Interpreting the asymmetric interaction between yeast and acetic acid bacteria in kefir grains from a metabolic perspective.

Acetic acid bacteria (AAB, family Acetobacteraceae) are obligate aerobic microorganisms characterized by a highly efficient oxidative metabolism driven by membrane-bound dehydrogenases. Their ability to incompletely oxidize ethanol and various carbohydrates underlies the production of key food-related metabolites, including acetic acid, gluconic acids, and bacterial cellulose. This review summarizes current knowledge on AAB physiology, metabolic pathways, and ecological adaptations, with emphasis on their relevance to food biotechnology and value-added bioprocesses. AAB plays a central role in traditional and modern food fermentations, contributing to the production of vinegar, cocoa, coffee, kombucha, and other fermented beverages. Their metabolites influence food preservation, sensory attributes, and texture formation, supporting the development of clean-label and sustainable food products. In addition, AAB are increasingly applied in environmental biotechnology, including biodegradation and wastewater treatment, owing to their tolerance to acidic and oxidative stress conditions. Recent advances in metabolic and genetic engineering have enhanced the efficiency, robustness, and product specificity of industrial AAB strains, enabling improved production of organic acids, bacterial cellulose, and other high-value compounds. Emerging evidence also highlights the potential probiotic and postbiotic functions of selected AAB strains, including modulation of gut microbiota, production of bioactive metabolites, and support of intestinal barrier integrity, although these properties remain less explored than in lactic acid bacteria. Despite significant progress, challenges persist in strain standardization, genetic accessibility, and process optimization. Future research should focus on developing advanced engineering tools, improving large-scale fermentation strategies, and further elucidating the functional and health-related properties of AAB. Overall, AAB represents versatile microbial platforms with expanding applications in food science, biotechnology, and sustainable bioprocessing.

Backgroundprp operon usually comprises prpB, prpC, prpD and prpE genes, encoding 2-methylisocitrate (2-MIC) lyase, 2-methylcitrate (2-MC) synthase, 2-MC dehydratase and propionyl-CoA synthetase, respectively, which constitute the pathway 2-methylcitrate cycle (2-MCC) well-known for microbial propionate metabolism. Acetic acid bacteria (AAB) represent a group of obligate aerobic and Gram-negative microorganisms. Attributed to the robust ethanol-oxidating and acetic acid-resisting abilities, vinegar production stands for an important AAB’s industrial application. Previously, we unveiled the first AAB’s prp operon in Acetobacter pasteurianus CGMCC 1.41—a vinegar-brewing strain, and its up-regulation during acetic acid fermentation, suggesting the genes’ potential contribution to the adaptive processes of this strain. In this study, the prevalence of prp genes among AAB were analyzed, followed by constructing six markerless deletion mutants to investigate the genes’ role in carbon source metabolism and acetic acid resistance in A. pasteurianus CGMCC 1.41.ResultsThe prp gene cluster lacking prpE was found widespread in the genus Acetobacter. A gene encoding PrpE-homologous acetyl-CoA synthetase, designated prpE’, was discovered to participate the formation of the putative operon in many AAB genomes. Growth experiments revealed the ability to utilize propionate as a carbon source of A. pasteurianus CGMCC 1.41, which simultaneously required the function of PrpB, PrpC and PrpD. A role of PrpD or PrpB in promoting ethanol assimilation was observed, whereas PrpE’ appeared to be important for metabolizing both ethanol and acetic acid as carbon sources. PrpB, PrpC and PrpD also exerted a positive effect on glycerol metabolism. Moreover, the integrity of the 2-MCC pathway exhibited a great importance upon the initiation of the bioreaction catalyzed by PrpC. Finally, PrpE’ was assumed to be critical for an acetic acid-resisting process—acetic acid overoxidation, which might be potentially enhanced by PrpD as well.ConclusionThe present work expands the knowledge to the prp operon in the context of AAB, which also indicates the significance of gaining deeper insight into the specific roles of relevant genes in this group of bacteria.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-026-05213-2.

ABSTRACTAcetobacter indonesiensis UNPADCC 01‐5 is a recently discovered acetic acid bacterium isolated from fermented food, oncom merah. With its current lack of polyphasic approach in classification, this study aims to provide the first morphological, physiological, biochemical and molecular analyses. These analyses are achieved by morphological and physiological characterization, Kirby–Bauer antibiotic susceptibility, biochemical profiling, gas chromatography–mass spectrometry and whole‐genome sequencing (WGS). The results found that A. indonesiensis UNPADCC 01‐5, a non‐motile cell, is a gram‐negative, short rod‐shaped cell that formed cream‐white colonies with 0.3–1.3 in diameter. It exhibited environmental resilience, including acid‐tolerant, halotolerant and thermotolerant. Against antibiotics, it showed resistance towards chloramphenicol. Biochemical tests revealed its ability to ferment glucose, rhamnose, melibiose and arabinose, as well as to produce bioactive metabolites such as acetoin and acetic acid. WGS identified potential gene expressions associated with acetic acid production, stress tolerance, nitrogen fixation, hydrocarbon degradation and heavy‐metal resistance. WGS analysis revealed no detectable genes associated with human virulence factors or pathogenic secretion systems. The results suggested that A. indonesiensis UNPADCC 01‐5 from oncom merah possessed adaptive traits which, with future assessments and industrial‐scale tests, hold potential as a functional/adjunct culture for acidic fermentations and a biofunctional agent.

Characterization and application of indigenous microbial strains in mixed-culture fermentation of cashew apple juice

Impact of minerals on the microbial species diversity and metabolite dynamics during spontaneous backslopped wheat sourdough productions.

Kombucha quality is largely governed by polyphenol transformation during fermentation. However, interaction between substrate composition and microbial communities regulating phenolic transformation and quality formation remains unclear. In this study, six tea substrates (white, green, yellow, black, oolong, and mint tea) were fermented using three defined microbial communities (SMC1-SMC3) and a traditional symbiotic culture of bacteria and yeast (SCOBY) to evaluate carbon metabolism, phenolic transformation, antioxidant activity, and sensory quality. After 10 d of fermentation, SMC2 and SMC3, containing acetic acid bacteria, showed stronger acidification (pH 2.2–2.5) and lower ethanol (0.34–0.52 mg/mL) than SMC1 (13.09–15.88 mg/mL). Phenolic transformation was substrate-dependent: total phenolics and flavonoids decreased in green tea, both increased in white tea, while flavonoids increased in oolong and black tea. Meanwhile, rutin decreased in white and green tea, whereas gallic acid accumulated in yellow, black, and oolong teas and was positively correlated with antioxidant activity. Sensory evaluation showed SMC3 achieved higher overall acceptability in most substrates, whereas SCOBY performed best in mint tea. These findings indicate substrate-microbiota interactions play a key role in phenolic transformation and quality formation in kombucha. Rational matching of tea substrates with defined microbial communities enables coordinated optimization of antioxidant activity, ethanol control, and sensory quality.

Lignocellulosic and cellulose-derived carriers in biofilm-based food fermentations: Food-contact design, hydrodynamics and validation.

Background: Acetification is a complex process driven by acetic acid bacteria (AAB), in which high ethanol and acidity levels require strong microbial metabolic adaptation. Although the microbiota involved in vinegar production has been described, the functional mechanisms that enable these bacteria to maintain metabolic activity remain poorly understood. In this study, the functional dynamics of AAB during Verdejo vinegar acetification were analyzed using a quantitative metaproteomic approach. Methods: Acetification was performed in submerged culture under semi-continuous conditions, and samples were collected at four stages of the cycle (S1–S4). Results: LC-MS/MS analysis led to the identification of 1626 proteins, of which 1409 were assigned to the Acetobacteraceae family. Komagataeibacter europaeus was the dominant species (73.7%). Hierarchical clustering revealed four protein abundance patterns, and differential analysis identified 350 proteins with increased abundance and 169 with decreased abundance, with the greatest changes observed between S1 and S4. Functional annotation and protein–protein interaction analyses indicated that the main metabolic adaptations involve pathways related to energy metabolism, amino acid biosynthesis, membrane-associated functions, cellular homeostasis, and acid stress response. Conclusions: Overall, the results show that K. europaeus concentrates most of the metabolic activity during acetification and that proteome reorganization reflects key molecular strategies for adaptation and survival under high-acidity conditions.

The present study aimed to investigate the fermentation temperature for mulberry leaf bioferment (MLB) using a symbiotic culture of bacteria and yeast (SCOBY) and characterize its physicochemical, microbiological and antioxidant composition. MLB was prepared at 25, 30, 37 and 45 ± 2 °C, respectively, for 7 days. Fourier transform infrared (FTIR) spectroscopy was employed for functional group identification. Antioxidant activities were evaluated based on 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and total phenolic content (TPC). The phenolic profile was characterized chromatographically, and microbial loads were determined. All of the MLB showed low alcohol concentration (<0.5%). The changes of functional groups revealed the broken down of carbohydrates and transformation of phenolic compounds after the fermentation. MLB at 30-37 °C supported high microbial activity, with 30 ± 2 °C showing the highest culturable counts for acetic acid bacteria and lactic acid bacteria, and 37 ± 2 °C producing the lowest pH (3.48) and highest volatile acidity, indicating distinct community dynamics across temperatures. Antioxidant capacity (DPPH and ABTS) and TPC increased up to two-fold at 37-45 ± 2 °C compared with unfermented controls. Ultra-HPLC quantification indicated a 131.4% increase in total quantified phenolics 37 ± 2 °C, with catechin being the most abundant identified phenolic (2.59 mg L-1), followed by vanillic, gallic, caffeic, chlorogenic, 4-hydroxybenzoic, p-coumaric and protocatechuic acids. The findings show that fermentation temperature regulates microbial activity and phenolic biotransformation in SCOBY-fermented mulberry leaves. Fermentation at 37 ± 2 °C most effectively enhances phenolic content and antioxidant potential, supporting the potential of MLB as a functional nutraceutical ingredient. © 2026 Society of Chemical Industry.

Kombucha is traditionally produced from sweetened Camellia sinensis tea fermented by a symbiotic culture of bacteria and yeasts (SCOBY). However, limited information is available on the effects of replacing tea with Coffea arabica infusion on fermentation parameters, microbial ecology, and overall quality of the beverage. This study evaluated the effects of substituting green tea with C. arabica infusion (25%-100%, v/v) during kombucha fermentation on fermentation kinetics, microbial populations, SCOBY structure, and physicochemical, functional, and sensory attributes. Coffee substitution reduced sugar consumption rates without significantly affecting final pH (2.8-3.2) or titratable acidity (∼0.4). Increasing coffee proportions markedly reshaped microbial populations, reducing acetic acid bacteria and increasing lactic acid bacteria, thereby shifting metabolism toward lactic acid production. Structural analyses revealed preserved SCOBY integrity, with a more porous cellulose network in coffee-based formulations. Total phenolic content decreased after fermentation in most treatments, whereas the 100% coffee kombucha remained stable and maintained antioxidant capacity. GC-MS analysis identified 111 volatile compounds, and multivariate and machine learning approaches revealed coffee-associated biomarkers associated with lactic acid and aroma-active compounds. Overall, C. arabica infusion proved to be a suitable alternative substrate for kombucha fermentation, enabling substrate-driven modulation of microbial dynamics and metabolic profiles while maintaining product safety and functional potential. PRACTICAL APPLICATIONS: Replacing green tea with C. arabica infusion in kombucha production enables the development of beverages with differentiated microbial, chemical, and sensory profiles while maintaining fermentation performance and safety. Coffee-based kombucha promotes a shift toward lactic acid-oriented fermentation, distinct aroma signatures, and functionality without requiring significant changes to conventional SCOBY-based processes. From a practical perspective, coffee infusion is a feasible strategy for product diversification, allowing manufacturers to modulate fermentation outcomes through raw material selection and to support the development of innovative, scalable, and consumer-oriented kombucha beverages.

Kombucha is a traditional fermented beverage produced through the fermentation of sugared tea by a symbiotic culture of bacteria and yeasts (SCOBY). In recent years, the valorisation of plant-based by-products as fermentation substrates has gained attention as a sustainable approach to improving both the nutritional and economic efficiency of fermented beverages. The present study investigated the production of kombuchas supplemented with pineapple, fennel, and carrot by-products during the secondary fermentation phase, aiming to evaluate their influence on fermentation dynamics, microbial ecology, and the chemical and aromatic profiles of the final products. The experimental design integrated culture-dependent and culture-independent approaches, including amplicon sequencing, to characterize microbial community composition and evolution throughout fermentation. Chemical profiling was carried out using gas chromatography coupled with quadrupole mass spectrometry (GC-qMS) and high-performance liquid chromatography equipped with diode-array and refractive index detectors (HPLC-DAD/RI). The fermentation process was monitored during both the primary and secondary stages, and a shelf-life assessment was conducted over 14days of refrigerated storage (4°C) to evaluate product stability. Microbiological results indicated a predominance of Schizosaccharomyces spp., while Komagataeibacter spp. was the only bacterial genus identified. A significant reduction in α-diversity was observed over time, suggesting selective adaptation of the microbial community to the fermentation environment. β-diversity analysis revealed clear differences among samples collected after 8 and 22days, reflecting the combined influence of time and substrate composition on microbial succession. Chemical analyses demonstrated an increase in acetic acid concentration and a progressive decline in pH throughout fermentation, consistent with the metabolic activity of acetic acid bacteria. Among volatile organic compounds (VOCs), alcohols and organic acids were the most abundant chemical classes detected. Several VOCs were associated with minor yeast genera, including Hannaella, Galactomyces, Aureobasidium, and Millerozyma, whereas Schizosaccharomyces spp. showed a strong correlation with specific aroma-active compounds, highlighting its key role in defining the sensory characteristics of the beverage. Overall, this study provides new evidence on how different vegetable by-products and microbial consortia influence the development of chemical and aromatic compounds in kombucha. The findings highlight the potential of using by-products as a sustainable, value-added strategy for producing fermented beverages, while also supporting the principles of the circular economy and resource-efficient food systems.

Antimicrobial resistance in microorganisms associated with fermented foods is increasingly recognized, yet rapid methods to characterize antibiotic response dynamics remain limited. This study evaluates antibiotic susceptibility and physiological response patterns of kombucha-associated acetic acid bacteria and motile Escherichia coli using optical nanomotion detection (ONMD), a label-free technique that quantifies single-cell mechanical activity. Two cellulose-producing species (Komagataeibacter xylinus and K. rhaeticus), one non-cellulose-producing species (K. melaceti), and E. coli were exposed to ampicillin, ciprofloxacin, and chloramphenicol. Minimum inhibitory concentrations (MICs) were determined prior to time-resolved ONMD analysis. Susceptible strains exhibited progressive suppression of confined nanomotion consistent with MIC-defined susceptibility, whereas resistant profiles maintained sustained mechanical activity. Chloramphenicol initially induced persistent or increased nanomotion at 120 min; however, extending the observation to 180 min revealed delayed suppression in susceptible strains, demonstrating that bacteriostatic antibiotics require longer observation windows for accurate ONMD classification. In motile E. coli, ONMD revealed both intracellular nanomotion puncta and swimming trajectories, which were progressively attenuated following antibiotic exposure. These findings demonstrate that ONMD complements conventional susceptibility testing by resolving time-dependent suppression of both translational motility and intracellular nanomechanical activity at the single-cell level.

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.

Gluconobacter cerinus is an important acetic acid bacterium identified in rotted tissues of sugarbeet root. Here, we announce the draft genome of a G. cerinus strain isolated from postharvest sugarbeet root, which was assembled into three contigs with a total length of 3,324,464 bp and a GC content of 56.1%.

Kombucha is a fermented beverage produced using a symbiotic consortium of acetic acid bacteria and yeasts, often marketed for its health-promoting properties. However, probiotic bacteria in kombucha are typically present at inconsistent levels and may not remain viable during fermentation. In this study, three Lactobacillus strains (Lacticaseibacillus rhamnosus ATCC 53103 (L. rhamnosus), Lactiplantibacillus plantarum subsp. plantarum ATCC 14917 (L. plantarum) and Lentilactobacillus hilgardii (L. hilgardii) isolate) were encapsulated in whey protein using the lyophilization method and added individually at the start of kombucha fermentation. Lactic acid bacteria (LAB)-enriched kombucha samples were evaluated for chemical composition (polyphenols, flavonoids, vitamin C and organic acids) and functional properties (antimicrobial, antiproliferative, antioxidant and anti-inflammatory activities) and compared to a traditionally obtained control kombucha, primarily demonstrating in vitro and experimental assessment. Encapsulation maintained LAB viability above 6-7 log CFU/mL throughout fermentation, producing kombucha with enhanced microbial stability. LAB-enriched samples exhibited increased L-lactic acid and antimicrobial activity. L. rhamnosus and L. hilgardii-enriched samples exhibited increased antiproliferative and anti-inflammatory activities, which may be associated with strain-dependent production of organic acids, polyphenol modulation and LAB-derived bioactive metabolites. Antioxidant activity varied depending on assay, and L. rhamnosus-enriched kombucha showed higher anti-inflammatory activity. These findings demonstrate that whey protein encapsulation can preserve LAB during fermentation, enhance specific bioactive properties and provide a platform for developing functional kombucha beverages with potential applications in the food industry.

This study optimizes sequential fermentation and predictive modeling for sustainable, high-quality vinegar production from unripe Kharak dates, an underutilized byproduct. Acetic acid bacteria (AAB) were isolated from Tehran and Gorgan samples (Iran), identified as Acetobacter pasteurianus, A. aceti, and A. tropicalis via morphological, biochemical, and PCR methods. Alcoholic fermentation was done by Saccharomyces cerevisiae at 20%, 30%, and 40% date syrup concentrations, followed by acetic fermentation with AAB. Optimal yeast growth and alcohol yield occurred at 20%, while A. pasteurianus showed robust performance across all levels, with 30% date syrup concentration yielding the best balance of growth, acidity (2.3% w/v), and pH stability. Gaussian, Gompertz, Rational, Sinusoidal Fit, and Logistic models effectively described microbial dynamics. This work enhances byproduct valorization and supports scalable, dietary-friendly vinegar production.

During the fermentation of cacao beans, a succession of microorganisms, including yeasts, lactic acid bacteria, acetic acid bacteria, and Bacillus, are involved in the generation of aroma and flavor precursors. These microbial consortia have been extensively studied, except for the genus Bacillus. Accordingly, this work aimed to isolate and select Bacillus isolates and evaluate their effect on the fermentation process of CCN51 genotype cacao beans. The isolates were obtained from a fermentation process using native cacao material. The isolates were selected based on growth performance and biological activity assays, including the production of pectinases, proteases, citrate lyases, and antifungal activity. Subsequently, their effect as a starter culture on the CCN51 genotype beans was evaluated. The isolates were individually evaluated at a concentration of 7 × 106 CFU/mL in a proportion of 1% starter culture (v/w) relative to the cacao mass. Temperature, pH, acidity, fermentation index, and cut test were monitored, showing higher fermentation index values (up to 1.5) and a reduction of insufficiently fermented beans (from 36% in the control to 13–20%) in inoculated treatments. The evaluation of the effect of Bacillus isolate on cacao fermentation indicated that the B. megaterium isolate improved the sensorial quality of cacao beans, decreased their bitterness and astringency, and highlighted flavors such as floral, fruity, nutty, and cane sugar-malt. It also registered antifungal activity against mycotoxin-producing fungi. It can be concluded that Bacillus has biotechnological potential as a producer of enzymes of interest for cacao processing and the food industry.

Effects of Acetic Acid Bacteria in Starter Culture on the Properties of Sourdough

Acetic acid bacteria convert environmental sugars and alcohols into acetic acid and various sugars through oxidative fermentation, resulting in the accumulation of these compounds at high concentrations in the culture medium. One such product is the rare sugar 5-keto-d-fructose (5-KF). In Gluconobacter species, 5-KF is transported into the cell and reduced to fructose in a single step by 5-ketofructose reductase, allowing entry into glycolysis. However, it remains unclear whether eukaryotic microorganisms can metabolize 5-KF or which genes are involved in this process. In this study, we investigated the ability of various yeasts to utilize 5-KF and identified genes involved in its metabolism. The model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were unable to grow on 5-KF, whereas the oleaginous yeast Lipomyces starkeyi efficiently metabolized this sugar. RNA-seq analysis of L. starkeyi grown on 5-KF revealed genes specifically upregulated in response to 5-KF. Based on gene annotation and expression profiles, a putative metabolic pathway was proposed. Gene knockout analyses showed that mutants deficient in specific steps of the pathway grew on downstream intermediates but failed to grow on upstream substrates, indicating loss of the corresponding enzymatic functions. These results suggest that L. starkeyi metabolizes 5-KF via a multistep pathway, 5-KF → l-sorbose → d-sorbitol → d-fructose. This study provides the first evidence of a 5-KF metabolic pathway in yeast, distinct from the single-step conversion to fructose observed in Gluconobacter species.