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.

High-fat diets induce inflammatory IMD/NFκB signaling via gut microbiota remodeling in Drosophila.

High-fat diets (HFDs), a prevailing daily dietary style worldwide, induce chronic low-grade inflammation in the central nervous system and peripheral tissues, promoting a variety of diseases including pathologies associated with neuroinflammation. However, the mechanisms linking HFDs to inflammation are not entirely clear. Here, using a Drosophila HFD model, we explored the mechanism of HFD-induced inflammation in remote tissues. We found that HFDs activated the IMD/NFκB immune pathway in the head through remodeling of the commensal gut bacteria. Removal of gut microbiota abolished such HFD-induced remote inflammatory response. Further experiments revealed that HFDs significantly increased the abundance of Acetobacter malorum in the gut, and the re-association of this bacterium was sufficient to elicit inflammatory response in remote tissues. Mechanistically, Acetobacter malorum produced a greater amount of peptidoglycan (PGN), a well-defined microbial molecular pattern that enters the circulation and remotely activates an inflammatory response. Our results thus show that HFDs trigger inflammation mediated by a bacterial molecular pattern that elicits host immune response.

Microbiome-derived acidity protects against microbial invasion in Drosophila.

Microbial invasions underlie host-microbe interactions resulting in pathogenesis and probiotic colonization. In this study, we explore the effects of the microbiome on microbial invasion in Drosophila melanogaster. We demonstrate that gut microbes Lactiplantibacillus plantarum and Acetobacter tropicalis improve survival and lead to a reduction in microbial burden during infection. Using a microbial interaction assay, we report that L. plantarum inhibits the growth of invasive bacteria, while A. tropicalis reduces this inhibition. We further show that inhibition by L. plantarum is linked to its ability to acidify its environment via lactic acid production by lactate dehydrogenase, while A. tropicalis diminishes the inhibition by quenching acids. We propose that acid from the microbiome is a gatekeeper to microbial invasions, as only microbes capable of tolerating acidic environments can colonize the host. The methods and findings described herein will add to the growing breadth of tools to study microbe-microbe interactions in broad contexts.

Enhancing citric acid tolerance of Acetobacter tropicalis using chemical and physical mutagenesis and adaptive evolution to improve the quality of lemon fruit vinegar.

The high concentration of citric acid in lemons limits the production of lemon fruit vinegar because it inhibits the metabolism of acetic acid bacteria and reduces the utilization of raw materials. This study aimed to enhance the citric acid tolerance of Acetobacter tropicalis by using complex mutagenesis and adaptive laboratory evolution (ALE) and improving the quality of lemon fruit vinegar. After mutagenesis and ALE, A. tropicalis JY-135 grew well under 40 g/L citric acid, and it showed high physiological activity and excellent fermentation performance under high concentrations of citric acid. The survival rate and ATP content of JY-135 were 15.27 and 9.30 times higher than that of the original strain J-2736. In the fermentation of lemon fruit vinegar, the acid production and the number of aroma-active compounds were 1.61-fold and 2.17-fold than J-2736. In addition, we found that citric acid tolerance of JY-135 is related to the respiratory electron-transport chain and the tricarboxylic acid (TCA) cycle. This work is of great significance for the production of high-quality lemon fruit vinegar and the enrichment of seed resources of acetic acid bacteria.

Deeply analyzing dynamic fermentation of highland barley vinegar: Main physicochemical factors, key flavors, and dominate microorganisms.

Highland barley vinegar, as a solid-state fermentation-type vinegar emerged recently, is well-known in Qinghai-Tibet plateau area of China. This work aimed to explore the main physicochemical factors, key flavor volatile compounds, and dominate microorganisms of highland barley vinegar during fermentation. The results showed that the decrease trend of reducing sugar, pH and the increase trend of amino acid nitrogen were associated with the metabolism of dominate bacteria, especially Lactobacillus and Acetobacter. Totally, 35 volatile compounds mainly including 20 esters, 10 alcohols, 2 aldehydes, 1 ketone and 2 pyrazines and 7 organic acids were identified. Especially, isoamyl acetate, acetyl methyl carbinol, ethyl caprylate, 1,2-propanediol, 3-methyl-1-butanol and ethyl isovalerate with high odor activity values were confirmed as key aroma compounds. Meanwhile, the relative average abundance of bacteria at genus level decreased significantly as fermentation time goes on. Among these microbes, Lactobacillus were the dominate bacteria at alcohol fermentation stage, Lactobacillus and Acetobacter were dominate at acetic acid fermentation stage. Furthermore, the correlations between dominate bacteria and the key volatile compounds were revealed, which highlighted Lactobacillus and Acetobacter were significantly correlated with key volatile compounds (|r| > 0.5, P < 0.01). The fundings of this study provide insights into the flavor and assist to improve the production quality of highland barley vinegar.

Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3.

IMPORTANCE: Acetobacter pasteurianus, an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP, dctD, and glnD, were sufficient to reproduce the increased thermotolerance of A. pasteurianus. These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.

Alcohol degradation, learning, and memory-enhancing effect of Acetobacter pasteurianus BP2201 in Caenorhabditis elegans model.

AIMS: This study aimed to investigate the probiotic effects of Acetobacter pasteurianus BP2201, isolated from brewing mass, for the treatment of alcohol-induced learning and memory ability impairments in a Caenorhabditis elegans model. METHODS AND RESULTS: Acetobacter pasteurianus BP2201 was examined for probiotic properties, including acid and bile salt resistance, ethanol degradation, antioxidant efficacy, hemolytic activity, and susceptibility to antibiotics. The strain displayed robust acid and bile salt tolerance, efficient ethanol degradation, potent antioxidant activity, and susceptibility to specific antibiotics. Additionally, in the C. elegans model, administering A. pasteurianus BP2201 significantly improved alcohol-induced learning and memory impairments. CONCLUSIONS: Acetobacter pasteurianus BP2201 proves to be a promising candidate strain for the treatment of learning and memory impairments induced by alcohol intake.

Acetobacter pomorum in the Drosophila gut microbiota buffers against host metabolic impacts of dietary preservative formula and batch variation in dietary yeast.

Gut microbiota are fundamentally important for healthy function in animal hosts. Drosophila melanogaster is a powerful system for understanding host-microbiota interactions, with modulation of the microbiota inducing phenotypic changes that are conserved across animal taxa. Qualitative differences in diet, such as preservatives and dietary yeast batch variation, may affect fly health indirectly via microbiota, and may potentially have hitherto uncharacterized effects directly on the fly. These factors are rarely considered, controlled, and are not standardized among laboratories. Here, we show that the microbiota's impact on fly triacylglyceride (TAG) levels-a commonly-measured metabolic index-depends on both preservatives and yeast, and combinatorial interactions among the three variables. In studies of conventional, axenic, and gnotobiotic flies, we found that microbial impacts were apparent only on specific yeast-by-preservative conditions, with TAG levels determined by a tripartite interaction of the three experimental factors. When comparing axenic and conventional flies, we found that preservatives caused more variance in host TAG than microbiota status, and certain yeast-preservative combinations even reversed effects of microbiota on TAG. Preservatives had major effects in axenic flies, suggesting either direct effects on the fly or indirect effects via media. However, Acetobacter pomorum buffers the fly against this effect, despite the preservatives inhibiting growth, indicating that this bacterium benefits the host in the face of mutual environmental toxicity. Our results suggest that antimicrobial preservatives have major impacts on host TAG, and that microbiota modulates host TAG dependent on the combination of the dietary factors of preservative formula and yeast batch. IMPORTANCE Drosophila melanogaster is a premier model for microbiome science, which has greatly enhanced our understanding of the basic biology of host-microbe interactions. However, often overlooked factors such as dietary composition, including yeast batch variability and preservative formula, may confound data interpretation of experiments within the same lab and lead to different findings when comparing between labs. Our study supports this notion; we find that the microbiota does not alter host TAG levels independently. Rather, TAG is modulated by combinatorial effects of microbiota, yeast batch, and preservative formula. Specific preservatives increase TAG even in germ-free flies, showing that a commonplace procedure in fly husbandry alters metabolic physiology. This work serves as a cautionary tale that fly rearing methodology can mask or drive microbiota-dependent metabolic changes and also cause microbiota-independent changes.

Genomic Plasticity of Acid-Tolerant Phenotypic Evolution in Acetobacter pasteurianus.

Acetic acid bacteria have a remarkable capacity to cope with elevated concentrations of cytotoxic acetic acid in their fermentation environment. In particular, the high-level acetate tolerance of Acetobacter pasteurianus that occurs in vinegar industrial settings must be constantly selected for. However, the improved acetic acid tolerance is rapidly lost without a selection pressure. To understand genetic and molecular biology of this acquired acetic acid tolerance in A. pasteurianus, we evolved three strains A. pasteurianus CICIM B7003, CICIM B7003-02, and ATCC 33,445 over 960 generations (4 months) in two initial acetic acids of 20 g·L-1 and 30 g·L-1, respectively. An acetic acid-adapted strain M20 with significantly improved specific growth rate of 0.159 h-1 and acid productivity of 1.61 g·L-1·h-1 was obtained. Comparative genome analysis of six evolved strains revealed that the genetic variations of adaptation were mainly focused on lactate metabolism, membrane proteins, transcriptional regulators, transposases, replication, and repair system. Among of these, lactate dehydrogenase, acetolactate synthase, glycosyltransferase, ABC transporter ATP-binding protein, two-component regulatory systems, the type II toxin-antitoxin system (RelE/RelB/StbE), exodeoxyribonuclease III, type I restriction endonuclease, tRNA-uridine 2-sulfurtransferase, and transposase might collaboratively contribute to the improved acetic acid tolerance in A. pasteurianus strains. The balance between repair factors and transposition variations might be the basis for genomic plasticity of A. pasteurianus strains, allowing the survival of populations and their offspring in acetic acid stress fluctuations. These observations provide important insights into the nature of acquired acetic acid tolerance phenotype and lay a foundation for future genetic manipulation of these strains.

Role of bacterial community succession in flavor formation during Sichuan sun vinegar grain (Cupei) fermentation.

Sichuan sun vinegar (SSV) is a traditional Chinese vinegar with a unique flavor and it is fermented with bran as the main raw material. In the present study, we explored the bacterial community succession in fermented grains (Cupei) during SSV production. High-throughput sequencing results showed that bacterial community richness and diversity peaked on day 7 of fermentation. Lactobacillus and Acetobacter were the dominant bacteria throughout the fermentation process. However, Acetobacter, Cupriavidus, Sphingomonas, Pelomonas, and Lactobacillus were the most abundant genera in the late phase of fermentation on day 17. The boundaries of trilateral co-fermentation were determined through cluster analysis. Days 1-3 were considered the early fermentation stage (starch saccharification), days 5-11 were the middle fermentation stage (alcoholic fermentation), and days 13-17 represented the late fermentation stage (acetic acid fermentation). Changes in flavor compounds during Cupei fermentation were subsequently analyzed and a total of 86 volatile compounds, 9 organic acids, and 17 amino acids were detected. Although acetic acid, lactic acid, alcohols, and esters were the main metabolites, butyrate was also detected. Correlation analysis indicated that 20, 21, and 28 microorganisms were positively correlated with the abundance of amino acids, organic acids, and volatile flavor compounds, respectively. We further explored the microbial and metabolic mechanisms associated with the dominant volatile flavor compounds during SSV fermentation. Collectively, the findings of the current study provide detailed insights regarding the fermentation mechanisms of SSV, which may prove relevant for producing high-quality fermented products.

Dynamic changes of quality and flavor characterization of Zhejiang rosy vinegar during fermentation and aging based on untargeted metabolomics.

Zhejiang Rosy Vinegar (ZRV) is a traditional condiment in Southeast China. This study aimed to track the physicochemical, microbiological, sensory changes, and metabolomic profiles of ZRV during fermentation and aging. The increase of acidity and decrease of reducing sugar were associated with the dominant growth of Lactobacillus and Acetobacter. The total 35 volatile compounds were identified in ZRV, mainly containing alcohols, esters, acids, aldehydes, ketones acids, phenols and nitrogen-containing. Compared to phenethyl acetate with sweet aroma in fresh vinegar, the compound with high odor activity values was isoamyl acetate with fruity aromas in aged vinegar. Furthermore, 1309 types of non-volatile components were identified, and histidine metabolism and arginine biosynthesis were revealed as main pathways during fermenting and aging. Concurrently, various bioactive substances in ZRV were identified. This study enriched the knowledge on the components and flavor of ZRV, and assist to improve the production quality of vinegar.

Exploitation of strong constitutive and stress-driven promoters from Acetobacter pasteurianus for improving acetic acid tolerance.

Acetobacter pasteurianus is an excellent cell factory for production of highly-strength acetic acid, and attracts an increasing attention in metabolic engineering. However, the available well-characterized constitutive and inducible promoters are rather limited to adjust metabolic fluxes in A. pasteurianus. In this study, we screened a panel of constitutive and acid stress-driven promoters based on time-series of RNA-seq data and characterized in A. pasteurianus and Escherichia coli. Nine constitutive promoters ranged in strength from 1.7-fold to 100-fold that of the well-known strong promoter Padh under non-acetic acid environment. Subsequently, an acetic acid-stable red fluorescent visual reporting system was established and applied to evaluate acid stress-driven promoter in A. pasteurianus during highly-acidic fermentation environment. PgroES was identified as acid stress-driven strong promoters, with expression outputs varied from 100% to 200% when acetic acid treatment. To assess their application potential, ultra-strong constitutive promoter Ptuf and acid stress-driven strong promoter PgroES were selected to overexpress acetyl-CoA synthase and greatly improved acetic acid tolerance. Notably, the acid stress-driven promoter displayed more favorable for regulating strain robustness against acid stress by overexpressing tolerance gene. In summary, this is the first well-characterized constitutive and acid stress-driven promoter library from A. pasteurianus, which could be used as a promising toolbox for metabolic engineering in acetic acid bacteria and other gram-negative bacteria.

Combined effects of fermentation starters and environmental factors on the microbial community assembly and flavor formation of Zhenjiang aromatic vinegar.

Microbial ecosystems of fermented foods are largely interfered by human activities in myriad ways. The aim of this study was to illuminate the impacts of various starters and environmental variables on the fermentation process of Zhenjiang aromatic vinegar (ZAV), one of the four representative cereal vinegars in China. The effects of environmental variables (e.g., ethanol, total acidity, temperature) and starters (e.g., jiuqu, maiqu, seed pei) on the profiles of microbiome and metabolome (e.g., organic acids, amino acids and volatiles) during fermentation process of ZAV were analyzed. Amongst the four fermentation stages, acetic acid fermentation was the main stage for the accumulation of flavor substances, and subsequently, the contents of acids (mainly acetic, lactic and citric acids) and volatile metabolites (e.g., 2,3-butanedione, acetoin, etc.) continued to enrich in sealed fermentation stage. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) showed that the fungal and bacterial community structures of four fermentation stages were significantly different. As for bacterial community, the dominant OTUs with average relative abundance over 10% in at least one fermentation stage were assigned to the genera Acetilactobacillus, Acetobacter, Acinetobacter, Aeromonas, Lactobacillus, and Pseudomonas. The dominant fungal populations in each fermentation stage were obviously divergent, including Wickerhamomyces, Saccharomyces, Alternaria, Fusarium, etc. SourceTracker analysis demonstrated that jiuqu and seed pei provided microorganisms to initiate starch saccharification and acetic acid fermentation stages, respectively, and maiqu was mainly the donor of enzymes in alcohol fermentation. Spearman correlation coefficients revealed positive relationships between fungal community and various flavor metabolites, indicating the essential role of fungi in the flavor formation of ZAV. This study systematically reveals the effects of fermentation starters and environmental variables on vinegar production and deepens the understanding of the traditional production craft.

Exploring of seasonal dynamics of microbial community in multispecies fermentation of Shanxi mature vinegar.

The constituents of fermentation foods vary seasonally and the microbiota plays a crucial role in metabolites formation. Here, the diversity and succession of microbiota of Shanxi mature vinegar produced with solid-solid fermentation craft have been investigated by Illumina Hiseq sequencing in both summer and winter. Obvious differences were observed in the structure of microbiota between summer and winter, and the bacterial community showed a significant difference (P < 0.05). Alpha diversity analysis showed that the diversity and richness of bacterial community were basically higher than that of fungal community in both summer and winter. For bacterial community, Lactobacillus and Limosilactobacillus were the two major group bacteria in the fermentation process of Shanxi mature vinegar in summer, and they dominated in acetic acid fermentation and alcoholic fermentation stages, respectively. Lactobacillus and Acetobacter were the two major group bacteria during the fermentation of Shanxi mature vinegar in winter. Saccharomyces, Saccharomycopsis, and Issatchenkia were the main yeasts in both seasons, while the dominant mould was Rhizopus in summer and Monascus in winter, respectively. The diversity of yeasts and moulds in winter was far greater than that in summer, especially in alcoholic fermentation stage. Collectively, our work revealed critical insights into effect of seasonal variation on the structure of microbiota of Shanxi mature vinegar, and was relevant in understanding the relationships between environmental change and microbiota.

Significance of LED lights in enhancing the production of vinegar using Acetobacter pasteurianus AP01.

Vinegar is a common food additive produced by acetic acid bacteria (AAB) during fermentation process. Low yield and long incubation time in conventional vinegar fermentation processes has inspired research in developing efficient fermentation techniques by the activation of AAB for acetic acid production. The present study intends to enhance vinegar production using acetic acid bacteria and light emitting diode (LED). A total of eight acetic acid bacteria were isolated from Korean traditional vinegar and assessed for vinegar production. Isolate AP01 exhibited maximum vinegar production and was identified as Acetobacter pasteurianus based on the 16S rRNA sequences. The optimum fermentation conditions for the isolate AP01 was incubation under static condition at 30 °C for 10 days with 6% initial ethanol concentration. Fermentation under red LED light exhibited maximum vinegar production (3.6%) compared to green (3.5%), blue (3.2%), white (2.2%), and non-LED lights (3.0%). Vinegar produced using red LED showed less toxicity to mouse macrophage cell line (RAW 264.7) and high inhibitory effects on nitric oxide and IL-6 production. The results confirmed that red LED light could be used to increase the yield and decrease incubation time in vinegar fermentation process.

An integrated host-microbiome response to atrazine exposure mediates toxicity in Drosophila.

The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.

Effect of fermentation time on the content of bioactive compounds with cosmetic and dermatological properties in Kombucha Yerba Mate extracts.

Kombucha is a beverage made by fermenting sugared tea using a symbiotic culture of bacteria belonging to the genus Acetobacter, Gluconobacter, and the yeasts of the genus Saccharomyces along with glucuronic acid, which has health-promoting properties. The paper presents the evaluation of ferments as a potential cosmetic raw material obtained from Yerba Mate after different fermentation times with the addition of Kombucha. Fermented and unfermented extracts were compared in terms of chemical composition and biological activity. The antioxidant potential of obtained ferments was analyzed by evaluating the scavenging of external and intracellular free radicals. Cytotoxicity was determined on keratinocyte and fibroblast cell lines, resulting in significant increase in cell viability for the ferments. The ferments, especially after 14 and 21 days of fermentation showed strong ability to inhibit (about 40% for F21) the activity of lipoxygenase, collagenase and elastase enzymes and long-lasting hydration after their application on the skin. Moreover, active chemical compounds, including phenolic acids, xanthines and flavonoids were identified by HPLC/ESI-MS. The results showed that both the analyzed Yerba Mate extract and the ferments obtained with Kombucha may be valuable ingredients in cosmetic products.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

Thermal adaptation of acetic acid bacteria for practical high-temperature vinegar fermentation.

Thermotolerant microorganisms are useful for high-temperature fermentation. Several thermally adapted strains were previously obtained from Acetobacter pasteurianus in a nutrient-rich culture medium, while these adapted strains could not grow well at high temperature in the nutrient-poor practical culture medium, "rice moromi." In this study, A. pasteurianus K-1034 originally capable of performing acetic acid fermentation in rice moromi was thermally adapted by experimental evolution using a "pseudo" rice moromi culture. The adapted strains thus obtained were confirmed to grow well in such the nutrient-poor media in flask or jar-fermentor culture up to 40 or 39 °C; the mutation sites of the strains were also determined. The high-temperature fermentation ability was also shown to be comparable with a low-nutrient adapted strain previously obtained. Using the practical fermentation system, "Acetofermenter," acetic acid production was compared in the moromi culture; the results showed that the adapted strains efficiently perform practical vinegar production under high-temperature conditions.

Application of Glyceric Acid to Bio-related Functional Materials and Improvement of Microbial Production.

Glyceric acid (GA) is an oxidative product of glycerol, and its d-isomer is obtained as a phytochemical from tobacco leaves and fruits of some plants. However, the production and applications of GA have not yet been fully investigated. In this review, recent developments in the microbial production of GA and its application to bio-related materials are summarized. The sodium salt of diacylated GA showed superior surface tension-lowering activity and antitrypsin activity. GA and its glucosyl derivative had positive effects on the viability and collagen production of skin cells in vitro, respectively. Glucosyl derivatives of GA showed protective effects against heat-induced protein aggregation. In addition, the microbial production of GA using raw glycerol as the starting material was investigated. The effect of methanol, a major impurity in raw glycerol, on GA production was investigated, and mutant strains to tolerate methanol in the culture were constructed. Enantioselective production of GA using newly isolated microbial strains has also been developed.