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

1Kefir 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. 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. Kefir grains are a kind of yogurt starter, which are white to yellow – white, 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 (108 CFU/g), yeast (106–107 CFU/g), and acetic acid bacteria (105CFU/g) that stick to a polysaccharide matrix. After successive fermentations, kefir grains can break up to new generation grains, which have the same characteristics as the old ones.

2Commercial 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. 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. 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.

3Traditionally, 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. 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. The consumption of this fermented milk has been related to a variety of health benefits not only linked to its microflora, but also due to the presence of some metabolic products as organic acids. In addition, kefir cultures have the ability to assimilate cholesterol in milk. 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.

4In 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. It has been demonstrated that kefiran improves the viscosity and viscoelastic properties of acid milk gels, 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.

5Compared with other polysaccharides, kefiran has outstanding advantages such as antitumor, antifungal, antibacterial properties immunomodulation or epithelium protection, anti-inflammatory, healing, and antioxidant activity.

6This 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.

7Kefir grains have a complex composition of microbial species such as the predominance of lactic acid bacteria, acetic bacteria, yeasts, and fungi. This microbial species are classified into four groups: homofermentative and heterofermentative lactic acid bacteria and lactose and non-lactose assimilating yeast. In that way, Lactobacillus paracasei ssp. paracasei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus plantarum, and L. kefiranofaciens are predominant species. However, these species represent only 20% of the Lactobacillus in the final fermented beverage, with the remainder consisting of Lactobacillus kefiri (80%;; ). Acetobacter aceti and A. rasens have also been isolated, such as the fungus Geotrichum candidum. More than 23 different yeast species have been isolated from kefir grains and from fermented beverages of different origins. However, the predominant species are Saccharomyces cerevisiae, S. unisporus, Candida kefyr, and Kluyveromyces marxianus ssp. marxianus (;;; Table 1).

8The 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, 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.

9examined 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 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.

10After 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. Table 1 demonstrates the microbial composition, which has been isolated from kefir and kefir grains of different origins.

11Due to its composition, kefir is mainly considered a probiotic resource. “Probiotics are microbial cell preparations or components of microbial cells with a beneficial effect on the health of the host”. Some studies suggest that probiotic bacteria in kefir consumers’ gut are abundant and are correlated with health improvement; in that way, it had been demonstrated that the cell-free fraction of kefir enhances the ability to digest lactose relieving symptoms.

12Another 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.

13The microorganisms in the kefir grains produce lactic acid, antibiotics and bactericides, which inhibit the development of degrading and pathogenic microorganisms in kefir milk. Kefir acts against the pathogenic bacteria Salmonella, Helicobacter, Shigella, Staphylococcus, Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Bacillus subtilis, Micrococcus luteus, Listeria monocytogenes, Streptococcus pyrogenes,, Streptococcus faecalis KR6, Fusarium graminearum CZ1, 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. 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.

14It had been proved that many species of lactobacilli present in kefir have S-layer proteins. Surface layers (S-layers) can be aligned in unit cells on the outermost surface of many prokaryotic microorganisms. 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.

15However, there are other important bioactivities that have been tested with kefir grains, the cell-free fraction of kefir or acid lactic bacteria isolated from kefir, such as antitumoral, anti-inflammatory, antimicrobial immunoregulatory, antiallergenic, wound healing, antidiabetic antimutagenic, and antigenotoxic. In that way, it had been demonstrated that kefir cell-free fraction has antiproliferative effects on human gastric cancer SGC7901 cells, colon adenocarcinoma cells, HuT–102 malignant T lymphocytes, sarcoma 180 in mice, Lewis lung carcinoma and human mammary cancer, and reduce oxidative stress. Another study has shown that suspensions after 24 h fermentation and mechanically disintegrated kefir grains cause a significant inhibition of granuloma tissue formation and a 43% inhibition of the inflammatory process.

16Nevertheless, 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.

17The 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. 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.

18The 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.

19Many 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. 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.

20The EPS kefiran is produced by Lactobacillus kefiranofaciens from kefir grains, which are composed of proteins, polysaccharides, and a complex symbiotic microbial mixture. 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.

21Lactic acid bacteria can synthesize homopolysaccharides or heteropolysaccharides. The synthesized homopolysaccharides are glucans or fructans, which are composed of only one type of monosaccharide (glucose or fructose, respectively;; ), whereas the heteropolysaccharides contain different types of monosaccharides in different proportions (mainly glucose, galactose, and rhamnose),.

22Similarly to lactic acid bacteria, Lactobacillus sp. also produces glucan and fructan. The homopolysaccharides show a much higher performance compared with heteropolysaccharide production.

23The 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.

24Lactobacillus 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.

25Kefiran 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. 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. 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.

26The first study about kefiran structure was published by, 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 and nuclear magnetic resonance (NMR; ). The kefiran structure, according to them, is shown in Figure 1.

27Nowadays, 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.

28Other 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.

29Kefir, 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.

30Authors want to thank CNPq and CAPES for the financial support.

31The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.