kefir: a multifaceted fermented dairy product
TRANSCRIPT
Kefir: A Multifaceted Fermented Dairy Product
Barbara Nielsen • G. Candan Gurakan •
Gulhan Unlu
Published online: 27 September 2014
� Springer Science+Business Media New York 2014
Abstract Kefir is a fermented dairy beverage produced
by the actions of the microflora encased in the ‘‘kefir grain’’
on the carbohydrates in the milk. Containing many bacte-
rial species already known for their probiotic properties, it
has long been popular in Eastern Europe for its purported
health benefits, where it is routinely administered to
patients in hospitals and recommended for infants and the
infirm. It is beginning to gain a foothold in the USA as a
healthy probiotic beverage, mostly as an artisanal bever-
age, home fermented from shared grains, but also recently
as a commercial product commanding shelf space in retail
establishments. This is similar to the status of yogurts in
the 1970s when yogurt was the new healthy product. Sci-
entific studies into these reported benefits are being con-
ducted into these health benefits, many with promising
results, though not all of the studies have been conclusive.
Our review provides an overview of kefir’s structure,
microbial profile, production, and probiotic properties. Our
review also discusses alternative uses of kefir, kefir grains,
and kefiran (the soluble polysaccharide produced by the
organisms in kefir grains). Their utility in wound therapy,
food additives, leavening agents, and other non-beverage
uses is being studied with promising results.
Keywords Kefir � Kefir grain � Kefiran � Probiotic �Lactic acid bacteria � Fermented dairy product
Well before the advent of microbiology, humans learned
that certain foods, encouraged to ferment, would not spoil
as quickly and thus could be prepared in times of plenty for
use when food was scarce. Often these foods would also
develop pleasing aromas, flavors, and textures, as well as
enhanced nutritional traits. Milk is a commonly fermented
commodity. Fermented milks are popular worldwide, with
many world regions enjoying their own particular varieties.
A few of these cultured milks have broken from their
regional confines and now enjoy worldwide acceptance.
The most notable of these is yogurt [1] found in grocery
stores almost anywhere in the world. But kefir, a fermented
dairy beverage long popular in Eastern Europe, with its
roots in the Caucasus mountain region of central Asia [2–
4], is gaining new acceptance worldwide.
Kefir: An Introduction
The name ‘‘kefir’’ is likely derived from the Turkish word
‘‘keyif’’ which means ‘‘good feeling’’ [2]. Kefir is an
acidic, viscous, somewhat effervescent, slightly alcoholic
milk beverage produced by the actions of bacteria and
yeast embedded in a resilient, insoluble protein and poly-
saccharide matrix known as a ‘‘kefir grain’’ [5–7]. While
other fermented milks are produced using the practice of
back slopping, or adding a sample of fermented milk as
inoculum to fresh milk to produce more of the fermented
milk product (the common fermentation start for yogurts,
viili, filmjolk, and other traditional fermented milks), tra-
ditional kefir requires inoculating fresh milk with the entire
kefir grain and allowing fermentation to occur [3, 4]. This
is because of the complex symbiotic interactions between
the organisms in the kefir grain in their production of kefir,
rendering a beverage with a differing microbial profile than
B. Nielsen � G. Unlu (&)
School of Food Science, University of Idaho,
875 Perimeter Drive, Moscow, ID 83844-2312, USA
e-mail: [email protected]
G. C. Gurakan
Department of Food Engineering, Middle East Technical
University, Universiteler Mah., Dumlupınar Blv. No: 1,
06800 Cankaya, Ankara, Turkey
123
Probiotics & Antimicro. Prot. (2014) 6:123–135
DOI 10.1007/s12602-014-9168-0
that found in the kefir grain [8–11]. After fermentation, the
grain is filtered out to use as the inoculum for the next
batch. Theoretically, the grain as inoculum for subsequent
batches should be effective for infinite batches of kefir,
given the proper environmental conditions.
Though cow’s milk is most common, kefir can be made
from any type of milk. For dairy kefir, cow, goat, or sheep
milk are all commonly used [3]. Kefir is best made with
milk containing fat. As there is an established relationship
between many health problems and the consumption of
saturated fats and cholesterol, a non-fat choice in kefir is
desirable; however, non-fat milk makes a kefir with sig-
nificantly lower quality. Ertekin and Guzel-Seydim [12]
experimented with non-fat milk supplemented with the fat
substitutes inulin and Dairy-Lo� to improve the quality of
kefir made with skim milk. They found that while kefir
grains fermenting whole fat milk resulted in the best-
quality kefir, the fat substitutes did improve the quality of
the non-fat kefir fermentations. Kefir can also be prepared
using non-dairy beverages such as walnut milk [13], cocoa-
pulp beverage [14], soy milk [3, 15, 16], coconut milk [3],
rice milk [3], and peanut milk [17]. Supplementing the
alternative milk with 1 % glucose, lactose, or sucrose
helped stimulate lactic acid bacteria (LAB) and yeast
growth and the production of lactic acid and ethanol [15].
Non-dairy ‘‘milks,’’ while they do ferment and produce a
fermented product with probiotic properties, tend to leave
the kefir grain in a weakened state. After a few fermenta-
tion cycles in a non-dairy product, the grains should be
returned to a dairy milk containing fat to strengthen the
grain.
Kefir as a traditional beverage predates written record
[18]. It originated in the Caucasus mountains in Central
Asia 1,000s of years ago [3]. Legends have arisen around
kefir’s origin. Legend has it that the original kefir grains
were given to the Orthodox Christians of the region by the
prophet Muhammad with the strict instruction to never
share them [19]. Other tales of deception and intrigue
explain how the grains finally became more widely avail-
able [20].
In whatever manner the grains originated and were
disseminated, and kefir grains and the resultant kefir bev-
erage product can now be found all over the world. Grains
in active commercial or artisanal use are found all over
Europe (Bulgaria [8], Portugal [21, 22], Ireland (Buttermilk
plant) [23–25], Austria, Germany [6, 26], Poland [27],
France [22], Italy [22], Spain [22, 28–30], Sweden [7], and
Denmark [5]); Eurasia (Turkey [6, 22, 31] and Russia
[22]); and Asia (Iran [32, 33], China [34], Tibet [35, 36],
Japan [37], Taiwan [38, 39]); as well as in artisanal use in
South America (Brazil [40, 41], Argentina [42]), and
Africa (South Africa [43, 44]). Indeed, researchers study-
ing kefir often cite the source of their grains as being from
private households or local dairies in their various coun-
tries. Though there are similarities and in some cases direct
evidence and/or legend linking the grains [24, 25], it is not
clear whether all can trace their origins back to the Cau-
casus region [18, 45]. Grain formation may have happened
several times and in differing locations over the history of
man storing milk [45]. Many of the grains show regional
differences in grain structure [5, 6, 23] and microbial
profile [6, 7, 18, 31, 45]. These differences may be due to
the differing sources of the kefir grains, different tech-
niques employed during processing, differing ambient
temperatures globally, and the local LAB finding a niche in
the grains [5, 21, 24, 45, 46].
Kefir grains are a fascinating biological entity. They are
irregular, with an appearance of cauliflower, coral, cottage
cheese, or popcorn, off-white to pale yellow, and range in
size from several mm to a few cm or more [4, 7, 27, 47].
They are a complex community of around 30 species of
LAB and yeast [27] embedded in a polysaccharide and
protein matrix. Simova et al. [8] describe kefir grains as
behaving ‘‘as biologically vital organisms.’’ They grow,
propagate, and pass their properties on to the following
generations of new grains. Cui et al. [13] reported the kefir
grains as ‘‘hav(ing) a specified structure and behave(ing) as
biologically vital organisms.’’ Dr. Lynn Margulis had an
interesting observation in her study of kefir and evolution.
In her essay appearing in ‘‘Scientific American’’ in 1994,
then expanded and published in several essay collections,
she noted that the kefir grain ‘‘arose from the physical
association of 30 different kinds of microbes… remain(ing)
together in precise relationships as each divides, main-
taining the integrity of the individual curd.’’ In short,
Margulis maintains that ‘‘Kefir is a new individual, more
complex than its components…. A sparkling demonstration
that integration processes by which our cells evolved still
occur’’ [19, 47].
Kefir Structure
Kefir grains are made up of bacteria and fungi embedded in
a resilient insoluble polysaccharide matrix composed of
glucose and galactose known as kefiran [5, 23]. This car-
bohydrate is of bacterial origin, produced by some of the
lactobacilli embedded in the matrix [5]. The arrangement
of the microflora within the grains is still a subject of
debate. In some cases, scanning electron microscopy
(SEM) has shown the lactobacilli mainly near the exterior
of the kefir grain and the yeasts mainly toward the center
[48]. In areas where yeasts predominate, there are few
bacteria; where lactobacilli predominate, there are few
types of yeast [5, 48]. In other preparations, the SEM
revealed lactobacilli and yeast in comparable ratios
124 Probiotics & Antimicro. Prot. (2014) 6:123–135
123
between the exterior and interior of the grain, though there
were fewer total organisms in the interior [23, 49]. Another
research group found rod-shaped bacteria in both the inner
and outer grain portions of three Brazilian kefir grains with
yeasts most frequent in the outer portion [50]. One study, in
contrast to the others, observed a variety of lactobacilli but
no yeast in the interior [31].
Kefir grains appear to start out as thin sheet-like struc-
tures, developing into mature grains with the sheets folding
themselves into scrolls and rolls [5]. In observations by
Marshall et al. [5], one side of this sheet appears to be
smooth and flat; the other side is convoluted and rough.
The microflora of the kefir, as examined under SEM, is not
indiscriminately intermingled, but has a particular
arrangement in the grain yeasts, and short lactobacilli are
predominantly on the convoluted side, short lactobacilli on
smooth side. The zone in the polysaccharide matrix
between the smooth side and the rough side shows large
number of long, curved bacteria. These bacteria may be the
ones creating the kefiran composing the matrix [5]. The
structure of the grains suggests that grains arise from
curling of flat sheet-like structures with subsequent folding
and refolding, the grain size growing with the carbohy-
drate/microflora increase. The yeasts are predominantly
found in the interior because they adhere to the convoluted
side and thus fold to the interior [5]. Wang et al. [51]
suggested a further possible mechanism for grain folding
structure: Most LAB are hydrophilic and have a negative
charge on their cell surface; Lact. kefiranofaciens HL1 and
Lact. kefir HL2 are hydrophobic and have a positively
charged cell surface, allowing self-aggregation. Proteins in
the bacterial cell wall surface and polysaccharides in the
yeast cell wall play important roles in co-aggregation and
microbial adhesion [34]. The yeast involved enhances
aggregation, adhesion, and survival in harsh conditions
[34]. Yamin et al. [6] studied some kefir grains from
Germany that had pouches incorporated into the kefir grain
structure, a feature not seen in other samples. The outsides
of these pouches were rough, while the inner sides were
smooth. These German grains were much larger than the
Turkish grain samples they studied. Perhaps these have a
different folding mechanism.
Kefir grains cannot be synthesized artificially. They do
not form spontaneously when pure cultures of the organ-
isms involved are placed together in a test tube. But under
the proper conditions, kefir grains can apparently be
encouraged to form and grow in traditional ways. The
traditional way to ferment milk for kefir was in goatskin
bags. Fermentation of milk in skin bags as a way of pre-
serving the milk led to the first kefir grains and started the
long tradition of producing kefir [26]. These bags would
traditionally be hung by entrances to peoples’ homes,
where people entering or leaving would kick or hit the bags
to agitate the contents [52]. Bags could also be carried as
people traveled, the bumpiness of the ride mixing the
contents. Motaghi et al. [32] tested this hypothesis with
some success when they filled a goat-hide bag with pas-
teurized milk and intestinal flora from sheep, incubating at
24–26 �C, shaking hourly, and replacing 75 % of the milk
at a time as it coagulated. After 12 weeks, a polysaccharide
layer had formed on the surface of the hide. This was
removed and propagated in milk. From this, they were
apparently able to obtain kefir grains.
Kefir grains appear to be very hardy. Kolakowski and
Ozimkiewicz [27] subjected the grains to homogenization,
rinsing, freezing in liquid nitrogen, frozen storage, cool
storage, and freeze-drying and milling. They found that, in
unfavorable conditions, grain growth is disturbed, their
appearance deteriorates, and they lose their resilience.
They shrink and their microbial balance is disrupted. When
favorable conditions return, after multiple passes, they
retrieve their typical appearance, physiological functions,
and technological properties, with the exception of the
freeze-dried and milled samples. Those grains never
reformed. Studies have been done on the bacterial popu-
lations and activity of kefir grains through freeze-drying
the grains [43] or the LAB isolated from the grains [53],
but there has been no report of the grains reforming. Many
commercial companies offer freeze-dried ‘‘Kefir starters,’’
which will not form grains and do not seem to remain
stable through more than a few fermentation cycles. These
do, however, produce a product that, while different from
the traditional product, is more uniform, making produc-
tion less laborious and ensuring a longer shelf life of the
product [54]. This is desirable, as a viable commercial
product needs to be uniform in culture and remain stable in
storage. Traditional kefir culturing is at a commercial dis-
advantage, as the uniformity and shelf life cannot be
guaranteed. The lactic acid concentration and the acetal-
dehyde, acetoin, and gas production increase upon storage
of traditional kefir, reaching peak acceptability levels in the
first 2 days [29]. A uniform freeze-dried kefir grain with
optimized viability of kefir organisms would be desirable
for the commercial market [55].
Kefir’s Microbial Profile
As mentioned earlier, kefir grains are a complex commu-
nity of around 30 species (or more) of LAB and yeast [27].
Early attempts at isolation were hampered by the fastidious
nature of the organisms involved. The LAB is aerotolerant
anaerobes with exacting nutritional requirements. Critical
organisms for the production of kefir may have remained
undiscovered due to missing nutritional components in the
culture media. Even with these limitations, many varieties
Probiotics & Antimicro. Prot. (2014) 6:123–135 125
123
of yeasts (Saccharomyces sp., Kluyveromyces sp., Candida
sp., Mycotorula sp., Torulaspora sp., Cryptococcus sp.,
Pichia sp. etc.) and LAB (Lactobacillus sp., Lactococcus
sp., Leuconostoc sp., etc.) have been isolated and identified
from kefir and kefir grains using established biochemical
profiles.
In last decade, culture-independent identification tech-
niques, in which cultivation in growth media is not
required, have received more attention. Several molecular
techniques varying in discriminatory power, reproducibil-
ity, and required effort have been developed. Among those
techniques, some molecular techniques such as denaturing
gradient gel electrophoresis (DGGE) and/or analysis of the
16S rRNA gene libraries were extremely useful to assess
the complex microbial population and diversity of strains
in probiotic preparations, kefir, and Coffea arabica [56–
60]. As Lactobacillus species are found to be the prevalent
group in the final kefir product, many studies have been
carried out to identify and type isolates of Lactobacilus
species in kefir grains.
The 16S rRNA gene and the 16S–23S intergenic spacer
region are successfully used for identification of Lactoba-
cillus isolates at the species level. PCR amplification and
DNA sequencing of variable regions of the 16S rRNA gene
such as the V1 region [61], a 500-bp region including the
V1 and V2 regions [62], the V2–V3 region [63], and the
1,500-bp region [63] of the 16S rRNA gene have allowed
species-specific identification, also in combination with
some techniques such as DGGE and amplified ribosomal
DNA restriction analysis (ARDRA).
Molecular identification of Lactobacillus isolates from
kefir grains by analysis of the 1,500-bp section of the 16S
rRNA gene and ARDRA was reported [60]. In this study,
the researchers discriminated the bacterial isolates at the
species level using the 16S–23S rRNA region. Moreover,
genotyping of Lactobacillus isolates from kefir grains was
performed using random amplified polymorphic DNA-
PCR (RAPD-PCR) analysis with four different primers. A
similar study using ARDRA and analysis of the 16S rRNA
internal spacer region was reported on identification of
homofermentative lactobacilli from kefir grains [65]. They
confirmed that all the homofermentative Lactobacillus
strains were plantarum species and showed desirable pro-
biotic properties. Lactobacillus species present in the gas-
trointestinal tract were differentiated and identified using a
combination of DGGE and species-specific primers for the
16S–23S rRNA intergenic spacer region or the V2–V3
region of 16S rRNA [63].
Pyrosequencing data using the V4 variable regions of
the 16S rRNA in a recent study have indicated that mic-
robiota of kefir milk and the starter grain are quite different
and that microbial diversity of the starter grain varies due
to the interior structure of the kefir starter grain [66].
Sequencing-based analysis of kefir grains and their milk-
based fermentation products have recently yielded detailed
bacterial and fungal composition profiles, identifying sev-
eral genera and species not previously identified in kefir
[46]. PCR amplification of the V3 region of the 16S rRNA
gene was used for pyrosequencing in addition to PCR-
DGGE fingerprint analysis of the microbial communities in
Brazilian kefir grains [49, 60]. Group-specific primers were
used for the detection of LAB [56, 60].
Lactobacillus acidophilus is one of the predominant
Lactobacillus species found in many kefir grains [40, 67].
However, identification and/or differentiation of six sepa-
rate species in Lact. acidophilus complex, including Lact.
acidophilus, Lact. amylovorous, Lact. crispatus, Lact. ga-
linarium, Lact. gasseri, and Lact. jonsonii, is difficult even
by molecular methods [68]. This differentiation was
achieved using multiplex PCR with species-specific prim-
ers from the 16S–23S intergenic spacer region and the
flanking region of the 23S rRNA gene in the study of Song
et al. [69]. Kullen et al. [62] have also differentiated these
strains by amplification and sequencing of the 500-bp
region of the 16S rRNA gene containing the V1 and V2
variable regions.
At the subspecies level, strain typing of certain species
was best achieved by the PFGE technique [68]. Strains of
Lact. acidophilus complex, including Lact. delbrueckii
subspecies (Lact. delbrueckii subsp. bulgaricus, Lact. del-
brueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis),
Lact. plantarum, Lact. fermentum, Lact. rhamnosus, and
Lact. sakei, were analyzed by PFGE [68]. The RAPD [64],
AFLP, and ribotyping are some other methods used for
molecular typing of kefir isolates of certain species.
Yeast strains in kefir grains also play crucial role in
fermentation and in forming the flavor and aroma in the
final product. Thus, identification studies of yeast strains
from kefir grains have been carried out. Kluyveromyces
maxianus, Torulaspora delbrueckii, Saccharomyces cere-
visiae, Candida kefir, Saccharomyces unisporus, Pichia
fermentans, Kazachastania aerobia, Lachanceae meyersii,
Yarrowia lipolytica, and Kazachstania unispora are found
as major yeast populations [8, 60, 70, 71]. Similar methods
for identification of LAB are also used for identification of
yeast isolates. Among molecular identification techniques,
restriction fragment length polymorphism (RFLP), DNA/
DNA hybridization, and PCR-DGGE are some that are
widely used.
Kefir Production
Traditional kefir production does not lend itself to large-
scale production, as the volumes required would make
fermentation uneven and grain recovery laborious and
126 Probiotics & Antimicro. Prot. (2014) 6:123–135
123
impractical [72]. Pure culture starts and lyophilized starts
have been developed, eliminating the need to recover
grains [9, 72], but the product does not stay true through
additional fermentation cycles. Russian-style kefir is made
by taking the traditional kefir product, removing grains,
and inoculating it into pasteurized milk at a concentration
of 1–3 %, and then subjecting it to incubation and matu-
ration. Industrial kefir is made by then taking Russian-style
kefir and inoculating it into pasteurized milk at a concen-
tration of 2–3 % and then subjecting it to incubation and
maturation. Every pass results in a change in the microbial
composition of the kefir and a decline in the quality of the
beverage [8]. After the cycle leading to industrial kefir, the
product has lost most of its kefir characteristics.
Any kefir product prepared for widespread commercial
distribution would have to be consistent and defined. As
grains vary by origin [45], consistency is hard to control
[73]. In a review on innovations in production of kefir,
Sarkar [74] concluded that a scientifically developed
defined starter culture would be desirable in improving the
quality and consistency of commercial kefir. Much
research has been done to determine the microbial popu-
lations of kefir grains in the attempt to develop a pure
culture inoculum. Beshkova et al. [9] optimized a starter
culture using selected microorganisms isolated from kefir
grains and varying culture conditions to produce a kefir-
like beverage with very good sensory properties. They
concluded that a standardized production was possible.
Chen et al. [38] experimented on making a synthetic ‘‘kefir
grain,’’ entrapping bacteria and yeasts in two different
microspheres in which the entrapment ratio of the strains
was based on the distribution ratio found in kefir grains.
They prepared yeast microspheres and bacterial micro-
spheres, then made kefir using the entrapped culture starter,
passing it through 28 fermentation cycles. Nambou et al.
[73] combined six pure microbial strains in varying con-
centrations, finding one that closely approximated the
characteristics of traditional kefir.
Kefir as a Probiotic
Kefir has long been used in Eastern Europe for its pur-
ported health benefits. But, like most of the foods touted as
health promoting in other countries and cultures, its ben-
efits are accepted as common knowledge, with, until fairly
recently, little peer-reviewed scientific evidence to support
the claims [18]. Farnworth [18] gives the example that in
Russia, a daily serving of kefir is standard practice in many
hospitals because it is believed to be a ‘‘general health
promoter,’’ particularly good in the recovery from digestive
maladies, and is recommended to mothers to use during
weaning. Most opinion on the beneficial effects of kefir
was, and to a certain degree, still is based on anecdotal
evidence and personal experience. The Internet is full of
testimonials not backed up by scientific research. But as the
interest in kefir’s health effects grows, the amount of
research has grown to where there is now and offers some
solid evidence as to kefir’s health benefits.
Kefir has been touted for use as a probiotic. There are
many current scientific studies and some excellent reviews
in the current literature dealing with the nutritional and
probiotic characteristics of kefir [50, 75, 76]. The term
‘‘probiotic’’ was defined by Fuller as ‘‘a live microbial feed
supplement that beneficially affects the host beyond cor-
recting for nutritional deficiencies by improving the intes-
tinal balance’’ [77]. This definition was broadened at the
Joint FAO/WHO Expert Consultation on Evaluation of
Health and Nutritional Properties of Probiotics in Food
Including Powder Milk with Live Lactic Acid Bacteria
held in 2001. The FAO/WHO defined a probiotic as ‘‘Live
microorganisms which when administered in adequate
amounts confer a health benefit on the host’’ [78]. Effective
probiotics are required to have these properties: must
adhere to cells; must exclude or reduce pathogenic adher-
ence; must persist and multiply; must produce acids, per-
oxide, and bacteriocins antagonistic to pathogen growth;
must be safe, noninvasive, non-carcinogenic, and non-
pathogenic; must co-aggregate to form a normal, balanced
flora [79, 80]. Also, probiotics need to be able to survive
the harsh acid/bile conditions in the digestive tract [65].
The concept of probiotics was pioneered by Elie
Metchnikoff in his work, The Prolongation of Life: Opti-
mistic Studies, originally published in 1908. Dr. Metch-
nikoff theorized that intestinal putrification shortened life.
He observed that humans who consumed fermented foods
showed remarkable health benefits, ‘‘absorb(ing) quantities
of lactic microbes by consuming in the uncooked condition
substances such as soured milk, kefir, sauer-kraut, or salted
cucumbers which have undergone lactic fermentation. By
these means they have unknowingly lessened the evil
consequences of intestinal putrification.’’ He reported on
many races making copious use of soured milk with ben-
efits of health and longevity. Cautioning that ‘‘in a question
so important, the theory must be tested by direct observa-
tion,’’ he left it to the ‘‘future, near or remote, that we shall
obtain exact information upon what is one of the chief
problems of humanity.’’ Dr. Metchnikoff, however, did not
approve of kefir due to its alcohol content and varied
microbial flora [81].
Dr. Metchnikoff’s views notwithstanding, today kefir is
valued for its health benefits. Since Dr. Metchnikoff’s
groundbreaking studies, and especially over the last couple
of decades, serious kefir research has increased. Today
there is significant research available on the use of kefir as
a probiotic. Several of the microorganisms that make up
Probiotics & Antimicro. Prot. (2014) 6:123–135 127
123
kefir are known probiotics, for example: Lact. acidophilus,
Lact. casei, Lact. paracasei, Lact. fermentum, and Sac-
charomyces cerevisiae [80]. Other organisms, known and
unknown, that may be found in kefir may yet be found to
have probiotic properties.
The properties kefir exhibits indicate it may be useful as
a probiotic. Golowczyc showed that several bacteria iso-
lated from kefir showed a high resistance to bile and low
pH conditions and were able to adhere to intestinal epi-
thelium [65]. Yeasts present in the kefir have been shown
to enhance aggregation and adhesion of LAB to the epi-
thelial cells; they also strengthen LAB gastrointestinal
tolerance [26, 34]. Organisms isolated from kefir grains
have also been shown to produce substances antagonistic to
pathogen growth, such as organic acids and bacteriocins
[41, 43, 82]. The bacteria have shown competitive adhesion
interfering with the adhesion of pathogenic bacteria [22,
34]. These bioactive properties of kefir may have various
causes. They may be due to the action of microorganisms
themselves (either dead or alive). They may be due to
metabolites of the organism formed during fermentation.
Finally, they may be due to the actions of the breakdown
products of the foods involved [4].
Reported probiotic activity of kefir includes protection
from toxins. In recent studies, a kefir isolate Lactococcus
lactis subsp. lactis was shown to inhibit the cytotoxic effect
of Clostridium difficile on eukaryotic cells in vitro [83].
Another study showed protection of Vero cells from type II
shiga toxin from Escherichia coli O157:H7 using Lact.
plantarum [84, 85]. The cell surface adhesion proteins of
Lact. plantarum appear to be critical for the protection of
cells against injury from E. coli [86]. The expression of
functional cell wall proteins may be involved as cell sur-
face proteins may mimic the receptors on any specific
target for pathogens and toxins [84].
The antimicrobial probiotic aspects of kefir are also
noteworthy. The organisms in kefir produce many known
antimicrobials, including lactic acid, acetic acid, carbon
dioxide, hydrogen peroxide, ethanol, diacetyl, and antimi-
crobial peptides such as bacteriocins [4]. Golowczyc et al.
[65] found that several lactobacilli in kefir exhibited pro-
biotic potential, surviving bile salts and stomach acid and
some of them adhering to Caco-2 cells moderately well.
Many isolates were antagonistic to pathogens, an effect not
seen in artificially acidified media, suggesting that the
production of organic acids was not the inhibitory factor.
Powell et al. found a bacteriocin (bacST8KF) produced by
Lact. plantarum isolated from kefir that showed inhibition
of both Gram-positive and Gram-negative bacteria [44].
Silva et al. [41] demonstrated antimicrobial activity against
several pathogens during kefir fermentations using various
sugar broths. Sezer and Guven [82] isolated a bacteriocin-
producing lactic acid bacterium. They partially purified a
bacteriocin that showed strong antimicrobial activity
against both Gram-positive and Gram-negative bacteria.
Santos et al. [22] investigated several Lactobacillus iso-
lates against six pathogenic bacteria and found about 75 %
showed antimicrobial activity against E. coli and Yersinia
enterocolitica, 64 % showed inhibition against Shigella
flexneri, 50 % showed inhibition against Listeria mono-
cytogenes, 40 % showed inhibition against Salmonella
enteritidis, and 19 % showed inhibition against Salm. ty-
phi. This seems to be due to secreted antimicrobial sub-
stances. Kefir isolates may also demonstrate antimicrobial
activity due to bacterial interference as Lactobacillus
adheres to receptor sites in the gut. The inhibition of the
attachment of Salm. typhimurium to Caco-2 cells appears to
be directly related to the adhesion capacities of the Lac-
tobacillus isolates [22]. Kefir shows inhibition of one
bacterium of particular interest, Helicobacter pylori, which
has been linked to chronic gastritis, ulcers, and gastric
cancer [87]. Oh et al. [88] isolated two yeasts and several
strains of lactobacilli from a traditional Tibetan kefir-like
yogurt, which, in combination, showed near 100 % bacte-
ricidal activity against H. pylori, mediated by soluble
factors in the kefir. Zubillaga et al. [89] report that kefir has
a stimulatory effect on the motor and emptying function of
the gastric stump, which would also have a beneficial effect
in the control and treatment of H. pylori.
Kefir affects blood pressure through angiotensin-con-
verting enzyme (ACE) inhibition. ACE is one of the main
molecules responsible for increasing blood pressure
because it is necessary for the conversion of angiotensin I
to angiotensin II, a potent vasoconstrictor. ACE also
inactivates bradykinin, a vasodilator [90]. Nakamura et al.
[91] isolated peptides from Calpis sour milk (a traditional
Japanese milk fermented with Lact. helveticus and S. cer-
evasiae) where both the orally administered milk and the
peptides inhibited ACE. Quiros et al. [90] found similar
ACE activity in a commercial kefir made from caprine
milk. They were able to isolate several low molecular mass
peptides, of which two showed potent ACE inhibitory
properties [90].
Studies on the benefits of kefir on cholesterol reduction
have shown mixed results. Early studies on fermented
milks looked promising, with studies showing bacteria
apparently removing cholesterol from media in vitro [92,
93]. This effect was later found to be at least partially due
to the cholesterol precipitating out of solution in the pre-
sence of bile salts, and not totally due to bacterial assimi-
lation [94, 95]. Cholesterol consumption or removal
in vitro is not a good index of its cholesterol-lowering
potential in vivo [96]. Testing has been done on kefir,
bacterial isolates of kefir, and kefiran in mice, rats, ham-
sters, and humans with mixed results. St. Onge et al. [97]
studied the effects of drinking 500 ml kefir/day over a
128 Probiotics & Antimicro. Prot. (2014) 6:123–135
123
4-week period in a double-blind experiment and found no
significant total cholesterol, HDL cholesterol, LDL cho-
lesterol, or triglyceride lowering effect. Others performed
similar experiments using rats and hamsters with mixed
results [30, 98]. These mixed results may be due to dosage
inequities and/or the variability in kefir composition. The
human study used 500 ml kefir made with 2 % milk, while
one animal study used 0.42 % by weight whole milk kefir
in their feed, resulting in slightly elevated serum triacyl-
glycerol, total cholesterol, and high-density lipoprotein
cholesterol [30], and another animal study used 10 %
lyophilized skim milk or 10 % soyamilk kefir in their feed,
resulting in slightly reduced levels of serum cholesterol
[98]. The milk fat involved in these three studies may have
influenced the results, as well as the gross bacterial count
differences, relative body size/dose, soy versus dairy milk,
and the variable mixed culture nature of the kefir itself.
Other studies used kefir fractions, screening for bacteria
that show the best effect and using pure cultures of those
selected bacteria [35, 36, 99] or using purified kefir poly-
saccharide, kefiran [100, 101]. Some bacterial isolates
showed significant decreases in total cholesterol, triglyc-
erides, LDL, and HDL [35, 36, 92] as did the kefiran trials
[100, 101]. It appears that large numbers of the correct
probiotic bacteria isolates from kefir, as well as doses of
purified kefiran, may be therapeutic in the treatment of high
cholesterol.
Vinderola et al. [102] have shown that kefir has an
immunomodulating effect. They have shown that the
introduction of kefir in varying dilutions to mice increases
the number of IgA? cells in the intestinal and bronchial
mucosa. They conclude that different components of kefir
have an in vivo role as bio-therapeutic substances capable
of stimulating immune cells of the innate immune system,
to downregulate the Th2 immune phenotype or to promote
cell-mediated immune responses against tumors and also
against intracellular pathogenic infections [103]. Romanin
et al. [104] tested various isolates of yeast and bacteria
from kefir and determined that probiotic yeasts were able to
regulate intestinal epithelial innate response even better
than lactobacilli.
Because of its immunomodulating effects, Kefir may
play a role in reducing allergic responses in food allergy.
Food allergies are of worldwide concern and seem to be
occurring with increased frequency. As allergen-specific
IgE is directly involved in the mediation of many allergic
reactions, its inhibition would be desirable in the treatment
of allergic response. Liu et al. [39] showed that kefir
increases Th1 response in mice, which inhibits IgE pro-
duction by secreting interferon. The IgE and IgG1 levels go
down, while the IgG2 levels remain constant. Chen et al.
[105] demonstrated that a strain of Lact. kefiranofaciens
isolated from kefir reduced intestinal inflammation disease.
Their isolate significantly inhibited the pro-inflammatory
production of IL-1b and TNF-a and increase the anti-
inflammatory production of cytokine IL-10. This may
restore barrier function and reduce the permeability of the
intestine [105], reducing allergic stimulus. A study done in
a mouse asthma model showed that kefir displays anti-
inflammatory and anti-allergenic effects, possibly becom-
ing an avenue for treatment of allergic bronchial asthma
[106].
Kefir and kefir fractions have been shown to be effective
in killing cancer cells in vitro and in slowing cancer growth
in vivo. Shiomi et al. [37] showed that solid tumor growth
was inhibited significantly in mice fed a purified polysac-
charide fraction derived from kefir grains. Liu et al. [16]
studied the anti-mutagenic properties of milk kefir and
soymilk kefir and found that they both possess significant
anti-mutagenic and antioxidant activity. De Moreno de
LeBlanc et al. [107] studied immune cells using kefir and
kefir fractions in mice to better understand the mechanisms
involved. A 2-day cyclical kefir treatment proved more
effective than a seven-day cyclical treatment. Orally
administered LAB increased the number of IgA cells, not
only in the intestine, but also in distant mucosal sites.
Further, the 2-day cyclic treatment showed significantly
increased cellular apoptosis, compared to the tumor con-
trol, at 20 days. By 27 days, however, apoptosis in all
groups except the kefir fraction group was statistically
similar. The 2-day cyclic administration of the kefir frac-
tion was best at inducing the activation of apoptosis in the
tumor, resulting in tumors of lower volume than those of
other groups [107]. Maalouf et al. [108] found that a cell-
free fraction of kefir was effective in inhibiting prolifera-
tion and inducing apoptosis of malignant T-lymphocytes
through the downregulation of TGF-a and the upregulation
of TGF-b1, though it did not affect the mRNA expression
of metalloproteinases needed for the invasion of leukemic
cell lines. A similar study showed apoptosis of gastric
cancer cells in vitro, affected through upregulation of bax
(a apoptosis promoter) and downregulation of bcl-2 (an
apoptosis inhibitor) [109]. It appears that the kefiran frac-
tion of kefir has significant anti-tumoral activity.
Kefir has been used for many years to promote good
health and has been touted anecdotally for its curative
properties. It only seems natural for people in dire need of
kefir’s purported benefits to supplement their traditional
medical treatments with kefir. According to one study,
more than a third of patients with cancer use complemen-
tary and alternative medicine, and the use of kefir by
patients undergoing chemotherapy to help them with the
gastrointestinal side effects has increased [110]. Unfortu-
nately, the consumption of kefir did not help alleviate the
gastrointestinal symptoms, though some felt that they were
sleeping better [110]. Another study focused on testing
Probiotics & Antimicro. Prot. (2014) 6:123–135 129
123
kefir’s protective effect against mouth lesions in chemo-
therapy, again with negative results [111].
A large percentage of the world’s population is affected
by some form of lactose maldigestion. The percentage of
population and the relative severity of symptoms vary
regionally, with the lowest instances in Scandinavia and the
highest in Asia [112]. Lactose should be digested in the
small intestine due to the action of the enzyme beta-
galactosidase (the enzyme responsible for the hydrolysis of
lactose into galactose and glucose) on dietary lactose.
Lactose maldigestion symptoms occur when undigested
lactose leaves the small intestine, to then be subject to
fermentation by colonic bacteria, resulting in the genera-
tion of hydrogen gas [113]. Symptoms of lactose maldi-
gestion may range from mild asymptotic fermentation, with
gasses diffusing into the blood, to full lactose intolerance,
with its accompanying abdominal pain, flatulence, bloat-
ing, nausea, or diarrhea [113]. Milk fermented by the
action of kefir grains shows a 30 % reduction in lactose
over non-fermented milk, allowing for greater tolerance
[112]. The lactose in milk is degraded into lactic acid
during fermentation [26]. Moreover, virtually all of the
lactic acid produced in kefir is L (?) lactic acid, the type
most easily metabolized [114]. Kefir grains show beta-
galactosidase activity, while the kefir product does not
[115]. Consumption of a mixture of kefir and kefir grains
appears to aid in the digestion of lactose, reducing the
symptoms of lactose maldigestion and intolerance. Kefir
appears to have buffering capacity, allowing survival of the
beta-galactosidase activity through the gastric juices in the
stomach (activity is irreversibly inactivated at pH 2.0;
stable at pH 4.0) [115]. The buffering action of kefir allows
some of the bacterial cells of the kefir grain to survive into
the small intestine at which point it appears that bile acids
play a part in making beta-galactosidase available for
further lactose digestion, possibly either by lysing bacterial
cells, thus releasing the beta-galactosidase, or perhaps by
altering the permeability of the cell membranes so lactose
can easily enter into the cells [113]. While kefir contains
different organisms, and likely differing enzymatic activi-
ties and sensitivities, evidence supports that plain kefir
improves lactose digestion as well as that shown by plain
yogurt [113]. More research on this would be desirable.
EFSA published series of recent reports indicating that
health claims of some probiotics were not proven by
human intervention studies [116]. In one of these scientific
opinions, EFSA reported that species identification by
DNA–DNA hybridization or the 16S rRNA gene sequence
analysis and/or sequence analysis of other relevant genetic
markers as well as strain identification by DNA macrore-
striction followed by pulsed-field gel electrophoresis,
RAPD analysis, or other internationally accepted genetic
typing molecular methods should be performed in order to
characterize the bacterium sufficiently. This characteriza-
tion is required for each bacterium in case that combina-
tions of several bacteria are used. Otherwise, EFSA panel
considers that food constituents that are bacteria are not
sufficiently characterized [117]. Therefore, the strains from
kefir grains with high potential as probiotics should be
clinically tested to provide evidence for their beneficial
effects and sufficiently characterized.
Kefir as a probiotic has a few disadvantages. As noted
previously, kefir defies standardization due to its variable
nature. A second disadvantage is that not everyone is
willing or able to consume the drink as a probiotic regimen.
A team of scientists in Taiwan prepared a chewable kefir
candy with high probiotic activity to extend options con-
sumers may have to enjoy the health benefits of kefir and
help resolve the difficulty of kefir commercialization [118].
Another way that kefir may prove useful in the probiotic
field is as a delivery system for viable health-promoting
organisms to the gut [46]. Marsh et al. [46] found that
natural kefir was capable of hosting several health-associ-
ated organisms, suggesting that it could theoretically be
altered to incorporate pre-established and certified probi-
otic strains with minimal sensory impact. This hypothesis
was supported by the recent results of Serafini et al. [119]
who found that Bifidobacterium bifidum PRL2010 could
utilize dietary glycogens in kefiran and at least temporarily
colonize kefir milk. They showed that the kefir matrix
modulated the expression of particular PRL2010 genes
demonstrated to play a role in host-interaction, possibly
enhancing the effects of probiotic administration. Thus the
kefiran component of kefir may also act as a prebiotic,
supporting the growth and expression of known probiotic
bacteria.
Other Uses for Kefir and Kefir Products
Kefir and kefir-related products have potential outside of
their use as a probiotic beverage. They are attractive to
industry due in part to their LAB having the status ‘‘gen-
erally recognized as safe’’ (GRAS). This allows kefir and
its functional exopolysaccharides to escape the rigorous
toxicological testing and marketing required of other pro-
ducts that may be useful in industrial applications [120].
Possible other uses for kefir grains, kefir, and kefir products
such as kefiran include exploiting its antimicrobial and
anti-inflammatory properties in both medical and industrial
applications, as well as multiple applications in the food
industry for gelling, texturizing, rheology, increased
nutrition, packaging, and leavening.
The antibacterial activity of kefir has led to the inves-
tigation of kefir and its polysaccharide kefiran as a potential
antimicrobial agent for topical therapy. Rodrigues et al.
130 Probiotics & Antimicro. Prot. (2014) 6:123–135
123
[121] found that a 70 % kefir gel (using either dehydrated
kefir grain or lyophilized kefiran extract) applied to a
wound inoculated with Staph. aureus was effective in
healing and good scar formation, with better results than
even the neomycin-clostebol positive control. Another
study by Rodrigues et al. [122] showed that kefir (and to a
lesser extent purified kefiran extract) inhibited inflamma-
tion and exhibited a significant antimicrobial response.
Huseini et al. [123] found that kefir gels were very effec-
tive in the treatment of severe burns, with less inflamma-
tion and better epithelization and scar formation than the
silver sulfadiazine (Silvadene) positive control. The anti-
inflammatory properties of kefir and kefiran also enhance
wound healing [121–123].
The LAB, such as the ones found in kefir, are known to
produce extracellular polysaccharides. These polysaccha-
rides have been used in the food industry as thickening,
viscosifying, emulsifying, or gelling agents [42, 124].
Kefiran, the exopolysaccharide produced by the LAB in
kefir, has been studied for use as a bioactive food-grade
additive. It enhances rheological properties of chemically
acidified milks [125]. It has great gelling properties
attractive for gelled foods as it gels at freezing tempera-
tures and melts at mouth temperatures [21, 125]. Kefiran
can also be considered a functional additive, due to its
antimicrobial, antibacterial, and immunomodulating prop-
erties [60, 125].
To efficiently utilize kefiran as an additive in the food
industry (or in any industrial application), an efficient way to
produce kefir grains and extract kefiran from the grains
would need to be developed. Rimada and Abraham
[42] developed a method to optimize the production of
bacterial exopolysaccharides using kefir grains and depro-
teinized (DP)-whey (this resulted in a value-added product,
as the disposal of DP-whey represents an environmental
problem causing concern for the dairy industry). Kefir grains
were able to grow and produce exopolysaccharides in DP-
whey from lactose, suggesting that whey proteins were not
required for this process. Also, the reduction of fermentable
sugar in the whey reduced the biological oxygen demand
(BOD), reducing the environmental impact of the disposal of
the used whey. Piermaria et al. [125] found a method for
isolation and quantification of kefiran that is simple with a
good yield, making kefiran ideal for the consideration of
further application. Zajsek et al. [126] further optimized
kefiran production, customizing the milk media with addi-
tional nutrients, and studying the effects of temperature and
agitation. They found that supplementing UHT full fat milk
with 5 % lactose, 0.1 % thiamine, and 0.1 % FeCl3, at the
fermentation temperature of 25 �C with an agitation rate of
80 RPM gave optimal kefiran production.
The increasing popularity of kefir-containing products
has led to the use of kefir starters in cheese making [45,
127, 128]. Kefir culture used as a starter culture in cheese
manufacturing shows promise, adding to the structure,
flavor, and shelf life of the resultant cheese [129]. The
extension of the shelf life through antimicrobial action and
increased acidity of the cheeses is especially attractive to
manufacturers as there is increasing pressure on them to
use more ‘‘natural’’ alternatives to chemical preservatives
in their products [127].
Kefiran shows promise as a component in biodegradable
edible films. These films are important because health-
conscious consumers and environmentally conscious con-
sumers (and therefore the food industry) demand products
utilizing fewer artificial preservatives in their preparation
and less petroleum-based products in their packaging,
while still insisting on high-quality products that resist
spoilage. Kefiran is an attractive choice over other poly-
saccharides due to its immunomodulation, antibacterial,
antifungal, and antitumor properties [130]. These proper-
ties may produce packaging that is naturally resistant to
contamination. Kefiran has the added advantage that, due
to its health-promoting properties, it can be considered a
functional additive [125]. Consumers would find naturally
derived packaging products with enhanced safety and
nutritional qualities very attractive when compared to the
traditional petroleum-based plastic films.
Films based on kefiran (or any polysaccharide) alone are
relatively stiff and brittle, so plasticizers such as water,
oligosaccharides, polyols, and lipids are necessary to
facilitate handling [131]. A kefiran biofilm prepared with a
glucose plasticizer rendered a film with the lowest per-
meability. The film with the best mechanical properties
was obtained with glycerol as the plasticizer [132]. Bio-
degradable edible film made from kefiran, prepared with
glycerol as plasticizer, shows promise as a barrier to con-
trol the transfer of moisture, oxygen, lipids, and flavors,
increasing shelf life and preventing quality deterioration
[33, 131, 132]. Motedayen et al. [130] prepared composite
films made from a blend of kefiran and starch, using
glycerol as plasticizer. They were able to prepare a film
incorporating the strengths of both film producers, with
kefiran’s good mechanical properties overcoming the
weaknesses of starch’s mechanical properties.
Kefir grains have been studied for use as a bakers’ yeast
in bread baking. The researchers found that, while the
leavening rate was slower than with the control (using
traditional baker’s yeast), the kefir grains performed well as
a leavening agent, producing loaves of good quality,
resembling traditional sourdough bread. The bread was
moister, firmer textured, had lower acidity and retained its
freshness longer compared to bread made with baker’s
yeast [133]. When the kefir was immobilized on brewery
spent grains (BSG), the resulting loaves again exhibited
good rising good overall quality, better flavor, and a
Probiotics & Antimicro. Prot. (2014) 6:123–135 131
123
doubling of the shelf life over baker’s yeast samples, with
the added nutrition of the added nutritional value (in
addition to utilization of a value-added product) of the BSG
[134]. Another study showed better-retained freshness in
the kefir sourdough, with no appearance of rope spoilage
caused by Bacillus ssp. for 15 days, compared with control
samples of sourdough with wild microflora, which showed
spoilage by day 7 [135].
Kefir is proving to be a remarkable commodity for
study. At the very least, it is an enjoyable healthy dairy
beverage. When the potential probiotic activity is consid-
ered, kefir’s value increases. Adding the potential alterna-
tive uses for kefir, kefir grains, and kefir by-products
(especially kefiran) makes kefir even more attractive.
Further research on these and other aspects of kefir would
be beneficial.
Acknowledgments Gulhan Unlu thanks (1) J. William Fulbright
Scholarship Board; (2) Institute of International Education—Council
for International Exchange of Scholars (IIE-CIES); and (3) Fulbright
Commission for Educational Exchange between the USA and Turkey
for their generous support of her work as a Fulbright Scholar
(2012–2013) at Middle East Technical University, Ankara, Turkey.
Conflict of interest The authors declare that they have no conflict
of interest.
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