IngredientsLactobacillus kefiranofaciens is a species of slime-forming, homofermentative, rod-shaped lactic acid bacteria first isolated from kefir grains, hence its name. It is part of the Lactobacillus genus and the Firmicutes phylum. A polyphasic approach based on various criteria — including core genome phylogeny, pairwise average amino acid identity, clade-specific signature genes, physiological criteria, and ecology — was used recently for the re-assessment of the taxonomy of the families Lactobacillaceae and Leuconostocaceae, resulting in the reclassification of the genus Lactobacillus into 25 genera including an emended genus Lactobacillus. The nomenclature of L. kefiranofaciens in the emended genus remained unchanged and it is still taxonomically assigned to the genus Lactobacillus.
L. kefiranofaciens comprises two subspecies, namely kefiranofaciens and kefirgranum, but only the first one is responsible for the production of kefiran, the water-soluble polysaccharide which is a basic component of the kefir grain. DNA–DNA binding values of over 79% and analogous DNA G+C contents of 37–38 mol% showed that the two earlier-named taxa belonged to one species: L. kefirgranum was determined to be a later synonym of L. kefiranofaciens. An emended description was proposed, and it was proposed that L. kefirgranum should be reclassified as L. kefiranofaciens subsp. kefirgranum.
L. kefiranofaciens subsp. kefiranofaciens, with a genome size of 2.26 Mbp and mol% G+C content of DNA 37.2 for the type strain LMG 19149T, is the polysaccharide kefiran-producing subspecies and is a prominent member of the kefir microbiota. L. kefiranofaciens subsp. kefirgranum, with a genome size of 2.10 Mbp and mol% G+C content of DNA 37.5 for the type strain LMG 15132T, is part of the core microbiota of kefir grains.
Strains of both subspecies are Gram-positive, non-motile, capsulated, non-spore-forming rods (generally 0.8 to 1.2 μm by 3.0 to 20.0 μm) that occur as single cells, in pairs, or occasionally in short chains. The isolated strains were characterized as slime-forming, homofermentative, facultatively anaerobic, and rod-shaped lactic acid bacteria, which differed from all the validly described homofermentative species of the genus Lactobacillus in the carbohydrate fermentation pattern.
One of the main lactic acid bacterial species found in the kefir grain ecosystem worldwide is Lactobacillus kefiranofaciens, exhibiting strong auto-aggregation capacity and, therefore, being involved in the mechanism of grain formation. It comprises two subspecies and has been isolated not only from kefir grains but from other fermented dairy products as well, such as koumiss, hurunge, and tarag. Kefiran-producing, encapsulated Lactobacillus kefiranofaciens was located all over the kefir grain and increased toward the center, while Lactobacillus kefiri populated only a small region at the surface layers.
Kefir beverages can be produced by fermenting cow, goat, buffalo, sheep, camel, mare, and donkey milk with kefir grains according to the "Russian method," which is essentially a "back-slopping" procedure that can be repeated ad infinitum. In Russia, kefir has also been produced by inoculating pasteurized milk with kefir beverage, a mother culture prepared by carrying out traditional kefir fermentation and sieving the grains. A newer technique to produce kefir using immobilized starter cultures isolated from kefir grains — among them L. kefiranofaciens — has been developed, in which various LAB and yeast strains entrapped in microspheres are used for kefir beverage production that is microbiologically similar to the original kefir beverage. Kefir grains are also industrially produced and commercialized by various companies worldwide, and L. kefiranofaciens appears to be one of the key microbial species of commercial blends.
Kefir is a traditional fermented milk beverage that originates from the Caucasus region and Eastern Europe, and was used in ancient times. Its origins predate written records, and it has been consumed for centuries as part of the daily diet and valued for its perceived health-promoting properties. Kefir is likely of central Eurasian origin and has a significant history in the Caucasus Mountains. Nomadic goat and cattle herders in the Caucasus Mountains likely created the first versions of kefir thousands of years ago.
Kefir is a viscous, slightly carbonated dairy beverage, which has its origins in the Caucasian, Tibetan, and Mongolian mountains. It comprises a complex microbial consortium of mainly lactic acid bacteria, acetic acid bacteria, and yeasts, and is considered a functional dairy product associated with a wide range of health benefits.
Genomic research found that the L. kefiranofaciens used in fermentation comprised two clades. The first mainly consisted of strains from Europe (e.g., modern Germany) and coastal areas and islands in Asia (e.g., modern Guangdong, Taiwan Island, Japan, and Singapore). This distribution conformed to the dispersal route from the Caucasus to Europe and to the coastal areas of Asia and Southeast Asia. The other clade mainly consisted of strains distributed in inland East Asia (including Tibet). The reconstructed ancient strains are located at the base of the clade, suggesting an additional route of diffusion of kefir production technology from Xinjiang to inland East Asia through techno-cultural exchange.
Traditional kefir is fermented at ambient temperatures, generally overnight. Fermentation of the lactose yields a sour, carbonated, slightly alcoholic beverage, with a consistency and taste similar to drinkable yogurt. Lactose conversion is accomplished by lactic acid-producing Streptococcus and Lactobacillus bacteria, including L. kefiranofaciens and several other species. Kefir was traditionally consumed as a nutritive daily food and also used in the broader Caucasian folk-medicine tradition for its perceived digestive and fortifying properties; however, these traditional attributed uses pre-date formal scientific verification.
The origins of kefir predate written records, and it has been consumed for centuries as part of the daily diet and valued for its perceived health-promoting properties. Owing to this long history of consumption, kefir has attracted increasing scientific interest as a functional food, prompting clinical investigations into its potential health effects.
Formally, Lactobacillus kefiranofaciens was first identified in 1967 in Russia through studying kefir granules. Fujisawa et al. obtained and analyzed four strains of the species, namely WT-2B, WT-6A, WT-7, and WT-8, isolated from kefir grains; they formally described L. kefiranofaciens sp. nov., with WT-2B (ATCC 43761) being assigned as the type strain. This formal species description was published in the International Journal of Systematic Bacteriology in 1988.
In kefir grains, the main polysaccharide is kefiran, which is a heteropolysaccharide composed of equal proportions of glucose and galactose and is mainly produced by Lactobacillus kefiranofaciens. This biofilm matrix is a heteropolysaccharide called kefiran, which is composed of equal proportions of glucose and galactose. Infrared spectra of kefiran reveal the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide.
Kefiran shows an average molecular weight (Mw) of approximately 534 kDa and a number-average molecular weight (Mn) of 357 kDa in some preparations, though kefiran from certain extractions has shown molecular weights as high as approximately 3,000 kDa, with pseudoplastic rheological behavior.
Kefiran, produced from the kefir fermentation by L. kefiranofaciens, is water-soluble and biodegradable. Compared with other polysaccharides, kefiran has outstanding advantages such as antitumor, antifungal, and antibacterial properties, as well as immunomodulation or epithelium protection, anti-inflammatory activity, and wound-healing properties.
During milk fermentation by kefir grains, many functional compounds like bioactive peptides — with antihypertensive, antioxidative, antiallergenic, antitumor, antimicrobial, anti-inflammatory, and cholesterol-lowering activities — as well as antimicrobial compounds (organic acids, alcohols, carbon dioxide, and bacteriocins) and heteropolysaccharides such as kefiran with potential prebiotic activity are formed.
Kefir grains are initially created by auto-aggregations of Lactobacillus kefiranofaciens and Maudiozyma turicensis or M. humilis, where multiple biofilm producers cause surfaces to adhere, forming a three-dimensional microcolony. The strong auto-aggregation capacity of L. kefiranofaciens is partly attributable to kefiran itself, which acts as the matrix "glue" of the kefir grain. An enhancing effect on the production of interferon β, cortisol, and noradrenaline in human cell lines has been reported, and a possible use as a stress-reducing food component has been hypothesized.
L. kefiranofaciens M1 could strengthen epithelial barrier function in vitro by increasing the transepithelial electrical resistance (TEER) and significantly upregulating the level of the chemokine CCL-20 at both the apical and basolateral sites. In vitro studies show that kefir metabolites upregulate tight junction proteins and mucin secretion, thereby reinforcing epithelial integrity.
The exopolysaccharide kefiran produced by L. kefiranofaciens has been investigated in vivo for its immunomodulating capacity by analyzing the profile of cytokines and immunoglobulins induced at the intestinal mucosa level, in intestinal fluid, and in blood serum, using BALB/c mice that received the exopolysaccharide for 2, 5, or 7 consecutive days. There was an increase of IgA+ cells in the small and large intestine lamina propria, without change in the number of IgG+ cells in the small intestine.
Bacterial colonization, the Th1/Th2 cytokine profile of the mice's splenocytes, and the anti-colitis effect of L. kefiranofaciens M1 were investigated. The strongest response in terms of splenic Th1 cytokine IFN-γ and IL-12 production upon TLR activation was detected in the continuous treatment group.
The putative receptor for the protective effects of L. kefiranofaciens M1 was toll-like receptor 2 (TLR2), which was involved in probiotic-induced cytokine production in vitro and in attenuation of the bleeding score and colon length shortening in vivo.
Research has demonstrated that L. kefiranofaciens M1 exerts anti-allergic activity by regulating the Th1/Th2 balance, increasing the proportion of CD4+CD25+ regulatory T cells, and decreasing the activation of CD19+ B cells. In animal studies, body weight and thymus index returned to normal levels; allergy scores, serum OVA-sIgE, IL-4, IL-5, and IL-10 expression decreased; and IFN-γ and IL-2 increased significantly in the ZW3-treated group compared with the allergy group.
Lactobacillus kefiranofaciens, a lactic acid bacterium isolated from kefir grains, has various probiotic functions, including antioxidant activity, improvement of intestinal health, and immunomodulation. Investigation into the ZW18 postbiotic found that it increased the phagocytosis of RAW264.7 cells and promoted the secretion of cytokines including NO, IL-6, and TNF-α, thereby activating the immune response in macrophages in vitro.
Colitis (Animal Models): L. kefiranofaciens M1, isolated from and identified in Taiwanese milk kefir grain, has demonstrated immune-modulating activity. Its effects on intestinal epithelial cells in vitro and on dextran sodium sulfate (DSS)-induced colitis in vivo were investigated. Production of proinflammatory cytokines was decreased and that of the anti-inflammatory cytokine IL-10 was increased in DSS-treated mice given L. kefiranofaciens M1. Continuous inoculation with L. kefiranofaciens M1 was found to ameliorate the symptoms of DSS-induced colitis in germ-free mice, though L. kefiranofaciens M1 failed to colonize the host, suggesting the organism acts directly on the host rather than through microbiota regulation.
Intestinal Mucosal Immunity (Animal and In Vitro): The probiotic effects ascribed to lactic acid bacteria and their fermented dairy products arise not only from whole microorganisms and cell wall components but also from peptides and extracellular polysaccharides produced during the fermentation of milk. There is an acknowledged lack of knowledge concerning the immune mechanisms induced by exopolysaccharides, which would allow a better understanding of the functional effects described to them.
Evidence Strength: Evidence in this area is primarily preclinical — derived from murine (mouse) models and in vitro cell-line experiments. No randomized controlled trials in human subjects have been published specifically examining L. kefiranofaciens and intestinal inflammation.
The immunoregulatory, anti-allergic, anti-asthmatic, and anti-colitis abilities of L. kefiranofaciens M1 have been demonstrated in a number of in vitro and in vivo experiments. A study evaluated the anti-allergic effects of L. kefiranofaciens ZW3 in ovalbumin-induced allergic mice, with mice divided into groups including food allergy, positive control (L. rhamnosus GG), and low-, mid-, and high-dose ZW3 groups. Mice were sensitized by intraperitoneal injection of OVA/complete Freund's adjuvant, and probiotics were administered orally once every two days. Outcomes measured included allergic score, serum OVA-sIgE, body mass, thymus and spleen indexes, and inflammatory cytokine mRNA expression. Results showed that body weight and thymus index returned to normal levels; allergy scores, serum OVA-sIgE, IL-4, IL-5, and IL-10 expression decreased; and IFN-γ and IL-2 increased significantly. ZW3 also decreased Muribaculaceae and Ruminococcaceae abundance while increasing Lachnospiraceae abundance in the intestinal flora. In summary, ZW3 induced anti-allergic effects by increasing Th1 cytokines and decreasing Th2 cytokines, thereby ameliorating the symptoms of ovalbumin-induced food allergy.
Evidence Strength: Preliminary. All data are from ovalbumin-induced animal models; no human clinical trials have tested L. kefiranofaciens for allergy or asthma outcomes specifically.
L. kefiranofaciens, although very demanding concerning its growth conditions, can be involved in mechanisms affecting intestinal health, immunomodulation, control of blood lipid levels, hypertension, antimicrobial action, and protection against diabetes and tumors.
The L. kefiranofaciens ZW18 postbiotic was investigated for immunomodulatory activities both in vitro and in vivo, finding that it increased phagocytosis and cytokine secretion in macrophages and upregulated abundance of bacteria associated with immune activation. Specifically, it promoted the secretion of cytokines including NO, IL-6, and TNF-α, thereby activating the immune response in macrophages in vitro.
Evidence Strength: Preclinical only. Evidence for immunomodulation in humans is limited to outcomes attributed to kefir broadly; no clinical trials have isolated L. kefiranofaciens as the active agent in human immunomodulation studies.
The oral administration of kefiran in mice shows that kefiran possesses antitumor activities and modulates delayed-type hypersensitivity reactions. Kefiran is a striking option over other exopolysaccharides due to its antitumor, antibacterial, antifungal, and immunomodulation activities that have been extensively studied.
Evidence Strength: Evidence is limited to animal studies and in vitro cell culture assays. No controlled human clinical trials have been performed to evaluate antitumor effects attributable specifically to L. kefiranofaciens or kefiran.
Fermentation of milk by kefir grains produces many functional compounds, including bioactive peptides with antihypertensive, antioxidant, antiallergic, antitumor, anti-inflammatory, and cholesterol-lowering activities. Evidence indicates kefir could have numerous health benefits including regulating blood lipid levels and high blood pressure, along with antimicrobial properties.
The antihypertensive effects associated with kefir fermented by L. kefiranofaciens-containing grain communities are believed to stem from angiotensin-converting enzyme (ACE)-inhibitory peptides generated during proteolysis of milk proteins during fermentation. Studies targeting kefir have come to public attention because of kefir's potential health-promoting effects, which include antimicrobial activity, anticarcinogenic properties, antimutagenic properties, improved gastrointestinal functioning, cholesterol-lowering effects, and immunoregulatory effects.
Evidence Strength: Cholesterol-lowering and antihypertensive evidence is largely derived from animal studies and in vitro bioassays examining kefir-derived peptides and kefiran broadly. The contribution of L. kefiranofaciens specifically, as opposed to the complex kefir microbiome, has not been isolated in clinical human trials. Data from human clinical trials apply to kefir as a whole functional food product, not to the bacterium in isolation.
Kefiran has been reported to have antibacterial, antifungal, and anti-inflammatory activities and to serve as a cicatrizing (wound-healing) agent for use against a variety of infections. Kefiran also protects Caco-2 cells from cytopathic effects induced by Bacillus cereus infection.
Evidence Strength: Antimicrobial evidence is based on in vitro assays and cell-line experiments. No prospective human trials have been reported specifically attributing antimicrobial outcomes to L. kefiranofaciens.
The modern commercialization of kefir has driven the need for high-quality research into its impact on the human microbiome and associated health outcomes; however, there is currently very limited scientific evidence supporting effects of kefir consumption on the human oral and gut microbiome. High-quality human clinical trials are essential to establish the safety and effectiveness of kefir before it can be advised for use in treating conditions linked to the oral and gut microbiota or metabolic health.
In rodent studies, L. kefiranofaciens ZW3, especially in combination with collagen II/glutamine supplementation, increased beneficial bacteria of Ruminococcaceae and Lachnospiraceae abundances and elevated short-chain fatty acid (SCFA) levels in intestinal contents in osteoarthritic rats.
Whether the effects of L. kefiranofaciens M1 are elicited directly on the host or act by regulating the host's microbiota was studied using germ-free mice. Four-week-old female germ-free mice were inoculated intragastrically with 2×108 CFU/mouse of L. kefiranofaciens M1 once or at 2-day intervals for 14 days. Continuous inoculation with L. kefiranofaciens M1 ameliorated symptoms of DSS-induced colitis in germ-free mice; however, L. kefiranofaciens M1 failed to colonize the host, suggesting the organism acts directly on the host rather than via microbiota regulation.
L. kefiranofaciens is not typically standardized as an isolated probiotic supplement in the way that, for example, Lactobacillus acidophilus or Bifidobacterium longum preparations are. It is primarily consumed as an active constituent of fermented kefir beverages. However, several studies have used specific strains at defined dosages:
No established human clinical dosing regimen specific to isolated L. kefiranofaciens has been published in the peer-reviewed literature identified in this review.
Lactobacillus species are part of the microbiota of humans and animals, are found in a variety of food products, and have been studied extensively as fermentation starter and/or adjunct cultures and probiotics. They are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) and belong to the Qualified Presumption of Safety (QPS) list of the European Food Safety Authority (EFSA). L. kefiranofaciens, which is from a safe source and has multiple biological activities, has been permitted to be used in functional foods in many countries, including the European Union countries, Japan, Korea, Canada, and China.
There is no evidence of pathogenicity nor toxicity from kefir, leading to L. kefiranofaciens being widely regarded as safe for ingestion and for assisting in fermentation. Overall, L. kefiranofaciens M1 did not induce adverse effects on hematology, serum biochemistry, and urinalysis parameters. Gross and microscopic histopathology of the organs revealed no toxicity effect.
In recent years, lactobacilli have received considerable attention owing to their potential involvement in the spread of antibiotic resistance. Intrinsic (as opposed to acquired) resistance has minimal potential for horizontal spread and therefore usually poses no risk in non-pathogenic bacteria. Most lactobacilli show intrinsic resistance to aminoglycosides (gentamicin, kanamycin, neomycin, and streptomycin), vancomycin, ciprofloxacin, and trimethoprim, while Lactobacillus species are generally susceptible to β-lactams (penicillin and ampicillin) and inhibitors of protein synthesis (tetracycline, erythromycin, chloramphenicol, and linezolid). The significance of acquired versus intrinsic resistance in individual L. kefiranofaciens strains must be assessed on a strain-by-strain basis according to EFSA breakpoint guidelines.
While Lactobacillus species are generally recognized as GRAS and belong to the EFSA QPS list, several species behave as opportunistic pathogens in certain contexts. Certain Lactobacillus strains have been associated with cases of sepsis, endocarditis, or bacteremia, mostly in association with a severe underlying disease. No specific cases involving L. kefiranofaciens were identified in the sources reviewed, though this general caveat pertains to the broader genus.
It is hard to standardize the composition and probiotic effects of kefir because the microbiota population of kefir grains is dynamic in nature and can be affected by the source of the milk, the production process, and various other factors that may influence the composition of the kefir grains. Furthermore, L. kefiranofaciens M1 failed to colonize the host in germ-free mouse studies, suggesting the organism acts directly on the host rather than via permanent microbiota establishment. This transient-colonization characteristic may have implications for the duration and frequency of dosing in any intended probiotic application.
L. kefiranofaciens, although very demanding concerning its growth conditions, can be involved in mechanisms affecting intestinal health and other areas. Its fastidious nutritional requirements mean that standardized production and stable supplemental formulations pose technical challenges, and the organism may not survive in the same concentrations across all commercially prepared kefir beverages.
The scientific literature on L. kefiranofaciens is predominantly preclinical. There is currently very limited scientific evidence supporting effects of kefir consumption on the human oral and gut microbiome. High-quality human clinical trials are essential to establish the safety and effectiveness of kefir before it can be advised for use in treating specific conditions. The vast majority of mechanistic data come from murine models and cell-line experiments; the results of these studies are promising across several biological areas but cannot be extrapolated to established human clinical outcomes without appropriately powered and designed clinical trials.
A further complication is that kefir is a complex multi-species consortium; studies using kefir as a whole product cannot attribute observed effects exclusively to L. kefiranofaciens. Research using isolated strains (M1, ZW3, ZW18, OSU-BDGOA1) addresses this limitation but introduces strain-specificity concerns — findings from one strain may not apply to another. Different strains of L. kefiranofaciens have demonstrated different health benefits, and the genetic factors exerting diverse functionalities in different strains, as well as potential niche-specific genes and pathways, remain an area of ongoing investigation.
Health conditions that Lactobacillus kefiranofaciens may help support.
Body systems that Lactobacillus kefiranofaciens may help support.
Go back