Milk kefir: composition, microbial cultures, biological activities, and related products

In recent years, there has been a strong focus on beneficial foods with probiotic microorganisms and functional organic substances. In this context, there is an increasing interest in the commercial use of kefir, since it can be marketed as a natural beverage that has health promoting bacteria. There are numerous commercially available kefir based-products. Kefir may act as a matrix in the effective delivery of probiotic microorganisms in different types of products. Also, the presence of kefir’s exopolysaccharides, known as kefiran, which has biological activity, certainly adds value to products. Kefiran can also be used separately in other food products and as a coating film for various food and pharmaceutical products. This article aims to update the information about kefir and its microbiological composition, biological activity of the kefir’s microflora and the importance of kefiran as a beneficial health substance.


INTRODUCTION
Kefir is an acidic-alcoholic fermented milk product with little acidic taste and creamy consistency that was originated in the Balkans, in Eastern Europe, and in the Caucasus (Fontán et al., 2006;Serafini et al., 2014). Kefir can be produced by fermenting milk with commercial freeze-dried kefir starter cultures, traditional kefir grains, and the product that remains after the removal of kefir grains (Bensmira et al., 2010). Kefir grains are a kind of yogurt starter, which are white to yellowwhite, gelatinous, and variable in size (varying from 0.3-3.5 cm in diameter) and are composed by a microbial symbiotic mixture of lactic acid bacteria (10 8 CFU/g), yeast (10 6 -10 7 CFU/g), and acetic acid bacteria (10 5 CFU/g) that stick to a polysaccharide matrix (Garrote et al., 2010;Chen et al., 2015). After successive fermentations, kefir grains can break up to new generation grains, which have the same characteristics as the old ones (Gao et al., 2012).
Commercial kefir is produced by two methods: The "Russian method" and the pure cultures. In the "Russian method" kefir is produced on a larger scale, using a series fermentation process, beginning with the fermentation of the grains and using the percolate. The other method employs pure cultures isolated from kefir grains or commercial cultures (Leite et al., 2013). Also, the industrial or commercial process uses direct-to-vat inoculation (DVI) or direct-to-vat set (DVS) kefir starter cultures. In addition, Bifidobacterium sp., Lactobacillus sp. and probiotic yeast (Saccharomyces boulardii) may be used as adjunct cultures when blended with kefir grains or kefir DVI cultures (Wszolek et al., 2006). On the other hand, whey may be a practical base for kefir culture production, and fermented whey has shown to be a suitable cryoprotective medium during freeze-drying. The freeze-dried culture retains a high survival rate and shows good metabolic activity and fermentation efficiency, indicating a good potential for its use as a value-added starter culture in dairy technology. All of these studies have shown promising perspectives for the application of kefir grains in whey valorization strategies (Bensmira et al., 2010;Cheirsilp and Radchabut, 2011).
Traditionally, kefir is manufactured using cow, ewe, goat, or buffalo milk. However, in some countries, animal milk is scarce, expensive, or minimally consumed due to dietary constraints, preferences, or religious customs. Therefore, there have been many attempts to produce kefir from a variety of food sources such as soy milk (Botelho et al., 2014). Historically, kefir has been linked with health, for example, in Soviet countries, kefir has been recommended for consumption by healthy people to restrain the risk of some diseases (Saloff-Coste, 1996;St-Onge et al., 2002;Farnworth and Mainville, 2003). The consumption of this fermented milk has been related to a variety of health benefits (Vujičič et al., 1992;McCue and Shetty, 2005;Rodrigues et al., 2005a) not only linked to its microflora, but also due to the presence of some metabolic products as organic acids (Garrote et al., 2001;Ismaiel et al., 2011). In addition, kefir cultures have the ability to assimilate cholesterol in milk (Yanping et al., 2009). On the other hand, there is a growing commercial interest in using kefir as a suitable food matrix for supplementation with health-promoting bacteria. Kefir may not only be a natural probiotic beverage, but also acts as an effective matrix for the delivery of probiotic microorganisms Medrano et al., 2008;Oliveira et al., 2013).
In kefir grains the main polysaccharide is kefiran, which is a heteropolysaccharide composed by equal proportions of glucose and galactose and is mainly produced by Lactobacillus kefiranofaciens (Zajšek et al., 2011). It has been demonstrated that kefiran improves the viscosity and viscoelastic properties of acid milk gels (Rimada and Abraham, 2006), and is able to form gels that have interesting viscoelastic properties at low temperatures, because of that, kefiran can also be used as an additive in fermented products. Besides, kefiran can enhance the rheological properties of chemically acidified skim milk gels increasing their apparent viscosity (Zajšek et al., 2013).
This review presents the most recent advances about kefir and kefiran, their production and microbial cultures involved, biological activities and potential applications in health and food industries.
The microbial composition may vary according to kefir origin, the substrate used in the fermentation process and the culture maintenance methods. Tibetan kefir, which is used in China, is composed of Lactobacillus, Lactococcus, and yeast. Additionally, acetic acid bacteria have been identified in Tibetan kefir, depending on the region in China from where it was obtained (Gao et al., 2012), additionally, Tibetan kefir composition differs from that of Russian kefir, Irish kefir, Taiwan kefir, Turkey fermented beverage with kefir; however, it is known that this microbial diversity is responsible for the physicochemical features and biological activities of each kefir (Jianzhong et al., 2009;Kabak and Dobson, 2011;Gao et al., 2012;Altay et al., 2013). Wang et al. (2012) examined a section of a whole kefir grain and found in the outer layer of the grain, lactococci, and yeasts, and, in the inner layer of the grain, the quantity of lactobacilli were much higher and more yeasts cells were found. There are little information about the mechanism of grain formation, so the same authors, proposed a hypothesis to explain that. "Initially, Lactobacillus kefiranofaciens and Saccharomyces turicensis start to auto-aggregate and co-aggregated to small granules." The aggregation is enhanced when the pH drops. The biofilm producers, Lactobacillus kefiri, Kluyveromyces marxianus HY1, and Pichia fermentans HY3 then adhere to the surface of these small granules due to their cell surface properties and their strong aggregation ability, which gives rise to thin biofilms. After biofilm formation, the kefir yeasts and Lactobacillus continue to co-aggregated with the granule strains and associate with the granule biofilm to become a three dimensional microcolony. As the cell density due to the growth of kefir yeasts and Lactobacillus increases, cells and milk components that are present in the liquid phase accumulate on the granule surface   Antimicrobial activity Rodrigues et al., 2005a Modulation of the intestinal immune system and protection of epithelial cells against Bacillus cereus exocellular factors Medrano et al., 2008;Piermaria et al., 2010 and the kefir grains are formed. There is a symbiotic relation between the microorganisms present in kefir grains, wherein the bacteria and yeast survive and share their bioproducts as power sources and microbial growth factors. This microorganism association is responsible for lactic and alcoholic fermentation (Witthuhn et al., 2005;Wang et al., 2012;Hamet et al., 2013). After receiving its actual/present denomination, some of the microorganisms isolated and identified in kefir cultures were classified using the product name, as in Lactobacillus kefiri, L. kefiranofaciens, L. kefirgranum, Lactobacillus parakefir, and Candida kefyr (Wyder et al., 1999;Kwon et al., 2003;Yang et al., 2007;Kok-Tas et al., 2012). Table 1 demonstrates the microbial composition, which has been isolated from kefir and kefir grains of different origins.

BIOLOGICAL ACTIVITY OF KEFIR
Due to its composition, kefir is mainly considered a probiotic resource (Nalbantoglu et al., 2014). "Probiotics are microbial cell preparations or components of microbial cells with a beneficial effect on the health of the host" (Lopitz et al., 2006). Some studies suggest that probiotic bacteria in kefir consumers' gut are abundant and are correlated with health improvement (Ahmed et al., 2013;Zheng et al., 2013); in that way, it had been demonstrated that the cell-free fraction of kefir enhances the ability to digest lactose relieving symptoms (Farnworth, 2005;Rizk et al., 2009).
Another reason for the increased interest in probiotic strains from kefir is its capacity to lower cholesterol levels. There are different ways in which bacteria can alter serum cholesterol: (i) through the binding to and absorption into the cell before it can be absorbed into the body; (ii) producing free and deconjugating bile acids; (iii) inhibiting the enzyme HMG-CoA reductase (Yanping et al., 2009).
The microorganisms in the kefir grains produce lactic acid, antibiotics and bactericides, which inhibit the development of degrading and pathogenic microorganisms in kefir milk (Liu et al., 2002). Kefir acts against the pathogenic bacteria Salmonella, Helicobacter, Shigella, Staphylococcus, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Bacillus subtilis, Micrococcus luteus, Listeria monocytogenes, Streptococcus pyrogenes, (Lopitz et al., 2006), Streptococcus faecalis KR6, Fusarium graminearum CZ1 (Ismaiel et al., 2011), and the fungus Candida albicans. On the other hand, it has been demonstrated that a mixture of kefir isolated bacteria and yeast is able to prevent diarrhea and enterocolitis triggered by Clostridium difficile (Bolla et al., 2013). Besides, kefir showed good efficacy in inhibiting spore formation and aflatoxin B1 produced by the fungus Aspergillus flavus, which is a toxic compound formed either in the field or during food storage. Therefore, kefir appears as a promising safe alternative natural food preservative offering protection against intoxication with aflatoxin B1 (Ismaiel et al., 2011).
It had been proved that many species of lactobacilli present in kefir have S-layer proteins. Surface layers (Slayers) can be aligned in unit cells on the outermost surface of many prokaryotic microorganisms (Mobili et al., 2009). It has been demonstrated that these S-layer proteins can apply a protective action inhibiting the grown of Salmonella enterica serovar Enteritidis in Caco-2 cells, and also have the ability to antagonize the effects of toxins from Clostridium difficile on eukaryotic/eukaryotic cells in vitro (Carasi et al., 2012).
Nevertheless, there are other important studies performed with some microorganisms isolated from different types of kefir. Some microorganisms with their biological activities and origin are shown in Table 2.

KEFIRAN, A POTENTIAL EXOPOLYSACCHARIDE
The increased search for natural polysaccharides has been very significant due to their use in the food, pharmaceutical, and cosmetic industries as additives, bio-absorbents, metal removal agents, bioflocculants, and medicine delivery agents, among other functions (De Vuyst et al., 2001;Welman and Maddox, 2003;Badel et al., 2011). Many microorganisms, such as bacteria, fungi, and weeds, have the capacity/ability to synthesize and excrete extracellular polysaccharides, and these polysaccharides can be either soluble or insoluble (Wang et al., 2010;Badel et al., 2011).
The polysaccharides that are commonly used as food additives are xanthan, dextran, gellan, and alginates, while the exopolysaccharides (EPSs) produced by lactic acid bacteria  show good physicochemical characteristics for their use as food additives. In addition to these characteristics, EPSs are obtained from microorganisms classified as GRAS (generally recognized as safe), such as lactic acid bacteria Saija et al., 2010;Badel et al., 2011). Many reports have demonstrated that the quantity and properties of EPSs depend on the microorganisms used in the fermentation process and on the fermentation conditions and the composition of the culture media (Kim et al., 2008). EPSs have physicochemical and rheological properties that make them suitable as additives, which can be used as stabilizers, emulsifiers, gelling agents, and viscosity improvers. Additionally, EPSs possess biological properties suggesting their use as antioxidants, antitumor agents, antimicrobial agents, and immunomodulators, among other roles (Suresh Kumar et al., 2008;Bensmira et al., 2010;Piermaria et al., 2010).
The EPS kefiran is produced by Lactobacillus kefiranofaciens (Kooiman, 1968;Wang et al., 2010) from kefir grains, which are composed of proteins, polysaccharides, and a complex symbiotic microbial mixture (Witthuhn et al., 2005;Jianzhong et al., 2009). These microorganisms grow in kefiran, which is a polysaccharide matrix consisting of glucose and galactose. Despite good kefiran production by L. kefiranofaciens alone, it has been observed that the addition of Saccharomyces sp. to the culture improves the net quantity of kefiran, illustrating the importance of the symbiosis between the bacteria and yeast that are present in kefir (Cheirsilp et al., 2003).
Similarly to lactic acid bacteria, Lactobacillus sp. also produces glucan and fructan. The homopolysaccharides show a much higher performance compared with heteropolysaccharide production (Welman and Maddox, 2003;Badel et al., 2011).
The heteropolysaccharides excreted by Lactobacillus delbrueckii, Lactobacillus bulgaricus, Lactobacillus rhamnosus, and Lactobacillus helveticus contain galactose, glucose, and rhamnose as the main monosaccharides, with other monosaccharides being present in smaller concentrations. They are also highly branched with different types of linkages, and their denominations are complex and generally dependent on the main monosaccharide (De Vuyst and Degeest, 1999;Badel et al., 2011).
Lactobacillus plantarum isolated from Tibetan kefir excretes EPS classified as heteropolysaccharides composed of galactose, glucose, and mannose. This EPS has the capacity/ability to reduce blood cholesterol and form a biofilm shape (Zhang et al., 2009;Wang et al., 2010).
Kefiran is an EPS classified as a heteropolysaccharide comprising glucose and galactose in high concentrations, and it is classified as a water-soluble glucogalactan, which makes it suitable to be used as an additive (Wang et al., , 2010. Kefiran has excellent rheological properties and can significantly improve the viscosity of lacteous products by favoring and maintaining gel properties and avoiding the loss of water during storage (Rimada and Abraham, 2006). With respect to the biological activity of kefiran, several studies have demonstrated that this EPS can be used as a nutraceutical, as described in Table 3.
The first study about kefiran structure was published by Kooiman (1968), who proposed a structure composed of two units: kefiran (polysaccharide) and kefirose (pentasaccharide). Then, some authors analyzed the polysaccharide structure with current techniques such chromatography and infrared spectroscopy Chen et al., 2015) and nuclear magnetic resonance (NMR; Ghasemlou et al., 2012). The kefiran structure, according to them, is shown in Figure 1.

KEFIR-BASED PRODUCTS
Nowadays, the interest in developing functional foods is increasing because people want to improve their health and prevent diseases. Keeping in mind that kefir is a beverage with high probiotic activity, among other bioactivities, new companies are emerging around the world. One of the biggest kefir companies known is Lifeway, which started in 1986; their products can be obtained in the United States, Canada, and Great Britain, all of them based in kefir beverages, frozen, and cheese.
Other companies are Evolve Kefir with its principal product, a smoothie; Wallaby Yogurt Company with Low Fat Kefir; and CocoKefir LLC, which provides drinks/beverages based mainly on coconut water cultured with a comprehensive blend of probiotics. Table 4 summarizes the products provided these companies with some general information about each one.

CONCLUSION
Kefir, the traditional beverage, is now recognized as a potential source of probiotics and molecules with highly interesting healthy properties. The careful and detailed characterization of kefir composition has helped the scientific community to find new possibilities for its application. Kefiran, the EPS of kefir, has very important physicochemical and rheological properties. Besides, its biological properties suggest its use as antioxidant, antitumor agent, antimicrobial agent, and immunomodulator, among other roles. Research is constantly being conducted to consolidate kefir and kefiran properties for the development of new important products to preserve consumer's health.

ACKNOWLEDGMENT
Authors want to thank CNPq and CAPES for the financial support.