Probiotics & Antimicro. Prot. DOI 10.1007/s12602-014-9168-0
Kefir: A Multifaceted Fermented Dairy Product Barbara Nielsen • G. Candan Gu¨rakan ¨ nlu¨ Gu¨lhan U
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 bacterial 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 beverage, 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. Scientific studies into these reported benefits are being conducted 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 ¨ nlu¨ (&) B. Nielsen G. U School of Food Science, University of Idaho, 875 Perimeter Drive, Moscow, ID 83844-2312, USA e-mail: firstname.lastname@example.org G. C. Gu¨rakan Department of Food Engineering, Middle East Technical ¨ niversiteler Mah., Dumlupınar Blv. No: 1, University, U 06800 C¸ankaya, Ankara, Turkey
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  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’’ . 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 polysaccharide 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, filmjo¨lk, and other traditional fermented milks), traditional 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
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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 . 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 significantly lower quality. Ertekin and Guzel-Seydim  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 bestquality 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 , cocoapulp beverage , soy milk [3, 15, 16], coconut milk , rice milk , and peanut milk . 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 . 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 fermentation 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 . It originated in the Caucasus mountains in Central Asia 1,000s of years ago . 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 . Other tales of deception and intrigue explain how the grains finally became more widely available . In whatever manner the grains originated and were disseminated, and kefir grains and the resultant kefir beverage product can now be found all over the world. Grains in active commercial or artisanal use are found all over Europe (Bulgaria , Portugal [21, 22], Ireland (Buttermilk plant) [23–25], Austria, Germany [6, 26], Poland , France , Italy , Spain [22, 28–30], Sweden , and Denmark ); Eurasia (Turkey [6, 22, 31] and Russia ); and Asia (Iran [32, 33], China , Tibet [35, 36], Japan , Taiwan [38, 39]); as well as in artisanal use in South America (Brazil [40, 41], Argentina ), and Africa (South Africa [43, 44]). Indeed, researchers studying kefir often cite the source of their grains as being from
private households or local dairies in their various countries. 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 Caucasus region [18, 45]. Grain formation may have happened several times and in differing locations over the history of man storing milk . 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 techniques 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  embedded in a polysaccharide and protein matrix. Simova et al.  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.  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, maintaining 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 carbohydrate is of bacterial origin, produced by some of the lactobacilli embedded in the matrix . 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 . 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
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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 . One study, in contrast to the others, observed a variety of lactobacilli but no yeast in the interior . Kefir grains appear to start out as thin sheet-like structures, developing into mature grains with the sheets folding themselves into scrolls and rolls . In observations by Marshall et al. , 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 . 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 carbohydrate/microflora increase. The yeasts are predominantly found in the interior because they adhere to the convoluted side and thus fold to the interior . Wang et al.  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 . The yeast involved enhances aggregation, adhesion, and survival in harsh conditions . Yamin et al.  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 organisms 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 preserving the milk led to the first kefir grains and started the long tradition of producing kefir . 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 . Bags could also be carried as people traveled, the bumpiness of the ride mixing the contents. Motaghi et al.  tested this hypothesis with some success when they filled a goat-hide bag with pasteurized 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  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 populations and activity of kefir grains through freeze-drying the grains  or the LAB isolated from the grains , 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 production less laborious and ensuring a longer shelf life of the product . 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 disadvantage, as the uniformity and shelf life cannot be guaranteed. The lactic acid concentration and the acetaldehyde, acetoin, and gas production increase upon storage of traditional kefir, reaching peak acceptability levels in the first 2 days . A uniform freeze-dried kefir grain with optimized viability of kefir organisms would be desirable for the commercial market .
Kefir’s Microbial Profile As mentioned earlier, kefir grains are a complex community of around 30 species (or more) of LAB and yeast . 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
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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 techniques, in which cultivation in growth media is not required, have received more attention. Several molecular techniques varying in discriminatory power, reproducibility, 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 Lactobacillus isolates at the species level. PCR amplification and DNA sequencing of variable regions of the 16S rRNA gene such as the V1 region , a 500-bp region including the V1 and V2 regions , the V2–V3 region , and the 1,500-bp region  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 . 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 DNAPCR (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 . They confirmed that all the homofermentative Lactobacillus strains were plantarum species and showed desirable probiotic properties. Lactobacillus species present in the gastrointestinal 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 . Pyrosequencing data using the V4 variable regions of the 16S rRNA in a recent study have indicated that microbiota 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 .
Sequencing-based analysis of kefir grains and their milkbased fermentation products have recently yielded detailed bacterial and fungal composition profiles, identifying several genera and species not previously identified in kefir . PCR amplification of the V3 region of the 16S rRNA gene was used for pyrosequencing in addition to PCRDGGE 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 separate species in Lact. acidophilus complex, including Lact. acidophilus, Lact. amylovorous, Lact. crispatus, Lact. galinarium, Lact. gasseri, and Lact. jonsonii, is difficult even by molecular methods . This differentiation was achieved using multiplex PCR with species-specific primers from the 16S–23S intergenic spacer region and the flanking region of the 23S rRNA gene in the study of Song et al. . Kullen et al.  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 . Strains of Lact. acidophilus complex, including Lact. delbrueckii subspecies (Lact. delbrueckii subsp. bulgaricus, Lact. delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis), Lact. plantarum, Lact. fermentum, Lact. rhamnosus, and Lact. sakei, were analyzed by PFGE . The RAPD , 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 cerevisiae, 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 largescale production, as the volumes required would make fermentation uneven and grain recovery laborious and
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impractical . 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 maturation. Industrial kefir is made by then taking Russian-style kefir and inoculating it into pasteurized milk at a concentration 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 . 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 , consistency is hard to control . In a review on innovations in production of kefir, Sarkar  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 populations of kefir grains in the attempt to develop a pure culture inoculum. Beshkova et al.  optimized a starter culture using selected microorganisms isolated from kefir grains and varying culture conditions to produce a kefirlike beverage with very good sensory properties. They concluded that a standardized production was possible. Chen et al.  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 microspheres, then made kefir using the entrapped culture starter, passing it through 28 fermentation cycles. Nambou et al.  combined six pure microbial strains in varying concentrations, finding one that closely approximated the characteristics of traditional kefir.
Kefir as a Probiotic Kefir has long been used in Eastern Europe for its purported health benefits. But, like most of the foods touted as health promoting in other countries and cultures, its benefits are accepted as common knowledge, with, until fairly recently, little peer-reviewed scientific evidence to support the claims . Farnworth  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 correcting for nutritional deficiencies by improving the intestinal balance’’ . 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’’ . Effective probiotics are required to have these properties: must adhere to cells; must exclude or reduce pathogenic adherence; must persist and multiply; must produce acids, peroxide, and bacteriocins antagonistic to pathogen growth; must be safe, noninvasive, non-carcinogenic, and nonpathogenic; 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 . The concept of probiotics was pioneered by Elie Metchnikoff in his work, The Prolongation of Life: Optimistic Studies, originally published in 1908. Dr. Metchnikoff 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 benefits of health and longevity. Cautioning that ‘‘in a question so important, the theory must be tested by direct observation,’’ 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 . 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
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kefir are known probiotics, for example: Lact. acidophilus, Lact. casei, Lact. paracasei, Lact. fermentum, and Saccharomyces cerevisiae . 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 isolated from kefir showed a high resistance to bile and low pH conditions and were able to adhere to intestinal epithelium . Yeasts present in the kefir have been shown to enhance aggregation and adhesion of LAB to the epithelial 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 . 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 . 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 . The expression of functional cell wall proteins may be involved as cell surface proteins may mimic the receptors on any specific target for pathogens and toxins . 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 antimicrobial peptides such as bacteriocins . Golowczyc et al.  found that several lactobacilli in kefir exhibited probiotic 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 . Silva et al.  demonstrated antimicrobial activity against several pathogens during kefir fermentations using various sugar broths. Sezer and GuÂ¨ven  isolated a bacteriocinproducing lactic acid bacterium. They partially purified a
bacteriocin that showed strong antimicrobial activity against both Gram-positive and Gram-negative bacteria. Santos et al.  investigated several Lactobacillus isolates 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 monocytogenes, 40 % showed inhibition against Salmonella enteritidis, and 19 % showed inhibition against Salm. typhi. This seems to be due to secreted antimicrobial substances. 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 Lactobacillus isolates . Kefir shows inhibition of one bacterium of particular interest, Helicobacter pylori, which has been linked to chronic gastritis, ulcers, and gastric cancer . Oh et al.  isolated two yeasts and several strains of lactobacilli from a traditional Tibetan kefir-like yogurt, which, in combination, showed near 100 % bactericidal activity against H. pylori, mediated by soluble factors in the kefir. Zubillaga et al.  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-converting 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 . Nakamura et al.  isolated peptides from Calpis sour milk (a traditional Japanese milk fermented with Lact. helveticus and S. cerevasiae) where both the orally administered milk and the peptides inhibited ACE. QuiroÂ´s et al.  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 . 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 presence of bile salts, and not totally due to bacterial assimilation [94, 95]. Cholesterol consumption or removal in vitro is not a good index of its cholesterol-lowering potential in vivo . Testing has been done on kefir, bacterial isolates of kefir, and kefiran in mice, rats, hamsters, and humans with mixed results. St. Onge et al.  studied the effects of drinking 500 ml kefir/day over a
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4-week period in a double-blind experiment and found no significant total cholesterol, HDL cholesterol, LDL cholesterol, 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 triacylglycerol, total cholesterol, and high-density lipoprotein cholesterol , and another animal study used 10 % lyophilized skim milk or 10 % soyamilk kefir in their feed, resulting in slightly reduced levels of serum cholesterol . 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 polysaccharide, kefiran [100, 101]. Some bacterial isolates showed significant decreases in total cholesterol, triglycerides, 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.  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 . Romanin et al.  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.  showed that kefir increases Th1 response in mice, which inhibits IgE production by secreting interferon. The IgE and IgG1 levels go down, while the IgG2 levels remain constant. Chen et al.  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 antiinflammatory production of cytokine IL-10. This may restore barrier function and reduce the permeability of the intestine , reducing allergic stimulus. A study done in a mouse asthma model showed that kefir displays antiinflammatory and anti-allergenic effects, possibly becoming an avenue for treatment of allergic bronchial asthma . 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.  showed that solid tumor growth was inhibited significantly in mice fed a purified polysaccharide fraction derived from kefir grains. Liu et al.  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.  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 control, 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 fraction was best at inducing the activation of apoptosis in the tumor, resulting in tumors of lower volume than those of other groups . Maalouf et al.  found that a cellfree fraction of kefir was effective in inhibiting proliferation 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) . It appears that the kefiran fraction 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 kefirâ€™s purported benefits to supplement their traditional medical treatments with kefir. According to one study, more than a third of patients with cancer use complementary and alternative medicine, and the use of kefir by patients undergoing chemotherapy to help them with the gastrointestinal side effects has increased . Unfortunately, the consumption of kefir did not help alleviate the gastrointestinal symptoms, though some felt that they were sleeping better . Another study focused on testing
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kefir’s protective effect against mouth lesions in chemotherapy, again with negative results . 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 . Lactose should be digested in the small intestine due to the action of the enzyme betagalactosidase (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 generation of hydrogen gas . Symptoms of lactose maldigestion may range from mild asymptotic fermentation, with gasses diffusing into the blood, to full lactose intolerance, with its accompanying abdominal pain, flatulence, bloating, nausea, or diarrhea . Milk fermented by the action of kefir grains shows a 30 % reduction in lactose over non-fermented milk, allowing for greater tolerance . The lactose in milk is degraded into lactic acid during fermentation . Moreover, virtually all of the lactic acid produced in kefir is L (?) lactic acid, the type most easily metabolized . Kefir grains show betagalactosidase activity, while the kefir product does not . 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) . 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 . While kefir contains different organisms, and likely differing enzymatic activities and sensitivities, evidence supports that plain kefir improves lactose digestion as well as that shown by plain yogurt . 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 . 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 macrorestriction 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 characterization is required for each bacterium in case that combinations of several bacteria are used. Otherwise, EFSA panel considers that food constituents that are bacteria are not sufficiently characterized . 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 consumers may have to enjoy the health benefits of kefir and help resolve the difficulty of kefir commercialization . Another way that kefir may prove useful in the probiotic field is as a delivery system for viable health-promoting organisms to the gut . Marsh et al.  found that natural kefir was capable of hosting several health-associated organisms, suggesting that it could theoretically be altered to incorporate pre-established and certified probiotic strains with minimal sensory impact. This hypothesis was supported by the recent results of Serafini et al.  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 ‘‘generally recognized as safe’’ (GRAS). This allows kefir and its functional exopolysaccharides to escape the rigorous toxicological testing and marketing required of other products that may be useful in industrial applications . 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 investigation of kefir and its polysaccharide kefiran as a potential antimicrobial agent for topical therapy. Rodrigues et al.
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 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.  showed that kefir (and to a lesser extent purified kefiran extract) inhibited inflammation and exhibited a significant antimicrobial response. Huseini et al.  found that kefir gels were very effective in the treatment of severe burns, with less inflammation and better epithelization and scar formation than the silver sulfadiazine (Silvadene) positive control. The antiinflammatory 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 polysaccharides 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 . It has great gelling properties attractive for gelled foods as it gels at freezing temperatures and melts at mouth temperatures [21, 125]. Kefiran can also be considered a functional additive, due to its antimicrobial, antibacterial, and immunomodulating properties [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  developed a method to optimize the production of bacterial exopolysaccharides using kefir grains and deproteinized (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 DPwhey 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.  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. Zajsˇek et al.  further optimized kefiran production, customizing the milk media with additional 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 . 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 . Kefiran shows promise as a component in biodegradable edible films. These films are important because healthconscious consumers and environmentally conscious consumers (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 polysaccharides due to its immunomodulation, antibacterial, antifungal, and antitumor properties . These properties 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 . 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 . A kefiran biofilm prepared with a glucose plasticizer rendered a film with the lowest permeability. The film with the best mechanical properties was obtained with glycerol as the plasticizer . Biodegradable edible film made from kefiran, prepared with glycerol as plasticizer, shows promise as a barrier to control the transfer of moisture, oxygen, lipids, and flavors, increasing shelf life and preventing quality deterioration [33, 131, 132]. Motedayen et al.  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 . When the kefir was immobilized on brewery spent grains (BSG), the resulting loaves again exhibited good rising good overall quality, better flavor, and a
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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 . 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 . 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 considered, kefir’s value increases. Adding the potential alternative 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. ¨ nlu¨ thanks (1) J. William Fulbright Acknowledgments Gu¨lhan U 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 of interest.
The authors declare that they have no conflict
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