kefir: Keﬁr – a complex probiotic

......................................................................................................................................................................
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 exopolysacchar-
ides 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 fermenta-
tion, 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
known than yoghurt; however, an analysis of its composi-
1. Introduction
tion indicates that it may contain bioactive ingredients that
Archaeological evidence has indicated that the process of give it unique health beneﬁts, which means that keﬁr may
fermentation in foods was discovered accidentally thou- be an important probiotic product (Farnworth 1999).
sands of years ago. However, over time, it soon became
apparent that many fermented foods had longer storage
2. Origins of keﬁr
lives and improved nutritional values compared to their
Keﬁr is a viscous, slightly carbonated dairy beverage that
unfermented equivalents, making this form of food proces-
contains small quantities of alcohol and, like yoghurt, is
sing a popular technique. It is not surprising, therefore, to
believed to have its origins in the Caucasian mountains of
ﬁnd that many foods including vegetables, fruits, cereals,
the former USSR. It is also manufactured under a variety
meat and ﬁsh have all been converted into desirable food
of names including kephir, kiaphur, kefer, knapon, kepi
products by fermentation and are still being consumed
and kippi (Koroleva 1988a), with artisanal production of
throughout the world today (Farnworth 2004).
keﬁr occurring in countries as widespread as Argentina,
Certain bacteria, either alone or through the changes
Taiwan, Portugal, Turkey and France (Thompson et al.
they bring about during fermentation, have been shown to
1990; Angulo et al. 1993; Lin et al. 1999; Garrote et al.
have positive effects on health as well as resistance to dis-
2001; Santos et al. 2003; Gulmez and Guven 2003). It is
ease. Interest in such probiotic species has increased in
not clear whether all keﬁrs originate from a single original
recent years as more is learned about the microorganisms
starter culture, since microbial analyses of keﬁr samples
used in the fermentation process, and the possibility of
taken from different locations indicate microﬂora popula-
adding beneﬁcial bacteria to food products. Furthermore,
tion differences.
consumers are increasingly looking to improve their health
The FAO/WHO (2001) have proposed a deﬁnition of
and increase their resistance to disease through dietary
keﬁr based on the microbial composition of both keﬁr
means.
grains (the starter culture used to produce keﬁr) and the
Fermented dairy products from milk from a variety of
ﬁnal keﬁr product (see Table 1).
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 3. Keﬁr manufacture
is familiar to consumers. Keﬁr, meanwhile, is less well
Although commercial keﬁr is traditionally manufactured
from cows’ milk, it has also been made from the milk of
Food Science and Technology Bulletin: Functional Foods 2 (1) 1–17
ewes, goats and buffalos. Moreover, keﬁr produced using
DOI: 10.1616/1476-2137.13938. Published 4 April 2005
ISSN 1476-2137 # IFIS Publishing 2005. All Rights Reserved soy milk has also been recently reported (Ismail et al.
Keﬁr – a complex probiotic E.R. Farnworth
2
lactic acid production and ethanol production, compared
Table 1. Codex Alimentarius description of keﬁr*
to keﬁr produced from soy milk alone (Liu and Lin 2000).
Deﬁnition
The grains used in this study were found to have a-galac-
Starter culture prepared from keﬁr grains, Lactobacillus keﬁri, tosidase activity that helped explain how these keﬁr grains
and species of the genera Leuconostoc, Lactococcus and
were able to use the galactose-based carbohydrates which
Acetobacter growing in a strong speciﬁc relationship. Keﬁr
occur in soy milk.
grains constitute both lactose-fermenting yeasts (Kluyveromyces
Keﬁr grains are key to keﬁr production, and it has been
marxianus) and non-lactose-fermenting yeasts (Saccharomyces
unisporus, Saccharomyces cerevisiae and Saccharomyces found that the ﬁnished product has a different microbiolo-
exiguus). gical proﬁle from the grains and therefore cannot be used
to inoculate a new batch of milk (Simova et al. 2002).
Composition
Grains have been shown to possess a dynamic and com-
Milk protein (% w/w) min. 2.8
plex ﬂora which is not conducive to commercial produc-
<10
Milk fat (% m/m)
tion of a uniform, stable product; this has prompted
Titratable acidity, expressed as % of lactic acid min. 0.6
groups to try to produce keﬁr from a mixture of pure cul-
(% m/m)
tures (Petersson et al. 1985). Duitschaever et al. (1987,
Ethanol (% vol./w) not stated
1988a) combined a yoghurt culture with three other lactic
min. 107
Sum of speciﬁc microorganisms constituting the
acid bacteria and Saccharomyces cerevisiae (a non-lactose
starter culture (cfu/g, in total)
fermenting yeast) to produce a fermented milk with keﬁr
min. 104
Yeasts (cfu /g)
characteristics (which produced CO2 and contained etha-
*From Codex Standard for Fermented Milks CODEX STAN 243-2003
nol) 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 manu-
´
1983; Mann 1985; Zourari and Anifantakis 1988; Halle facture keﬁr under a continuous process. More recently,
et al. 1994; Kuo and Lin 1999). Traditionally, keﬁr is pro- Beshkova et al. (2002) produced a starter consisting of
duced by adding keﬁr grains (a mass of proteins, polysac- two bacteria (Lactobacillus helveticus and Lactococcus
charides, mesophilic, homofermentative and hetero- lactis subsp. lactis,) and one yeast (S. cerevisiae) isolated
fermentative lactic acid streptococci, thermophilic and from keﬁr grains and combined with two yoghurt strains
mesophilic lactobacilli, acetic acid bacteria, and yeast) to (Lactobacillus delbrueckii subsp. bulgaricus, and Strepto-
´
a quantity of milk (Koroleva 1982; Halle et al. 1994; coccus thermophilus). Yeast was added to the starter with
Tamime et al. 1999). The size of the initial keﬁr grain sucrose either at the beginning, or after lactic acid fermen-
inoculum affects the pH, viscosity and microbiological tation. The two resulting keﬁrs produced were found to
proﬁle of the ﬁnal product (Koroleva and Bavina 1970; have high numbers of viable cocci and lactobacilli and
Garrote et al. 1998). Koroleva (1991) reported that grain had chemical and organoleptic properties that were similar
to milk ratios of 1:30 to 1:50 were optimum. In some to traditional keﬁr. A commercial keﬁr is being produced
manufacturing procedures, a perculate of the grains from a in the United States using a mixture of deﬁned microor-
coarse sieve is used as the mother culture to inoculate ganisms rather than keﬁr grains. This starter culture mix-
fresh milk. Fermentation of the milk by the inoculum pro- ture has been reported to contain Streptococcus lactis, L.
ceeds for approximately 24 hours, during which time plantarum, Streptococcus cremoris, L. casei, Streptococcus
homofermentative lactic acid streptococci grow rapidly, diacetylactis, Leuconostoc cremoris and Saccharomyces
initially causing a drop in pH. This low pH favours the ﬂorentinus (Hertzler and Clancy 2003).
growth of lactobacilli, but causes the streptococci numbers Starter cultures containing freeze-dried lactic acid bac-
to decline. The presence of yeasts in the mixture, together teria and yeasts from keﬁr grains are now available com-
with fermentation temperature (21–238C), encourages the mercially; some are supplemented with additional microor-
growth of aroma-producing heterofermentative strepto- ganisms to impart desirable characteristics in the ﬁnished
cocci. As fermentation proceeds, growth of lactic acid keﬁr product (Piotr Kolakowski, private communication).
bacteria is favoured over growth of yeasts and acetic acid It is evident that the ﬁnal product, as produced from keﬁr
bacteria (Koroleva 1982). grains, will have a larger number and variety of microor-
Taiwanese researchers have shown that the lactic acid ganisms than keﬁr produced from a mixture of a small
bacteria from keﬁr grains grow more slowly in soy milk number of pure cultures.
compared to cows’ milk (Liu and Lin 2000). This may be Keﬁr is still most familiar to consumers in Eastern Eur-
due, in part, to the slower production of growth factors at ope, although commercial production now occurs in North
the beginning of fermentation when soy milk is the America. However, several patents can be found relating
substrate rather than cows’ milk. Addition of carbohydrate to commercial keﬁr production worldwide (Klupsch 1984;
(e.g. 1% glucose) to soy milk increases yeast numbers, Dmitrovskaya 1986; Tokumaru et al. 1987; Kabore 1992).
Keﬁr – a complex probiotic E.R. Farnworth 3
Production/consumption ﬁgures for keﬁr are not readily acceptability of keﬁr, compared to traditionally made keﬁr
(Duitschaever et al. 1991; Muir et al. 1999).
available since statistics for fermented dairy products are
not always broken down into separate items such as Acetaldehyde and acetoin have received particular
yoghurt and keﬁr (Mann 1989; Libudzisz and Piatkiewicz attention with regard to their roles during keﬁr manufac-
1990; Serova 1997; Zimovetz and Boyko 2000). A survey ture because of their contribution to taste; both have been
of keﬁr products purchased on the retail market in War- found to increase in concentration during keﬁr fermenta-
saw, Poland showed that 73% of products contained 107– tion. During storage, acetaldehyde increases in concentra-
109 cfu bacteria/g, and that 97% of samples were coli- ¨
tion and acetoin decreases (Guzel-Seydim et al. 2000a,
˘
¨
form-free (Molska et al. 2003). However, 48% of samples 2000b). Yuksekgag et al. (2004a), in their study of 21 iso-
did not meet FAO/WHO requirements for yeast numbers lates of lactic acid bacteria from various sources of Turk-
(FAO/WHO 2001). ish keﬁr, were able to show that all 21 isolates produced
acetaldehyde (0.88–4.40 mg/ml) when added to milk.
A whey beverage with an acceptable ﬂavour has
4. Characteristics of keﬁr
recently been developed using keﬁr yeasts (Athanasiadis
et al. 2004), especially when fructose was added to fresh
The ﬂavour, viscosity and microbial/chemical composition
milk before fermentation, and ﬁnal pH of the beverage
of the ﬁnal keﬁr product can be affected by the size of the
inoculum added to the milk, the occurrence of any agita- was 4.1. Fructose was found to increase production of sev-
tion during fermentation, and the rate, temperature and eral ﬂavour volatiles, but did not increase fermentation
time.
duration of the cooling and ripening stages following fer-
mentation (Koroleva 1988b). Natural keﬁr has a refresh-
ing, yeasty taste and a ‘sparkling’ mouth feel (Kemp
5. Keﬁr grains
1984).
Keﬁr grains resemble small cauliﬂower ﬂorets: they mea-
Modern manufacturing procedures for keﬁr result in
sure 1–3 cm in length, are lobed, irregularly shaped, white
ethanol levels in the ﬁnished product of 0.01–0.1% (Koro-
to yellow-white in colour, and have a slimy but ﬁrm tex-
leva 1982), although keﬁr with ethanol concentrations as
`
ture (La Riviere et al. 1967; Kosikowski and Mistry 1997;
high as 0.25% have been produced from grains in the
laboratory (Kuo and Lin 1999; Simova et al. 2002; Besh- see Figure 1). Grains are kept viable by transferring them
kova et al. 2002). The amounts of ethanol and CO2 pro- daily into fresh milk and allowing them to grow for
duced during fermentation of keﬁr depend on the produc- approximately 20 hours; during this time, the grains will
´
have increased their mass by 25% (Halle et al. 1994).
tion conditions used. CO2 content of keﬁr has been said to
be ‘comparatively low’ in relation to other fermented Grains must be replicated in this way to retain their viabi-
drinks (Koroleva 1982); values of 0.85–1.05 g/l have been lity, since old and dried keﬁr grains have little or no abil-
`
ity to replicate (La Riviere et al. 1967). Keﬁr grains repli-
reported for keﬁr produced from keﬁr grains (Beshkova
et al. 2002; Simova et al. 2002) and 1.7 g/l for keﬁr pro-
duced from puriﬁed cultures (Gobbetti et al. 1990) How-
ever, the generation of CO2 during keﬁr manufacture,
especially after packaging, presents some practical pro-
blems, since the microorganisms (particularly yeasts) in
the keﬁr continue to grow following packaging. The con-
tainer 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 Figure 1. Keﬁr grains. Reproduced with permission from
ﬂavour, or modiﬁcation of the fermentation process (e.g. Handbook of Fermented Functional Foods, Farnworth,
E.R. editor. Copyright CRC Press.
addition of lactococci, lactobacilli or yeasts) increased the
Keﬁr – a complex probiotic E.R. Farnworth
4
Table 2. Microorganisms* in keﬁr grains, mother
culture and keﬁr drink
Lactococci Lactobacilli Yeasts
Keﬁr grains 7.37 8.94 8.30
Mother culture 8.43 7.65 5.58
(wash of grains)
Keﬁr drink 8.54 7.45 5.24
*log CFU/g
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 pro-
duct are not the same (see Table 2). This, in part, explains
why production of keﬁr must start with keﬁr grains, since
Figure 2. Electron micrograph of a keﬁr grain.
the ﬁnal drink does not have the number or complexity of
microorganisms as the grains, preventing the drink from
cated in milk ‘at home with daily changes of milk’ and
being used as a starter culture for a new batch of keﬁr.
stored for three months either at room temperature or at
Kandler and Kunath (1983) reported similar results when
48C had microbiological proﬁles that were different to
they compared the microﬂora of keﬁr, inoculated milk
those of fresh grains (Pintado et al. 1996). In addition,
before incubation, and a mixture of keﬁr grains.
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
6. Microbiology of keﬁr grains
daily transfers and should only be replaced if their ability
to ferment milk becomes impaired. (Koroleva 1982). Low
6.1 Bacteria
temperature storage appears to be the best way to maintain
keﬁr grains for long periods. Garrote et al. (1997) showed The microbial population found in keﬁr grains has been
that storage of keﬁr grains at À80 or À208C for 120 days used as an example of a symbiotic community (Margulis
did not change their fermentation properties compared to 1995); this symbiotic nature has made identiﬁcation and
grains that had not been stored; however, grains stored at study of the constituent microorganisms within keﬁr grains
À48C did not produce acceptable keﬁr after thawing. Keﬁr difﬁcult. Koroleva (1991) stated that keﬁr bacteria and
grains replicated in soy milk have been reported to be yeasts, when separated as pure cultures, either do not grow
smaller in size compared to grains replicated in cows’ in milk or have a decreased biochemical activity, which
milk (Liu et al. 2002). There have been no reports of suc- further complicates the study of the microbial population
cessful production of keﬁr grains from pure cultures. of keﬁr grains. Several media have been proposed for the
While early studies of keﬁr grains employed light isolation and identiﬁcation of bacteria in keﬁr grains
microscopy, later investigations used electron microscopy (Kojima et al. 1993). Linossier and Dousset (1994)
to describe the complex microbial community of which showed that Lactobacillus keﬁr grew better when the yeast
they were comprised (Ottogalli et al. 1973; Bottazzi and Candida keﬁr was added to the milk. Garrote et al. (2004)
Bianchi 1980; Molska et al. 1980; Marshall et al. 1984; reported a similar observation when they attempted to
Duitschaever et al. 1988b; Toba et al. 1990; Neve 1992; grow L. keﬁr in milk. In general, lactic acid bacteria are
more numerous (108–109) than yeasts (105–106) and acetic
Bottazzi et al. 1994; Rea et al. 1996). Figure 2 shows an
acid bacteria (105–106) in keﬁr grains, although fermenta-
electron micrograph of keﬁr grains obtained from the
Moscow Dairy Institute. Ottogalli et al. (1973) showed tion conditions can affect this pattern (Koroleva 1991;
that the chemical and microbiological compositions of Garrote et al. 2001) Table 3 shows a list of the various
keﬁr grains from four different sources were different, bacteria that have been reported in keﬁr and keﬁr grains
making comparisons between results published by differ- from around the world.
ent laboratories difﬁcult. Garrote et al. (2004) carried out several in vitro tests to
The microbial population that makes up keﬁr grains try to explain how the bacteria in keﬁr grains function.
appears to be relatively constant over time, although sea- They showed that two of the heterofermentative lactoba-
sonal variations in the grain ﬂora have been noted which cilli, L. keﬁr and L. parakeﬁr, possessed S-layer proteins
`
can affect the ﬁnal product consistency (La Riviere et al. that can be used to explain in part their auto-aggregation
Keﬁr – a complex probiotic E.R. Farnworth 5
the yeasts in keﬁr grains provide an environment for the
Table 3. Bacteria found in keﬁr grains and keﬁr
growth of keﬁr bacteria, producing metabolites that contri-
Lactobacilli bute to the ﬂavour and mouthfeel of keﬁr (Clementi et al.
keﬁr a,c,j,n,o,p,r Lactobacillus delbrueckii a,h,p
Lactobacillus
1989; Kwak et al. 1996; Simova et al. 2002). Table 4 lists
keﬁranofaciens l,n,p Lactobacillus rhamnosus a,r
Lactobacillus
the various yeasts that have been reported in keﬁr grains.
keﬁrgranum n Lactobacillus casei h
Lactobacillus
To prevent excessive CO2 production (particularly after
parakeﬁr n,o Lactobacilli paracasei p
Lactobacillus fermentation), Kwak et al. (1996) suggested a two stage
brevis g,h,p,r Lactobacillus fructivorans k
Lactobacillus fermentation process starting with a non-lactose ferment-
plantarum o,p Lactobacillus hilgardii k
Lactobacillus ing yeast such as Saccharomyces cerevisiae.
helveticus a,b,h Lactobacillus fermentum r
Lactobacillus The properties of yeasts found in keﬁr grains vary. For
acidophilus g,p,r Lactobacillus viridescens r example, some of the yeasts found in keﬁr grains are cap-
Lactobacillus
able of fermenting lactose, while some are not. Also, it
Lactococci
has been observed that some types of yeasts are located at
Lactococcus lactis subsp. lactis a,c,e,f,g,h,k,o,r
the surface of the grain, while others inhabit the interior.
Lactococcus lactis subsp. cremoris a,e,f
It may be that yeasts located at different locations in the
Streptococci keﬁr grains play different roles in the fermentation pro-
Streptococcus thermophilus e,h cess. (Iwasawa et al. 1982; Wyder et al. 1997). Iwasawa
et al. (1982) showed that the electrophoretic pattern of the
Enterococci
yeast Torulopsis holmii isolated from Danish keﬁr grains
**
Enterococcus durans d ,e
demonstrated patterns indicating the presence of ten differ-
(reported as Streptobacterium durans in ref. d; reported as
Streptococcus durans in ref. e) ent enzymes. Wyder et al. (1997) used restriction analysis
of the two ITS regions to show that yeasts from ﬁve keﬁr
Leuconostocs
grain samples of different origins had unique patterns,
Leuconostoc sp. r
indicating the presence of different yeast species in keﬁr
*
Leuconostoc mesenteroides a,b,g ,o
grains from different origins. Like keﬁr bacteria, the pro-
(reported as Leuconostoc keﬁr in ref. g)
ﬁle of yeasts is different in keﬁr grains when compared to
Acetic acid bacteria
the ﬁnal keﬁr product (Wyder et al. 1997). Abraham and
Acetobacter sp. o De Antoni (1999) showed that the yeast population in
*
Acetobacter pasteurianus g keﬁr produced from cows’ milk using grains was two logs
(reported as Acetobacter rancens in ref. g)
higher than when the same grains were added to soy milk.
Acetobacter aceti a,d
Other bacteria
7. Other uses of keﬁr grains
Bacillus sp. r Micrococcus sp. r
Bacillus subtilis g Escherichia coli r The ability of keﬁr grains to grow in milk whey prompted
Rimada and Abraham (2001) to study whether keﬁr grains
a b c d
Koreleva 1991; Lin et al. 1999; Pintado et al. 1996; Rosi 1978; ref.
could be added to whey produced as a by-product of the
e f g
¨
Yuksekdag et al. 2004; Dousset and Caillet 1993; Ottogalli et al. 1973;
dairy industry in Argentina, thereby producing a value-
h j k
Simova et al. 2002; Kandler and Kunath 1983; Yoshida and Toyoshima
l n o
1994; Fujisawa et al. 1988; Takizawa et al. 1994; Garrote et al. 2001; added product called keﬁran. Keﬁran was produced at a
p r
Santos et al. 2003; Angulo et al. 1993.
rate of 103 mg/l following fermentation at 438C for 120 h,
with an inoculation rate of 100 g grains per litre of milk.
Athanasiadis et al. (1999) showed that keﬁr yeast cells
and haemagglutination properties. In addition, these
that had been immobilized on de-ligniﬁed cellulose were
two bacteria were also shown to adhere to Caco-2 cells,
capable of producing commercially important quantities of
raising the possibility that these bacteria would be good
ethanol from glucose over a wide variety of temperatures
probiotics.
(5–308C). Production of volatiles (e.g. ethanal, ethyl acet-
ate, propanol-1, isobutyl alcohol and amyl alcohols) was
found to depend on fermentation temperature. Ethyl acet-
6.2 Yeasts
ate content did not change as fermentation temperature
It has been recognized that yeasts play an important role decreased, although contents of total volatiles during fer-
in the preparation of fermented dairy products, where they mentations at 58C were 38% of those carried out at 308C.
can provide essential growth nutrients such as amino acids Using this system, it was shown that glucose produced the
and vitamins, alter the pH, secrete ethanol and produce fastest fermentation compared to fructose or sucrose,
CO2 (Viljoen 2001). The yeasts in keﬁr have been less although glucose-based fermentations also yielded lower
well studied than keﬁr bacteria, although it is obvious that concentrations of amyl alcohols, ethyl acetate and ethanol
Keﬁr – a complex probiotic E.R. Farnworth
6
Table 4. Yeasts found in keﬁr grains and keﬁr Table 5. Deﬁnitions of functional foods and probiotics
* *
Kluyveromyces marxianus a,b,f ,g,h,i,j,k,m ,n Candida friedrichii n Functional foods
(reported as Saccharomyces lactis in A functional food is one that is consumed as part of a usual diet,
ref. f; reported as Kluyveromyces lactis and is demonstrated to have physiological beneﬁts and/or reduce
in ref. m) the risk of chronic disease beyond basic nutritional functions.
Saccharomyces sp. k Candida (Health Canada 2004)
pseudotropicalis f
Probiotics
*
Saccharomyces cerevisiae a,d,e,f ,g,j,m,n Candida tenuis f
Live microorganisms that, when administered in adequate
(reported as Saccharomyces carlbergensis
amounts, confer a health beneﬁt on the host. (FAO/WHO 2002).
in ref. f)
Report of a Joint FAO/WHO Working Group, ‘Guidelines for
Saccharomyces unisporus c,h,j,m Candida the Evaluation of Probiotics in Food’, London, Ontario, Canada,
inconspicua g 2002.
*
Saccharomyces exiguus l Candida maris g
(reported as Torolopsis holmii in ref. l)
Saccharomyces turicensis h Candida lambica j (1982b) who reported decreases in biotin, vitamin B12 and
Saccharomyces delbrueckii d Candida pyridoxine, and signiﬁcant increases in folic acid, as com-
tannotelerans e pared to non-fermented milk.
Saccharomyces dairensis n Candida valida 6 e
Torulaspora delbrueckii a,h,m Candida kefyr a,j,n
9. Bioactive ingredients in keﬁr
Brettanomyces anomalus h Candida holmii j,m
Issatchenkia occidentalis j Pichia The area of functional foods (see Table 5 for deﬁnition)
fermentans b,m,n has attracted a great deal of interest since it is now recog-
nized that many foods contain bioactive ingredients which
a b c d e
Koreleva 1991; Lin et al. 1999; Pintado et al. 1996; Rosi 1978; Dous-
g h
offer health beneﬁts or disease resistance. A subset of
set and Caillet 1993; ref. fOttogalli et al. 1973; Simova et al. 2002; Wyder
i j
and Puhan 1997, 1999; Yoshida and Toyoshima 1994; Engel et al. 1986; functional foods is probiotic foods, from which there are
k m n
Garrote et al. 2001; ref. lIwasawa et al. 1982; Angulo et al. 1993; Rohm
several possible sources of bioactive ingredients. The
et al. 1992
microorganisms themselves (dead or alive), metabolites of
the microorganisms formed during fermentation (including
(Athanasiadis et al. 2001). The de-ligniﬁed cellulose mate-
antibiotics or bactericides), or breakdown products of the
rial supporting keﬁr yeast cells were able to ferment a
food matrix, such as peptides, may be responsible for
mixture of whey and raisins to produce a fermented pro-
these beneﬁcial effects (Ouwehand and Salminen 1998;
duct with an alcohol content of 4.4% v/v.
Farnworth 2002; see Figure 3). Keﬁr has a long tradition
of offering health beneﬁts, especially in eastern Europe
8. Composition of keﬁr ´
(Halle et al. 1994). There are several compounds in keﬁr
that may have bioactive properties.
The composition of keﬁr depends greatly on the type of
milk that was fermented (Kneifel and Mayer 1991). How-
ever, during the fermentation, changes in composition of
9.1 Exopolysaccharides
nutrients and other ingredients have also been shown to
occur. (Bottazzi et al. 1994). L(þ) lactic acid is the Exopolysaccharides of differing structures and composi-
tions are produced by a variety of lactic acid bacteria
organic acid in highest concentrations after fermentation
including Lactobacillus, Streptococcus, Lactococcus and
and is derived from approximately 25% of the original
Leuconostoc (De Vuyst and Degeest 1999; Ruas-Madiedo
lactose in the starter milk (Alm 1982d; Dousset and Cail-
et al. 2002.). These cell-surface carbohydrates confer pro-
let 1993). The amino acids valine, leucine, lysine and ser-
tective and adaptive properties on their bacterial produ-
ine are formed during fermentation, while the quantities of
cers; since they are often loosely bound to the cell mem-
alanine and aspartic acid increase when compared to raw
milk (Alm 1982e). Bottazzi et al. (1994) reported the brane, they are, therefore, easily lost to their environment
(Jolly et al. 2002). In food products, exopolysaccharides
occurrence of acetic acid in their keﬁr, although others
¨ often contribute to organoleptic and stability characteris-
reported that no acetic acid was present (Guzel-Seydim
et al. 2000a, 2000b). tics. A unique polysaccharide called keﬁran has been
found in keﬁr grains; grains may also contain other exopo-
Kneifel and Mayer (1991) found that appreciable
lysaccharides.
amounts of pyridoxine, vitamin B12, folic acid and biotin
Keﬁran contains D-glucose and D-galactose only in a
were synthesized during keﬁr production, depending on
ratio of 1:1. Hydrolysis reactions followed by NMR ana-
the source of keﬁr grains used, while thiamine and riboﬂa-
lyses have been used to determine the chemical structure
vin levels were reduced. These results contrast with Alm
Keﬁr – a complex probiotic E.R. Farnworth 7
exopolysaccharides in different media (Grobben et al.
Probiotic
1995; Van Geel-Schutten et al. 1999). Furthermore, Santos
(Kefir) 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% concentra-
Fermentation Bacteria, Yeasts Metabolites
´
tion (La Riviere et al. 1967). Carboxymethyl keﬁran has a
Products
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).
Intestinal Microbial Digestion, Metabolism,
Keﬁran can form weak gels when added to k-carrageenan
Population Immune Status,
Disease Resistance (1% 1:4 keﬁran/k-carrageenan), which have gelation tem-
peratures and melting temperatures similar to those of
Figure 3. Probiotic effects on metabolism and health.
guar/k-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
of keﬁran (see Figure 4). The proposed structure is a
from keﬁr grains which have been obtained from several
branched hexa- or heptasaccharide repeating unit that is
´
sources (La Riviere and Kooiman 1967; Toba et al. 1987;
itself composed of a regular pentasaccharide unit to which
Mukai et al. 1990; Hosono et al. 1990; Yokoi et al. 1991;
one or two sugar residues are randomly linked (Kooiman
Pintado et al. 1996; Mitsue et al. 1999; Micheli et al.
1968; Micheli et al. 1999). Subsequent methylation/hydro-
1999; Santos et al. 2003). Whether in fact the bacteria
lysis studies have shown that the structure of keﬁran may
reported are the same has not been studied, nor has any
be more complex than ﬁrst thought (Mukai et al. 1988;
deﬁnitive identiﬁcation been published to fully character-
Mukai et al. 1990). Methylation and NMR analyses have
ize those bacteria reported as keﬁran producers.
also been used to verify the production of keﬁran by new
Bacteria which produce exopolysaccharides are often
bacterial strains (Yokoi et al. 1991). A closer examination
found in milk or milk products, although studies have
of chemical data published by Mukai et al. (1990) raises
shown that maximum production of exopolysaccharide
the question if, in fact, two exopolysaccharides are being
may occur in chemically deﬁned media (containing a
produced that have very similar chemical structure and
´ carbohydrate source, mineral salts, amino acids/peptides,
properties. La Riviere et al. (1967) reported that their
vitamins and nucleic acids) at a constant pH (Mozzi
keﬁran had a 1:1 glucose to galactose ratio and an optical
rotation of þ68.08, while Mukai et al. (1990) isolated a et al. 1996; Dupont et al. 2000). The potential health
properties of keﬁran have prompted several groups to
keﬁran with a glucose to galactose ratio of 0.9:1.1 and an
optical rotation of þ548. Examples can be found in the lit- develop media and growing conditions that optimize
keﬁran production (Toba et al. 1987; Yokoi et al. 1990;
erature where the same bacterial strain produced different
→ 6)-β-D-Gp-{1→2(6)}-β-D-Galp-(1→4)-α-D-Galp-(1→3)-β -D-Galp-(1→4)-β-D-Gp-(1
6(2)
↑
21
1
β-D-Gp
Figure 4. Proposed chemical structure of keﬁran. Reprinted from Carbohydrate Research 7, Kooiman, P. The chemical
structure of keﬁran, the water-soluble polysaccharide of the keﬁr grain, pp 200–211. Copyright 1968 with permission
from Elsevier.
8
Delta O.D. /cfu/ml(x10-12)
8
IM
Lc. lactis
7
6
5
4
3
2
1
0
10
1
IM
10
5
IM
00
2
IM
00
5
IM
00
8
IM
01
1
IM
02
2
IM
02
3
IM
01
4
IM
01
5
IM
08
2
Keﬁr – a complex probiotic E.R. Farnworth
Lb. kefirgranum
Lb. helveticus
Lb. kefir
Ln. mesenteroides
Lc. cremoris (Prt-)
Kefir Strains Reference Strains
Figure 5. Proteinase activity of bacteria from keﬁr and keﬁr grains.
9.2 Bioactive peptides
Yokoi and Watanabe 1992; Micheli et al. 1999; Mitsue
et al. 1999). Media based on lactic acid whey have been
Many organisms possess enzymes (e.g. proteinases and
found to be optimum for keﬁran production. A batch
peptidases) that are able to hydrolyse the protein in a med-
procedure using a modiﬁed MRS media (MRSL) was
ium, thereby supporting growth of the organism by liberat-
reported by Micheli et al. 1999 to produce consistent
ing peptides and amino acids (Thomas and Pritchard
yields of 2 g/l of keﬁran. The best keﬁran yields, how-
1987; Matar et al. 1996). The action of proteinase and
ever, have been reported by Mitsue et al. (1999) when
peptidase enzymes on milk proteins can theoretically
they combined the keﬁran producing bacterium, Lactoba-
result in a very large number of possible peptides. An ana-
cillus keﬁranofaciens, with the yeast Torulaspora del-
lysis of the proteinase activity of keﬁr grain bacterial iso-
brueckii. When these two organisms were grown in a 50
lates has shown that several isolates have high proteinase
l fermentor in a fed-batch protocol, a yield of 3740 mg/l
activities (see Figure 5), which increases the possibility
was obtained over a 7 day period.
that bioactive peptides may be present in keﬁr. In their
No measurements have been reported with regard to
˘
¨
study of lactic acid bacteria in Turkish keﬁr, Yuksekdag
keﬁran concentration in the ﬁnal keﬁr product. However,
et al. (2004b) showed that 13 out of 21 lactococci strains
a comparison of the carbohydrate content of milk
had measurable proteolytic activity.
(USDA 2004) and that of keﬁr shows a more than dou-
Initial studies on the peptide content of keﬁr drink have
bling of the carbohydrate content; how much of this is
shown that keﬁr contains a large number of peptides and
keﬁran is not known. Abraham and De Antoni (1999)
that the majority of keﬁr peptides have molecular weights
did show that the polysaccharide content of keﬁr from
of 5000 kDa (Farnworth 2005, unpublished results).
cows’ milk was almost twice that of keﬁr produced
from soy milk.
Keﬁr grains grown in soy milk produce an exopolysac-
10. Health beneﬁts of keﬁr
charide that Liu et al. (2002) have shown to be primarily
composed of D-glucose and D-galactose (ratio 1.00: 0.43), Keﬁr has had a long history of being beneﬁcial to health
with a molecular weight of approximately 1.7 Â 106 Da. in Eastern European countries, where it is associated with
Keﬁr – a complex probiotic E.R. Farnworth 9
general wellbeing. It is easily digested (Alm 1982c) and is the polysaccharide stimulated PP cells, causing them to
often the ﬁrst weaning food received by babies. Many of secrete water-soluble factors that, in turn, enhanced the
the studies regarding health beneﬁts of keﬁr have been mitogenic response of thymocytes and splenocytes in nor-
published in Russian and Eastern European journals and mal mice.
therefore are not easily accessible to Western science
(Batinkov 1971; Ormisson and Soo 1976; Evenshtein
10.2 Inhibition of tumour growth
1978; Safonova et al. 1979; Ivanova et al. 1981; Sukhov
et al. 1986; Besednova et al. 1997; Oleinichenko et al. Shiomi et al. (1982) were the ﬁrst to report the antitumour
effects of a water-soluble polysaccharide (approximate
1999). However, the health beneﬁts of keﬁr were demon-
molecular weight 1 000 000 Da) isolated from keﬁr grains.
strated in Canada as early as 1932 (Rosell 1932).
Whether given orally or intraperitoneally, the polysacchar-
ide was able to inhibit the growth of Ehrlich carcinoma or
10.1 Stimulation of the immune system
Sarcoma 180 compared to control mice receiving no keﬁr-
derived polysaccharide (Shiomi et al. 1982; Murofushi
It has been proposed that stimulation of the immune sys-
et al. 1983). The mechanism of action was not clear, since
tem may be one mechanism whereby probiotic bacteria
in vitro incubation of the two cancer cell lines with the
may exert many of their beneﬁcial effects (De Simone
et al. 1991; Gill 1998); this may be a direct effect of the polysaccharide showed low cytotoxicity during 42 hours of
bacteria themselves (Cross 2002). However, peptides incubation. This group then went on to show that this
formed during the fermentation process or during diges- water-soluble polysaccharide was able to reach the spleen
tion have also been shown to be bioactive, and demon- and thymus of mice and, based on the response to thymus-
strate a variety of physiological activities, including stimu- dependent and thymus-independent antigens, concluded
lation of the immune system in animal models (LeBlanc that oral immune enhancement was mediated through T-
et al. 2002; Matar et al. 2003). cell, but not B-cell activity. (Murofushi et al. 1986). More
Thoreux and Schmucker (2001) fed keﬁr produced from recently, a water soluble polysaccharide fraction from keﬁr
grains to young (6 months) and old (26 months) rats and grains was shown to inhibit pulmonary metastasis of Lewis
found an enhanced mucosal immune response in the lung carcinoma, whether the keﬁr-derived polysaccharide
young animals, as shown by a higher anti-cholera toxin was given orally before or after tumour transplantation.
Murofushi et al. (1983) also reported the antitumour effec-
(CT) IgA response compared to controls. Both young and
old rats had signiﬁcantly increased total non-speciﬁc IgG tiveness of keﬁr grain polysaccharides regardless of the
blood levels, and a decreased systemic IgG response to time of administration, although they cautioned that larger
CT. Taken together, Thoreux and Schmucker concluded doses may only be more effective if administered after
that keﬁr, like other probiotics, was exerting an adjuvant establishment of the tumours. A water-insoluble fraction
effect on the mucosal immune system, perhaps produced containing keﬁr grain microorganisms, rather than the
by bacterial cell wall components. water-soluble polysaccharide fraction, signiﬁcantly inhib-
Stimulation of the immune system may also occur due ited metastasis of highly colonized B16 melanoma. (Furu-
kawa et al. 1993; Furukawa et al. 2000). It was suggested
to the action of exopolysaccharides found in keﬁr grains.
Murofushi et al. (1983, 1986) used the method of La Riv- that the water-soluble polysaccharide suppressed tumour
´
iere et al. (1967) for the extraction of keﬁran from keﬁr growth by means of the lymphokine activated macrophage
grains to produce a water-soluble polysaccharide fraction (Mf) via the gut-associated lymphoid tissue, while the
that they fed to mice. The reduction in tumour growth that water-insoluble microorganism fraction acted through an
they observed was linked to a cell-mediated response, and increase of NK cell activity.
it appeared that the total dose of the polysaccharide deter- Feeding keﬁr itself (2 g/kg body weight by intubation)
mined its effectiveness. Furukawa et al. (1992) have also was more effective in inhibiting tumour (Lewis lung carci-
shown that a water-soluble fraction of keﬁr grains may act noma) growth than yoghurt, when given for 9 days after
tumour inoculation (Furukawa et al. 1990). It was also
as a modulator of the immune response.
The effect of keﬁr exopolysaccharides on the immune shown that mice receiving keﬁr had an improved delayed-
system may be dependent on whether the host is healthy type hypersensitivity response compared to tumour-bearing
or has developed any tumours. Furukawa et al. (1996) mice receiving no keﬁr, although the mean survival time
was not affected (Furukawa et al. 1991). Kubo et al.
incubated keﬁr grain polysaccharides with Peyer’s Patch
(PP) cells from tumour-bearing mice and found that the (1992) also reported that feeding keﬁr (100–500 mg/kg
supernatant of this mixture enhanced proliferation of sple- body weight) inhibited the proliferation of Ehrlich ascites
nocytes from normal mice and increased the mitogenic carcinoma. In addition, keﬁr, from which the grains had
activities of lipopolysaccharides (LPS) and phytohaemag- been removed by ﬁltration, were shown to kill or arrest
glutinin-P (PHA-P) in splenocytes. They concluded that the growth of fusiform cell sarcomas induced by 7,12-
Keﬁr – a complex probiotic E.R. Farnworth
10
10.3 Keﬁr and lactose intolerance
dimethylbenzanthracene in mice when the keﬁr was
injected intraperitoneally (Cevikbas et al. 1994). Examina-
A proportion of the global population is unable to digest
tion of tissue in keﬁr-treated mice showed a small amount
lactose (the major sugar found in milk), because of insufﬁ-
of mitosis, some stromal connections and, in some cases,
cient intestinal b-galactosidase (or lactase) activity (Alm
disappearance of tumour necrosis.
1982a). Research has shown, however, that lactose maldi-
Hosono et al. (1990) showed that isolates of Streptococ-
gestors are able to tolerate yoghurt, providing the number
cus, Lactobacillus and Leuconostoc in Mongolian keﬁr all
of live bacteria present in the yoghurt consumed is high
showed strong in vitro binding to amino acid pyrolysates
enough (Pelletier et al. 2001). It is believed that the bac-
which are believed to be mutagens and are commonly
teria in the yoghurt matrix are protected by the buffering
found in food. Similarly, Miyamoto et al. (1991) reported
effect of the yoghurt. Bacterial cells remain viable, and
that three slime-producing strains of Streptococcus lactis
the bacterial cell walls remain intact, and thus the b-galac-
subsp. cremoris found in German keﬁr had strong desmu-
tosidase enzyme contained in the yoghurt-producing bac-
tagenic properties, which they attributed to the ability of
teria (L. acidophilus) is protected during transit through
such strains to bind to a known mutagen. Using an Ames
the stomach until it arrives at the upper gastrointestinal
test, Yoon et al. (1999) showed that Lactobacillus spp.
tract (Montes et al. 1995; De Vrese et al. 2001). It has
isolated from keﬁr and yoghurt had antimutagenic proper-
also been shown that fermented milk products have a
ties against the mutagen 2-nitroﬂuorene.
slower transit time than milk, which may further improve
Liu et al. (2002) studied the effects of soy milk and
lactose digestion (Vesa et al. 1996; Labayen et al. 2001).
cows’ milk fermented with keﬁr grains on the growth of
Some keﬁr grains have been shown to possess b-galac-
tumours in mice, using freeze-dried keﬁr (produced from
tosidase activity which remains active when consumed
either soy or cows’ milk) from which the grains had been
(De Vrese et al. 1992). A recent study has shown that a
removed following fermentation. Mice were injected with
commercial keﬁr produced using a starter culture contain-
0.2 Â 108 Sarcoma 180 cells one week prior to the start
ing six bacteria (but not L. acidophilus) and one yeast was
of the feeding portion of the experiment. Tumour growth
equally as effective as yoghurt in reducing breath hydro-
(volume) was estimated for up to 30 days, after which
gen in adult lactose maldigestors (Hertzler and Clancy
tumours were removed and weighed. Both soy milk keﬁr
2003). Severity of ﬂatulence in this group was also
(À70.9%) and cows’ milk keﬁr (À64.8%) signiﬁcantly
reduced when either yoghurt or keﬁr was consumed com-
inhibited tumour growth, compared to mice in the positive
pared to milk.
control group. Microscopic examination of the tumours
De Vrese et al. (1992) showed that when pigs were fed
indicated that apoptosis may have been responsible for
keﬁr containing fresh grains, their plasma galactose con-
reduced tumour growth. Similar effects of yoghurt on
centrations rose signiﬁcantly higher than pigs given keﬁr
apoptosis have been reported (Rachid et al. 2002). Mice
containing heated grains. The diet containing keﬁr and
fed unfermented soy milk did not have reduced tumour
fresh grains had a b-galactosidase activity of 4.4 U/l, which
volumes at day 30, and Liu et al. (2002) concluded that
was identiﬁed as being responsible for the hydrolysis of
either the microorganisms themselves or any polysacchar-
lactose in the intestine, thus yielding galactose that can be
ides formed during fermentation by the keﬁr grains micro-
absorbed. Keﬁr itself contains no galactose (Alm 1982).
ﬂora were responsible for the antitumour response. Genis-
tein itself has been shown to inhibit tumours (Murrill
et al. 1996; Constantinou et al. 1996), although in this
10.4 Antimicrobial properties of keﬁr
study genistein levels did not change during the fermenta-
There are data to show that many lactobacilli are capable
tion process. Mice consuming keﬁr samples also had sig-
niﬁcantly increased levels of IgA in their small intestines of producing a wide range of antimicrobial compounds,
compared to control animals, and it was proposed that the including organic acids (lactic and acetic acids), carbon
dioxide, hydrogen peroxide, ethanol, diacetyl and peptides
PP tissue was increasing IgA secretion into the intestine in
(bacteriocins) that may be beneﬁcial not only in the reduc-
response to food antigens.
¨
Guven et al. (2003) proposed an alternative suggestion tion of foodborne pathogens and spoilage bacteria during
as to how keﬁr may protect tissues. They showed that food production and storage, but also in the treatment and
prevention of gastrointestinal disorders and vaginal infec-
mice exposed to carbon tetrachloride (a hepatotoxin to
tions (Tahara and Kanatani 1997; Zamﬁr et al. 1999;
induce oxidative damage) and given keﬁr by gavage had
´
decreased levels of liver and kidney malondialdehyde, Bonade et al. 2001; Messens and De Vuyst 2002; Jamuna
indicating that keﬁr was acting as an antioxidant. Further- and Jeevaratnam 2004).
Garrote et al. (2000) tested the inhibitory activity of a
more, their data showed that keﬁr was more effective than
supernatant of cows’ milk fermented with keﬁr grains,
vitamin E (which is well known to have antioxidative
properties) in protecting against oxidative damage. against Gram-negative and Gram-positive bacteria. Gram-
Keﬁr – a complex probiotic E.R. Farnworth 11
10.5 Behaviour of keﬁr bacteria in the
positive microorganisms were inhibited to a greater extent
than Gram-negative microorganisms; moreover, both lactic gastrointestinal tract
and acetic acids were found in the supernatants. Garrote
One of the criteria for probiotic bacteria is that they
et al. (2000) showed that milk supplemented with lactic
should be able to withstand the harsh conditions of the
acid or lactic acid plus acetic acid at the concentrations
gastrointestinal tract, including extreme pH conditions pre-
found in the keﬁr supernatant also had inhibitory activity
sent in the stomach and the action of bile salts and diges-
against E. coli 3. They concluded that organic acids pro-
tive enzymes (Lee and Salminen 1995). It is also believed
duced during keﬁr fermentation could have important bac-
that one way in which probiotic bacteria could protect
teriostatic properties even in the early stages of milk fer-
against pathogenic bacteria would be to compete with or
mentation. Cevikbas et al. (1994) found similar results
displace pathogenic bacteria by adhering to intestinal
against Gram-positive coccus, staphylococcus, and Gram-
epithelial cells. (Kirjavainen et al. 1998; Fujiwara et al.
positive bacillus, and noted that keﬁr grains were more
2001; Gibson and Rastall 2003).
effective with regard to their antibacterial properties than
No results from human feeding trials have been pub-
the ﬁnal keﬁr product.
lished with regard to the ability of the microorganisms
Keﬁr grains themselves have inhibitory power against
found in keﬁr to traverse the upper GI tract in large num-
bacteria that can be preserved during lyophilization, parti-
bers and arrive at the large intestine. Keﬁr, because it is
cularly when glycerol is added as a cryopreservative
milk based, is able to buffer the pH of the stomach when
(Brialy et al. 1995). Fresh keﬁr grains were found to inhi-
ingested and thereby provide time for many of the bacteria
bit the growth of the bacteria Streptococcus aureus, Kleb-
to pass through to the upper small intestine (Farnworth
siella pneumoniae and Escherichia coli, but not the yeasts
et al. 2003). Santos et al. (2003) isolated 58 strains of
Candida albicans and Saccharomyces cerevisiae. Leuco-
Lactobacillus spp. and isolates of L. paracasei, L. plan-
nostoc mesenteroides and Lactobacillus plantarum, iso-
tarum, L. delbrueckii, L. acidophilus and L. keﬁranofa-
lated from keﬁr grains, have both been shown to produce
ciens from different sources of keﬁr grains and exposed
antimicrobial compounds that are present in keﬁr. Both
them to an MRS medium at pH 2.5 and MRS containing
inhibit Gram-positive and Gram-negative bacteria, have a
0.3% Oxgall (bile salts). They found that all strains sur-
molecular weight of approximately 1000 kDa and are heat
vived 4 h incubation at pH 2.5, but did not grow. Eighty-
stable, although their antimicrobial properties are reduced
ﬁve percent of isolates showed high resistance to Oxgall,
after exposure to proteolytic enzymes (Serot et al. 1990).
but had delayed growth.
Santos et al. (2003) showed that lactobacilli isolated from
The caco-2 cell assay has been used to show that many
keﬁr grains had antimicrobial activities against E. coli (43/
of the lactobacilli isolated from keﬁr grains are able to
58 strains), Listeria monocytogenes (28/58 strains), Salmo-
bind to enterocyte-like cells (Santos et al. 2003), although
nella typhimurium (10/58 strains), S. enteritidis (22/58
the authors also cautioned that results using this model
strains), S. ﬂexneri (36/58 strains) and Yersinia enterocoli-
might not necessarily apply in vivo.
tica (47/58 strains). Bacteriocins were thought to be
Human studies of the effects of diet on intestinal micro-
responsible, although they were not identiﬁed.
ﬂora are limited to the analysis of faecal samples,
In a study in which foodborne bacterial pathogens (E.
although no detailed human study has been published in
coli O157:H7, L. monocytogenes 4b, Y. enterocolitica 03)
which keﬁr has been used. Marquina et al. (2002) used
were added at the beginning of yoghurt or keﬁr fermenta-
mice to study the effect of consuming keﬁr (source not
tion, both keﬁr and yoghurt failed to inhibit pathogenic
deﬁned) in a feeding study that lasted 7 months. They
bacterial growth. For keﬁr, this was explained as being
were able to show that the numbers of lactic acid bacteria
due to the slow acid development during fermentation.
in the mouse small and large intestines increased signiﬁ-
Interestingly, fermentations of keﬁr and yoghurt combina-
cantly. Streptococci increased by 1 log, while sulﬁte-redu-
tions proved to be more effective at pathogen suppression
cing clostridia decreased by 2 logs.
than single fermentation (Gulmez and Guven 2003)
Hydrogen peroxide is another metabolite produced by
˘
¨
some bacteria as an antimicrobial compound. Yuksekdag
et al. (2004a) showed that all 21 isolates of lactic acid
10.6 Keﬁr and cholesterol metabolism
bacteria from Turkish keﬁr produced hydrogen peroxide
Positive effects of yoghurt consumption on cholesterol
(0.04–0.19 ug/ml). In a later paper, they reported that 11
out of 21 strains of keﬁr lactococci produced hydrogen metabolism have been reported (Kiessling et al. 2002;
˘
¨
peroxide (Yuksekdag et al. 2004b). All lactococci strains Xiao et al. 2003), although a review of the literature
were effective in inhibiting growth of Streptococcus aur- reveals that the results are at best moderate, and are often
inconsistent (Taylor and Williams 1998; St-Onge et al.
eus, but were less effective against E. coli NRLL B-704
and Pseudomonas aeruginosa. 2000; Pereira and Gibson 2002).
Keﬁr – a complex probiotic E.R. Farnworth
12
Several hypotheses have been proposed regarding the found in keﬁr. Furthermore, there is evidence to show that
possible mechanism of action employed by bacteria to keﬁr consumption not only affects digestion, but also
reduce cholesterol levels (St. Onge et al. 2002). Vujicic inﬂuences metabolism and immune function in humans.
et al. (1992) showed that keﬁr grains from Yugoslavia,
Hungary and the Caucase region were able to assimilate 12. References
cholesterol in milk either incubated at 208C for 24 h
Abraham, A.G. and De Antoni, G.L. 1999. Characterization of
(reductions of up to 62%) or incubated and stored at 108C keﬁr grains grown in cows’ milk and in soya milk. Journal of
for 48 h (reductions of up to 84%). These authors claimed Dairy Research 66: 327-333.
that their results indicated that keﬁr grains had a choles- Alm, L. 1982a. Effect of fermentation on lactose, glucose, and
galactose content in milk and suitability of fermented milk
terol-degrading enzyme system. Similar results were
products for lactose intolerant individuals. Journal of Dairy
reported for 27 lactic acid bacterial strains. However, it
Science 65: 346-352.
was pointed out that isolates from dairy products had Alm, L. 1982b. Effect of fermentation on B-vitamin content of
lower cholesterol-assimilating capacity than strains iso- milk in Sweden. Journal of Dairy Science 65: 353-359.
lated from infant faeces (Xanthopoulos et al. 1998). Alm, L. 1982c. Effects of fermentation on curd size and digest-
ibility of milk proteins in vitro of Swedish fermented milk
In a clinical trial in which 13 subjects were fed 500 ml/
products. Journal of Dairy Science 65: 509-514.
day of keﬁr for 4 weeks in a placebo-controlled design, per-
Alm, L. 1982d. Effect of fermentation on L(þ) and D(À) lactic
centage changes in serum triglycerides compared to base- acid in milk. Journal of Dairy Science 65: 515-520.
line levels were lower (although not signiﬁcantly) than Alm, L. 1982e. Effect of fermentation on proteins of Swedish
when subjects consumed unfermented milk; the percentage fermented milk products. Journal of Dairy Science 65: 1696-
1704.
serum high-density lipoprotein (HDL) cholesterol change
Angulo, L., Lopez, E. and Lema, C. 1993. Microﬂora present in
compared to baseline increased (although not signiﬁcantly)
keﬁr grains of the Galician region (North-West of Spain).
when subjects consumed keﬁr compared to milk (St. Onge Journal of Dairy Research 60: 263-267.
et al. 2002). Similarly, Kiessling et al. (2002) found that Arihara, K., Toba, T. and Adachi, S. 1990. Immunoﬂuorescence
HDL levels increased after 6 months of feeding yoghurt microscopic studies on distribution of Lactobacillus keﬁranofa-
ciens and Lactobacillus keﬁr in keﬁr grains. International
supplemented with Lactobacillus acidophilus and Biﬁdo-
Journal of Food Microbiology 11: 127-134.
bacterium longum, thereby producing an improved low-
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