Microbiological Exploration of Different Types of Kefir Grains

Many studies have been published lately verifying the probiotic character of kefir grains. Most of them focused on the benefits to human health through the consumption of fermented food with kefir grains. However, the challenge is to characterize and isolate specific probiotic microorganisms involved in the kefir microbiota. The main reason for this is that the food industry prefers to apply isolated probiotic microorganisms from kefir grains rather than kefir grains in order to produce respective fermented products with added value. Thus, modern molecular techniques such as polymerase chain reaction (PCR)-based amplification, new generation sequencing (NGS) or denaturing gradient gel electrophoresis (DGGE) analyses have been applied. Furthermore, this review emphasizes the latest outcomes regarding the health benefits of the consumption of foods fermented with kefir grains and particularly the isolation of microorganisms from kefir grains worldwide, some of them exhibiting probiotic properties.


Introduction
Kefir is a traditional fermented dairy beverage, first consumed in Caucasus Mountains of Russia centuries ago.Nowadays, its popularity has been expanded worldwide and it is considered as a healthy product with high nutritional value in Europe, Asia, and South and North America [1,2].It is a stimulating and self-carbonated beverage produced through fermentation of different types of milk such as sheep, cow, goat, etc.However, currently, alternative kefir drinks have been proposed, substituting milk and applying other carbohydrate sources, such as fruit juices, molasses, etc. [3,4].
The fermented kefir beverage contains white or yellow kefir grains which are insoluble in water; their size is irregular and the grains tend to stay on the bottom of the fermentation broth [5,6].The size of kefir grains ranges between 0.3-3.5 cm in diameter and their typical chemical composition is: water (89-90% w/w), lipids (0.2% w/w), proteins (3.0% w/w), sugar (6.0% w/w), and ash (0.7% w/w) [7].Kefir grains consist of a consortium of several exopolysaccharides, named kefiran, as well as various microorganisms [8].Kefiran is a heteropolysaccharide (EPS) comprising glucose and galactose in high concentrations, and it is classified as a water-soluble glucogalactan exhibiting very good rheological properties and enhancing gel properties [9,10] A wide variety of microorganisms, sharing symbiotic relationships, have been isolated from kefir microflora, including yeasts (Kluyveromyces, Candida, Saccharomyces and Pichia), lactic acid bacteria (LAB) (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus), and occasionally acetic acid bacteria [11,12].
Kefir grains are considered a multi-functional starter culture in the food industry since they have been applied in various fermented systems such as bread, cheese and milk with very promising results.It is noteworthy that kefir grains are feasible for upgrading cheese whey, which is a widely abundant waste from the dairy industry with a high organic load [13].In addition, there are studies revealing that kefir grains can be successfully cultured in cheese whey in appropriate conditions, which results in the reduction of the cost of kefir grain culturing substituting synthetic mediums or even milk [4].Likewise, the application of kefir grains as a starter culture is more attractive in the food industry since they can be easily cultured in cheese whey at a low cost [14].
The overall objective of this review, hence, is to find and assess data available in the literature regarding: (i) the main microorganisms that are isolated and characterized from different types of kefir grains worldwide with various methods; and (ii) the probiotic properties and health benefits of kefir grains.

Microbiology of Kefir Grains and Origin of Kefir Grains Worldwide
Many origins of kefir grains are reported in the literature, including Brazilian, Argentinean, Irish and Turkish kefir grains (Table 1).Even though, at first glance, the differences seem to be minor, in the last year noticeable differences have been revealed in the microbiological microflora through molecular analysis.Belgian and Irish kefir grains exhibit greater variety in dominant LAB, followed by Chinese and Brazilian grains.Lactobacillus kefiri and Lactobacillus kefiranofaciens spp.are two common dominant LAB in Ireland, Malaysia and Belgium.
China (Tibet) and Brazil are next on the list with dominant LAB to be Lactobacillus species (helveticus, casei, kefiri).Kefir grains from other countries (Slovenia, Italy, South Africa, Russia, Bulgaria, Turkey, Taiwan and Argentina) present their own microbial profiles with different percentages in dominant LABs and with reduced strain variety.
As far as dominant yeasts are concerned, Taiwan and Slovenia have a greater number of different yeasts, with Kluyveromyces marxianus and S. cerevisiae being the most common and dominant over others such as S. unisporous, Rodosporidium kratochvilovae and Kazachstania exigua.
South Africa, Belgium, Brazil, Italy and Bulgaria follow on the list with different dominant yeasts, such as Candida spp., Cryptococcus spp., Dekkera anomala, etc.

Microorganisms Isolated from Different Types of Kefir Grains Worldwide
In recent years, plenty of scientific surveys were published regarding the isolation and characterization of microorganisms from kefir grains all over the world, some of them exhibiting probiotic properties.All the updated data are represented in Tables 1 and 2. Gao et al. selected and evaluated four samples of Tibetan kefir, namely TK-ZJUJ01 from Tibet, TK-ZJUJ02 from Sichuan, TK-ZJUJ03 from Xinjiang and TK-ZJUJ04 from Qinghain.The aim was to identify bacteria and yeasts through conventional methods using HANGWEI trae biochemical reaction tube kits and through molecular methods using PCR amplification [16].Regarding the TK-ZJU0Q1 sample, Bacillus subtilis, Lactococcus lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae were identified, whereas in the TK-ZJUJ024 sample Lactobacillus kefiri, Pichia kudriavzevii, Kluyveromyces marxianus, Kazachstania unispora were present.Analysis of the TK-ZJUJ03 sample led to the identification of Leuconostoc lactis, Lactococcus lactis, Lactobacillus plantarum, Kazachstania unispora, Kluyveromyces marxianus, and from the TK-ZJUJ04 sample, Lactobacillus plantarum, Acetobacter fabarum, Kazachstania unispora, Pichia guilliermondii were detected.The yeasts Pichia kudriavzevii and Pichia guilliermondii were isolated from kefir grains for the first time.
It is noteworthy that lactic acid bacteria contained in the TK-ZJUS01 sample constitute over 50% of the microflora in contrast to TK-ZJUJ04, in which the majority of microorganisms were yeasts [16].In another study, three different Tibetan kefir grains were analyzed by culture-independent methods.The most frequently isolated species were Pseudomonas sp., Leuconostoc mesenteroides, Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei, Kazachstania unispora, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania exigua.Among the three different Tibetan kefir grains, the percentage of similarities was 78%-84% in bacteria and 80%-92% in yeasts [28].Other studies in Tibetan kefir showed that Lactococcus lactis was the most commonly identified bacteria [16].Furthermore, Leuconostoc mesenteroides, another heterofermentative lactic acid bacterium, was found to be prevalent.
In Turkey, three kefir grains and four kefir beverages were investigated for their microbial diversity using culture-independent and culture-dependent methods.According to the results, Lactobacillus kefiranofaciens was the most predominant species in kefir grains, while Lactococcus lactis predominated in kefir beverages.Lactococcus lactis, Lactobacillus acidophilus and Streptococcus thermophilus were also detected with both methods [18].
Culture-dependent and culture-independent methods were applied in order to analyze the microflora of tibico (sugary kefir) grains from eight different states in Brazil.In particular, through the application of amplified ribosomal DNA restriction analysis (ARDRA) and/or 16S rRNA gene sequencing, 108 isolates from both substrates were identified.The identified bacteria belonged to the genera Lactobacillus, Acetobacter, Gluconobacter and Bacillus, whereas the yeasts identified were Pichia, Saccharomyces, Kazachstania, Candida, Zygosaccharomyces and Yarrowia.It should be underlined that the bacterial species Cluconobacter liquefaciens and Bacillus sereus, as well as the yeasts Pichia cecembensis, Pichia caribbica and Zygosaccharomyces fermentati, were detected for the first time in tibico grains [2,29].In another study, in eight Brazilian kefir grains, four of which were propagated in cow's milk and the rest in brown sugar, the dominant species were Lactobacillus casei, Lactobacillus kefiri, Lactobacillus perolens, Lactobacillus kefiranofaciens, Lactobacillus satsumensis, Lactobacillus mali, Lactobacillus parafarraginis, and Lactobacillus diolivorans [30].Additionally, another study investigated the bacterial composition of a Brazilian kefir beverage using phenotyping and genotyping methods.The most frequently isolated species were Lactobacillus paracasei, Lactobacillus parabuchneri, Lactobacillus casei, Lactobacillus kefiri, Lactococcus lactis, Acetobacter lovaniensis, Kluyveromyces lactis, Kazachstania aerobia, Saccharomyces cerevisiae and Lachancea meyersii.It should be underlined that the presence of Lachancea meyercii and Kazachstania aerobia was confirmed for the first time in Brazilian kefir [19].
Argentinean kefir grains were also analyzed for their microbial composition.Yeast species were isolated from four milky and three sugary kefir grains and identified through classical microbiological and molecular-genetic methods.The microbial composition obtained consisted of Zygosaccharomyces cerevisiae, Zygosaccharomyces unisporus, Issatchenkia occidentalis and Kluyveromyces marxianus.Thirteen of these strains were tolerant to low pH and bile salts.Thus, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8118 were selected for further tests in order to investigate their ability to adhere to intestinal epithelial cells.As a result, both yeast strains were not detected in the gut after five days [15].In another study, 11 isolated homofermentative lactobacilli were examined for potential probiotic activity.Restriction analysis of amplified ribosomal DNA (ARDNA) and analysis of the 16S rRNA internal spacer region were used to confirm that all 11 lactobacilli pertained to the species Lactobacillus plantarum.RAPD-PCR analysis showed that they belonged to five groups and each was tested for potential probiotic activity.All strains showed high tolerance to bile salts.Regarding their antimicrobial activity, all strains were effective against Salmonella typhimurium, Escherichia coli and Salmonella enterica strains.In addition, most of the strains were effective against Salmonella gallinarum.It was also concluded that L. plantarum CIDCA 8327 had the highest potential probiotic ability [9].
In Slovenia, culture-dependent and culture-independent methods were used to characterize and evaluate the stability of lactobacilli and yeast microbial flora in kefir grains.The results showed that the dominant bacterial species were Lactobacillus kefiranofaciens ssp.Kefirgranum, Lactobacillus parakefiri and Lactobacillus kefiri.Regarding the yeasts, the most frequent species were Kluyveromyces marxianus, Kazachstania exiqua and Rhodosporidium kratochvilovae.For the first time, it the presence of Rhodosporidium kratochvilovae was reported.Culture-independent analysis indicated that the majority of the microorganisms were Lactobacillus kefiranofaciens ssp.kefirgranum, Kluyveromyces marxianus and Kazachstania exigua, but there was no detection of Lactobacillus parakefiri, Lactobacillus kefir and Rhodosporidium kratochvilovae.The authors concluded that it is important to use a combination of methods for more reliable and detailed results [20].
The composition of Lactobacillus spp. in kefir grains from France, Italy, Portugal, Russia, Turkey and Spain was studied and the strains were tested for adherence to human anterocyte-like Caco2-cells, resistance to acidic pH and bile acid, antimicrobial activities against enteropathogenic bacteria and inhibition of Salmonella typhimurium attachment to Caco-2 cells.The conduction of in vitro tests showed that L. acidophilus CYC 10051 and Lactobacillus kefiranofaciens CYC 10058 exhibited the best results.Lactobacillus kefiranofaciens CYC 10058 produced kefiran-related exopolysaccharides that retard tumor growth in vivo when administered orally [19].The results suggested that this strain is of major interest due to its kefiran-related polysaccharide production.It is possible that the good health properties attributed to kefir may also be related to but not restricted by the therapeutic properties of kefiran.In other words, this study has clearly shown that the Lactobacillus strains isolated from kefir clearly showed probiotic properties [3].
In Taiwan, Lactobacillus kefiranofaciens M1, isolated and identified from Taiwanese milk kefir grains, demonstrated immune-modulating activity.In this study, an anticolitis effect was clearly demonstrated.The underlying mechanisms seem to affect intestinal epithelial cells directly.Lactobacillus kefiranofaciens M1 could restore barrier function and reduce the permeability of the intestine against DSS-induced damage by (i) increasing chemokine CCL-20; (ii) up-regulating the production of anti-inflammatory cytokine IL-10; and (iii) suppressing the secretion of proinflammatory cytokines through TLR2.The anticolitis effect of Lactobacillus kefiranofaciens M1 was investigated in vitro and in vivo.Based on these results, it was found that Lactobacillus kefiranofaciens M1 has the potential to be used in fermented milk products as an alternative therapy for intestinal disorders [27].In another study, various yeasts were isolated from kefir and those exhibiting tolerance to acidic conditions and bile salts were selected for further study of their adhesion to epithelial cells and their transit through the mouse gut.More specifically, from milky and sugary kefir grains, 34 yeast strains were isolated and identified through classical microbiological and molecular-genetic methods (internal transcribed spacer amplification, whole-cell protein pattern, and analysis of restriction fragment length polymorphisms).Fifteen strains of Saccharomyces cerevisiae, six strains of Saccharomyces unisporus, four strains of Issatchenkia occidentalis, and nine strains of Kluyveromyces marxianus were isolated.The strains that were further studied were Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112.Both strains were able to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice.At the end of the administration, both yeast strains were absent from the gut within five days [15].A combination of conventional microbiological cultivation was used with PCR-DGGE and resulted in the identification of four yeast species from both cultures in Taiwan.Specifically, Kluveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with 76%, 22%, and 2% distribution, respectively, while Kluveromyces marxianus, Saccharomyces unisporus, and Pichia fermentans were identified with 58%, 11%, and 31% distribution in viili starters, respectively.In addition, a culture-independent method was applied to identify the viili and kefir yeasts applying DGGE analysis.Pichia fermentans in kefir grains and Saccharomyces unisporus in viili starters were not identified.This result confirms that only the predominant yeast populations in these starters could be identified.This is the first report demonstrating that a combination of conventional microbiological cultivation, PCR-DGGE, and sequencing methods could successfully identify species of yeast and facilitate the study of their distribution in kefir grains [9].
In Italy, six different milk kefir grains were investigated and the dominant species were Lactobacillus kefiranofaciens and Dekkera anomala.The presence of the sub-dominant species ascribed to Sreptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted.In addition, Lactococcus lactis, Enterococcus spp., Bacillus spp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community [23].
In addition, interesting findings were obtained through the application of rapid amplified ribosomal DNA restriction analysis (ARDRA) as a second culture-independent technique to evaluate the data obtained using the sequencing approach.These studies detected the presence of bifidobacteria as part of the sugary kefir consortium for the first time [31].
The microbiological consortium of communities in kefir grains of different origin have also been studied by the pyrosequencing of rDNA amplicons [32].The outcome showed the prevalent strains were Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus parakefiri, Lactobacillus buchneri, while species of Acetobacter and Lactococcus were identified as minor bacterial constituents [17,33].
Next generation sequencing (NGS) was applied to investigate the predominant species in Malaysian kefir grains obtained locally.The taxonomic results analyses obtained when using BaseSpace (Illumina) and MEGAN were compared.The outcome showed that the predominant species was Lactobacillus kefiranofaciens (81.45%-91.93%),while Lactobacillus kefiri (2.01%-2.47%)was the second in abundance [26].

Health Benefits of Kefir and Kefir Grains
The complex microbiological consortium of kefir grains is responsible for the high numbers of produced metabolites that contribute to a wide range of health-promoting effects.The main established health benefits of kefir grains are represented in Table 3 and include antimutagenic, anticolitis, hypocholesterolemic and anti-atherogenic effects, as well as antimicrobial and antioxidant activities, protection against C. difficile infection, and inhibition of tumor growth and β-galactosidase activity.

Biological Activity Reference
antimutagenic effects against 2,3-dimethyl-4-aminobiphenyl (DMAB) and against acting N-methyl-N-nitro-N-nitrosoguanidine (MNNG) [27] hypocholesterolemic and anti-atherogenic effects [34,35] inhibition of tumor growth [36] antimicrobial activity against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and S. enteritidis [3] antioxidant activity antifungal, antibacterial properties and epithelium protection due to kefiran [9,37,38] antimicrobial activity, all strains were effective against Salmonella typhimurium and Escherichia coli, and against S. enterica strains [21] anticolitis effect [27] protect against C. difficile infection [39] β-Galactosidase Activity [40] Kefir grains can inhibit the development of undesirable and pathogenic microorganisms, since the microbes contained in grains are capable of producing lactic and acetic acids, ethanol, peptides (bacteriocins), and other biologically active components [12].Many studies have shown that species of lactobacilli contained in kefir grains can be successfully used in the treatment of vaginal infections, as they are able to produce a range of anti-microbial compounds [41].More specifically, it has been reported that milk fermented with kefir grains exhibits antimutagenic effects against 2,3-dimethyl-4-aminobiphenyl (DMAB) and against N-methyl-N-nitro-N-nitrosoguanidine (MNNG).Both of them are of the most potent mutagenic chemicals known [27].In recent research data, the consumption of fermented yogurt and milk with kefir grains showed hypocholesterolemic and anti-atherogenic effects that protect against atherogenesis and the formation of plaques in arteries [34,35].Liu et al. (2003) demonstrated that oral administration of kefir grains generated from milk or soy milk results in the significant inhibition of tumor growth [36].It should also be mentioned that there are positive effects on the cholesterol metabolism [34].
Moreover, a bacteriocin (lacticin 3147), produced by Lactococcus lactis strain DPC3147 isolated from kefir grains, exhibited antimicrobial activity against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Salmonella enteritidis [3].On the other hand, new research evidence revealed that the health benefits of kefir grains are also attributed to kefiran.Specifically, kefiran presents antioxidant activity [37], antifungal and antibacterial properties [9] and epithelium protection [38].
According to Bolla et al. (2013), a mixture of kefir-isolated microorganisms (Lactobacillus plantarum, Lactobacillus kefir, Lactococcus lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae) was administered to hamsters infected with Clostridium difficile and resulted in milder symptoms of Clostridium difficile-associated diarrhea (CDAD) and extinction of death among the hamsters examined [39].
The comparison was conducted between a group of mice that consumed the mixture of probiotic strains and another group administered clindamycin.The antibiotic showed more aggressive symptoms of diarrhea and almost all of the hamsters ended up dead.These results demonstrated that kefir-isolated bacteria and yeasts may contribute to protection against C. difficile-induced enterocolitis in hamsters.
In addition, kefir grains can be successfully applied to a specific population suffering from lactose intolerance.The enzyme β-galactosidase is naturally present in kefir grains and reduces the lactose content, leading to a product suitable for lactose-intolerant persons.Likewise, the reduced lactose concentration and higher β-galactosidase activity in kefir make it suitable for lactose-intolerant persons by improving lactose digestion [40].

Future Prospective
Lately, kefir grains have been applied in the production of non-dairy probiotic beverages.Likewise, interesting results have been presented regarding the fermentation of apple (Malus domestica Borkh, cv Gala), quince (Cydonia oblonga Mill., cv Del Portogallo), grape (Vitis vinifera L., white-berry cv Italia), kiwifruit (Actinidia chinensis PL., cv Hayward), prickly pear (Opuntia ficus-indica L., cv Sanguigna) and pomegranate (Punica granatum L., cv Dente di cavallo) juices with kefir grains [42].The products were of high added value and can be characterized as functional foods.
In the same manner, the fermentation of vegetable juices, such as carrot, onion, tomato and strawberry juices, was carried out by water kefir microorganisms.Likewise, the new products might represent important foods providing live microorganisms to vegetarians with a limited availability of fermented products [43].Lately, mixtures of pomegranate and orange juice as well as Cornus mas L. juice were successfully fermented with kefir grains and isolated lactic acid bacteria from kefir grains, respectively, leading to low-alcohol beverages with high nutritional value and probiotic properties [44,45].
Taking into account that kefir grains obviously represent probiotic features, the aforementioned scientific studies might show the way for the future employment of kefir grains in the fermentation industry, either as a whole mixed culture or as single cultures isolated from kefir grains, leading to products with high added value that are ready to be consumed.

Table 1 .
Dominant LAB and yeasts isolated from kefir grains worldwide.

Table 2 .
Fully characterized probiotic LAB and yeasts isolated from kefir grains.

Table 3 .
Presumed health benefits of kefir grains.