefir%20-%20A%20Complex%20Probiotic.pdf

Food Science & Technology Bulletin: Functional Foods 13 May 2005

http://www.ifis.org/fsc/bulletin-ff-free

© IFIS Publishing 2006 - All Rights Reserved

Kefir -- a complex probiotic

Edward R. Farnworth

Food Research and Development Centre, Agriculture and Agri-food Canada, St. Hyacinthe, Quebec,

Canada J2S 8E3. Tel. 450-773-1105. Fax 450-8461. E-mail farnworthed@agr.gc.ca

Abstract

Kefir is a fermented milk drink produced by the actions of bacteria and yeasts contained in kefir grains,

and is reported to have a unique taste and unique properties. During fermentation, peptides and

exopolysaccharides are formed that have been shown to have bioactive properties. Moreover, in vitro and

animal trials have shown kefir and its constituents to have anticarcinogenic, antimutagenic, antiviral and

antifungal properties. Although kefir has been produced and consumed in Eastern Europe for a long period

of time, few clinical trials are found in the scientific literature to support the health claims attributed to

kefir. The large number of microorganisms in kefir, the variety of possible bioactive compounds that could

be formed during fermentation, and the long list of reputed benefits of eating kefir make this fermented

dairy product a complex probiotic.

Keywords: kefir, probiotics, kefir grains, kefiran, human health, bioactive ingredients

1. Introduction

Archaeological evidence has indicated that the process of fermentation in foods was discovered

accidentally thousands of years ago. However, over time, it soon became apparent that many fermented

foods had longer storage lives and improved nutritional values compared to their unfermented

equivalents, making this form of food processing a popular technique. It is not surprising, therefore, to

find that many foods including vegetables, fruits, cereals, meat and fish have all been converted into

desirable food products by fermentation and are still being consumed throughout the world today

(Farnworth 2004).

Certain bacteria, either alone or through the changes they bring about during fermentation, have been

shown to have positive effects on health as well as resistance to disease. Interest in such probiotic species

has increased in recent years as more is learned about the microorganisms used in the fermentation

process, and the possibility of adding beneficial bacteria to food products. Furthermore, consumers are

increasingly looking to improve their health and increase their resistance to disease through dietary

means.

Fermented dairy products from milk from a variety of animals are perhaps the most common fermented

foods worldwide. Yoghurt, which is known by many different names in different countries, is a fermented

product which is familiar to consumers. Kefir, meanwhile, is less well known than yoghurt; however, an

analysis of its composition indicates that it may contain bioactive ingredients that give it unique health

benefits, which means that kefir may be an important probiotic product (Farnworth 1999).

2. Origins of kefir

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 (Koroleva

1988a), with artisanal production of kefir occuring in countries as widespread as Argentina, Taiwan,

Portugal, Turkey and France (Thompson et al. 1990; Angulo et al. 1993; Lin et al. 1999; Garrote et al.

2001; Santos et al. 2003; Gulmez and Guven 2003). 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.

The FAO/WHO (2001) have proposed a definition of kefir based on the microbial composition of both kefir

grains (the starter culture used to produce kefir) and the final kefir product (see Table 1).

3. Kefir manufacture

Although commercial kefir is traditionally manufactured from cows` 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

(Ismail et al. 1983; Mann 1985; Zourari and Anifantakis 1988; Hallé et al. 1994; Kuo and Lin 1999).

Traditionally, kefir is produced by adding kefir grains (a mass of proteins, polysaccharides, mesophilic,

homofermentative and heterofermentative lactic acid streptococci, thermophilic and mesophilic lactobacilli,

acetic acid bacteria, and yeast) to a quantity of milk (Koroleva 1982; Hallé et al. 1994; Tamime et al.

1999). The size of the initial kefir grain inoculum affects the pH, viscosity and microbiological profile of the

final product (Koroleva and Bavina 1970; Garrote et al. 1998). Koroleva (1991) reported that grain to milk

ratios of 1:30 to 1:50 were optimum. In some manufacturing procedures, a perculate 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 (Koroleva 1982).

Taiwanese researchers have shown that the lactic acid bacteria from kefir grains grow more slowly in soy

milk compared to cows` milk (Liu and Lin 2000). This may be due, in part, to the slower production of

growth factors at the beginning of fermentation when soy milk is the substrate rather than cows’ milk.

Addition of carbohydrate (e.g. 1% glucose) to soy milk increases yeast numbers, lactic acid production

and ethanol production, compared to kefir produced from soy milk alone (Liu and Lin 2000). The grains

used in this study were found to have α-galactosidase activity that helped explain how these kefir

grains were able to use the galactose-based carbohydrates which occur in soy milk.

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

(Simova et al. 2002). 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 groups to try to

produce kefir from a mixture of pure cultures (Petersson et al. 1985). Duitschaever et al. (1987, 1988a)

combined a yoghurt culture with three other lactic acid bacteria and Saccharomyces cerevisiae (a nonlactose

fermenting yeast) to produce a fermented milk with kefir characteristics (which produced CO2 and

contained ethanol) under a variety of conditions. Rossi and Gobbetti (1991) produced a multistarter

culture using four bacteria and two yeasts isolated from kefir grains in order to manufacture kefir under a

continuous process. More recently, Beshkova et al. (2002) produced a starter consisting of two bacteria

(Lactobacillus helveticus and Lactococcus lactis subsp. lactis,) and one yeast (S. cerevisiae) isolated from

kefir grains 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, L.

plantarum, Streptococcus cremoris, L. casei, Streptococcus diacetylactis, Leuconostoc cremoris and

Saccharomyces florentinus (Hertzler and Clancy 2003).

Starter cultures containing freeze-dried lactic acid bacteria and yeasts from kefir grains are now available

commercially; some are supplemented with additional microorganisms to impart desirable characteristics

in the finished kefir product (Piotr Kolakowski, private communication). It is evident that the final product,

as produced from kefir grains, will have a larger number and variety of microorganisms than kefir

produced from a mixture of a small number of pure cultures.

Kefir is still most familiar to consumers in Eastern Europe, although commercial production now occurs in

North America. However, several patents can be found relating to commercial kefir production worldwide

(Klupsch 1984; Dmitrovskaya 1986; Tokumaru et al. 1987; Kabore 1992). Production/consumption figures

for kefir are not readily available since statistics for fermented dairy products are not always broken down

into separate items such as yoghurt and kefir (Mann 1989; Libudzisz and Piatkiewicz 1990; Serova 1997;

Zimovetz and Boyko 2000). A survey of kefir products purchased on the retail market in Warsaw, Poland

showed that 73% of products contained 107–109 cfu bacteria/g, and that 97% of samples were coliformfree

(Molska et al. 2003). However, 48% of samples did not meet FAO/WHO requirements for yeast

numbers (FAO/WHO 2001).

4. 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 (Koroleva 1988b).

Natural kefir has a refreshing, yeasty taste and a ‘sparkling’ mouth feel (Kemp 1984).

Modern manufacturing procedures for kefir result in ethanol levels in the finished product of 0.01–0.1%

(Koroleva 1982), although kefir with ethanol concentrations as high as 0.25% have been produced from

grains in the laboratory (Kuo and Lin 1999; Simova et al. 2002; Beshkova et al. 2002). 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 (Koroleva

1982); values of 0.85–1.05 g/l have been reported for kefir produced from kefir grains (Beshkova et al.

2002; Simova et al. 2002) and 1.7 g/l for kefir produced from purified cultures (Gobbetti et al. 1990).

However, the generation of CO2 during kefir manufacture, especially after packaging, presents some

practical problems, since the microorganisms (particularly yeasts) in the kefir continue to grow following

packaging. The container used to package kefir must therefore be either strong enough to withstand any

pressure build up (e.g. glass) or flexible enough to contain the volume of gas produced (e.g. plastic with

an aluminium foil top (Kwak et al. 1996).

The distinctive taste of kefir results from the presence of several flavour compounds which are produced

during fermentation (Beshkova et al. 2003). Kefir produced from pure cultures did not receive high

sensory evaluation scores in Canada unless it was sweetened (Duitschaever et al. 1987, 1991);

Duitschaever et al. (1987) also showed that only about 40% of people tasting natural kefir for the first

time gave it a positive taste rating. Addition of peach flavour, or modification of the fermentation process

(e.g. addition of lactococci, lactobacilli or yeasts) increased the acceptability of kefir, compared to

traditionally made kefir (Duitschaever et al. 1991; Muir et al. 1999).

Acetaldehyde and acetoin have received particular attention with regard to their roles during kefir

manufacture because of their contribution to taste; both have been found to increase in concentration

during kefir fermentation. During storage, acetaldehyde increases in concentration and acetoin decreases

(Güzel-Seydim et al. 2000a, 2000b). Yüksekgağ et al. (2004a), in their study of 21 isolates of lactic

acid bacteria from various sources of Turkish kefir, were able to show that all 21 isolates produced

acetaldehyde (0.88–4.40 μg/ml) when added to milk.

A whey beverage with an acceptable flavour has recently been developed using kefir yeasts (Athanasiadis

et al. 2004), especially when fructose was added to fresh milk before fermentation, and final pH of the

beverage was 4.1. Fructose was found to increase production of several flavour volatiles, but did not

increase fermentation time.

5. Kefir grains

Kefir grains 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 (La Rivière et al. 1967;

Kosikowski and Mistry 1997; see Figure 1). 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% (Hallé et al. 1994). Grains must be replicated in this way to retain their

viability, since old and dried kefir grains have little or no ability to replicate (La Rivière et al. 1967). Kefir

grains replicated in milk ‘at home with daily changes of milk’ and stored for three months either at room

temperature or at 4°C had microbiological profiles that were different to those of fresh grains (Pintado et

al. 1996). 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. (Koroleva 1982). Low

temperature storage appears to be the best way to maintain kefir grains for long periods. Garrote et al.

(1997) showed that 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. Kefir grains replicated in soy milk have been reported to be smaller in size

compared to grains replicated in cows` milk (Liu et al. 2002). There have been no reports of successful

production of kefir grains from pure cultures.

While early studies of kefir grains employed light microscopy, later investigations used electron

microscopy to describe the complex microbial community of which they were comprised (Ottogalli et al.

1973; Bottazzi and Bianchi 1980; Molska et al. 1980; Marshall et al. 1984; Duitschaever et al. 1988b;

Toba et al. 1990; Neve 1992; Bottazzi et al. 1994; Rea et al. 1996). Figure 2 shows an electron

micrograph of kefir grains obtained from the Moscow Dairy Institute. Ottogalli et al. (1973) showed that

the chemical and microbiological compositions of kefir grains from four different sources were different,

making comparisons between results published by different laboratories difficult.

The microbial population that makes up kefir grains appears to be relatively constant over time, although

seasonal variations in the grain flora have been noted which can affect the final product consistency (La

Rivière et al. 1967; Koroleva et al. 1978). Analysis has shown that the microbial profiles of the grains

themselves, a percolate taken from the grains (mother culture), and the final product are not the same

(see Table 2). This, in part, explains why production of kefir must start with kefir grains, since the final

drink does not have the number or complexity of microorganisms as the grains, preventing the drink from

being used as a starter culture for a new batch of kefir. Kandler and Kunath (1983) reported similar

results when they compared the microflora of kefir, inoculated milk before incubation, and a mixture of

kefir grains.

6. Microbiology of kefir grains

6.1 Bacteria

The microbial population found in kefir grains has been used as an example of a symbiotic community

(Margulis 1995); this symbiotic nature has made identification and study of the constituent

microorganisms within kefir grains difficult. Koroleva (1991) stated that kefir bacteria and yeasts, when

separated as pure cultures, either do not grow in milk or have a decreased biochemical activity, which

further complicates the study of the microbial population of kefir grains. Several media have been

proposed for the isolation and identification of bacteria in kefir grains (Kojima et al. 1993). Linossier and

Dousset (1994) showed that Lactobacillus kefir grew better when the yeast Candida kefir was added to the

milk. Garrote et al. (2004) reported a similar observation when they attempted to grow L. kefir in milk. In

general, lactic acid bacteria are more numerous (108–109) than yeasts (105-106) and acetic acid bacteria

(105–106) in kefir grains, although fermentation conditions can affect this pattern (Koroleva 1991;

Garrote et al. 2001). Table 3 shows a list of the various bacteria that have been reported in kefir and kefir

grains from around the world.

Garrote et al. (2004) carried out several in vitro tests to try to explain how the bacteria in kefir grains

function. They showed that two of the heterofermentative lactobacilli, L. kefir and L. parakefir, possessed

S-layer proteins that can be used to explain in part their auto-aggregation and haemagglutination

properties. In addition, these two bacteria were also shown to adhere to Caco-2 cells, raising the

possibility that these bacteria would be good probiotics.

6.2 Yeasts

It has been recognized that yeasts play an important role in the preparation of fermented dairy products,

where they can provide essential growth nutrients such as amino acids and vitamins, alter the pH, secrete

ethanol and produce CO2 (Viljoen 2001). The yeasts in kefir 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 (Clementi et al.

1989; Kwak et al. 1996; Simova et al. 2002). Table 4 lists the various yeasts that have been reported in

kefir grains. To prevent excessive CO2 production (particularly after fermentation), Kwak et al. (1996)

suggested a two stage fermentation process starting with a non-lactose fermenting yeast such as

Saccharomyces cerevisiae.

The properties of yeasts found in kefir grains vary. For example, some of the yeasts found in kefir grains

are capable of fermenting lactose, while some are not. Also, it has been observed that some types 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. (Iwasawa

et al. 1982; Wyder et al. 1997). Iwasawa et al. (1982) showed that the electrophoretic pattern of the

yeast Torulopsis holmii isolated from Danish kefir grains demonstrated patterns indicating the presence of

ten different enzymes. Wyder et al. (1997) used restriction analysis of the two ITS regions to show that

yeasts from five kefir grain samples of different origins had unique patterns, indicating the presence of

different yeast species in kefir grains from different origins. Like kefir bacteria, the profile of yeasts is

different in kefir grains when compared to the final kefir product (Wyder et al. 1997). Abraham and De

Antoni (1999) showed that the yeast population in kefir produced from cows’ milk using grains was two

logs higher than when the same grains were added to soy milk.

7. Other uses of kefir grains

The ability of kefir grains to grow in milk whey prompted Rimada and Abraham (2001) to study whether

kefir grains could be added to whey produced as a by-product of the dairy industry in Argentina, thereby

producing a value-added product called kefiran. Kefiran was produced at a rate of 103 mg/l following

fermentation at 43°C for 120 h, with an inoculation rate of 100 g grains per litre of milk.

Athanasiadis et al. (1999) showed that kefir yeast cells that had been immobilized on de-lignified cellulose

were capable of producing commercially important quantities of ethanol from glucose over a wide variety

of temperatures (5–30°C). Production of volatiles (e.g. ethanal, ethyl acetate, propanol-1, isobutyl alcohol

and amyl alcohols) was found to depend on fermentation temperature. Ethyl acetate content did not

change as fermentation temperature decreased, although contents of total volatiles during fermentations

at 5°C were 38% of those carried out at 30°C. Using this system, it was shown that glucose produced the

fastest fermentation compared to fructose or sucrose, although glucose-based fermentations also yielded

lower concentrations of amyl alcohols, ethyl acetate and ethanol (Athanasiadis et al. 2001). The delignified

cellulose material supporting kefir yeast cells were able to ferment a mixture of whey and raisins

to produce a fermented product with an alcohol content of 4.4% v/v.

8. Composition of kefir

The composition of kefir depends greatly on the type of milk that was fermented (Kneifel and Mayer

1991). However, during the fermentation, changes in composition of nutrients and other ingredients have

also been shown to occur. (Bottazzi et al. 1994). 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 (Alm 1982d; Dousset and Caillet 1993). The amino acids valine, leucine, lysine and serine are

formed during fermentation, while the quantities of alanine and aspartic acid increase when compared to

raw milk (Alm 1982e). Bottazzi et al. (1994) reported the occurrence of acetic acid in their kefir, although

others reported that no acetic acid was present (Güzel-Seydim et al. 2000a, 2000b).

Kneifel and Mayer (1991) found that 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. These results contrast with Alm (1982b) who reported decreases in

biotin, vitamin B12 and pyridoxine, and significant increases in folic acid, as compared to non-fermented

milk.

9. Bioactive ingredients in kefir

The area of functional foods (see Table 5 for definition) has attracted a great deal of interest since it is

now recognized that many foods contain bioactive ingredients which offer health benefits or disease

resistance. A subset of functional foods is probiotic foods, from which there are several possible sources of

bioactive ingredients. The microorganisms themselves (dead or alive), metabolites of the microorganisms

formed during fermentation (including antibiotics or bactericides), or breakdown products of the food

matrix, such as peptides, may be responsible for these beneficial effects (Ouwehand and Salminen 1998;

Farnworth 2002; see Figure 3). Kefir has a long tradition of offering health benefits, especially in eastern

Europe (Hallé et al. 1994). There are several compounds in kefir that may have bioactive properties.

9.1 Exopolysaccharides

Exopolysaccharides of differing structures and compositions are produced by a variety of lactic acid

bacteria including Lactobacillus, Streptococcus, Lactococcus and Leuconostoc (De Vuyst and Degeest

1999; Ruas-Madiedo et al. 2002.). 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 (Jolly et al. 2002). 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. Hydrolysis reactions followed by NMR

analyses have been used to determine the chemical structure of kefiran (see Figure 4). The proposed

structure is a branched hexa- or heptasaccharide repeating unit that is itself composed of a regular

pentasaccharide unit to which one or two sugar residues are randomly linked (Kooiman 1968; Micheli et

al. 1999). Subsequent methylation/hydrolysis studies have shown that the structure of kefiran may be

more complex than first thought (Mukai et al. 1988; Mukai et al. 1990). Methylation and NMR analyses

have also been used to verify the production of kefiran by new bacterial strains (Yokoi et al. 1991). A

closer examination of chemical data published by Mukai et al. (1990) raises the question if, in fact, two

exopolysaccharides are being produced that have very similar chemical structure and properties. La

Rivière et al. (1967) reported that their kefiran had a 1:1 glucose to galactose ratio and an optical rotation

of +68.0°, while Mukai et al. (1990) isolated a kefiran with a glucose to galactose ratio of 0.9:1.1 and an

optical rotation of +54°. Examples can be found in the literature where the same bacterial strain produced

different exopolysaccharides in different media (Grobben et al. 1995; Van Geel-Schutten et al. 1999).

Furthermore, Santos et al. (2003) recently reported that they have also isolated an exopolysaccharide

closely related to kefiran.

Kefiran dissolves slowly in cold water and quickly in hot water, and forms a viscous solution at 2%

concentration (La Rivière et al. 1967). Carboxymethyl kefiran has a viscosity that is 14 times that of

kefiran, although this is still much lower than those of other thickening agents used in the food industry,

thus limiting any practical uses of kefiran or carboxymethyl kefiran (Mukai et al. 1990). Kefiran can form

weak gels when added to κ-carrageenan (1% 1:4 kefiran/κ-carrageenan), which have

gelation temperatures and melting temperatures similar to those of guar/κ-carrageenan gels

(Pintado et al. 1996).

Since its initial isolation, it has been reported that kefiran may be produced by a variety of bacteria

isolated from kefir grains which have been obtained from several sources (La Rivière et al. 1967; Toba et

al. 1987; Mukai et al. 1990; Hosono et al. 1990; Yokoi et al. 1991; Pintado et al. 1996; Mitsue et al.

1999; Micheli et al. 1999; Santos et al. 2003). Whether in fact the bacteria reported are the same has not

been studied, nor has any definitive identification been published to fully characterize those bacteria

reported as kefiran producers.

Bacteria which produce exopolysaccharides are often found in milk or milk products, although studies have

shown that maximum production of exopolysaccharide may occur in chemically defined media (containing

a carbohydrate source, mineral salts, amino acids/peptides, vitamins and nucleic acids) at a constant pH

(Mozzi et al. 1996; Dupont et al. 2000). The potential health properties of kefiran have prompted several

groups to develop media and growing conditions that optimize kefiran production (Toba et al. 1987; Yokoi

et al. 1990; Yokoi and Watanabe 1992; Micheli et al. 1999; Mitsue et al. 1999). Media based on lactic acid

whey have been found to be optimum for kefiran production. A batch procedure using a modified MRS

media (MRSL) was reported by Micheli et al. (1999) to produce consistent yields of 2 g/l of kefiran. The

best kefiran yields, however, have been reported by Mitsue et al. (1999) when they combined the kefiran

producing bacterium, Lactobacillus kefiranofaciens, with the yeast Torulaspora delbrueckii. When these

two organisms were grown in a 50 l fermentor in a fed-batch protocol, a yield of 3740 mg/l was obtained

over a 7 day period.

No measurements have been reported with regard to kefiran concentration in the final kefir product.

However, a comparison of the carbohydrate content of milk (USDA 2004) and that of kefir shows a more

than doubling of the carbohydrate content; how much of this is kefiran is not known. Abraham and De

Antoni (1999) did show that the polysaccharide content of kefir from cows’ milk was almost twice that of

kefir produced from soy milk.

Kefir grains grown in soy milk produce an exopolysaccharide that Liu et al. (2002) have shown to be

primarily composed of D-glucose and D-galactose (ratio 1.00: 0.43), with a molecular weight of

approximately 1.7 x 106 Da.

9.2 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 (Thomas

and Pritchard 1987; Matar et al. 1996). 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 (see Figure

5), which increases the possibility that bioactive peptides may be present in kefir. In their study of lactic

acid bacteria in Turkish kefir, Yüksekdağ et al. (2004b) showed that 13 out of 21 lactococci strains

had measurable proteolytic activity.

Initial studies on the peptide content of kefir drink have shown that kefir contains a large number of

peptides and that the majority of kefir peptides have molecular weights of ≤5000 kDa (Farnworth

2005, unpublished results).

10. Health benefits 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 (Alm 1982c) and is often the first weaning food

received by babies. Many of the studies regarding health benefits of kefir have been published in Russian

and Eastern European journals and therefore are not easily accessible to Western science (Batinkov 1971;

Ormisson and Soo 1976; Evenshtein 1978; Safonova et al. 1979; Ivanova et al. 1981; Sukhov et al.

1986; Besednova et al. 1997; Oleinichenko et al. 1999). However, the health benefits of kefir were

demonstrated in Canada as early as 1932 (Rosell 1932).

10.1 Stimulation of the immune system

It has been proposed that stimulation of the immune system may be one mechanism whereby probiotic

bacteria may exert many of their beneficial effects (De Simone et al. 1991; Gill 1998); this may be a

direct effect of the bacteria themselves (Cross 2002). However, 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 (LeBlanc et al. 2002;

Matar et al. 2003).

Thoreux and Schmucker (2001) fed kefir produced from grains to young (6 months) and old (26 months)

rats and found an enhanced mucosal immune response in the young animals, as shown by a higher anticholera

toxin (CT) IgA response compared to controls. Both young and old rats had significantly increased

total non-specific IgG blood levels, and a decreased systemic IgG response to CT. Taken together,

Thoreux and Schmucker concluded that kefir, like other probiotics, was exerting an adjuvant effect on the

mucosal immune system, perhaps produced by bacterial cell wall components.

Stimulation of the immune system may also occur due to the action of exopolysaccharides found in kefir

grains. Murofushi et al. (1983, 1986) used the method of La Riviére et al. (1967) for the extraction of

kefiran from kefir grains to produce a water-soluble polysaccharide fraction that they fed to mice. The

reduction in tumour growth that they observed was linked to a cell-mediated response, and it appeared

that the total dose of the polysaccharide determined its effectiveness. Furukawa et al. (1992) have also

shown that a water-soluble fraction of kefir grains may act as a modulator of the immune response.

The effect of kefir exopolysaccharides on the immune system may be dependent on whether the host is

healthy or has developed any tumours. Furukawa et al. (1996) incubated kefir grain polysaccharides with

Peyer’s Patch (PP) cells from tumour-bearing mice and found that the supernatant of this mixture

enhanced proliferation of splenocytes from normal mice and increased the mitogenic activities of

lipopolysaccharides (LPS) and phytohaemagglutinin-P (PHA-P) in splenocytes. They concluded that the

polysaccharide stimulated PP cells, causing them to secrete water-soluble factors that, in turn, enhanced

the mitogenic response of thymocytes and splenocytes in normal mice.

10.2 Inhibition of tumour growth

Shiomi et al. (1982) were the first to report the antitumour effects of a water-soluble polysaccharide

(approximate molecular weight 1 000 000 Da) isolated from kefir grains. Whether given orally or

intraperitoneally, the polysaccharide was able to inhibit the growth of Ehrlich carcinoma or Sarcoma 180

compared to control mice receiving no kefir-derived polysaccharide (Shiomi et al. 1982; Murofushi et al.

1983). The mechanism of action was not clear, since in vitro incubation of the two cancer cell lines with

the polysaccharide showed low cytotoxicity during 42 hours of incubation. This group then went on to

show that this water-soluble polysaccharide was able to reach the spleen and thymus of mice and, based

on the response to thymus-dependent and thymus-independent antigens, concluded that oral immune

enhancement was mediated through T-cell, but not B-cell activity. (Murofushi et al. 1986). More recently,

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. Murofushi et al. (1983) also reported the antitumour effectiveness of kefir grain

polysaccharides regardless of the time of administration, although they cautioned that larger doses may

only be more effective if administered after establishment of the tumours. A water-insoluble fraction

containing kefir grain microorganisms, rather than the water-soluble polysaccharide fraction, significantly

inhibited metastasis of highly colonized B16 melanoma. (Furukawa et al. 1993; Furukawa et al. 2000). It

was suggested that the water-soluble polysaccharide suppressed tumour growth by means of the

lymphokine activated macrophage (Mφ) via the gut-associated lymphoid tissue, while the waterinsoluble

microorganism fraction acted through an increase of NK cell activity.

Feeding kefir itself (2 g/kg body weight by intubation) was more effective in inhibiting tumour (Lewis lung

carcinoma) growth than yoghurt, when given for 9 days after tumour inoculation (Furukawa et al. 1990).

It was also shown that mice receiving kefir had an improved delayed-type hypersensitivity response

compared to tumour-bearing mice receiving no kefir, although the mean survival time was not affected

(Furukawa et al. 1991). Kubo et al. (1992) also reported that feeding kefir (100–500 mg/kg body weight)

inhibited the proliferation of Ehrlich ascites carcinoma. In addition, kefir, from which the grains had been

removed by filtration, were shown to kill or arrest the growth of fusiform cell sarcomas induced by 7,12-

dimethylbenzanthracene in mice when the kefir was injected intraperitoneally (Cevikbas et al. 1994).

Examination of tissue in kefir-treated mice showed a small amount of mitosis, some stromal connections

and, in some cases, disappearance of tumour necrosis.

Hosono et al. (1990) showed that isolates of Streptococcus, Lactobacillus and Leuconostoc in Mongolian

kefir all showed strong in vitro binding to amino acid pyrolysates which are believed to be mutagens and

are commonly found in food. Similarly, Miyamoto et al. (1991) reported that three slime-producing strains

of Streptococcus lactis subsp. cremoris found in German kefir had strong desmutagenic properties, which

they attributed to the ability of such strains to bind to a known mutagen. Using an Ames test, Yoon et al.

(1999) showed that Lactobacillus spp. isolated from kefir and yoghurt had antimutagenic properties

against the mutagen 2-nitrofluorene.

Liu et al. (2002) studied the effects of soy milk and cows’ milk fermented with kefir grains on the growth

of tumours in mice, using freeze-dried kefir (produced from either soy or cows’ milk) from which the

grains had been removed following fermentation. Mice were injected with 0.2 x 108 Sarcoma 180 cells one

week prior to the start of the feeding portion of the experiment. Tumour growth (volume) was estimated

for up to 30 days, after which tumours were removed and weighed. Both soy milk kefir (–70.9%) and

cows’ milk kefir (–64.8%) significantly inhibited tumour growth, compared to mice in the positive control

group. Microscopic examination of the tumours indicated that apoptosis may have been responsible for

reduced tumour growth. Similar effects of yoghurt on apoptosis have been reported (Rachid et al. 2002).

Mice fed unfermented soy milk did not have reduced tumour volumes at day 30, and Liu et al. (2002)

concluded that either the microorganisms themselves or any polysaccharides formed during fermentation

by the kefir grains microflora were responsible for the antitumour response. Genistein itself has been

shown to inhibit tumours (Murrill et al. 1996; Constantinou et al. 1996), although in this study genistein

levels did not change during the fermentation process. Mice consuming kefir samples also had significantly

increased levels of IgA in their small intestines compared to control animals, and it was proposed that the

PP tissue was increasing IgA secretion into the intestine in response to food antigens.

Güven et al. (2003) proposed an alternative suggestion as to how kefir may protect tissues. They showed

that mice exposed to carbon tetrachloride (a hepatotoxin to induce oxidative damage) and given kefir by

gavage had decreased levels of liver and kidney malondialdehyde, indicating that kefir was acting as an

antioxidant. Furthermore, their data showed that kefir was more effective than vitamin E (which is well

known to have antioxidative properties) in protecting against oxidative damage.

10.3 Kefir and lactose intolerance

A proportion of the global population is unable to digest lactose (the major sugar found in milk), because

of insufficient intestinal β-galactosidase (or lactase) activity (Alm 1982a). Research has shown,

however, that lactose maldigestors are able to tolerate yoghurt, providing the number of live bacteria

present in the yoghurt consumed is high enough (Pelletier et al. 2001). It is believed that the bacteria in

the yoghurt matrix are protected by the buffering effect of the yoghurt. Bacterial cells remain viable, and

the bacterial cell walls remain intact, and thus the β-galactosidase enzyme contained in the

yoghurt-producing bacteria (L. acidophilus) is protected during transit through the stomach until it arrives

at the upper gastrointestinal tract (Montes et al. 1995; De Vrese et al. 2001). It has also been shown that

fermented milk products have a slower transit time than milk, which may further improve lactose

digestion (Vesa et al. 1996; Labayen et al. 2001).

Some kefir grains have been shown to possess β-galactosidase activity which remains active when

consumed (De Vrese et al. 1992). A recent study has shown that a commercial kefir produced using a

starter culture containing six bacteria (but not L. acidophilus) and one yeast was equally as effective as

yoghurt in reducing breath hydrogen in adult lactose maldigestors (Hertzler and Clancy 2003). Severity of

flatulence in this group was also reduced when either yoghurt or kefir was consumed compared to milk.

De Vrese et al. (1992) showed that when pigs were fed kefir containing fresh grains, their plasma

galactose concentrations rose significantly higher than pigs given kefir containing heated grains. The diet

containing kefir and fresh grains had a β-galactosidase activity of 4.4 U/l, which was identified as

being responsible for the hydrolysis of lactose in the intestine, thus yielding galactose that can be

absorbed. Kefir itself contains no galactose (Alm 1982).

10.4 Antimicrobial properties of kefir

There are data to show that 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 foodborne

pathogens and spoilage bacteria during food production and storage, but also in the treatment and

prevention of gastrointestinal disorders and vaginal infections (Tahara and Kanatani 1997; Zamfir et al.

1999; Bonadè et al. 2001; Messens and De Vuyst 2002; Jamuna and Jeevaratnam 2004).

Garrote et al. (2000) tested the inhibitory activity of a supernatant of cows’ milk fermented with kefir

grains, against Gram-negative and Gram-positive bacteria. Gram-positive microorganisms were inhibited

to a greater extent than Gram-negative microorganisms; moreover, both lactic and acetic acids were

found in the supernatants. Garrote et al. (2000) showed that milk supplemented with lactic acid or lactic

acid plus acetic acid at the concentrations found in the kefir supernatant also had inhibitory activity

against E. coli 3. They concluded that organic acids produced during kefir fermentation could have

important bacteriostatic properties even in the early stages of milk fermentation. Cevikbas et al. (1994)

found similar results against Gram-positive coccus, staphylococcus, and Gram–positive bacillus, and noted

that kefir grains were more effective with regard to their antibacterial properties than the final kefir

product.

Kefir grains themselves have inhibitory power against bacteria that can be preserved during lyophilization,

particularly when glycerol is added as a cryopreservative (Brialy et al. 1995). Fresh kefir grains were

found to inhibit the growth of the bacteria Streptococcus aureus, Klebsiella pneumoniae and Escherichia

coli, but not the yeasts Candida albicans and Saccharomyces cerevisiae. Leuconostoc mesenteroides and

Lactobacillus plantarum, isolated from kefir grains, have both been shown to produce antimicrobial

compounds that are present in kefir. Both inhibit Gram-positive and Gram-negative bacteria, have a

molecular weight of approximately 1000 kDa and are heat stable, although their antimicrobial properties

are reduced after exposure to proteolytic enzymes (Serot et al. 1990). Santos et al. (2003) showed that

lactobacilli isolated from kefir grains had antimicrobial activities against E. coli (43/58 strains), Listeria

monocytogenes (28/58 strains), Salmonella typhimurium (10/58 strains), S. enteritidis (22/58 strains),

S. flexneri (36/58 strains) and Yersinia enterocolitica (47/58 strains). Bacteriocins were thought to be

responsible, although they were not identified.

In a study in which foodborne bacterial pathogens (E. coli O157:H7, L. monocytogenes 4b, Y.

enterocolitica 03) were added at the beginning of yoghurt or kefir fermentation, both kefir and yoghurt

failed to inhibit pathogenic bacterial growth. For kefir, this was explained as being due to the slow acid

development during fermentation. Interestingly, fermentations of kefir and yoghurt combinations proved

to be more effective at pathogen suppression than single fermentation (Gulmez and Guven 2003).

Hydrogen peroxide is another metabolite produced by some bacteria as an antimicrobial compound.

Yüksekdağ et al. (2004a) showed that all 21 isolates of lactic acid bacteria from Turkish kefir

produced hydrogen peroxide (0.04–0.19 ug/ml). In a later paper, they reported that 11 out of 21 strains

of kefir lactococci produced hydrogen peroxide (Yüksekdağ et al. 2004b). All lactococci strains were

effective in inhibiting growth of Streptococcus aureus, but were less effective against E. coli NRLL B-704

and Pseudomonas aeruginosa.

10.5. Behaviour of kefir bacteria in the gastrointestinal tract

One of the criteria for probiotic bacteria is that they should be able to withstand the harsh conditions of

the gastrointestinal tract, including extreme pH conditions present in the stomach and the action of bile

salts and digestive enzymes (Lee and Salminen 1995). It is also believed that one way in which probiotic

bacteria could protect against pathogenic bacteria would be to compete with or displace pathogenic

bacteria by adhering to intestinal epithelial cells. (Kirjavainen et al. 1998; Fujiwara et al. 2001; Gibson

and Rastall 2003).

No results from human feeding trials have been published with regard to the ability of the microorganisms

found in kefir to traverse the upper GI tract in large numbers and arrive at the large intestine. Kefir,

because it is milk based, is able to buffer the pH of the stomach when ingested and thereby provide time

for many of the bacteria to pass through to the upper small intestine (Farnworth et al. 2003). Santos et

al. (2003) isolated 58 strains of Lactobacillus spp. and isolates of L. paracasei, L. plantarum, L.

delbrueckii, L. acidophilus and L. kefiranofaciens from different sources of kefir grains and exposed them

to an MRS medium at pH 2.5 and MRS containing 0.3% Oxgall (bile salts). They found that all strains

survived 4 h incubation at pH 2.5, but did not grow. Eighty-five percent of isolates showed high resistance

to Oxgall, but had delayed growth.

The caco-2 cell assay has been used to show that many of the lactobacilli isolated from kefir grains are

able to bind to enterocyte-like cells (Santos et al. 2003), although the authors also cautioned that results

using this model might not necessarily apply in vivo.

Human studies of the effects of diet on intestinal microflora are limited to the analysis of faecal samples,

although no detailed human study has been published in which kefir has been used. Marquina et al.

(2002) used mice to study the effect of consuming kefir (source not defined) in a feeding study that lasted

7 months. They were able to show that the numbers of lactic acid bacteria in the mouse small and large

intestines increased significantly. Streptococci increased by 1 log, while sulfite-reducing clostridia

decreased by 2 logs.

10.6. Kefir and cholesterol metabolism

Positive effects of yoghurt consumption on cholesterol metabolism have been reported (Kiessling et al.

2002; Xiao et al. 2003), although a review of the literature reveals that the results are at best moderate,

and are often inconsistent (Taylor and Williams 1998; St-Onge et al. 2000; Pereira and Gibson 2002).

Several hypotheses have been proposed regarding the possible mechanism of action employed by bacteria

to reduce cholesterol levels (St. Onge et al. 2002). Vujicic et al. (1992) showed that kefir grains from

Yugoslavia, Hungary and the Caucase region were able to assimilate cholesterol in milk either incubated at

20°C for 24 h (reductions of up to 62%) or incubated and stored at 10°C for 48 h (reductions of up to

84%). These authors claimed that their results indicated that kefir grains had a cholesterol-degrading

enzyme system. Similar results were reported for 27 lactic acid bacterial strains. However, it was pointed

out that isolates from dairy products had lower cholesterol-assimilating capacity than strains isolated from

infant faeces (Xanthopoulos et al. 1998).

In a clinical trial in which 13 subjects were fed 500 ml/day of kefir for 4 weeks in a placebo-controlled

design, percentage changes in serum triglycerides compared to baseline levels were lower (although not

significantly) than when subjects consumed unfermented milk; the percentage serum high-density

lipoprotein (HDL) cholesterol change compared to baseline increased (although not significantly) when

subjects consumed kefir compared to milk (St. Onge et al. 2002). Similarly, Kiessling et al. (2002) found

that HDL levels increased after 6 months of feeding yoghurt supplemented with Lactobacillus acidophilus

and Bifidobacterium longum, thereby producing an improved low-density lipoprotein (LDL)/HDL

cholesterol ratio.

11. Conclusions

Many probiotic products have been formulated that contain small numbers of different bacteria. The

microbiological and chemical composition of kefir indicates that it is a much more complex probiotic, as

the large number of different bacteria and yeast found in it distinguishes it from other probiotic products.

Since the yeasts and bacteria present in kefir grains have undergone a long association, the resultant

microbial population exhibits many similar characteristics, making isolation and identification of individual

species difficult. 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. At least one exopolysaccharide has been identified in kefir, although

others may be present. Many bacteria found in kefir have been shown to have proteinase activity, and a

large number of bioactive peptides has been found in kefir. Furthermore, there is evidence to show that

kefir consumption not only affects digestion, but also influences metabolism and immune function in

humans.

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