Dynamic alterations of flavor, functional nutrients, and microbial community during fermentation of different animal milk kefirs.

Kefir is a traditional dairy beverage, usually made from cow or goat milk fermented with kefir grains, and has many health benefits. To elucidate the fermentation patterns of animal milk kefirs during the fermentation process and find the optimal milk types, cow, camel, goat, and donkey milk were fermented with kefir grains for 0, 1, 3, 5, and 7 days. Volatile and non-volatile metabolites and microbial changes were dynamically monitored. The results showed that volatile flavor substances were massively elevated in four kefirs on days 1-3. Lipids and carbohydrates gradually decreased, while amino acids, small peptides, and tryptophan derivatives accumulated during fermentation in four kefirs. Besides, four kefirs had similar alterations in Lactobacillus and Acetobacter, while some distinctions existed in low-abundance bacteria. Association analysis of microorganisms and volatile and non-volatile metabolites also revealed the underlying fermentation mechanism. This study found that appropriately extending the fermentation time contributed to the accumulation of some functional nutrients. Furthermore, goat and donkey milk could be the better matrices for kefir fermentation.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Whole Genome Analysis of Tibetan Kefir-Derived Lactiplantibacillus Plantarum 12-3 Elucidates Its Genomic Architecture, Antimicrobial and Drug Resistance, Potential Probiotic Functionality and Safety.

BACKGROUND: Lactiplantibacillus plantarum 12-3 holds great promise as a probiotic bacterial strain, yet its full potential remains untapped. This study aimed to better understand this potential therapeutic strain by exploring its genomic landscape, genetic diversity, CRISPR-Cas mechanism, genotype, and mechanistic perspectives for probiotic functionality and safety applications. METHODS: L. plantarum 12-3 was isolated from Tibetan kefir grains and, subsequently, Illumina and Single Molecule Real-Time (SMRT) technologies were used to extract and sequence genomic DNA from this organism. After performing pan-genomic and phylogenetic analysis, Average Nucleotide Identity (ANI) was used to confirm the taxonomic identity of the strain. Antibiotic resistance gene analysis was conducted using the Comprehensive Antibiotic Resistance Database (CARD). Antimicrobial susceptibility testing, and virulence gene identification were also included in our genomic analysis to evaluate food safety. Prophage, genomic islands, insertion sequences, and CRISPR-Cas sequence analyses were also carried out to gain insight into genetic components and defensive mechanisms within the bacterial genome. RESULTS: The 3.4 Mb genome of L. plantarum 12-3, was assembled with 99.1% completeness and low contamination. A total of 3234 genes with normal length and intergenic spacing were found using gene prediction tools. Pan-genomic studies demonstrated gene diversity and provided functional annotation, whereas phylogenetic analysis verified taxonomic identity. Our food safety study revealed a profile of antibiotic resistance that is favorable for use as a probiotic. Analysis of insertional sequences, genomic islands, and prophage within the genome provided information regarding genetic components and their possible effects on evolution. CONCLUSIONS: Pivotal genetic elements uncovered in this study play a crucial role in bacterial defense mechanisms and offer intriguing prospects for future genome engineering efforts. Moreover, our findings suggest further in vitro and in vivo studies are warranted to validate the functional attributes and probiotic potential of L. plantarum 12-3. Expanding the scope of the research to encompass a broader range of L. plantarum 12-3 strains and comparative analyses with other probiotic species would enhance our understanding of this organism's genetic diversity and functional properties.

The composition of Tibetan kefir grain TKG-Y and the antibacterial potential and milk fermentation ability of S. warneri KYS-164 screened from TKG-Y.

This study utilized high-throughput sequencing and SEM observation to elucidate the microbial composition of a Tibetan herder's homemade kefir grain named TKG-Y. Subsequently, S. warneri KYS-164 was isolated from TKG-Y, which can produce mixed protein substances with antibacterial activity, namely bacteriocin-like inhibitory substances (BLIS). BLIS can significantly reduce the growth rate of Escherichia coli 366-a, Staphylococcus aureus CICC 10384 and mixed strains at low concentrations (1 × MIC). The presence of the warnericin-centered gene cluster in KYS-164 may explain the antibacterial properties of the BLIS. Pepsin and an acidic environment can reduce the number of colonies of KYS-164 by 2.5 Log10 CFU mL-1 within 1 h, and reduce the antibacterial activity of BLIS by 21.48%. S. warneri KYS-164 showed no antibiotic resistance and biological toxicity after 80 subcultures, while BLIS produced by 40 generations of the strain retained their inhibitory efficacy against pathogenic bacteria. After 48-hour fermentation of milk with KYS-164, volatile compounds such as aldehydes, phenols, esters, and alcohols, giving it a floral, fruity, milky, oily, and nutty aroma, were released, enriching the sensory characteristics of dairy products. This study not only revealed the bacterial colony composition information of home-made kefir grain TKG-Y but also discovered and proved that S. warneri KYS-164 has the potential to inhibit bacteria and ferment dairy products. This will provide a basis for subsequent applied research on KYS-164.

Study and characterization of a product based on a vegetable extract of quinoa fermented with water kefir grains.

This work aimed to study and characterize a product based on vegetable extract of quinoa (WVEQ) fermented with water kefir grains. The effect of sucrose concentration (SC), inulin concentration (IC), and xanthan gum (XG) concentration were evaluated using a central composite design (CCD) 23. They were subsequently characterized regarding cellular growth of the grains, beverage yield, pH, soluble solids, carbon dioxide (CO2) production, lactic acid, and ethanol production. Therefore, for the final stage, two formulations (F1 and F8) of the CCD were chosen to be characterized in terms of proximate composition, microbiological composition of the kefir culture, analysis of organic compounds, sensory analysis, and enzymatic and microbiological characterization before and after simulation of in vitro gastrointestinal digestion. In the two chosen products, one can see that fermentation optimized the bioavailability of proteins due to the high proteolytic activity of the microorganisms in kefir and the increase in lipid content. In identifying microorganisms, there was a prevalence of Saccharomyces sp. yeasts. In the sensory analysis, the F8 formulation showed better results than the F1 formulation. In vitro, gastrointestinal digestion showed reduced lactic acid bacteria and yeast and increased acetic acid bacteria in the liquid phase for both formulations. In the enzymatic profile, there was a reduction in all enzymes analyzed for both formulations, except for amylase in F1, which went from 14.05 U/mL to 39.41 U/mL. Therefore, it is concluded that using WVEQ as a substrate for the product appears to be a viable alternative with nutritional and technological advantages for serving a specific market niche.

Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and life-threatening lung disease with high mortality rates. The limited availability of effective drugs for IPF treatment, coupled with concerns regarding adverse effects and restricted responsiveness, underscores the need for alternative approaches. Kefir peptides (KPs) have demonstrated antioxidative, anti-inflammatory, and antifibrotic properties, along with the capability to modulate gut microbiota. This study aims to investigate the impact of KPs on bleomycin-induced pulmonary fibrosis. METHODS: Mice were treated with KPs for four days, followed by intratracheal injection of bleomycin for 21 days. Comprehensive assessments included pulmonary functional tests, micro-computed tomography (µ-CT), in vivo image analysis using MMPsense750, evaluation of inflammation- and fibrosis-related gene expression in lung tissue, and histopathological examinations. Furthermore, a detailed investigation of the gut microbiota community was performed using full-length 16 S rRNA sequencing in control mice, bleomycin-induced fibrotic mice, and KPs-pretreated fibrotic mice. RESULTS: In KPs-pretreated bleomycin-induced lung fibrotic mice, notable outcomes included the absence of significant bodyweight loss, enhanced pulmonary functions, restored lung tissue architecture, and diminished thickening of inter-alveolar septa, as elucidated by morphological and histopathological analyses. Concurrently, a reduction in the expression levels of oxidative biomarkers, inflammatory factors, and fibrotic indicators was observed. Moreover, 16 S rRNA sequencing demonstrated that KPs pretreatment induced alterations in the relative abundances of gut microbiota, notably affecting Barnesiella_intestinihominis, Kineothrix_alysoides, and Clostridium_viride. CONCLUSIONS: Kefir peptides exerted preventive effects, protecting mice against bleomycin-induced lung oxidative stress, inflammation, and fibrosis. These effects are likely linked to modifications in the gut microbiota community. The findings highlight the therapeutic potential of KPs in mitigating pulmonary fibrosis and advocate for additional exploration in clinical settings.

Exploration adhesion properties of Liquorilactobacillus and Lentilactobacillus isolated from two different sources of tepache kefir grains.

Due to the distinctive characteristics of probiotics, it is essential to pinpoint strains originating from diverse sources that prove efficacious in addressing a range of pathologies linked to dysfunction of the intestinal barrier. Nine strains of lactic acid bacteria were isolated from two different sources of tepache kefir grains (KAS2, KAS3, KAS4, KAS7, KAL4, KBS2, KBS3, KBL1 and KBL3), and were categorized to the genus Lacticaseibacillus, Liquorilactobacillus, and Lentilactobacillus by 16S rRNA gene. Kinetic behaviors of these strains were evaluated in MRS medium, and their probiotic potential was performed: resistance to low pH, tolerance to pepsin, pancreatin, bile salts, antibiotic resistance, hemolytic activity, and adhesion ability. KAS7 strain presented a higher growth rate (0.50 h-1) compared with KAS2 strain, who presented a lower growth rate (0.29 h-1). KBS2 strain was the only strain that survived the in vitro stomach simulation conditions (29.3%). Strain KBL1 demonstrated significantly higher viability (90.6%) in the in vitro intestine simulation conditions. Strain KAS2 demonstrated strong hydrophilic character with chloroform (85.6%) and xylol (57.6%) and a higher percentage of mucin adhesion (87.1%). However, strains KBS2 (84.8%) and KBL3 (89.5%) showed the highest autoaggregation values. In terms of adhesion to the intestinal epithelium in rats, strains KAS2, KAS3 and KAS4 showed values above 80%. The growth of the strains KAS2, KAS3, KAS4, KBS2, and KBL3 was inhibited by cefuroxime, cefotaxime, tetracycline, ampicillin, erythromycin, and cephalothin. Strains KBS2 (41.9% and 33.5%) and KBL3 (42.5% and 32.8%) had the highest co-aggregation values with S. aureus and E. coli. The results obtained in this study indicate that lactic acid bacteria isolated from tepache can be considered as candidates for potentially probiotic bacteria, laying the foundations to evaluate their probiotic functionality in vivo and thus to be used in the formulation of functional foods.

Bibliometric analysis of water kefir and milk kefir in probiotic foods from 2013 to 2022: A critical review of recent applications and prospects.

Although milk kefir and water kefir have different physical, chemical and microbiological characteristics, several microbial species that make up kefir stand out with probiotic functions. Furthermore, because it is suitable for a variety of substrates, kefir and the species of probiotic microorganisms that make it up are seen as a promising alternative in the development of probiotic and health-promoting foods. The aim of this study was to carry out a bibliometric analysis of water kefir and milk kefir in probiotic foods and to critically analyze recent applications and prospects. Using the Scopus database, 202 documents published between 2013 and 2022 were identified and submitted to bibliometric analysis using the VOSviewer software. Regarding recent applications, 107 documents published between 2021 and June 2023 were identified. It was observed that, in the literature consulted, no study used bibliometric analysis to evaluate the use of water kefir and milk kefir in probiotic foods. Due to the presence of probiotic species, kefir has been listed as an alternative for the production of new probiotic food matrices that are beneficial to health. Recent applications show kefir's potential in the development of probiotic products based on fruit and fruit juice, whey beverages, fermented milks and derivatives, and alcoholic beverages such as beers.

Evaluation of antimicrobial activity of kefir drink against escherichia coli, salmonella typhimurium and shigella flexneri / Evaluación de la actividad antimicrobiana de la bebida de kéfir contra escherichia coli, salmonella typhimurium y shigella flexneri

Objetivo: Evaluar la actividad antimicrobiana de la bebida de Kéfir contra Escherichia coli, Salmonella typhimurium y Shigella flexneri. Método: El kéfir utilizado en la investigación fue adquirido en Toluca, Estado de México. Se realizó una reactivación del kéfir con leche pasteurizada y se analizaron 3 carbohidratos (miel, azúcar y piloncillo) en diferentes concentraciones y tiempos, 80, 100, 120% y 24, 48 y 72 horas respectivamente. Los microorganismos cultivables aislados se caracterizaron por técnicas fenotípicas, bioquímicas y de espectroscopia de masas. El pH inicial y final se determinaron durante el tiempo de estudio. La actividad antimicrobiana se realizó extrayendo los metabolitos presentes en el fermento con el método de Kirby-Bauer, además se evaluó el fermento directo, para determinar si hubo inhibición con las cepas de Escherichia coli ATCC 11229, Salmonella typhimurium ATCC 14028 y Shigella flexneri ATCC 12022. Resultados: Se observó que en los tres carbohidratos utilizados a una concentración de 120% y en un tiempo de 72 h, fue donde se obtuvo un pH menor (3,51 a 3,64) comparado con su concentración inicial (6,50 a 6,64). A partir de los metabolitos extraídos en los diferentes fermentos, no se obtuvo halo de inhibición con las cepas analizadas. Sin embargo, al usar fermentos directos, se observó que en los carbohidratos utilizados (azúcar, miel, piloncillo) existía la presencia de un halo de inhibición o el crecimiento de colonias distintas de las evaluadas. Los microorganismos cultivables aislados fueron: Pichia kudriavzevii (levadura); Enterococcus sp (coco grampositivo) y Lactobacillus sp (bacilo grampositivo). Conclusiones: Los fermentos de kéfir hechos con diferentes carbohidratos, llegaron a presentar un grado de inhibición solo como un consorcio contra microorganismos Gram-negativos analizados.Palabras Clave: Kéfir; fermentación microbiota; infección; actividad antimicrobiana.(AU)

Objective: Evaluate the antimicrobial activity of the Kefir drink against Escherichia coli, Salmonella typhimurium and Shigella flexneri. Method: The Kefir used in the investigation was acquired in Toluca, State of Mexico. We performed a reactivation of Kefir with pasteurized milk and analyzed 3 carbohydrates (honey, sugar and piloncillo) at different concentrations and times, 80, 100, 120% and 24, 48 and 72 hours respectively. Isolated cultivable microorganisms were characterized by phenotypic, biochemical and mass spectroscopy techniques. The initial and final pH were determined during the study time. The antimicrobial activity was carried out by extracting the metabolites present in the ferment with the Kirby-Bauer method, in addition the direct ferment was evaluated, to determine if there was inhibition with the Escherichia coli (ATCC 11229) strains, Salmonella typhimurium (ATCC 14028) and Shigella flexneri (ATCC 12022). Results: It was observed that in the three carbohydrates used at a concentration of 120% and at a time of 72 h, a lower pH was obtained (3.51 to 3.64) compared to their initial concentration (6.50 to 6.64). From the metabolites extracted in the different ferments, no inhibition halo was obtained with the strains analyzed. However, when using direct ferments, it was observed that in the carbohydrates used (sugar, honey, piloncillo) there was the presence of an inhibiting halo or the growth of colonies other than those evaluated. The isolated cultivable microorganisms were: Pichia kudriavzevii (yeast); Enterococcus sp (gram-positive coconut) and Lactobacillus sp (gram-positive bacillus). Conclusions : Kefir ferments made with different carbohydrates, came to present a degree of inhibition only as a consortium against Gram-negative microorganisms analyzed.(AU)

Determination of the Viability of Lactic Acid Bacteria by Dynamic In Vitro Gastrointestinal Model in Household and Industrial-Type Kefir Samples.

This study presents results based on differences in the antioxidant activity and lactic acid bacteria counts in different parts of the digestive tract following simulated gastrointestinal digestion of kefir samples. Statistically significant differences were observed in Lactobacillus counts in different kefir types including industrial (IK), starter culture (SCK), and kefir grains (KG). These differences were observed between the initial and second min in the mouth region (T = 3.968; p < 0.05); and between the initial, 60th, and 120th min in the stomach region (R = 11.146; p < 0.05). Additionally, a statistically significant difference was noted in the initial Lactobacillus levels among the IK, SCK, and KG in the stomach region (H = 7.205; p < 0.05). Also, significant differences were identified between the Lactococcus counts of IK across 0, 60, and 120 min in the stomach region (R = 10.236; p < 0.05). Notably, a statistically significant difference was noted in the Lactococcus levels in the KG between the initial and second min in the mouth region (T = 3.101; p < 0.05) and between 0, 60, and 120 min in the stomach region (R = 25.771; p < 0.001). These findings highlight the differences between the physicochemical characteristics of different kefir types. A decrease in lactic acid bacteria counts in kefir samples was observed throughout the dynamic in vitro gastrointestinal tract to reveal the significance of the digestive process when determining probiotic product capacity.

A robust nanoLC high-resolution mass spectrometry methodology for the comprehensive profiling of lactic acid bacteria in milk kefir.

Consumer attention to functional foods containing probiotics is growing because of their positive effects on human health. Kefir is a fermented milk beverage produced by bacteria and yeasts. Given the emerging kefir market, there is an increasing demand for new methodologies to certify product claims such as colony-forming units/g and bacterial taxa. MALDI-TOF MS proved to be useful for the detection/identification of bacteria in clinical diagnostics and agri-food applications. Recently, LC-MS/MS approaches have also been applied to the identification of proteins and proteotypic peptides of lactic acid bacteria in fermented food matrices. Here, we developed an innovative nanoLC-ESI-MS/MS-based methodology for profiling lactic acid bacteria in commercial and artisanal milk kefir products as well as in kefir grains at the genus, species and subspecies level. The proposed workflow enables the authentication of kefir label claims declaring the presence of probiotic starters. An overview of the composition of lactic acid bacteria was also obtained for unlabelled kefir highlighting, for the first time, the great potential of LC-MS/MS as a sensitive tool to assess the authenticity of fermented foods.

Milk kefir alters fecal microbiota impacting gut and brain health in mice.

Kefir is a fermented beverage made of a symbiotic microbial community that stands out for health benefits. Although its microbial profile is still little explored, its effects on modulation of gut microbiota and production of short-chain fatty acids (SCFAs) seems to act by improving brain health. This work aimed to analyze the microbiota profile of milk kefir and its effect on metabolism, oxidative stress, and in the microbiota-gut-brain axis in a murine model. The experimental design was carried out using C57BL-6 mice (n = 20) subdivided into groups that received 0.1 mL water or 0.1 mL (10% w/v) kefir. The kefir proceeded to maturation for 48 h, and then it was orally administered, via gavage, to the animals for 4 weeks. Physicochemical, microbiological, antioxidant analyzes, and microbial profiling of milk kefir beverage were performed as well as growth parameters, food intake, serum markers, oxidative stress, antioxidant enzymes, SCFAs, and metabarcoding were analyzed in the mice. Milk kefir had 76.64 ± 0.42% of free radical scavenging and the microbiota composed primarily by the genus Comamonas. Moreover, kefir increased catalase and superoxide dismutase (colon), and SCFAs in feces (butyrate), and in the brain (butyrate and propionate). Kefir reduced triglycerides, uric acid, and affected the microbiome of animals increasing fecal butyrate-producing bacteria (Lachnospiraceae and Lachnoclostridium). Our results on the brain and fecal SCFAs and the antioxidant effect found were associated with the change in the gut microbiota caused by kefir, which indicates that kefir positively influences the gut-microbiota-brain axis and contributes to the preservation of gut and brain health. KEY POINTS: • Milk kefir modulates fecal microbiota and SCFA production in brain and colon. • Kefir treatment increases the abundance of SCFA-producing bacteria. • Milk kefir increases antioxidant enzymes and influences the metabolism of mice.

Metagenomic analysis and antibacterial activity of kefir microorganisms.

The microbiota composition of kefir grain and milk kefir was assessed via a metagenomic approach. Significant microorganisms were isolated and identified using molecular methods. A safety assessment was conducted based on antibiotic susceptibility and blood hemolysis. Probiotic traits such as resistance to gastric tract conditions, surface characteristics, adhesion to intestinal cells, and antibacterial activity were also assessed. Metagenomic analysis revealed that kefir grains are a more stable community with clear dominant species as compared to milk kefir. Lactobacillus kefiranofaciens BDGO-A1, Lactobacillus helveticus BDGO-AK2, and Lactobacillu kefiri strains showed tolerance to acidic pH and the presence of bile salts, adhesion capability to Caco-2 cells, in vitro antibacterial activity, and the production of antibacterial proteins. In the metagenomic analysis, contigs associated with these species showed the presence of genes involved in exporting polyketide antibiotics and bacteriocin production. To fully exploit the potential probiotic properties of these microorganisms to help human health, further investigation is necessary to elucidate the mechanisms behind the biological activity and the genotypic characteristics of the isolated strains.

Use of kefir-derived lactic acid bacteria for the preparation of a fermented soy drink with increased estrogenic activity.

Fermented foods are receiving growing attention for their health promoting properties. In particular, there is a growing demand for plant-based fermented foods as dairy alternatives. Considering that soy is a vegetal food rich in nutrients and a source of the phytoestrogen isoflavones, the aim of this study was to select safe food microorganisms with the ability to ferment a soy drink resulting in a final product with an increased estrogenic activity and improved functional properties. We used milk kefir grains, a dairy source of microorganisms with proven health-promoting properties, as a starting inoculum for a soymilk. After 14 passages of daily inoculum in fresh soy drink, we isolated four lactic acid bacterial strains: Lactotoccus lactis subsp. lactis K03, Leuconostc pseudomesenteroides K05, Leuconostc mesenteroides K09 and Lentilactobacillus kefiri K10. Isolated strains were proven to be safe for human consumption according to the assessment of their antibiotic resistance profile and comparative genomics. Furthermore, functional characterization of the bacterial strains demonstrated their ability to ferment sugars naturally present in soybeans and produce a creamy texture. In addition, we demonstrated, by means of a yeast-based bioluminescence reporter system, that the two strains belonging to the genus Leuconostoc increased the estrogenic activity of the soybean drink. In conclusion, the proposed application of the bacterial strains characterized in this study meets the growing demand of consumers for health-promoting vegetal food alternatives to dairy products.

Yeast cell wall polysaccharides in Tibetan kefir grains are key substances promoting the formation of bacterial biofilm.

This study investigated the interaction among Kluyveromyces marxianus G-Y4 (G-Y4), Lacticaseibacillus paracasei GL1 (GL1) and Lactobacillus helveticus SNA12 (SNA12) that isolated from Tibetan kefir grains. Additionally, the effects of G-Y4 on the growth and biofilm formation of GL1 and SNA12 were determined. The results indicated that G-Y4 promoted the growth of GL1 and SNA12 and improved their biofilm-forming ability. Furthermore, the dead cells of G-Y4 were found that could enhance bacterial biofilm formation, and the cell wall polysaccharide (CWPS) produced by G-Y4 was performed to be key substances that promote the formation of bacterial biofilms. Moreover, the structure of soluble cell wall polysaccharides (SCWP) and insoluble cell wall polysaccharide (NCWP) of G-Y4 were studied to determine their contribution to biofilm formation. Results showed that G-Y4-SCWP was α-mannan with the main chain of a →6)-α-d-Manp-(1→ unit and the branch structure of →2)-α-d-Manp-(1. At the same time, G-Y4-NCWP was a glucan rich in ß-(1→3), ß-(1→2), or ß-(1→4) linkages.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition.

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

A Combined In Vitro and In Vivo Assessment of the Safety of the Yeast Strains Kluyveromyces marxianus A4 and A5 Isolated from Korean Kefir.

Kefir is a traditional fermented milk containing beneficial bacteria and yeasts. Despite Kluyveromyces marxianus, isolated from kefir, gaining increasing attention as a potential probiotic yeast owing to its biological function, Saccharomyces boulardii is the only species considered as a probiotic yeast. We evaluated the safety of K. marxianus strains A4 and A5, isolated from Korean kefir, in comparison with that of S. boulardii. Virulence attributes were preliminarily assessed in vitro including their ability of gelatin hydrolysis, pseudohyphae formation, and hemolysis. To evaluate in vivo safety, the strains were challenged in a healthy animal model, four-week-old female BALB/c mice. Mice were orally administered 0.2 mL of 0.9% sterilized saline (NC_S; n = 6), S. boulardii ATCC MYA-796 (high concentration, S.b_H; low concentration, S.b_L; n = 6 for each), K. marxianus A4 (high concentration, A4_H; low concentration, A4_L; n = 6 for each), or K. marxianus A5 (high concentration, A5_H; low concentration, A5_L; n = 6 for each) for 2 weeks. At study end, body weight, spleen and liver weights, and blood parameters were assessed. K. marxianus A4 and A5 were tested negative for gelatinase and hemolysis. Overall, hematological, plasma biochemical, and cytokine (interleukin-1ß and tumor necrosis factor-α) parameters were comparable between the experimental and negative control (NC) groups. Notably, the interleukin-6 level of the A5_H group was significantly lower than that of the NC group (p < 0.05), suggesting anti-inflammatory potential of K. marxianus A5.

Anthocyanin Addition to Kefir: Metagenomic Analysis of Microbial Community Structure.

The addition of anthocyanin to kefir for the production of more functional and bio-diversified kefir beverages has the potential to increase kefir's healthful activities. In the present study, anthocyanin extracts, obtained from black carrots, were added into kefir mixture during the fermentation process in different concentrations (1% and 5%, w/v). These kefir samples were then analyzed in terms of their microbiological qualities by metagenomic analysis. The results of the analyses show that the addition of anthocyanin has significant impacts on the community structure of kefir microbiome which in turn directly affects the expected health impacts of the beverage. Kefir with no anthocyanin included predominantly probiotic bacteria such as Lactococcus lactis (34%) and Lactobacillus kefiri (34%). On the other hand, kefir with 1% anthocyanin demonstrated a more balanced distribution of probiotic species like Lb. kefiri (17%), Leuconostoc mesenteroides (9%), and Lc. lactis (5%) at similar abundance rates. 5% anthocyanin kefir demonstrated the highest polarity in the community with a strong dominance of probiotic Lb. kefiri (72%), and distinctly less abundant bacteria such as Streptococcus salivarius subsp. thermophilus (3%). These findings provide that fortification with anthocyanins can be utilized to enhance the quality, composition, and beneficial functions of kefir.

Antibacterial Property and Metagenomic Analysis of Milk Kefir.

Milk kefir fermentation has been used in households for generations. Consumption of milk kefir has been associated with various health benefits, presumably from the probiotics of yeast and bacteria that make up the kefir grains. In addition, many of the microbes are known to produce novel antimicrobial compounds that can be used for other applications. The microbes living inside kefir grains differ significantly depending on geographical location and production methods. In this study, we aimed to use metagenomic analysis of fermented milk by using three different kefir grains (kefir 1, kefir 2, and kefir 3) from different US sources. We analyzed the microbial compositions of the three milk fermentation samples. This study revealed that each sample contains unique and distinct groups of microbes, kefir 1 showed the least diversity, and kefir 3 showed the highest diversity. Kefir 3 is rich in Proteobacteria while kefir 2 is dominated by the Firmicutes. Using bacterial indicator growth analyses carried out by continuous readings from microplate-based bioreactor assays suggested that kefir 2 fermentation filtrate has higher antibacterial property. We have screened 30 purified cultures of kefir 2 sample and isolated two lactic acid bacteria strains with higher antibacterial activities; the two strains were identified as Leuconostoc mesenteroides 28-1 and Lentilactobacillus kefiri 25-2 by 16S genomic PCR with confirmed antibacterial activities of fermentation filtrate after growing under both aerobic and anaerobic conditions.

Biopolymer from Water Kefir as a Potential Clean-Label Ingredient for Health Applications: Evaluation of New Properties.

The present work aimed to characterize the exopolysaccharide obtained from water kefir grains (EPSwk), a symbiotic association of probiotic microorganisms. New findings of the technological, mechanical, and biological properties of the sample were studied. The EPSwk polymer presented an Mw of 6.35 × 105 Da. The biopolymer also showed microcrystalline structure and characteristic thermal stability with maximum thermal degradation at 250 °C. The analysis of the monosaccharides of the EPSwk by gas chromatography demonstrated that the material is composed of glucose units (98 mol%). Additionally, EPSwk exhibited excellent emulsifying properties, film-forming ability, a low photodegradation rate (3.8%), and good mucoadhesive properties (adhesion Fmax of 1.065 N). EPSwk presented cytocompatibility and antibacterial activity against Escherichia coli and Staphylococcus aureus. The results of this study expand the potential application of the exopolysaccharide from water kefir as a potential clean-label raw material for pharmaceutical, biomedical, and cosmetic applications.