Exploring the Health Benefits of Yeast Isolated from Traditional Fermented Foods in Korea: Anti-Inflammatory and Functional Properties of Saccharomyces and Non-Saccharomyces Strains.

Traditional yeast (Saccharomyces cerevisiae) has been used for its benefits in various fermentation processes; the benefits of non-Saccharomyces yeast as a material for food, feed, and pharmaceuticals have been studied recently. This study evaluated the anti-inflammatory activity and extracellular functional characteristics of wild-type yeasts isolated from traditional fermented foods (doenjang (common name: soybean paste) and nuruk) in Korea. The viability of the yeast and lipopolysaccharide (LPS)-stimulated RAWBlue™ cells was improved, similar to unstimulated RAWBlue™ cells, and the isolates demonstrated NF-κB inhibitory activity. Yeast suppressed the nitric oxide production in LPS-stimulated RAWBlue™ cells, which was attributed to the inhibition of iNOS or COX-2 mRNA expression depending on the strain. Although there were differences depending on the strain, the production of anti-inflammatory cytokines was reduced in the yeast and LPS-stimulated RAWBlue™ cells, some of which were demonstrated at the mRNA level. In addition, the isolates exhibited high antioxidant and antihypertensive activities (similar to the positive control), which varied depending on the strain. This suggests that yeast can be used for fermentation with enhanced antioxidant and antihypertensive activities. Furthermore, the isolates inhibited the growth of pathogenic Gram-negative bacteria, indicating that yeast can inhibit food spoilage and the growth of pathogenic bacteria during fermentation. Consequently, utilizing raw materials to cultivate yeast strains could be a promising avenue for developing functional foods to prevent and treat inflammatory reactions; such foods may exhibit antioxidant, antihypertensive, and antibacterial properties.

Microbiome Analysis of Traditional Grain Vinegar Produced under Different Fermentation Conditions in Various Regions in Korea.

The fermentation of traditional vinegar is a spontaneous and complex process that involves interactions among various microorganisms. Here, we used a microbiome approach to determine the effects of networks, such as fermentation temperature, location, physicochemical and sensory characteristics, and bacterial profile, within traditional grain vinegar samples collected from various regions of Korea. Acetic acid and lactic acid were identified as the major metabolites of grain vinegar, and sourness and umami were determined as taste fingerprints that could distinguish between vinegar samples. Acetobacter ghanensis and Lactobacillus acetotolerans were the predominant bacterial species, and the functional composition of the microbiota revealed that the nucleotide biosynthesis pathway was the most enriched. These results reveal that vinegar samples fermented outdoors are more similar to each other than vinegar samples fermented at 30 °C, when comparing the distance matrix for comprehending bacterial networks among samples. This study may help obtain high-quality vinegar through optimized fermentation conditions by suggesting differences in sensory characteristics according to the fermentation environment.

Safety Evaluation of Weissella cibaria JW15 by Phenotypic and Genotypic Property Analysis.

Weissella cibaria is one of the bacteria in charge of the initial fermentation of kimchi and has beneficial effects such as immune-modulating, antagonistic, and antioxidant activities. In our study, we aimed to estimate the safety of W. cibaria JW15 for the use of probiotics according to international standards based on phenotypic (antibiotic resistance, hemolysis, and toxic metabolite production) and genotypic analysis (virulence genes including antibiotic resistance genes). The results of the safety assessment on W. cibaria JW15 were as follows; (1) antibiotic resistance genes (ARGs) (kanamycin and vancomycin etc.) were intrinsic characteristics; (2) There were no acquired virulence genes including Cytolysin (cylA), aggregation substance (asa1), Hyaluronidase (hyl), and Gelatinase (gelE); (3) this strain also lacked ß-hemolysis and the production of toxic metabolites (D-lactate and bile salt deconjugation). Consequently, W. cibaria JW15 is expected to be applied as a functional food ingredient in the food market.

Comparative Evaluation of Quality and Metabolite Profiles in Meju Using Starter Cultures of Bacillus velezensis and Aspergillus oryzae.

The production of good Meju soybean paste primarily depends on the selection of raw materials and fermenting microorganisms, which together influence its characteristic metabolites, taste, and aroma. In this study, we analyzed the relationship between properties and metabolites in Meju samples fermented by Aspergillus oryzae alone or with Bacillus velezensis. We developed fast-stable processing techniques to obtain Meju from A. oryzae and B. velezensis using the inoculation method, thereby ensuring safety in the production of soybean paste. The amino-type nitrogen content increased from an initial 180-228 mg% to a final 226-776 mg% during fermentation and was higher in Meju inoculated separately with the fungi and bacteria (C group) than in Meju co-inoculated with both the starters concurrently (D group). The levels of metabolites such as glucose, myo-inositol, glycerol, and fatty acids (palmitic, stearic, oleic, and linoleic acids) in Meju fermented by A. oryzae with B. velezensis were higher than those in Meju fermented by A. oryzae alone. Fungal growth was affected by the inoculated bacteria, which often occurs during the fermentation of co-inoculated Meju.

Short-term Cudrania tricuspidata fruit vinegar administration attenuates obesity in high-fat diet-fed mice by improving fat accumulation and metabolic parameters.

Previous studies have suggested that vinegar intake can help to reduce body fat and hyperglycemia. Therefore, this study aimed to evaluate the anti-obesity efficacy of vinegar fermented using Cudrania tricuspidata fruits (CTFV) and its main phenolic constituents and to analyze its molecular mechanism and changes in obesity-related metabolizing enzymatic activities. We found that HFD significantly caused hepatic steatosis; increases in body fats, feed efficiency, liver mass, lipids, insulin, oxidative parameters, cardiovascular-associated risk indices, lipase and α-amylase activities, whereas CTFV efficaciously attenuated HFD-induced oxidant stress, fat accumulation, obesity-related enzymatic activity, and the activation or reduction of obesity-related molecular reactions via improving metabolic parameters including phosphorylated insulin receptor substrate 1, protein tyrosine phosphatase 1B, phosphorylated phosphoinositide 3-kinase/protein kinase B, phosphorylated mitogen-activated protein kinases, sterol regulatory element-binding protein 1c, CCAAT/enhancer-binding protein, and fatty acid synthase; and decreases in adiponectin receptor 1, leptin receptor, adenosine monophosphate-activated protein kinase, acetyl-CoA carboxylase, and peroxisome proliferator-activated receptor, subsequently ameliorating HFD-induced obesity. Therefore, CTFV might provide a functional food resource or nutraceutical product for reducing body fat accumulation.

Anti-Inflammatory and Cytotoxicity Effects of Cudrania tricuspidata Fruits Vinegar in a Co-Culture System with RAW264.7 Macrophages and 3T3-L1 Adipocytes.

Vinegar has been found to have in vitro improvement effect on inflammatory biomarkers, and clinically used to improve inflammation and obesity-related diseases. This study was designed to analyze in vitro anti-inflammatory effects of Cudrania tricuspidata fruits vinegar (CTFV) in a co-culture system with macrophages and adipocytes. We analyzed the physicochemical properties and polyphenolic ingredients of CTFV, and investigated in vitro anti-inflammatory effects of CTFV in a co-culture system with macrophages and adipocytes. The cells were cultured in the presence of CTFV for 24 h in contact with each other, then, harvested. The levels of monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), nitric oxide (NO), and interleukin (IL)-6 were evaluated by using the Griess reagent, western blot, or enzyme-linked immunosorbent assay assays. We found that increasing levels for NO, iNOS, TNF-α, IL-6 and MCP-1 were caused by LPS treatment and co-culture using the contact method, whereas CTFV efficaciously attenuated inflammatory response by improving inflammatory parameters including NO, iNOS, TNF-α, IL-6 and MCP-1. The present study indicates that CTFV might provide a nutraceutical product or functional food resource for improving inflammation processed via the interaction of adipocytes and macrophages.

Metabolite Profile and Immunomodulatory Properties of Bellflower Root Vinegar Produced Using Acetobacter pasteurianus A11-2.

Fermented vinegar is prepared from grains and medicinal plants. Here, we produced vinegar from peeled and unpeeled roots of bellflowers (Platycodon grandiflorum) using Acetobacter pasteurianus A11-2 and analyzed bellflower vinegar (BV) samples using gas chromatography-mass spectrometry and quadrupole time-of-flight mass spectrometry over 15 days of fermentation to assess the quality. We also evaluated their antibacterial and immunoenhancing effects using RAW 264.7 macrophage cells. The major metabolites in BV are organic acids, with the main volatile compounds being ethyl acetate, isoamyl acetate, 1-pentanol, hydroxypropanoic acid, and malonic acid. When we fermented BV from unpeeled roots for 10 days with a starter culture, we observed significant antibacterial and immunoenhancing effects in macrophages. Therefore, we could determine the metabolite and functional differences in vinegar obtained from bellflower roots and proposed that bellflower roots with peel are an effective substrate for developing vinegar and healthy food products.

Acetobacter oryzoeni sp. nov., isolated from Korean rice wine vinegar.

A Gram-stain-negative, obligately aerobic bacterium, designated strain B6T, was isolated from rice wine vinegar in the Republic of Korea. Cells were non-motile and oval short rods showing catalase-positive and oxidase-negative activities. Growth was observed at 15-45 °C (optimum, 30 °C) and pH 3.5-8.0 (optimum, pH 5.5-6.5). Strain B6T contained summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1 ω6c), and C16 : 0 as major fatty acids and ubiquinone-9 was identified as the sole isoprenoid quinone. The G+C content of the genomic DNA calculated from the whole genome was 53.1 mol%. Strain B6T was most closely related to Acetobacter pasteurianus LMG 1262T with very high 16S rRNA gene sequence similarity (100 %) and the strains formed a very close phylogenetic lineage together in phylogenetic trees based on 16S rRNA gene sequences. However, relatedness analyses based on concatenated amino acid sequences of 354 core genes and whole-cell MALDI-TOF profiles showed that strain B6T may form a distinct phyletic lineage from Acetobacter species. In addition, average nucleotide identity and in silico DNA-DNA hybridization values between strain B6T and the type strains of Acetobacter species were less than 93.3 and 51.4 %, respectively. The genomic features of strain B6T were also differentiated from those of closely related Acetobacter type strains. Based on the phenotypic, chemotaxonomic and genomic features, strain B6T clearly represents a novel species of the genus Acetobacter, for which the name Acetobacter oryzoeni sp. nov. is proposed. The type strain is B6T (=KACC 21201T=JCM 33371T).

Effects of Maclura tricuspidata (Carr.) Bur fruits and its phytophenolics on obesity-related enzymes.

The purpose of the present study was to investigate whether several phytophenolic ingredients isolated from Maclura tricuspidata (Carr.) Bur fruits inhibit the activity of obesity-related enzymes including pancreatic lipase, α-amylase, ß-glucosidase, phosphodiesterase IV, alkaline phosphatase, and citrate synthase, and the compounds play as an inhibitor against the target enzymes in kinetic studies. The enzyme assays indicated that the fruit extract and its phytophenolic compounds inhibited significantly the enzymatic activity of the five target enzymes. The kinetic studies demonstrated that the inhibitory properties of p-hydroxybenzoic acid (4-HA), protocatechuic acid (PA), and isovanillic acid (IA) against pancreatic lipase, ß-glucosidase, citrate synthase, or alkaline phosphatase. Our results suggested that the compounds detected from Maclura tricuspidata (Carr.) Bur fruit extract may regulate carbohydrate/lipid/energy metabolism by obesity-related enzymes' inhibition. PRACTICAL APPLICATIONS: The obesity-related metabolizing enzymes affect (in)directly the metabolites absorption on carbohydrate/lipid/energy metabolism. Accordingly, it is an important strategy to treat obesity through target pathways and enzymes which include the reduction in energy intake and consumption. In our results, Maclura tricuspidata (Carr.) Bur fruit extract and its phytophenolic compounds inhibited significantly the enzymatic activity of the five target enzymes, in particular, 4-HA, PA, and IA have each specific inhibition type on pancreatic lipase, ß-glucosidase, citrate synthase, and alkaline phosphatase. Therefore, M. tricuspidata (Carr.) Bur fruit may be a strong candidate as a food material or therapeutic agent for obesity improvement.

Comparison of Bioactive Compounds and Antioxidant Activities of Maclura tricuspidata Fruit Extracts at Different Maturity Stages.

Abstract: Maclura tricuspidata fruit contains various bioactive compounds and has traditionally been used in folk medicine and as valuable food material in Korea. The composition and contents of bioactive compounds in the fruit can be influenced by its maturity stages. In this study, total phenol, total flavonoid, individual polyphenolic compounds, total carotenoids and antioxidant activities at four maturity stages of the fruit were determined. Polyphenolic compounds were analyzed using high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) and HPLC. Among 18 polyphenolic compounds identified in this study, five parishin derivatives (gastrodin, parishin A, B, C, E) were positively identified for the first time in this plant. These compounds were also validated and quantified using authentic standards. Parishin A was the most abundant component, followed by chlorogenic acid, gastrodin, eriodictyol glucoside, parishin C, parishin E and parishin B. The contents of all the polyphenolic compounds were higher at the immature and premature stages than at fully mature and overmature stages, while total carotenoid was found to be higher in the mature and overmature stages. Overall antioxidant activities by three different assays (DPPH, ABTS, FRAP) decreased as maturation progressed. Antioxidant properties of the fruit extract are suggested to be attributed to the polyphenols.

Overexpression of PkINO1 improves ethanol resistance of Pichia kudriavzevii N77-4 isolated from the Korean traditional fermentation starter nuruk.

The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.

p-Hydroxybenzyl alcohol inhibits four obesity-related enzymes in vitro.

Recently, antiobesity studies using the method of inhibiting enzymatic activity of obesity-related enzymes as targets have received considerable attention. The aims of the current study were to investigate whether p-hydroxybenzyl alcohol (HBA), identified from Cudrania tricuspidata fruits with antiobesity effects, inhibits the activity of digestive and obesity-related enzymes and acts as an inhibitor against four target enzymes in kinetic studies. In vitro enzyme assays showed HBA at the highest concentration significantly reduced the enzymatic activity of four targets: pancreatic lipase (IC50 = 2.34-3.70 µM), α-glycosidase (IC50 = 9.08 µM), phosphodiesterase IV (IC50 = 4.99 µM), and citrate synthase (IC50 = 2.07 µM) enzymes. Based on the results of kinetic assays, the types of inhibition were investigated. Our findings indicate that HBA could have antiobesity efficacy, and it deserves further study.

Effect of heat-treated Nuruk on the quality characteristics of aged Yakju.

Long-term aging of Yakju, a traditional Korean liquor made of rice and Nuruk (a fermentation agent), causes browning and odor and flavor development. This study investigated the effects of heat-treated Nuruk (50-80 °C, 30 min) on Yakju quality. The saccharogenic powers and glucoamylase, α-amylase, and carboxypeptidase activities were similar in non-heat-treated Nuruk and that treated at 50 °C. However, acidic protease and alcohol dehydrogenase decreased above 50 °C. The content of nitrogen-containing compounds was inversely proportional to the heat-treatment temperature. Compounds that cause off-flavors decreased at 50-60 °C, but increased at 70-80 °C, whereas compounds that provide fragrance increased at 50-60 °C. Sensory evaluation indicated that bad taste attributes were higher in Yakju produced using non-heat-treated Nuruk. Therefore, heat treatment of Nuruk at 50 °C can be adopted as a method for improving Yakju quality, as enzymatic activities that affect color, aroma, and taste are regulated.

Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov.

Twelve Acetobacter pasteurianus-related strains with publicly available genomes in GenBank shared high 16S rRNA gene sequence similarity (>99.59%), but average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values and multilocus sequence- and genome-based relatedness analyses suggested that they were divided into four different phylogenetic lineages. Relatedness analyses based on multilocus sequences, 1,194 core genes and whole-cell MALDI-TOF profiles supported that strains LMG 1590T and LMG 1591 (previously classified as the type strains of A. pasteurianus subsp. ascendens and paradoxus, respectively) and strain SLV-7T do not belong to A. pasteurianus. Strain SLV-7T, isolated from Korean traditional vinegar, shared low ANI (<91.0%) and in silico DDH (44.2%) values with all other Acetobacter type strains analyzed in this study, indicating that strain SLV-7T represents a new Acetobacter species. The phenotypic and chemotaxonomic analyses confirmed these results and therefore a new species named Acetobacter oryzifermentans sp. nov. is proposed with SLV-7T (=KACC 19301T=JCM 31096T) as the type strain. Strains LMG 1590T and LMG 1591 shared high ANI (99.4%) and in silico DDH (96.0%) values between them, but shared low ANI (<92.3%) and in silico DDH (<49.0%) values with other type strains analyzed in this study, indicating that strains LMG 1590T and LMG 1591 should be reclassified into a new single species that should be named Acetobacter ascendens sp. nov., comb. nov., with LMD 51.1T (=LMG 1590T=NCCB 51001T) as its type strain.

Characterization of brewing microorganisms isolated from Korean traditional nuruk for Cheongju production.

The objective of this study was to select and identify yeasts and molds isolated from traditional nuruk and to investigate their brewing characteristics for Cheongju production. The yeast strains Y190 (Accession ID-KACC 93251P), Y263 (Accession ID-KACC 93252P), and Y270 (Accession ID-KACC 93253P) showing high alcohol and flavor productivity were isolated and identified by phylogenetic inference based on an internal transcribed spacer 2 region sequence analysis. In addition, Aspergillus oryzae 83-10 (Accession ID-KACC 93254P) showing the highest enzyme activity was isolated. This study provides basic data for the production of Korean Cheongju and assesses the applicability of these three yeast strains and A. oryzae 83-10 isolated from traditional nuruk.

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity.

The brewing of makgeolli, one of Korea's most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

Optimization of air-blast drying process for manufacturing Saccharomyces cerevisiae and non-Saccharomyces yeast as industrial wine starters.

Wine yeast (Saccharomyces cerevisiae D8) and non-Saccharomyces wine yeasts (Hanseniaspora uvarum S6 and Issatchenkia orientalis KMBL5774) were studied using air-blast drying instead of the conventional drying methods (such as freeze and spray drying). Skim milk-a widely used protective agent-was used and in all strains, the highest viabilities following air-blast drying were obtained using 10% skim milk. Four excipients (wheat flour, nuruk, artichoke powder, and lactomil) were evaluated as protective agents for yeast strains during air-blast drying. Our results showed that 7 g lactomil was the best excipient in terms of drying time, powder form, and the survival rate of the yeast in the final product. Finally, 7 types of sugars were investigated to improve the survival rate of air-blast dried yeast cells: 10% trehalose, 10% sucrose, and 10% glucose had the highest survival rate of 97.54, 92.59, and 79.49% for S. cerevisiae D8, H. uvarum S6, and I. orientalis KMBL5774, respectively. After 3 months of storage, S. cerevisiae D8 and H. uvarum S6 demonstrated good survival rates (making them suitable for use as starters), whereas the survival rate of I. orientalis KMBL5774 decreased considerably compared to the other strains. Air-blast dried S. cerevisiae D8 and H. uvarum S6 showed metabolic activities similar to those of non-dried yeast cells, regardless of the storage period. Air-blast dried I. orientalis KMBL5774 showed a noticeable decrease in its ability to decompose malic acid after 3 months of storage at 4 °C.