Mechanistic insights into LuxS/AI-2 quorum sensing-regulated biofilm formation and its impact on the texture and flavor of kefir.

Paraprobiotics from water kefir grains probiotic strain: Obtainment and innovative approaches for functional fermented beverages

Kefiran is a bioactive exopolysaccharide produced by kefir grains, whose synthesis is strongly influenced by culture medium composition. In this study, cheese whey was evaluated as an alternative fermentation substrate for kefiran production, and the effect of supplementation with fermentable sugars (glucose, galactose, and lactose) and casein was assessed under controlled conditions. Kefir grains were cultivated in whey- and milk-based media, and kefiran production was quantified using an anthrone-based method, while grain growth and carbohydrate consumption were monitored. Supplementation with sugars and casein reduced kefiran production by up to 34.6% and did not improve yield, whereas unsupplemented whey supported the highest kefiran concentration (86.9 ± 3.7 mg/L), comparable to that obtained in semi-skimmed milk (84.0 ± 3.0 mg/L). The recovered polysaccharide was characterized by Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (1H NMR), X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), showing structural and physicochemical properties comparable to kefiran obtained from semi-skimmed milk. These results indicate that whey constitutes a feasible and simple fermentation medium for kefiran production, and that increased medium complexity does not necessarily improve process performance.

Polysaccharides from water kefir grains (WG) are functional, food-safe, and potential novel materials for functional food development. This study focused on the extraction process, structural characteristics, and in vitro biological activities of polysaccharides from WG (WPU), as well as the effects of WPU on goat yogurt (GY). WPU was optimally extracted from WG via ultrasound-assisted extraction (UE) under the conditions: 340W (ultrasonic power), 42min (ultrasonic time), 20mL/g (liquid-to-solid ratio), and 80°C (ultrasonic temperature), achieving a high yield of 27.64%. The monosaccharide composition of WPU-4 (the main purified fraction) was glucose (96.59mol%), arabinose (0.23mol%), galactose (0.66mol%), and mannose (2.52mol%). Its backbone was predominantly composed of 6-Glcp. Scanning electron microscopy (SEM) revealed that WPU-4 exhibited a sheet-like structure, with an uneven and loose porous network on its surface and a honeycomb-like morphology in its interior. In vitro assays showed WPU had superior antioxidant, α-glucosidase, and pancreatic lipase inhibitory activities compared to purified fractions. Adding 1.0mg/mL WPU to GY enhanced its antioxidant, antidiabetic, and hypolipidemic activities. This research provides an efficient WPU extraction method and confirms bioactivity potential, offering technical support for WPU industrial applications as functional ingredients in foods and pharmaceuticals.

Unraveling the microbial community restructuring and metabolic alterations from Tibetan kefir grains to fermented milk

Immunotherapy targeting the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) (PD-1/PD-L1) axis has revolutionized cancer treatment, yet its efficacy in hepatocellular carcinoma (HCC) remains limited. Emerging evidence suggests that gut microbiota plays a pivotal role in modulating tumor immune microenvironments (TIME), offering a novel avenue to enhance immunotherapy outcomes. This study investigates the regulatory effects of Lactobacillus kefiranofaciens (LK) on the TIME in HCC, focusing on its modulation of Suppressor of cytokine signaling 3 (SOCS3) expression and the Janus-activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, with the goal of improving responses to anti-PD-1/PD-L1 therapy. LK was isolated from kefir grains and identified through genomic sequencing. In vitro assays, including Cell Counting Kit-8 (CCK-8), 5-Ethynyl -2'- deoxyuridine (EdU) staining, colony formation, Transwell, and apoptosis detection, were conducted using Hepa1-6 HCC cells. In vivo, subcutaneous and orthotopic HCC mouse models were treated with LK to assess tumor progression. Single-cell RNA sequencing (scRNA-seq) and Bulk RNA-seq analyses were performed to identify key signaling pathways and therapeutic targets. SOCS3 expression was manipulated via lentiviral transfection to validate its role in immunotherapy enhancement. LK significantly inhibited HCC cell growth, migration, and invasion, while promoting apoptosis in vitro. In vivo, LK treatment reduced tumor size and improved immune cell infiltration, particularly T cells and NK cells. Transcriptomic analysis revealed that LK upregulates SOCS3, suppresses the JAK-STAT signaling pathway, and reduces PD-L1 expression, enhancing T cell-mediated immune responses. This study highlights the potential of gut microbiota modulation, specifically through LK, to enhance the efficacy of anti-PD-1/PD-L1 immunotherapy in HCC by targeting SOCS3 and the JAK-STAT pathway. These findings provide a new therapeutic approach for improving immunotherapy outcomes in HCC. Gut probiotics modulate the immune microenvironment to enhance the response of liver cancer patients to anti-PD-1/PD-L1 immunotherapy: molecular mechanisms.

Water Kefir is a plant-based fermented beverage, traditionally produced on a small scale by fermenting a sucrose solution with fresh or dried fruits, using water kefir grains as inoculum. The grains are relatively simple communities that consist of both eukaryotes and prokaryotes, rendering them a paradigm for studying microbial ecology and interspecies interactions. Recently, water kefir has attracted growing research and industrial interest due to its potential and perceived health benefits. Owing to its increasing popularity, there is a growing demand for controlled and standardised production on an industrial scale. However, industrial-scale production remains a challenge due to the limited knowledge of the biological interactions of the microbial consortia and the lack of defined starter cultures. This review examines the current understanding of microbial and metabolic complexity of water kefir obtained from various omics studies. It further investigates the potential of an integrated multi-omics approach to elucidate mechanisms of microbial interactions and provides a roadmap for conducting multi-omics studies on fermented foods using water kefir as an example. This review also explores the potential application of genome-scale metabolic modelling in the development of functional and defined microbial communities for food fermentation. It identifies key challenges associated with such modelling and provides perspectives to address them. Finally, this review briefly discusses the regulatory challenges associated with the use of defined communities in food systems.

Water kefir grains are complex probiotic granules that can efficiently ferment fruit and vegetable juices and significantly improve product flavor. However, the mechanisms of flavor formation remain unclear, which limits the process optimization of this technology. This study investigated the mechanisms involved in flavor formation during the fermentation of strawberry juice with water kefir grains. The results showed that as fermentation progressed, the total acidity increased, whereas the pH value and soluble solids content decreased. Additionally, the contents of citric acid and malic acid gradually decreased with fermentation, while the contents of lactic, acetic, and succinic acid increased, and three soluble sugars showed reduced levels. A total of 218 volatile compounds were identified. Eight dominant bacterial genera and one dominant yeast species were detected. Significant correlations between some key microorganisms and flavor compounds were observed. Specifically, Lactiplantibacillus was positively correlated with hexyl acetate. Meanwhile, Gluconobacter and Acetobacter were positively correlated with methyl (Z,Z)-9,12-octadecadienoate, isoamyl acetate, etc. In contrast, LAB such as Lacticaseibacillus and Schleiferilactobacillus showed the opposite correlations with these key flavor compounds. Saccharomyces showed a positive correlation with ethyl palmitate, ethyl propionate, phenylsuccinic acid, and 1-pentanol. The main flavor compound metabolic pathways were predicted and they were significantly related with yeasts, acetic acid bacteria, and lactic acid bacteria. Overall, this study offers a theoretical basis for the directional regulation and optimization of the flavor quality of strawberry juice fermented with water kefir.

Kefir grains are a valuable source of exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) with potential applications in fermented dairy products. In this study, LAB isolated from kefir grains originating from five regions were screened for EPS production and probiotic-related properties. Three strains, Lactiplantibacillus plantarum XZ61, Lactobacillus kefiranofaciens EG10, and Lentilactobacillus kefiri EG12, were selected based on high EPS yield, antimicrobial activity, antioxidant capacity, and tolerance to acidic and bile salt conditions. After optimization, the highest EPS yield (539.57 μg/mL) was obtained from strain EG10.The purified EPS consisted of two molecular weight fractions (≈1.4 and 23~25 kDa) and was rich in mannose (33.38~61.58%). Among the three EPS, EG10-EPS exhibited superior emulsifying and flocculating properties comparable to commercial stabilizers, as well as notable ABTS•+ and hydroxyl radical scavenging activities. Furthermore, co-fermentation of L. kefiranofaciens EG10 with conventional yogurt starter cultures significantly improved exopolysaccharide content, water-holding capacity, texture, and antioxidant activity of fermented milk, particularly in cow milk. These results demonstrate the potential of kefir-derived EPS-producing LAB as natural functional cultures for fermented dairy applications.

Dual-component kefir (grain and beverage) supplementation confers protective effects against Salmonella infection through immune modulation and pathogen inhibition.

Fermentation method outweighs geographic origin in shaping kefir's in vitro anti-obesity potential and probiotic properties.

Selenium bioenrichment and functional enhancement with antioxidants in Actinidia arguta juice by fermentation with selenium-enriched Tibetan kefir grains.

This study aimed to characterize the fatty acid composition and to evaluate lipid quality indices of kefir produced with varying ratios of chestnut milk and reconstituted skim milk, using both traditional and commercial starter cultures. Lactobacillaceae counts remained high in all samples (8.35-8.70 log cfu/g on MRS and 8.45-9.35 log cfu/g on M17), while intermediate chestnut milk substitution ratios promoted higher yeast growth than either 100% reconstituted or 100% plant-based formulations, particularly in samples fermented with grains. Chestnut milk samples fermented with kefir grains or commercial starter cultures exhibited distinct fatty acid profiles and changes in lipid quality during storage. The 20% chestnut milk sample had significantly higher levels of butyric, caproic, caprylic, capric, myristic, and palmitic acids and α-linolenic acid. In contrast, the 100% chestnut milk sample exhibited the highest concentrations of linoleic and γ-linolenic acids. Fermentation with kefir grains resulted in elevated butyric, caproic, capric, palmitic, margaric, and arachidic acids, as well as polyunsaturated linoleic acid. Samples fermented with a commercial starter culture contained higher levels of oleic, γ-linolenic, α-linolenic, nonadecylic, and gadoleic acids. Throughout storage, short- and medium-chain fatty acids and polyunsaturated fatty acids (ω-6 and ω-3) decreased, while palmitic, margaric, oleic, and arachidic acids increased. The 20% chestnut milk sample had the highest contents of saturated fatty acids, total ω-3 fatty acids, and short- and medium-chain fatty acids. The 100% chestnut milk sample had the highest levels of total unsaturated fatty acids, ω-6 fatty acids, and long-chain fatty acids. Lipid quality indices were most favorable in the 100% chestnut milk and commercial starter fermentations, showing lower atherogenic and thrombogenic indices. These results highlight chestnut milk-based kefir as a low-fat, functional dairy alternative with an enhanced bioactive fatty acid content and favorable lipid quality indices.

ABSTRACT This study investigates the potential for developing a dairy‐free, functional kefir using Sarıkılçık rice ( Oryza sativa L.), a traditional Turkish variety cultivated in Kastamonu. Kefir was produced by fermenting rice milk with water kefir grains at three inoculation levels (2%, 5%, and 9%) and was then evaluated in comparison with traditional milk kefir and a commercial kefir product. Comprehensive assessments included physicochemical measurements (pH, dry matter content, titratable acidity, and color), sensory evaluation, and metagenomic profiling via high‐throughput 16S rRNA sequencing. Among the rice kefirs, the 9% inoculated rice kefir exhibited the lowest pH of 3.56 and the highest lactic acid content, along with darker and more green‐blue color values. Although traditional and commercial kefirs had higher sensory acceptability, rice kefir with 9% inoculation emerged as the best plant‐based option. Metagenomic results showed Lactobacillus ghanensis dominated rice kefirs, while Lactococcus kefiranofaciens was abundant in commercial kefir. Microbial diversity was highest in traditional kefir, followed by commercial and rice kefirs. The highest diversity index among rice kefirs was found in those inoculated with 5%, 9%, and 2% water kefir, respectively. Rice milk showed the lowest microbial richness. These findings demonstrate the potential of water kefir fermentation as a promising approach for developing microbiologically diverse and plant‐based kefir alternatives. Integrating sensory, chemical, and microbial data provides valuable insight for future optimization and industrial applications.

Obesity is a complex metabolic disorder characterized by excessive accumulation of adipose tissue, resulting from an imbalance between energy intake and expenditure. It is associated with an increased risk of chronic diseases such as type 2 diabetes, cardiovascular disease, and cancer. Kefiran is a water-soluble exopolysaccharide produced by lactic acid bacteria, Lactobacillus kefiranofaciens, in kefir grains, composed primarily of glucose and galactose. It has garnered scientific interest due to its antioxidant, anti-inflammatory, and antimicrobial properties. Rice Kefiran (RK) is a functional food made with culturing L. kefiranofaciens in a medium containing rice. It is standardized to contain at least 5 mg/g of kefiran. This study investigated the anti-obesity effect of RK on a high-fat diet (HFD)-induced obese mouse model. HFD-fed mice exhibited marked increases in body weight gain (10.3 g vs. 2.0 g in controls) and adipose tissue mass (2.4 g vs. 0.4 g in controls). RK administration significantly attenuated weight gain to 8.3 g and 6.0 g at doses of 10 and 50 mg/kg, respectively, and reduced adipose tissue mass to 2.2 g (RK10) and 1.7 g (RK50). Oral glucose tolerance testing revealed impaired glucose clearance in HFD-fed mice, with blood glucose levels of 403.5 mg/dL at 15 min and 314.6 mg/dL at 120 min, compared with 348.8 mg/dL and 232.2 mg/dL in controls. RK treatment improved glucose tolerance, particularly at 50 mg/kg, reducing glucose levels to 359.0 mg/dL at 15 min and 263.8 mg/dL at 120 min. Biochemical analyses demonstrated that RK significantly reduced serum total cholesterol (213.6 mg/dL in HFD vs. 178.0 and 184.0 mg/dL in RK10 and RK50), triglycerides (379.0 mg/dL in HFD vs. 228.8 and 234.6 mg/dL), and non-esterified fatty acids (0.89 mEq/mL in HFD vs. 0.54 and 0.35 mEq/mL), while phospholipid levels remained unchanged. Furthermore, RK increased serum nicotinamide phosphoribosyltransferase (NAMPT) levels from 15.8 ng/mL in HFD-fed mice to 30.0 and 50.0 ng/mL in the RK10 and RK50 groups, respectively, and restored hepatic NAD+/NADH ratios toward control levels (1.78 µmol/L in HFD vs. 1.90 µmol/L and 2.07 µmol/L in RK10 and RK50). Gene expression analysis showed that RK increased Nampt mRNA expression and decreased the mRNA expression of adipogenic and lipogenic genes, including Srebp-1c, Acc-1, and Fas. These findings suggest that RK may ameliorate obesity-related metabolic disturbances and its associated metabolic dysfunctions by modulating lipid metabolism, glucose tolerance, and NAD+ biosynthesis pathways.

Background: The increasing interest in functional fermented dairy products has intensified research on lactic acid bacteria (LAB) as potential probiotics. Kefir, a symbiotic matrix of LAB and yeasts, represents a rich source of probiotic microorganisms, yet limited data exist on strains isolated from Indonesian goat milk kefir. Exploring the molecular identity and probiotic traits of these local isolates can contribute to the development of novel functional dairy cultures suited to regional production systems. Methods: Sixteen LAB isolates were procured from Indonesian kefir grains sub-cultured in goat milk and evaluated for probiotic characteristics. Molecular identification was conducted via 16S rRNA sequencing and in vitro experiments assessed acid and bile salt tolerance, carbohydrate utilization with 4% inulin and antibacterial efficacy against Staphylococcus aureus, Escherichia coli and Salmonella Typhi. Statistical analysis was conducted to ascertain strain-level variations in probiotic characteristics. Result: Twelve isolates were successfully identified, including eight Lactiplantibacillus plantarum and four Lactobacillus plantarum strains. Strains AN and QF showed high acid tolerance ( greater than 50% survival at pH 2.0 for 90 min) and strain NT exhibited strong bile salt resistance up to 1.5% (log 6.5 CFU/mL). All isolates metabolized inulin as the sole carbon source and inhibited pathogenic bacteria with inhibition zones of 3.7-6.0 mm, the strongest against S. aureus. L. plantarum strains obtained from goat milk kefir exhibited strong probiotic properties, validating their suitability as starting cultures for functional dairy fermentation and as possibilities for industrial probiotic formulations.

This study was carried out on the effect of different amounts of whey on the kefir production process and the bacterial microbiota of the beverages. Fermentation was carried out by mixing commercial kefir grains with cow, sheep, and goat milk with their whey at concentrations of 0% (control group), 25%, 50%, and 75%. The bacterial microbiota in the produced beverages and the relative abundances of seven microorganisms (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus delbrueckii, Lactococcus lactis, Streptococcus thermophilus, Leuconostoc mesenteroides, and acetic acid bacteria) were determined using metagenomic analysis targeting the V3-V4 region of 16 S rRNA gene, and quantitative polymerase chain reaction (qPCR), respectively. Lactococcus lactis and Leuconostoc mesenteroides were detected in higher abundance in whey-containing goat samples. While Lactobacillus was dominant in the cow milk samples containing 0%, 25%, and 50% whey, Enterococcus was dominant in the samples containing 75% whey. In sheep milk samples, Lactobacillus was dominant in samples with 75% and 50% whey, Lactococcus was dominant in samples with 25% whey, and Enterococcus was dominant in samples with no (0%) whey. Lactobacillus was dominant in all goat milk samples. In conclusion, high levels of lactic acid bacteria were shown to preserve their viability in the experimentally produced beverages with the addition of whey at different concentrations, and the whey could be used to produce kefir.

Kefir is a fermented dairy product that can be prepared through microbial fermentation using kefir grains. These grains consist of a symbiotic community of bacteria and yeasts that influence the chemical composition, texture, and sensory characteristics of fermented milk. The incorporation of fruit by-products during fermentation has been explored as a strategy to enhance the functional quality of kefir-based beverages. Among them, plantain (Musa paradisiaca) by-products represent a promising source of bioactive compounds with antioxidant potential and significant amounts of dietary fiber. This study aimed to optimize the fermentation conditions of milk kefir enriched with green plantain peel using response surface methodology and evaluate the microbial viability of the optimized beverage during 21 days of storage. Fermentation parameters were established through preliminary tests, employing UHT milk, sugar (8%), kefir grains, and green plantain peel, fermented for 4 h at 25 °C, using a central composite rotational design (CCRD). The CCRD included two independent variables (X1: green plantain peel 10%-30.0% and X2: Kefir grains, 5%-20%). The optimized formulation, containing 20% green plantain peel and 10% kefir grains, showed increased protein content and reduced carbohydrate levels compared to the control beverage. Although higher inoculum levels did not significantly enhance bioactive compound content, this was likely due to microbial utilization of these metabolites. Lactic acid bacteria (LAB) counts increased over storage, reaching ~104 CFU ml-1 after 21 days, demonstrating the stability of the core kefir microbiota. Sensory evaluation indicated an overall acceptability index of 81.29%. In conclusion, the enrichment of milk kefir with green plantain peel resulted in a nutritionally improved and sensorially accepted beverage, characterized by higher protein density and lower carbohydrate content. These findings highlight the potential of plantain peel as a functional ingredient for the development of enhanced fermented dairy products.

Kefiran, a polysaccharide from kefir grain, modulates gut microbiota and hepatic metabolism to mitigate metabolic dysfunction-associated steatotic liver disease.

ABSTRACTThis study aimed to evaluate the effects of milk kefir on parameters related to inflammatory bowel disease (IBD) in interleukin‐10 knockout (IL‐10−/−) mice. Sixteen C57BL/6J IL‐10−/− male mice were divided into two experimental groups. The control group (n = 8) received 0.4 mL of whole milk (UHT) and the kefir group (n = 8) received 0.4 mL of a fermented beverage made from milk kefir grains (2.4 × 108 colony‐forming units [CFU]), both administered via gavage for 4 weeks. Feces were collected and body weight was measured weekly. At the end of the study, the animals were anesthetized and euthanized, and the small intestine and cecum content were collected for analysis. Proximate composition of kefir and microbiological analysis were conducted. Histomorphometry measurements and quantification of short‐chain fatty acids (SCFAs) were conducted in small intestine. SFCAs were also evaluated in cecum content, and microbiota composition was evaluated in fecal samples. The kefir beverage improved histomorphometry characteristics of the small intestine, increased the number of goblet cells, reduced inflammatory infiltrates, increased Lactobacillus, and reduced Desulfovibrio, suggesting attenuation of the inflammatory process. Furthermore, the kefir beverage increased the concentration of butyrate, a short‐chain fatty acid with anti‐inflammatory properties, in the small intestine, which may also be associated with reduced inflammation in this group.