Apple polyphenols prevent patulin-induced intestinal damage by modulating the gut microbiota and metabolism of the gut-liver axis.

Patulin (PAT), a foodborne toxin, causes severe intestinal damage. To mitigate this health threat, mice were pretreated with apple polyphenols (AP) in their drinking water (0.01 % and 0.05 %) for eight weeks, followed by exposure to PAT during the last two weeks. Subsequently, histopathological and biochemical evaluations of intestinal tissues were conducted, alongside assessments of alterations in gut microbiota, colonic content metabolome, and hepatic metabolome. Consequently, AP alleviated PAT-induced villus and crypt injury, mucus depletion, GSH level decline, GSH-Px and SOD activity reduction, and MPO activity elevation. Notably, AP counteracted PAT-mediated microbiota disruptions and promoted the abundance of beneficial bacteria (Dubosiella, Akkermansia, Lachnospiraceae, and Lactobacillus). Furthermore, AP counteracted PAT-induced metabolic disorders in the colonic contents and liver. Ultimately, AP prevented intestinal injury by regulating the gut microbiota and amino acid, purine, butanoate, and glycerophospholipid metabolism in the gut-liver axis. These results underscore the potential of AP to prevent foodborne toxin-induced intestinal damage.

Untargeted metabolomics combined with chemometrics reveals distinct metabolic profiles across two sparkling cider fermentation stages.

This study aimed to examine the metabolic profiles of Saccharomyces cerevisiae yeasts (WLS21 and Y41) in two phases of sparkling cider making (normal and pressure fermentation) by combining untargeted metabolomic with chemometrics. The results showed that of the 634 nonvolatile metabolites identified using LC-MS and 83 volatile metabolites identified by GC-MS, the differential metabolites were 226 and 54, respectively. Metabolic pathway and correlation analyses showed that aspartic acid, phenylalanine and tyrosine, glutamic acid and purine metabolism were associated with flavor formation. The pressure fermentation process increased apigenin, naringenin, toxifolin, pyridoxine and thiamine contents in the final cider. These findings provide useful information and new research ideas for the formation of flavor in sparkling cider and the regulation of phenolic and vitamin production by microbial stress fermentation.

The dose-dependent mechanism behind the protective effect of lentinan against acute alcoholic liver injury via proliferating intestinal probiotics.

Acute alcoholic liver injury (AALI) is a widespread disease that can develop into hepatitis, liver fibrosis, and cirrhosis. In severe cases, it can be life-threatening, while drug treatment presents various side effects. This study characterized the structure of natural lentinan (LNT) from the Qinba Mountain area and investigated the protective mechanism of different LNT doses (100 mg kg-1, 200 mg kg-1, and 400 mg kg-1) on AALI. The results showed that LNT was a glucose-dominated pyran polysaccharide with a triple-helical structure and a molecular weight (Mw) of 7.56 × 106 Da. An AALI mouse model showed that all the LNT doses protected liver function, reduced hepatic steatosis, alleviated oxidative stress and inflammatory response, and stimulated probiotic proliferation. Low-dose LNT increased anti-oxidant-associated beneficial bacteria, medium-dose LNT improved liver swelling and promoted anti-oxidant-associated probiotics, and high-dose LNT increased the probiotics that helped protect liver function and anti-oxidant and anti-inflammatory properties. All the LNT doses inhibited pathogenic growth, including Oscillospiraceae, Weeksellaceae, Streptococcaceae, Akkermansiaceae, Morganellaceae, and Proteus. These results indicated that the protective effect of LNT against AALI was mediated by the proliferation of various intestinal probiotics and was related to the consumption doses. These findings offer new strategies for comprehensively utilizing Lentinula edodes from the Qinba Mountain area and preventing AALI using natural food-based substances.

Different polysaccharide-enhanced probiotic and polyphenol dual-functional factor co-encapsulated microcapsules demonstrate acute colitis alleviation efficacy and food fortification.

Probiotics and polyphenols have multiple bioactivities, and developing co-encapsulated microcapsules (CM) is a novel strategy to enhance their nutritional diversity. However, the development of CMs is challenged by complicated processing, single types, and unclear in vivo effects and applications. In this study, the co-microencapsulations of polyphenol and probiotic were constructed using pectin, alginate (WGCA@LK), and Fu brick tea polysaccharides (WGCF@LK), respectively, with chitosan-whey isolate proteins by layer-by-layer coacervation reaction, and their protective effects, in vivo effectiveness, and application potential were evaluated. WGCA@LK improved the encapsulation rate of polyphenols (42.41 %), and remained high viability of probiotics after passing through gastric acidic environment (8.79 ± 0.04 log CFU/g) and storage for 4 weeks (4.59 ± 0.06 log CFU/g). WGCF@LK exhibited the highest total antioxidant activity (19.40 ± 0.25 µmol/mL) and its prebiotic activity removed the restriction on probiotic growth. WGCA@LK showed strong in vitro colonic adhesion, but WGCF@LK promoted in vivo retention of probiotics at 48 h. WGCF@LK showed excellent anti-inflammatory effects and alleviated symptoms of acute colitis in mice. These findings provide unique insights into the fortification of probiotic-polyphenol CMs by different polysaccharides and the development of novel health foods with rich functional hierarchies and superior therapeutic effects.

Trimethylamine N-oxide promotes the proliferation and migration of hepatocellular carcinoma cell through the MAPK pathway.

Trimethylamine-n-oxide (TMAO) is a metabolite of intestinal flora following the consumption of phosphatidylcholine-rich foods. Clinical cohort studies have shown that plasma TMAO may be a risk factor for cancer development, including hepatocellular carcinoma (HCC), but fundamental research data supporting this hypothesis are lacking. In this study, HCC cells were treated with TMAO in vivo and in vitro to evaluate the effect on some indicators related to the malignancy degree of HCC, and the relevant molecular mechanisms were explored. In vitro, TMAO promoted the proliferation and migration of HCC cells and significantly upregulated the expression of proteins related to epithelial-mesenchymal transformation (EMT). In vivo, after HCC cells were inoculated subcutaneously in nude mice given water containing TMAO, the tumors grew faster and larger than those in the mice given ordinary water. The immunohistochemistry analysis showed that proliferation, migration and EMT-related proteins in the tumor tissues were significantly upregulated by TMAO. Furthermore, TMAO obviously enhanced the phosphorylation of MAPK signaling molecules in vivo and in vitro. In conclusion, TMAO promotes the proliferation, migration and EMT of HCC cells by activating the MAPK pathway.

Downregulation of C1R promotes hepatocellular carcinoma development by activating HIF-1α-regulated glycolysis.

C1R has been identified to have a distinct function in cutaneous squamous cell carcinoma that goes beyond its role in the complement system. However, it is currently unknown whether C1R is involved in the progression of hepatocellular carcinoma (HCC). HCC tissues were used to examine C1R expression in relation to clinical and pathological factors. Malignant characteristics of HCC cells were assessed through in vitro and in vivo experiments. The mechanism underlying the role of C1R in HCC was explored through RNA-seq, methylation-specific PCR, immuno-precipitation, and dual-luciferase reporter assays. This study found that the expression of C1R decreased as the malignancy of HCC increased and was associated with poor prognosis. C1R promoter was highly methylated through DNMT1 and DNMT3a, resulting in a decrease in C1R expression. Downregulation of C1R expression resulted in heightened malignant characteristics of HCC cells through the activation of HIF-1α-mediated glycolysis. Additionally, decreased C1R expression was found to promote xenograft tumor formation. We found that C-reactive protein (CRP) binds to C1R, and the free CRP activates the NF-κB signaling pathway, which in turn boosts the expression of HIF-1α. This increase in HIF-1α leads to higher glycolysis levels, ultimately promoting aggressive behavior in HCC. Methylation of the C1R promoter region results in the downregulation of C1R expression in HCC. C1R inhibits aggressive behavior in HCC in vitro and in vivo by inhibiting HIF-1α-regulated glycolysis. These findings indicate that C1R acts as a tumor suppressor gene during HCC progression, opening up new possibilities for innovative therapeutic approaches.

Prophylactic effects of Tibetan goat kefir on depression-like behaviors in chronic unpredictable stress model through the gut-brain axis.

BACKGROUND: Depression is a common psychological disorder, and traditional therapeutic drugs often result in side effects such as emesis, dry mouth, headache, dysentery and constipation. Probiotics and goat milk have garnered widespread attention for their ability to modulate immune function and regulate the endocrine system, and for their anti-inflammatory effects. In this work, the effects of Tibetan goat kefir on the behavior, immune status, neuroendocrine response and gut microbiological composition of chronic unpredictable mild stress (CUMS) mouse models were evaluated. RESULTS: The results indicated that Tibetan kefir goat milk significantly alleviated behavioral despair in mice. Furthermore, the results demonstrated that Tibetan kefir goat milk mitigated the inflammatory response in the mice and moderated the hyperactivity of the hypothalamic-pituitary-adrenal axis and the expression of brain-derived neurotrophic factor. Meanwhile, chronic stress-induced gut microbial abnormalities were restored. In addition, the correlation between gut microbiota and nervous system was evaluated. CONCLUSION: These results explained the potential mechanism of Tibetan kefir in the antidepressant effect on the CUMS model and enriched diets for depressed patients. © 2024 Society of Chemical Industry.

Characterization and subcellular localization of selenium in Limosilactobacillus fermentum Ln-9 obtained by intense pulsed light-ultraviolet combined mutagenesis.

Lactic acid bacteria (LAB) can convert inorganic selenium (Se) to organic Se and elemental forms with low toxicity and high bioavailability, but a comprehensive Se analysis of Se-enriched LAB is lacking. In this study, Limosilactobacillus fermentum Ln-9 was obtained by intense pulsed light-ultraviolet combined mutagenesis, and its characteristics and subcellular localization of Se were analyzed. The results displayed that Ln-9 accumulated 3.03 times Se that of the original strain. Under optimal fermentation conditions, the total Se content of Se-enriched Ln-9 (SeLn-9) reached 12.16 mg/g with 96.34% contained in Se nanoparticles (SeNPs), which was much higher than that of organic macromolecules. Furthermore, SeNPs were mainly localized outside the cell, Se-proteins were in the membrane and cytoplasmic fractions, and Se-polysaccharides were in the membrane fraction. Besides, SeLn-9 maintained a good morphology and gastrointestinal tolerance and had an enhanced antioxidant capacity. These findings make Ln-9 promising for applications in the food industry.

Accumulation and metabolism of selenium in the rare yeast Kazachstania unispora during the selenium enrichment process.

Selenium (Se)-enriched yeast is a good nutritional source for human being. Kazachstania unispora (K. unispora) has shown the positive physiological functionality for human health, whose potential for Se enrichment, however, remains elusive. This study demonstrated the ability of K. unispora to convert inorganic Se to organic Se, and then comprehensively investigated the accumulation and metabolism of Se in K. unispora. The results indicated that K. unispora can effectively accumulate organic Se, of which 95% of absorbed Se was converted to organic forms. Among these organic Se, 46.17% of them was bound to protein and 16.78% was combined with polysaccharides. In addition, some of the organic Se was metabolized to selenomethionine (30.26%) and selenocystine (3.02%), during which four low-molecular weight selenometabolites were identified in K. unispora. These findings expand the scope of Se-enriched yeast species, and provide useful knowledge for further investigation of Se enrichment mechanism in K. unispora.

Efficacy, kinetics, inactivation mechanism and application of cold plasma in inactivating Alicyclobacillus acidoterrestris spores.

As spores of Alicyclobacillus acidoterrestris can survive traditional pasteurization, this organism has been suggested as a target bacterium in the fruit juice industry. This study aimed to investigate the inactivation effect of cold plasma on A. acidoterrestris spores and the mechanism behind the inactivation. The inactivation effect was detected by the plate count method and described by kinetic models. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), the detection of dipicolinic acid (DPA) release and heat resistance detection, the detection and scavenging experiment of reactive species, and cryo-scanning electron microscopy were used to explore the mechanism of cold plasma inactivation of A. acidoterrestris. The results showed that cold plasma can effectively inactivate A. acidoterrestris spores in saline with a 3.0 ± 0.3 and 4.4 ± 0.8 log reduction in CFU/mL, for 9 and 18 min, respectively. The higher the voltage and the longer the treatment time, the stronger the overall inactivation effect. However, a lower gas flow rate may increase the probability of spore contact with reactive species, resulting in better inactivation results. The biphasic model fits the survival curves better than the Weibull model. SEM and TEM revealed that cold plasma treatment can cause varying degrees of damage to the morphology and structure of A. acidoterrestris spores, with at least 50 % sustaining severe morphological and structural damage. The DPA release and heat resistance detection showed that A. acidoterrestris spores did not germinate but died directly during the cold plasma treatment. 1O2 plays the most important role in the inactivation, while O3, H2O2 and NO3- may also be responsible for inactivation. Cold plasma treatment for 1 min reduced A. acidoterrestris spores in apple juice by 0.4 ± 0.0 log, comparable to a 12-min heat treatment at 95 °C. However, as the treatment time increased, the survival curve exhibited a significant tailing phenomenon, which was most likely caused by the various compounds in apple juice that can react with reactive species and exert a physical shielding effect on spores. Higher input power and higher gas flow rate resulted in more complete inactivation of A. acidoterrestris spores in apple juice. What's more, the high inactivation efficiency in saline indicates the cold plasma device provides a promising alternative for controlling A. acidoterrestris spores during apple washing. Overall, our study provides adequate data support and a theoretical basis for using cold plasma to inactivate A. acidoterrestris spores in the food industry.

An alginate-based edible coating containing lactic acid bacteria extends the shelf life of fresh strawberry (Fragaria × ananassa Duch.).

Edible coatings, formulated with sodium alginate and various strains of lactic acid bacteria, were evaluated for their effectiveness in extending the shelf life and mitigating microbial risks associated with strawberries. This study specifically employed strains of Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus plantarum as antimicrobial agents. Through physicochemical property analysis, the alginate-based antimicrobial coating proved most effective in reducing the strawberry weight loss rate, decay index, and ascorbic acid degradation. Over time, all treatments exhibited increased fungal growth. However, strawberries treated with alginate and lactic acid bacteria recorded lower final colony formation counts-6.82 log CFU/g for SA + LPC, 6.04 log CFU/g for SA + LGG, and 6.26 log CFU/g for SA + LP-compared to 8.73 log CFU/g in the control group. In terms of bacterial resistance under gastrointestinal conditions, L. paracasei demonstrated the highest survival rate post-simulated gastric fluid exposure, while L. plantarum showed the greatest resilience post-simulated intestinal fluid exposure. These findings underscore the efficacy of alginate-based antimicrobial coatings in not only enhancing the storage quality of strawberries but also ensuring microbial safety and potential benefits for gut health.

Biosafety measures for Alicyclobacillus spp. strains across various levels of biohazard.

Alicyclobacillus bacteria are important contaminants in the beverage industry because their spores remain in the product after usual pasteurization. At the same time, their impact on human health has yet to be characterized, as it is generally assumed to be low or non-existent. However, these bacteria are causing quality concerns mainly due to odor and taste changes of the product. Since potential health effects are not precisely known, an experimental assessment was performed, including a biosafety assessment of six viable and non-viable vegetative and spore forms of Alicyclobacillus spp. strains using cell cultures and rodent study. The monolayer of Caco-2 (Cancer coli-2) cells was investigated for its adsorption effect on the epithelium of the small intestine of mice. Lactate dehydrogenase leakage (LDH) and transepithelial electrical resistance (TEER) tests were used to ensure the integrity of the cell membrane and tight junctions. The methylthiazole tetrazolium bromide (MTT) assay examined in vitro cytotoxicity in Caco-2 and HepG2 cell lines. The hemolysis of erythrocytes was spectrophotometrically measured. The results showed negligible cytotoxicity or non-toxic response in mice. In conclusion, Alicyclobacillus spp. exhibited biocompatibility with negligible cytotoxicity and minimal safety concerns.

A versatile bilayer smart packaging based on konjac glucomannan/alginate for maintaining and monitoring seafood freshness.

This study introduces a novel multi-functional double-layer intelligent packaging. It focuses on developing a dual-function system capable of real-time monitoring and freshness preservation. Specifically, cellulose nanocrystalline (CNC) was obtained through acid hydrolysis, and then CNC/soybean protein isolate (CNC/SPI) complex colloid particles were prepared via antisolvent method. These particles served as stabilizers to prepare oil-in-water (O/W) cinnamon essential oil Pickering emulsion (CSCEO). The CSCEO was then integrated into the emulsified hydrophobic layer of a konjac glucomannan (Kgm) matrix through intermolecular hydrogen bonding. Finally, alginate (Alg) matrix containing alizarin (Al) as an indicator was added to construct the bilayer structure using a layer-by-layer casting strategy. The inner layer Alg/Al was the pH/NH3-responsive indicator layer, while the outer layer Kgm/CSCEO acted as the high-barrier bacteriostatic layer. The obtained dual-function, double-layer film (Alg/Al-Kgm/CSCEO), which possesses a sensitive, reversible and rapid response towards pH/NH3, shows exceptional antibacterial and antioxidant properties, as well as excellent mechanical property, light-blocking capability and hydrophobicity. For monitoring and maintaining the actual freshness of shrimp, such a bilayer packaging displays smallest change of ∆E and TVB-N (18.65 mg/100 g) even after 72 h, which further highlighting its potential in enhancing food safety and extending shelf life.

Fermented goat milk by selenium-enriched Lactobacillus paracasei alleviates depressive psychological disturbance.

Depression is a prevalent psychiatric disease with the characteristic of persistently gloomy mood. The treatment of depression with traditional therapeutic medications suffers from low efficacy and adverse side effects due to the extremely unpredictable courses and uneven responses to treatment. The goal of this paper was to investigate the preparation of selenium-enriched fermented goat milk and the potential mechanism of its intervention on the chronic unpredictable stress-induced depression mice model. The results showed that Se-Lactobacillus paracasei 20241 (Se-20241) significantly alleviated depressive behavior, reversed the upregulation of inflammatory factors, and attenuated glucocorticoid resistance. Meanwhile, the results showed a modulatory function on oxidative stress dysfunction in the liver, hippocampus, and prefrontal cortex. The change in abundance of Ileibacterium, Muribaculaceae, Turicibacter, Dubosiella, and Bifidobacterium was also modified. These results provided the theoretical groundwork for the development of psychoactive probiotic supplements for depressed patients and clarified the probable mechanism of Se-20241 for antidepressant impact on the CUMS model.

Immobilization of pancreatin based on ultrasound-assisted polydopamine functionalized magnetic porous chitosan for the detoxification of ochratoxin A in wine.

Ochratoxin A (OTA) is a mycotoxin that globally contaminates fruits and their products. Since OTA have a huge negative impact on health hazards and economic losses, it is imperative to establish an effective and safe strategy for detoxification. Here, pancreatin was immobilized on the surface of polydopamine functionalized magnetic porous chitosan (MPCTS@ PDA) for the degradation of OTA. Compared with free pancreatin, MPCTS@ PDA@ pancreatin displayed excellent thermal stability, acid resistance, storage stability and OTA detoxification in wine (>58%). Moreover, the MPCTS@ PDA@ pancreatin retained 43% initial activity after 8 reuse cycles. There was no significant change in the quality of wine after MPCTS@ PDA@ pancreatin treatment. Moreover, it did not exhibit cytotoxicity which facilitated its application in wine. These results demonstrated that MPCTS@ PDA@ pancreatin can be used as a highly effective biocatalysate for OTA detoxification in wine.

Regenerated SERS substrate based on Ag/AuNPs-TiO2-oxidized carbon cloth for detection of imidacloprid.

Imidacloprid (IMI) are widely used in modern tea industry for pest control, but IMI residues pose a great threat to human health. Herein, we propose a regeneration metal-semiconductor SERS substrate for IMI detection. We fabricated the SERS sensor through the in-situ growth of a nano-heterostructure incorporating a semiconductor (TiO2) and plasmonic metals (Au, Ag) on oxidized carbon cloth (OCC). Leveraging the high-density hot spots, the formed Ag/AuNPs-TiO2-OCC substrate exhibits higher enhancement factors (1.92 × 108) and uniformity (RSD = 7.68%). As for the detection of IMI on the substrate, the limit of detection was lowered to 4.1 × 10-6 µg/mL. With a hydrophobic structure, the Ag/AuNPs-TiO2-OCC possessed excellent self-cleaning performance addressing the limitation of single-use associated with traditional SERS substrates, as well as the degradation capability of the substrate under ultraviolet (UV) light. Accordingly, Ag/AuNPs-TiO2-OCC showcases outstanding SERS sensing and regenerating properties, making it poised for extensive application in the field of food safety assurance.

Detoxification of Mycotoxin Patulin by the Yeast Kluyveromyces marxianus YG-4 in Apple Juice.

Patulin (PAT) is a mycotoxin produced by Penicillium species, which often contaminates fruit and fruit-derived products, posing a threat to human health and food safety. This work aims to investigate the detoxification of PAT by Kluyveromyces marxianus YG-4 (K. marxianus YG-4) and its application in apple juice. The results revealed that the detoxification effect of K. marxianus YG-4 on PAT includes adsorption and degradation. The adsorption binding sites were polysaccharides, proteins, and some lipids on the cell wall of K. marxianus YG-4, and the adsorption groups were hydroxyl groups, amino acid side chains, carboxyl groups, and ester groups, which were combined through strong forces (ion interactions, electrostatic interactions, and hydrogen bonding) and not easily eluted. The degradation active substance was an intracellular enzyme, and the degradation product was desoxypatulinic acid (DPA) without cytotoxicity. K. marxianus YG-4 can also effectively adsorb and degrade PAT in apple juice. The contents of organic acids and polyphenols significantly increased after detoxification, significantly improving the quality of apple juice. The detoxification ability of K. marxianus YG-4 toward PAT would be a novel approach for the elimination of PAT contamination.

Tibetan kefir grains fermentation alters physicochemical properties and improves antioxidant activities of Lycium barbarum pulp polysaccharides.

There is a lack of research on how Tibetan kefir grains fermentation alters the physicochemical properties and biological activity of Lycium barbarum pulp polysaccharides, despite some reports that fermentation can affect the structure and activity of plant polysaccharides. This study demonstrated that, through fermentation, the molecular weight of polysaccharides decreased from 25.33 to 15.11 kg/mol while the contents of total sugar and uronic acid increased by 19.11% and 40.38%, respectively. Furthermore, after fermentation, the polysaccharides exhibited an uneven and rough surface along with a reduced number of branched chains and triple helix structures. Tibetan kefir grains fermentation enhanced the antioxidant activity of polysaccharides, which may be attributed to an increase in arabinose, galactose, and uronic acid content and a decrease in polysaccharide molecular weight. This research offers an alternative viewpoint on the potential application of Tibetan kefir grains-fermented Lycium barbarum pulp polysaccharides in functional foods.

Enzymatically green-produced bacterial cellulose nanoparticle-stabilized Pickering emulsion for enhancing anthocyanin colorimetric performance of versatile films.

To enhance the colorimetric performance of anthocyanin (Ant), a konjac glucomannan (KGM)-based multifunctional pH-responsive indicator film was fabricated by introducing enzymatically prepared bacterial nanocellulose (EBNC) stabilized camellia oil/camellia essential oil Pickering emulsion (BCCE). Specifically, optimized enzymatic hydrolysis time (36 h) was determined based on the particle size and microstructure. Then BCCE (containing 0.4% EBNC) was incorporated into Ant-containing KGM, and the novel active indicator film (KGM-Ant-BCCE) was constructed. Films with varying BCCE concentrations (3%-11%) exhibited enhanced UV shielding, thermal stability, mechanical strength, water vapor and oxygen permeability, hydrophobicity, and antioxidant performance. The pronounced color change of KGM-Ant-BCCE indicated its potential for visually detecting shrimp freshness. Moreover, the biodegradability (25 days) confirmed the environmentally benign property of the film. In summary, incorporating green-produced EBNC nanoparticle-stabilized BCCE offers an innovative pathway to improve the color indication capability of polysaccharide-based smart packaging.

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.