Antifungal mechanisms investigation of lactic acid bacteria against Aspergillus flavus: through combining microbial metabolomics and co-culture system.

AIMS: To develop antifungal lactic acid bacteria (LAB) and investigate their antifungal mechanisms against Aspergillus flavus in aflatoxin (AF) production. METHODS AND RESULTS: We isolated 179 LABs from cereal-based fermentation starters and investigated their antifungal mechanism against A. flavus through liquid chromatography-mass spectrometry and co-culture analysis techniques. Of the 179 isolates, antifungal activity was identified in Pediococcus pentosaceus, Lactobacillus crustorum, and Weissella paramesenteroides. These LABs reduced AF concentration by (i) inhibiting mycelial growth, (ii) binding AF to the cell wall, and (iii) producing antifungal compounds. Species-specific activities were also observed, with P. pentosaceus inhibiting AF production and W. paramesenteroides showing AF B1 binding activity. In addition, crucial extracellular metabolites for selecting antifungal LAB were involved in the 2',3'-cAMP-adenosine and nucleoside pathways. CONCLUSIONS: This study demonstrates that P. pentosaceus, L. crustorum, and W. paramesenteroides are key LAB strains with distinct antifungal mechanisms against A. flavus, suggesting their potential as biological agents to reduce AF in food materials.

Analytical factors for eight short-chain fatty acid analyses in mouse feces through headspace solid-phase microextraction-triple quadrupole gas chromatography tandem mass spectrometry.

This study developed a method for quantifying eight short-chain fatty acids (SCFAs) in mouse fecal samples using solid-phase microextraction (SPME) coupled with triple quadrupole gas chromatography tandem mass spectrometry. Furthermore, significant factors affecting SCFA analysis, including SPME fiber selection, pH, salting-out agent, and sample collection time, were investigated. Contrary to previous studies, we found that the CAR/PDMS fiber had the highest extraction efficiency for all SCFAs. The optimal extraction efficiency was observed at pH 2.0, particularly for low-molecular-weight SCFAs. NaH2PO4 showed a more effective extraction efficiency than NaCl, owing to its pH stability and less interference with the solvent matrix. Additionally, our results showed that the SCFA concentration increased over collection time. The composition ratio of the eight SCFAs was maintained for up to 24 h; thus, we concluded that samples should be collected within four hours to obtain reliable results. Our findings may improve laboratory methods for SCFA extraction and mouse fecal sample analysis.

Gellan gum prevents non-alcoholic fatty liver disease by modulating the gut microbiota and metabolites.

Gellan gum (GG) is an anionic polysaccharide used as an additive in the food industry. However, the effect of GG on gut microbiota regulation and nonalcoholic fatty liver disease (NAFLD) has not yet been investigated. In vitro fermentation experiments have demonstrated that GG promoted the growth of probiotic strains such as Lactiplantibacillus rhamnosus and Bifidobacterium bifidum, producing metabolites beneficial to gut health. In mice, GG reduced hepatic triglyceride content, serum biomarkers, and body fat mass and weight gain induced by a high fat diet. Additionally, GG regulated the gut microbiota including Desulfovibrionales, Deferribacterales, Bacteroidales, and Lactobacillales at the order level and also promoted short-chain fatty acid production. Moreover, GG improved the expression of proteins related to hepatic inflammation and lipid metabolism. Taken together, GG ameliorated NAFLD, possibly by acting on the gut-liver axis via improving the gut health, indicating its potential as a food supplement and/or prebiotic against NAFLD.

The Probiotic Effects of the Saccharomyces cerevisiae 28-7 Strain Isolated from Nuruk in a DSS-Induced Colitis Mouse Model.

Probiotics are microorganisms that can benefit host health when ingested in a live state, and lactic acid bacteria are the most common type. Among fungi, Saccharomyces boulardii (SB) is the only strain known to have a probiotic function with beneficial effects on colitis; however, information on other probiotic yeast strains is limited. Therefore, this study aimed to discover yeast strains expressing intestinal anti-inflammatory activities by exhibiting probiotic properties in dextran sodium sulfate (DSS)-induced colitis mice model. Nuruk (Korean traditional fermentation starter) containing various microbial strains was used as a source for yeast strains, and S. cerevisiae 28-7 (SC28-7) strain was selected with in vitro and in vivo characteristics to enable survival in the intestines. After 14 days of pretreatment with the yeast strains, DSS was co-administered for six days to induce colitis in mice. The results revealed that the disease activity index score was lowered by SC28-7 treatment compared to the DSS group, and the colon length and weight/length ratio were recovered in a pattern similar to that of the normal group. SC28-7 administration significantly reduced the secretion of pro-inflammatory cytokines in the serum and modified the mRNA expression of inflammatory cytokines (interleukin-1ß, transforming growth factor-ß, and interferon-γ) and proteins involved in gut barrier functions (mucin 2, mucin 3, zonula occludens-1, and occludin) in colon tissues. These results indicate that SC28-7 attenuates DSS-induced colon damage and inflammation, supporting its future use as a probiotic yeast for treating and preventing intestinal inflammatory diseases such as inflammatory bowel disease.

Untargeted Metabolomics reveals Doenjang metabolites affected by manufacturing process and microorganisms.

Doenjang is a traditional Korean fermented soybean-based food that is now produced industrially as traditional Doenjang and modernized Doenjang, depending on the conditions of the manufacturing process. In this study, the effect of the production process on Doenjang metabolites was analyzed using untargeted capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS). Of the 247 metabolites detected in 19 traditional and three modernized Doenjang samples, the level of 55 metabolites were significantly different between the two production groups. The S-plot of the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed that nine compounds (tryptamine, 2-phenylethylamine, citrulline, gamma-aminobutyric acid, putrescine, tyramine, 2-aminoisobutyric acid, stachydrine, and N5-ethylglutamine) were highly distributed in the traditional group, and six compounds (arginine, citric acid, choline, cytidine, hypoxanthine, and glucaric acid) were considered distinguishable metabolites of the modernized group. Microbial community analysis indicated that the levels of these metabolites were significantly altered by the presence of Bacillus spp., Enterococcus faecium, Tetragenococcus halophilus, Aspergillus oryzae, Penicillium spp., and Saccharomyces cerevisiae. These findings will give better understanding of the influence of the manufacturing process on Doenjang production in terms of both microbial activity and metabolite profiles.

Stachydrine derived from fermented rice prevents diet-induced obesity by regulating adipsin and endoplasmic reticulum homeostasis.

Makgeolli, a widely consumed traditional alcoholic beverage in Korea, is brewed mainly from rice using Nuruk as a fermentation starter, which contains fungi, yeast, and lactic acid bacteria. Among 58 Makgeolli samples brewed using various Nuruks, we found that one exhibited anti-obesity properties, with stachydrine shown to be responsible for these properties. Stachydrine promotes lipolysis and inhibits lipid accumulation in 3T3-L1 adipocytes; it also reduces weight gain and improves glucose tolerance and insulin resistance in a mouse model. Stachydrine dramatically suppresses adipsin mRNA levels in liver and adipose tissue, whereas serum adipsin levels were elevated in stachydrine-treated mice compared to mice fed a high-fat diet alone. Moreover, stachydrine recovers endoplasmic reticulum homeostasis and regulates adipsin expression. We highlight the potential use of stachydrine as a therapeutic agent for the treatment of obesity and insulin resistance and the use of Makgeolli fermented by Nuruk as a source of novel bioactive compounds.

Characterization, prebiotic and immune-enhancing activities of rhamnogalacturonan-I-rich polysaccharide fraction from molokhia leaves.

Plant-derived polysaccharides possess potential health benefits that improve intestinal health and the immune system. Molokhia leaves have a large amount of mucilage polysaccharide; in the present study, crude polysaccharide extract was prepared from molokhia leaves. The molecular weight of molokhia leaf polysaccharide fraction (MPF) was estimated to be 51.2 × 103 Da. Polysaccharide was methylated and the structure of MPF was mainly composed of rhamnogalacturonan-I structure with side chains, such as galactans and linear glucan (starch), as shown by GC-MS analysis. To study the biofunctional effects of MPF, its prebiotic and intestinal immune-enhancing activities were assayed in vitro. MPF exhibited good prebiotic activity, as shown by its high prebiotic scores, and increased contents of total short-chain fatty acids on five probiotic strains. In addition, MPF showed immune-enhancing activity on Peyer's patches, as revealed by the high bone marrow cell proliferating activity and production of immunoglobulin A and cytokines. These results demonstrate that MPF may be a potential beneficial prebiotic and intestinal immune-enhancer, which may have wide implications in the food industry.

Alteration of gut microbiota composition by short-term low-dose alcohol intake is restored by fermented rice liquor in mice.

Excessive alcohol consumption can increase gut permeability and alter the gut bacterial communities, influencing the pathogenesis of several disorders including alcoholic liver disease, endotoxemia, and systemic inflammation. Although recent studies have highlighted a pivotal role for gut microbiota in alcohol metabolism and alcoholic liver injury, the results have primarily been obtained from binge models treated with physiologically acute dose of alcohol. We sought to investigate the effect of low-dose (0.8 g/kg/day) and short-term (one week) consumption of alcohol on changes in gut microbiota composition in mice. The low-dose consumption of alcohol immediately altered fecal microbiota composition in mice after 1 and 7 days of treatment. We also evaluated the influence of alcoholic beverages containing various microbes using fermented rice liquors (FRLs, called Makgeolli in Korea) on gut microbiota and systemic inflammation. One week of FRLs consumption restored fecal microbiota compositions altered by alcohol administration in mice, with the abundance of Bacteroidetes and Firmicutes phyla recovered to levels of the control group. In addition, mice receiving FRLs exhibited increased fecal production of short-chain fatty acids (SCFA) such as butyric acid and propionic acid within 7 days, and reduced inflammatory responses induced by alcohol administration in the serum and colon. Taken together, these results suggest that short-term and low-dose alcohol intake induces alterations in fecal microbiota composition, and FRLs administration can restore microbial composition and suppress intestinal inflammation, highlighting potential benefits of FRLs as fermented foods.

Influence of aflatoxin in Nuruk on the safety of starch-based alcoholic beverage.

Nuruk is a fermentation agent used to manufacture alcoholic beverages, which contains a variety of microorganisms. Most microorganisms in Nuruk are useful for the production of alcoholic beverages; however, Nuruk can be infected with Aspergillus flavus, which produces aflatoxin (AF). Therefore, this study analyzed total AF concentrations in Nuruk, the transfer of AF from Nuruk to alcoholic beverages, and AF-producing microorganisms to determine the safety of alcoholic beverages with respect to this toxin. ELISA showed that total AF levels in 14 of 61 Nuruk samples exceeded 15 ppb, the Korean permissible level in cereal products. In alcoholic beverages produced with Nuruk containing AF levels >15 ppb, only AF G1 was detected, at a level of 0.3 ppb, and the transfer ratio of AF G1 was approximately 1.2% to 1.3%. The dominant genera in Nuruk were Aspergillus and Rhizopus. Among 30 strains belonging to Aspergillus, 10 produced only AF B1 at levels of 0.1 to 2.4 ppb after incubation at 25 °C for 8 days on potato dextrose agar plates. Although AF in Nuruk was rarely transferred to alcoholic beverages and the aflatoxigenic strains were found to possess poor AF-producing capacity, further efforts to reduce AF in Nuruk are needed to ensure the safety of alcoholic beverages.

In Vitro Antidiabetic and Antiobesity Activities of Traditional Kochujang and Doenjang and Their Components.

In this study we investigated the antidiabetic and antiobesity effects of aqueous ethanol extracts of traditional kochujang and doenjang. The average α-glucosidase inhibitory activity and adipogenesis inhibitory activity for the kochujang samples were 29.6% and 20.8%, respectively, while those of the doenjang samples were 46.3% and 11.6%, respectively. Therefore, antidiabetic activity is high in doenjang and antiobesity activity is high in kochujang. Kochujang and doenjang components responsible for suppressing the functional effects were investigated by metabolomic analysis. For kochujang, p-coumaric acid, N6,N6,N6-trimethyllysine, threonine, and methionine positively correlated with inhibition of adipogenesis activity, whereas for doenjang, betaine and betaine aldehyde were thought to be responsible for the antidiabetic effects. As p-coumaric acid and betaine were the most probable candidates with functional effects, these two compounds were selected for further analysis. Inhibition of adipogenesis was shown to be 14.0±1.85% for betaine chloride and 38.3±3.27% for p-coumaric acid, suggesting that p-coumaric acid is more effective than betaine against obesity. However, betaine exhibited higher α-glucosidase inhibitory activity than p-coumaric acid. Our results suggest that both kochujang and doenjang can be used against diabetes and obesity. However, clinical trials are necessary to support these results.

Surface Film Formation in Static-Fermented Rice Vinegar: A Case Study.

In the present study, we aimed to determine the cause of surface film formation in three rice vinegars fermented using the traditional static fermentation method. The pH and total acidity of vinegar were 3.0-3.3 and 3.0-8.7%, respectively, and acetic acid was the predominant organic acid present. Colonies showing a clear halo on GYC medium were isolated from the surface film of all vinegars. Via 16S rDNA sequencing, all of the isolates were identified as Acetobacter pasteurianus. Furthermore, field-emission scanning electron microscopy analysis showed that the bacterial cells had a rough surface, were rod-shaped, and were ∼1 × 2 µm in size. Interestingly, cells of the isolate from one of the vinegars were surrounded with an extremely fine threadlike structure. Thus, our results suggest that formation of the surface film in rice vinegar was attributable not to external contamination, to the production of bacterial cellulose by A. pasteurianus to withstand the high concentrations of acetic acid generated during fermentation. However, because of the formation of a surface film in vinegar is undesirable from an industrial perspective, further studies should focus on devising a modified fermentation process to prevent surface film formation and consequent quality degradation.

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, ß-amylase, protease, and ß-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, ß-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, ß-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.

Quantitative analysis of volatile flavor components in Korean alcoholic beverage and Japanese sake using SPME-GC/MS.

Quantitative analysis of the volatile flavor components in Korean alcoholic beverages (makgeolli and yakju) and Japanese sake was carried out using SPME-GC/MS. Fusel oils (n-butyl alcohol, isobutyl alcohol, isoamyl alcohol, and phenethyl alcohol), ethyl esters (ethyl acetate, isoamyl acetate, ethyl caproate, ethyl caprylate, and ethyl caprate) and aldehydes (furfural and benzaldehyde) were analyzed quantitatively by an 85 µm SPME fiber (carboxen/polydimethylsiloxane) using internal standards (1-pentanol-1-13C and methyl nonanoate). Phenethyl alcohol (85-216 ppm) and isoamyl alcohol (38-115 ppm) constituted the majority of fusel oils in all the samples. Acetic acid was detected in sour makgeolli at a high level (0.02-0.14 ppm) compared with yakju and sake. A very high level of total ethyl esters (ethyl acetate, isoamyl acetate, ethyl caprate, and phenethyl acetate), having fruit and flower flavor, was found in makgeolli. Processing the volatile flavor data by multivariate partial least squares discriminant analysis, makgeolli, yakju, and sake showed cluster separation.

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

Metabolomic unveiling of a diverse range of green tea (Camellia sinensis) metabolites dependent on geography.

Numerous factors such as geographical origin, cultivar, climate, cultural practices, and manufacturing processes influence the chemical compositions of tea, in the same way as growing conditions and grape variety affect wine quality. However, the relationships between these factors and tea chemical compositions are not well understood. In this study, a new approach for non-targeted or global analysis, i.e., metabolomics, which is highly reproducible and statistically effective in analysing a diverse range of compounds, was used to better understand the metabolome of Camellia sinensis and determine the influence of environmental factors, including geography, climate, and cultural practices, on tea-making. We found a strong correlation between environmental factors and the metabolome of green, white, and oolong teas from China, Japan, and South Korea. In particular, multivariate statistical analysis revealed strong inter-country and inter-city relationships in the levels of theanine and catechin derivatives found in green and white teas. This information might be useful for assessing tea quality or producing distinct tea products across different locations, and highlights simultaneous identification of diverse tea metabolites through an NMR-based metabolomics approach.

Qualitative and quantitative prediction of volatile compounds from initial amino acid profiles in Korean rice wine (makgeolli) model.

UNLABELLED: In Korean rice wine (makgeolli) model, we tried to develop a prediction model capable of eliciting a quantitative relationship between initial amino acids in makgeolli mash and major aromatic compounds, such as fusel alcohols, their acetate esters, and ethyl esters of fatty acids, in makgeolli brewed. Mass-spectrometry-based electronic nose (MS-EN) was used to qualitatively discriminate between makgeollis made from makgeolli mashes with different amino acid compositions. Following this measurement, headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (GC-MS) combined with partial least-squares regression (PLSR) method was employed to quantitatively correlate amino acid composition of makgeolli mash with major aromatic compounds evolved during makgeolli fermentation. In qualitative prediction with MS-EN analysis, the makgeollis were well discriminated according to the volatile compounds derived from amino acids of makgeolli mash. Twenty-seven ion fragments with mass-to-charge ratio (m/z) of 55 to 98 amu were responsible for the discrimination. In GC-MS combined with PLSR method, a quantitative approach between the initial amino acids of makgeolli mash and the fusel compounds of makgeolli demonstrated that coefficient of determination (R(2)) of most of the fusel compounds ranged from 0.77 to 0.94 in good correlation, except for 2-phenylethanol (R(2) = 0.21), whereas R(2) for ethyl esters of MCFAs including ethyl caproate, ethyl caprylate, and ethyl caprate was 0.17 to 0.40 in poor correlation. PRACTICAL APPLICATION: The amino acids have been known to affect the aroma in alcoholic beverages. In this study, we demonstrated that an electronic nose qualitatively differentiated Korean rice wines (makgeollis) by their volatile compounds evolved from amino acids with rapidity and reproducibility and successively, a quantitative correlation with acceptable R2 between amino acids and fusel compounds could be established via HS-SPME GC-MS combined with partial least-squares regression. Our approach for predicting the quantities of volatile compounds in the finished product from initial condition of fermentation will give an insight to food researchers to modify and optimize the qualities of the corresponding products.

Electronic tongue-based discrimination of Korean rice wines (makgeolli) including prediction of sensory evaluation and instrumental measurements.

A commercial electronic tongue was used to discriminate Korean rice wines (makgeolli) brewed from nine cultivars of rice with different amino acid and fatty acid compositions. The E-tongue was applied to establish prediction models with sensory evaluation or LC-MS/MS by partial least squares regression (PLSR). All makgeollis were classified into three groups by principal components analysis, and the separation pattern was affected by rice qualities and yeast fermentation. Makgeolli taste changed from the complicated comprising sweetness, saltiness, and umami to the uncomplicated, such as bitterness and then, sourness, with a decrease of amino acids and fatty acids in the rice. The quantitative correlation between E-tongue and sensory scores or LC-MS/MS by PLSR demonstrated that E-tongue could well predict most of the sensory attributes with relatively acceptable r(2), except for bitterness, but could not predict most of the chemical compounds responsible for taste attributes, except for ribose, lactate, succinate, and tryptophan.

Identification of Wild Yeast Strains and Analysis of Their ß-Glucan and Glutathione Levels for Use in Makgeolli Brewing.

Makgeolli, also known as Takju, is a non-filtered traditional Korean alcoholic beverage that contains various floating matter, including yeast cells, which contributes to its high physiological functionality. In the present study, we assessed the levels of ß-glucan and glutathione in various yeast strains isolated from traditional Korean Nuruk and selected a ß-glucan- and glutathione-rich yeast strain to add value to Makgeolli by enhancing its physiological functionality through increased levels of these compounds. Yeast ß-glucan levels ranged from 6.26% to 32.69% (dry basis) and were strongly species-dependent. Dried Saccharomyces cerevisiae isolated from Nuruk contained 25.53 µg/mg glutathione, 0.70 µg/mg oxidized glutathione, and 11.69 µg/g and 47.85 µg/g spermidine and L-ornithine monohydrochloride, respectively. To produce functional Makgeolli, a ß-glucan- and glutathione-rich yeast strain was selected in a screening analysis. Makgeolli fermented with the selected yeast strain contained higher ß-glucan and glutathione levels than commercial Makgeolli. Using the selected yeast strain to produce Makgeolli with high ß-glucan and glutathione content may enable the production of functional Makgeolli.

MicroRNA function in NK-cell biology.

The important role of microRNAs in directing immune responses has become increasingly clear. Here, we highlight discoveries uncovering the role of specific microRNAs in regulating the development and function of natural killer (NK) cells. Furthermore, we discuss the impact of NK cells on the entire immune system during global and specific microRNA ablation in the settings of inflammation, infection, and immune dysregulation.

Prognostic value of 18-fluorodeoxyglucose positron emission tomography-computed tomography in resectable colorectal cancer.

AIM: To assess the prognostic value of preoperative 18 fluorodeoxyglucose positron emission tomography (FDG-PET)/computed tomography (CT) in patients with resectable colorectal cancer. METHODS: One hundred sixty-three patients with resectable colorectal cancer who underwent FDG-PET/CT before surgery were included. Patient data including pathologic stage at presentation, histology, treatment, disease-free survival and the maximum standardized uptake value (SUVmax) of the primary tumor on FDG-PET/CT were retrospectively analyzed. Median follow up duration was 756 (range, 419-1355). The primary end point was disease-free survival. RESULTS: Twenty-five of 163 patients (15.3%) had recurrences. The median SUVmax values of the recurrence and no-recurrence groups were 8.9 (range, 5-24) and 8.2 (range, 0-23, P = 0.998). Receiver operating characteristic (ROC) curve analysis showed no significant association between SUVmax and recurrence (area under the curve = 0.5, P = 0.998, 95% CI: 0.389-0.611). Because a statistically significant value was not found, SUVmax was dichotomized at its median of 8.6. The disease-free survival curve was analyzed using the median SUVmax (8.6) as the cut off. Univariate and multivariate analysis did not provide evidence that disease-free survival rates for the subgroups defined by the median SUVmax were significantly different (P = 0.52, P = 0.25). CONCLUSION: Our study suggests that the high FDG uptake of primary mass in resectable colorectal cancer doesn't have a significant relationship with tumor recurrence and disease-free survival.