Mining and characterization of novel antimicrobial peptides from the large-scale microbiome of Shanxi aged vinegar based on metagenomics, molecular dynamics simulations and mechanism validation.

The study aimed to mine and characterize novel antimicrobial peptides (AMPs) from the Shanxi aged vinegar microbiome. Utilizing machine learning techniques, AlphaFold2 structure prediction and molecular dynamics simulations, six novel AMPs were innovatively mined from 98,539 peptides based on metagenomic data, of which one peptide secreted by Lactobacillus (named La-AMP) was experimentally validated to have remarkable bactericidal effects against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) with high stability and no hemolytic activity. Scanning electron microscopy revealed that La-AMP caused irreversible damage to cell membranes of S. aureus and E. coli, a finding further confirmed by calcein-AM/propidium iodide staining. Additionally, La-AMP induced nucleic acid leakage and reactive oxygen species accumulation in bacterial cells. It was found to bind to DNA gyrase through salt bridges, hydrogen bonds, and hydrophobic interactions, ultimately inducing apoptosis. Thus, La-AMP exhibited encouraging promise as a valuable bioactive component for the development of natural preservatives.

Rapid non-destructive monitoring and quality assessment of the fumigation process of Shanxi aged vinegar based on Vis-NIR hyperspectral imaging combined with multiple chemometric algorithms.

The quality of the grains during the fumigation process can significantly affect the flavour and nutritional value of Shanxi aged vinegar (SAV). Hyperspectral imaging (HSI) was used to monitor the extent of fumigated grains, and it was combined with chemometrics to quantitatively predict three key physicochemical constituents: moisture content (MC), total acid (TA) and amino acid nitrogen (AAN). The noise reduction effects of five spectral preprocessing methods were compared, followed by the screening of optimal wavelengths using competitive adaptive reweighted sampling. Support vector machine classification was employed to establish a model for discriminating fumigated grains, and the best recognition accuracy reached 100%. Furthermore, the results of partial least squares regression slightly outperformed support vector machine regression, with correlation coefficient for prediction (Rp) of 0.9697, 0.9716, and 0.9098 for MC, TA, and AAN, respectively. The study demonstrates that HSI can be employed for rapid non-destructive monitoring and quality assessment of the fumigation process in SAV.

Separation and Detection of Catechins and Epicatechins in Shanxi Aged Vinegar Using Solid-Phase Extraction and Hydrophobic Deep Eutectic Solvents Combined with HPLC.

This research presents a new, eco-friendly, and swift method combining solid-phase extraction and hydrophobic deep eutectic solvents (DES) with high-performance liquid chromatography (SPE-DES-HPLC) for extracting and quantifying catechin and epicatechin in Shanxi aged vinegar (SAV). The parameters, such as the elution solvent type, the XAD-2 macroporous resin dosage, the DES ratio, the DES volume, the adsorption time, and the desorption time, were optimized via a one-way experiment. A central composite design using the Box-Behnken methodology was employed to investigate the effects of various factors, including 17 experimental runs and the construction of three-dimensional response surface plots to identify the optimal conditions. The results show that the optimal conditions were an HDES (tetraethylammonium chloride and octanoic acid) ratio of 1:3, an XAD-2 macroporous resin dosage of 188 mg, and an adsorption time of 11 min. Under these optimal conditions, the coefficients of determination of the method were greater than or equal to 0.9917, the precision was less than 5%, and the recoveries ranged from 98.8% to 118.8%. The environmentally friendly nature of the analytical process and sample preparation was assessed via the Analytical Eco-Scale and AGREE, demonstrating that this method is a practical and eco-friendly alternative to conventional determination techniques. In summary, this innovative approach offers a solid foundation for the assessment of flavanol compounds present in SAV samples.

Melanoidin-like carbohydrate-containing macromolecules from Shanxi aged vinegar exert immunoenhancing effects on macrophage RAW264.7 cells.

Bioactive macromolecule mining is important for the functional chemome analysis of traditional Chinese vinegar. In this study, we isolated and characterized carbohydrate-containing macromolecules from Shanxi aged vinegar (CCMSAV) and evaluated their immunomodulatory activity. The isolation process involved ethanol precipitation, deproteinization, decolorization, and DEAE-650 M column chromatography, resulting in the acquisition of four sub-fractions. All sub-fractions exhibited a molecular weight range of 6.92 to 16.71 kDa and were composed of 10 types of monosaccharides. Comparative analysis of these sub-fractions with two melanoidins exhibited similarities in elemental composition, spectral signature, and pyrolytic characteristics. Immunological assays confirmed the significantly enhanced cell viability, phagocytic activity, and secretion of nitric oxide, tumor necrosis factor (TNF)-α and interleukin (IL)-6 in RAW264.7 cells by all four sub-fractions. Further investigation of the immunomodulatory mechanism revealed that SAV-RP70-X, the most potent purified sub-fraction, enhanced aerobic glycolysis in macrophages and activated Toll-like receptor 2 (TLR2), TLR4, mannose receptor (MR), scavenger receptor (SR), and the dendritic cell-associated C-type lectin-1 receptor (Dectin-1). Furthermore, the activation of macrophages was associated with the MyD88/PI3K/Akt/NF-κB signaling pathway. Methylation analysis revealed that 1,4-Xylp was the most abundant glycosidic linkage in SAV-RP70-X.

Determination of Flavonoid Compounds in Shanxi Aged Vinegars Based on Hydrophobic Deep Eutectic Solvent VALLME-HPLC Method: Assessment of the Environmental Impact of the Developed Method.

This research presents a novel, eco-friendly, vortex-assisted liquid-liquid microextraction (VALLME) approach, integrating hydrophobic deep eutectic solvents (DESs) with HPLC for the identification and quantification of nine specific flavonoids in Shanxi aged vinegar (SAV). The parameters of DES-VALLME, including the ratio of trioctylmethylammonium chloride to 1,4-butanediol (1:6), DES volume (150 µL), vortex duration (5 min), the concentration of NaCl (0.40 g), and centrifugation time (10 min), were optimized to achieve the maximum extraction efficiency of target substances. Under these optimal conditions, quantitative analyses performed via HPLC demonstrated a broad linear range of 0.20-50.00 µg/mL and correlation coefficients (r2) greater than 0.9944 for all nine calibration curves. The limits of detection (LOD) and limits of quantitation (LOQ) were 0.09-0.18 µg/mL and 0.30-0.60 µg/mL, respectively, ensuring high sensitivity. The relative standard deviations for intra-day and inter-day variability were within the acceptable range, 2.34-3.77% and 3.04-4.96%, respectively, demonstrating the method's reliability. The recovery rates ranged from 85.97% to 108.11%, underscoring the method's precision. This technique exhibited a significant enrichment effect (enrichment factor: 43 to 296) on SAV flavonoids. Notably, the eco-friendliness of this procedure was evaluated using the Analytical Eco-Scale, Green Analytical Procedure Index, and Analytical Greenness Metric. The results suggested that this technique is a viable green alternative to traditional flavonoid determination methods in SAV. In summary, this novel method provides a theoretical basis for assessing flavonoid content in SAV samples and tracing SAV products. This contribution has significant implications for enhancing analytical techniques in food chemistry and environmental science and the sustainable development of the food industry.

Extraction methods and compositions of polyphenols in Shanxi aged vinegar.

The extraction, purification, qualification, and quantification of polyphenols (PPs) in vinegar are challenging owing to the complex matrix of vinegar and the specific physicochemical and structural properties of PPs. This study aimed to develop a simple, efficient, low-cost method for enriching and purifying vinegar PPs. The enrichment and purification effects of five solid phase extraction (SPE) columns and five macroporous adsorption resins (MARs) for PPs were compared. The results show that SPE columns were more effective in purifying vinegar PPs than MARs. Among them, the Strata-XA column showed a higher recovery (78.469 ± 0.949%), yield (80.808 ± 2.146%), and purity (86.629 ± 0.978%) than other columns. In total, 48 PPs were identified and quantified using SPE and gas chromatography-mass spectrometry from the SPE column extracts; phenolic acids, such as 4-hydroxyphenyllactic acid, vanillic acid, 4-hydroxycinnamic acid, 4-hydroxybenzoic acid, protocatechuic acid, and 3-(4-Hydroxy-3-methoxyphenyl) propionic acid, occupy a major position in SAV. Furthermore, considering the potential applications of PPs, the concentrates were characterized based on their bioactive properties. They exhibited high total PP, flavonoid, and melanoidin contents and excellent anti-glycosylation and antioxidant activities. These results indicate that the established methodology is a high-efficiency, rapid-extraction, and environment-friendly method for separating and purifying PPs, with broad application prospects in the food, chemical, and cosmetic industries.

Polyphenols extracted from Shanxi-aged vinegar exert hypolipidemic effects on OA-induced HepG2 cells via the PPARα-LXRα-ABCA1 pathway.

Hyperlipidemia is one of the key risk factors causing many chronic diseases, and lowering blood lipid levels can prevent many diseases. In this paper, a hyperlipidemic cell model of oleic acid (OA) induced hepatocellular carcinoma cells (HepG2) was established using polyphenols extracted from Shanxi-aged vinegar (SAVEP). The effects of SAVEP on nuclear damage, mitochondrial membrane potential, apoptosis, cellular lipid deposition, and lipid metabolism protein expression in HepG2 hyperlipidemic cells were examined to investigate the lipid-lowering mechanism of SAVEP at the cellular level. The results showed that SAVEP could reduce the content of TC/TG index, repair the nuclear damage, reduce lipid accumulation and finally decrease the rate of apoptosis by up-regulating the expression of key proteins such as PPARα, LXRα, and ABCA1 in the process of lipid metabolism. PRACTICAL APPLICATIONS: In this thesis, the hypolipidemic activity of polyphenol extracts from Shanxi-aged vinegar was analyzed on the level of HepG2 cells. The hypolipidemic mechanism of oxidative stress, lipid metabolism and inflammatory stress was also elucidated. It provided a theoretical basis for the in-depth understanding of the hypolipidemic health effects of Shanxi-aged vinegar.

Elucidation and Regulation of Polyphenols in the Smoking Process of Shanxi Aged Vinegar.

Polyphenols (PPs) are the main contributors to the health functions of Shanxi aged vinegar (SAV) and are mainly produced during the smoking process. This study aimed to explore the feasibility of regulating the accumulation of total water-soluble PPs (TWSP) by changing environmental factors based on the distribution of PPs. A total of eleven PPs, such as vanillin, vanillic acid, and (e)-ferulic acid, were detected during the smoking process. During the smoking process, the content of TWSP gradually increased and was accompanied by changes in environmental factors. Spearman correlation analysis and verification experiments showed that temperature, amino acids, and reducing sugars, as the main influencing factors, promoted the accumulation of TWSP. The in situ regulation strategy of changing environmental factors significantly increased the accumulation of TWSP by 12.24%.

Polyphenols Extracted from Shanxi-Aged Vinegar Inhibit Inflammation in LPS-Induced RAW264.7 Macrophages and ICR Mice via the Suppression of MAPK/NF-κB Pathway Activation.

Shanxi-aged vinegar, a traditional Chinese grain-fermented food that is rich in polyphenols, has been shown to have therapeutic effects on a variety of diseases. However, there has been no comprehensive evaluation of the anti-inflammatory activity of polyphenols extracted from Shanxi-aged vinegar (SAVEP) to date. The anti-inflammatory activities of SAVEP, both in RAW 264.7 macrophages and mice, were extensively investigated for the potential application of SAVEP as a novel anti-inflammatory agent. In order to confirm the notion that polyphenols could improve inflammatory symptoms, SAVEP was firstly detected by gas chromatography mass spectrometry (GC-MS). In total, 19 polyphenols were detected, including 12 phenolic acids. The study further investigated the protective effect of SAVEP on lipopolysaccharide-induced inflammation in RAW264.7 macrophages and ICR mice. The results showed that compared with those of the model group, SAVEP could remarkably recover the inflammation of macrophage RAW264.7 and ICR mice. SAVEP can normalise the expression of related proteins via the suppression of MAPK/NF-κB pathway activation, inhibiting the expression of iNOS and COX-2 proteins, and consequently the production of inflammatory factors, thus alleviating inflammatory stress. These results suggest that SAVEP may have a potential function against inflammation.

Metabolic network of ammonium in cereal vinegar solid-state fermentation and its response to acid stress.

Shanxi aged vinegar (SAV), a Chinese traditional vinegar, is produced by various microorganisms. Ammonium is an important nitrogen source for microorganisms and a key intermediate for the utilization of non-ammonium nitrogen sources. In this work, an ammonium metabolic network during SAV fermentation was constructed through the meta-transcriptomic analysis of in situ samples, and the potential mechanism of acid affecting ammonium metabolism was revealed. The results showed that ammonium was enriched as the acidity increased. Meta-transcriptomic analysis showed that the conversion of glutamine to ammonia is the key pathway of ammonium metabolism in vinegar and that Lactobacillus and Acetobacter are the dominant genera. The construction and analysis of the metabolic network showed that amino acid metabolism, nucleic acid metabolism, pentose phosphate pathway and energy metabolism were enhanced to resist acid damage to the intracellular environment and cell structures. The enhancement of nitrogen assimilation provides nitrogen for metabolic pathways that resist acid cytotoxicity. In addition, the concentration gradient allows ammonium to diffuse outside the cell, which causes ammonium to accumulate during fermentation.

Comparison of two types of vinegar with different aging times by NMR-based metabolomic approach.

Shanxi vinegar (SV) is well known as the most famous vinegar for its distinctive processing technique in China. Aging is the most important process for SV, and we call the vinegar aging more than 1-year Shanxi aged vinegar (SAV) and less than 1-year Shanxi mature vinegar (SMV). In this study, the chemical compositions and in vivo difference between SAV and SMV were compared by NMR based metabolomic profiling technique and multivariate statistical analysis. The results indicated that most of the metabolites exhibited higher concentrations in SAV than those in SMV¸ and the amino acids showed higher concentration ratio than the other compounds. The changes of endogenous metabolites after treatment with SMV and SAV were also investigated. Compared with the chemical differences, the differences of metabolomic changes between SAV and SMV were relatively minor. This study provides a valuable approach for accurately evaluating the differences of food products. PRACTICAL APPLICATIONS: Vinegar is commonly consumed as the important seasoning. SV, which is made from several kinds of cereal by solid-state fermentation techniques, is one of the famous vinegars in China. Aging is the most important process for SV which dividing vinegar into SAV and SMV according to the aging time. Due to the long aging process, the price of SAV is much higher than that of SMV in the market place. However, no investigation was conducted to compare their differences in vivo.

Evaluation of Nutritional Compositions, Bioactive Compounds, and Antioxidant Activities of Shanxi Aged Vinegars During the Aging Process.

Shanxi aged vinegar (SAV) is known as famous fermented food in China. During the brewing process, a large number of nutritional substances and bioactive compounds are produced, which have beneficial effects on human body. In this study, the contents of nutritional compositions including proteins, fats, carbohydrates, organic acids, and amino acids were determined in SAV samples. The antioxidant compounds and properties of SAV with different aging time were also evaluated. The results showed that the contents of proteins, crude fats, and carbohydrates in SAV were not changed with the aging time. Moreover, Alanine was the main component of amino acids in SAV, and the total contents of amino acids were increased with the aging time. Acetic acid and lactic acid were the predominant organic acids in SAV. The contents of acetic acid and lactic acid accounted for more than 90% of the total organic acids in SAV, which were increased during the aging process of 5 years. Furthermore, total phenols, flavonoids contents, and browning index in SAV were also increased during the aging time. These antioxidant compounds showed a high correlation with the antioxidant activities of SAV measured by ferric reducing antioxidant power and 2,2'-azino-bis(3-ethylbenzthi azoline-6-sulfonic acid) assays. The contribution of polyphenolic fractions and high molecular weight melanoidins to total antioxidant activities was similar (about 49% and 48%, respectively). Our findings would provide new insights to further explore the development of new vinegar-based functional foods. PRACTICAL APPLICATION: The analysis of nutritional compositions, bioactive compounds, and antioxidant capacities in vinegars provides a theoretical basis for the function of SAV. It also provides references for further explore the development of new-type functional and healthy vinegars.

Shanxi Aged Vinegar Protects against Alcohol-Induced Liver Injury via Activating Nrf2-Mediated Antioxidant and Inhibiting TLR4-Induced Inflammatory Response.

Shanxi aged vinegar (SAV) is a typical fermented and antioxidant food, which has various health-promoting effects. This work aimed to explore the effects of SAV on alcohol-induced liver injury. A mice model of alcoholic liver injury was established to illuminate its potential mechanisms. All mice pretreated with SAV and then received an ethanol solution (50% w/v, 4.8 g/kg b.w.). The results showed that SAV ameliorated alcohol-induced histological changes and elevation of liver enzymes. SAV attenuated alcohol-induced oxidative stress by declining levels of hepatic oxidants, and restoring depletion of antioxidant enzyme activities in mice livers. Moreover, SAV alleviated alcohol-induced oxidative damage by activating nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated signal pathway. In addition, SAV prevented alcohol-induced inflammation by suppressing lipopolysaccharide (LPS) level and activities of pro-inflammatory enzymes, and regulating inflammatory cytokines. SAV inhibited alcohol-induced inflammation through down-regulating the expression of Toll-like receptor 4 (TLR4)-mediated inflammatory response. The findings provide crucial evidence for elucidating the hepatoprotective mechanisms of SAV and encourage the future application of SAV as a functional food for liver protection.

Succession sequence of lactic acid bacteria driven by environmental factors and substrates throughout the brewing process of Shanxi aged vinegar.

Lactic acid bacteria (LAB) are essential microbiota for the fermentation and flavor formation of Shanxi aged vinegar, a famous Chinese traditional cereal vinegar that is manufactured using open solid-state fermentation (SSF) technology. However, the dynamics of LAB in this SSF process and the underlying mechanism remain poorly understood. Here, the diversity of LAB and the potential driving factors of the entire process were analyzed by combining culture-independent and culture-dependent methods. Canonical correlation analysis indicated that ethanol, acetic acid, and temperature that result from the metabolism of microorganisms serve as potential driving factors for LAB succession. LAB strains were periodically isolated, and the characteristics of 57 isolates on environmental factor tolerance and substrate utilization were analyzed to understand the succession sequence. The environmental tolerance of LAB from different stages was in accordance with their fermentation conditions. Remarkable correlations were identified between LAB growth and environmental factors with 0.866 of ethanol (70 g/L), 0.756 of acetic acid (10 g/L), and 0.803 of temperature (47 °C). More gentle or harsh environments (less or more than 60 or 80 g/L of ethanol, 5 or 20 g/L of acetic acid, and 30 or 55 °C temperature) did not affect the LAB succession. The utilization capability evaluation of the 57 isolates for 95 compounds proved that strains from different fermentation stages exhibited different predilections on substrates to contribute to the fermentation at different stages. Results demonstrated that LAB succession in the SSF process was driven by the capabilities of environmental tolerance and substrate utilization.

Antioxidant Activity of Chinese Shanxi Aged Vinegar and Its Correlation with Polyphenols and Flavonoids During the Brewing Process.

One of the most famous Chinese vinegars, Shanxi aged vinegar (SAV), is produced with solid-state fermentation technology. Total antioxidant activity (TAC) is a special property for SAV. In this study, we investigate correlations between total antioxidant activity (TAC) and total polyphenol (TP) and total flavonoid (TF) contents of SAV, especially during the brewing process. For SAV, TAC, TP, and TF increased with the increase of aging time. The correlation coefficients between TAC and TP were 0.869 and 0.934, respectively, when analyzed with the method of ABTS and FRAP. They were 0.828 (ABTS) and 0.877 (FRAP) between the TAC and TF. In smoking pei stage that is a special technique for SAV different from other Chinese cereal vinegars, TAC increased by 120% (ABTS) and 111% (FRAP) mainly due to the increase of TP (89%) and TF (75%), which was more obvious than that during alcohol fermentation and acetic acid fermentation stages. Moreover, variation during brewing process of 8 main polyphenol compounds that were proved responsible for the TAC of SAV was analyzed. In addition to catechins and chlorogenic acid, gallic acid serves as one of the principal antioxidant ingredients in SAV. PRACTICAL APPLICATION: Total antioxidant activity (TAC) of Shanxi aged vinegar (SAV), which is highly correlated with total polyphenol and total flavonoid, increased with aging time, however, there is a little loss of total antioxidant after more than 8 y. During the brewing process smoking pei technique is important for enhancing the TAC of SAV suggesting critical controlled and thoroughly study of smoking pei stage are needed to improve the quality of SAV.

Determination of rheological property and its effect on key aroma release of Shanxi aged vinegar.

The rheological property of Shanxi aged vinegar (SAV) was determined by rheometer, and its effects on release in eight key aroma components of SAV was investigated by SPME-GC-MS. In order to simulate the change of rheological property of SAV, a modified SAV system was developed from a finished SAV using carboxymethylcellulose, pectin, glucose, fructose, sodium chloride and tannic acid at indicate levels. The consistency coefficients (K) of SAV ranged from 1.09e-5 to 0.0137, which was correlated to glucose, polyphenol, acids and oBx. SAV changed from shear-thickening to Newtonian fluid during long-time ageing. In the modified SAV system, the K values increased significantly, and two modified vinegar became quasi-Newtonian fluids too. Furthermore, release of the eight key aroma compounds decreased significantly and decreased was pronounced, for acetic acid, furfural and tetramethylpyrazine. The results demonstrated rheological property correlated to the concentrate of sugar, salt, polyphenol, acids and macromolecule, which significantly affected the release of major aroma compounds.

Aroma Constituents in Shanxi Aged Vinegar before and after Aging.

Shanxi aged vinegar is one of the most famous Chinese traditional cereal vinegars produced by spontaneous solid-state fermentation. However, the aroma composition of Shanxi aged vinegar is still ambiguous. The Shanxi vinegars before and after aging were both analyzed by solvent-assisted flavor evaporation combined with gas chromatography-mass spectrometry (GC-MS) as well as gas chromatography-olfactometry (GC-O) in aroma extract dilution analysis. A total of 87 odor-active regions were found by GC-O, and 80 odor-active compounds were identified. By GC-MS/MS, in selected reaction monitoring mode, 30 important identifications were quantitated using authentic standards. In comparison, the aroma molecules for the vinegars before and after aging were almost the same; only their levels were altered, with mostly the esters and some compounds that produce pungent smells being lost and the levels of those from the Maillard reaction, especially the pyrazines (e.g., tetramethylpyrazine), being greatly increased. As for the aged vinegar, the compounds found to have high flavor dilution factors (>128) were 3-(methylthio)propanal, vanillin, 2,3-butanedione, tetramethylpyrazine, 3-methylbutanoic acid, γ-nonalactone, guaiacol, 3-(methylthio)propyl acetate, dimethyl trisulfide, phenylacetaldehyde, 2-ethyl-6-methylpyrazine, 2-acetylpyrazine, 2,3-dimethylpyrazine, furfural, and 3-hydroxy-2-butanone. However, the aroma compounds found at high concentrations (>25 µg/L) in the aged vinegar were acetic acid, followed by 2,3-butanedione, furfural, 3-hydroxy-2-butanone, tetramethylpyrazine, furfuryl alcohol, and 3-methylbutanoic acid.

Development of a SPME-GC-MS method for the determination of volatile compounds in Shanxi aged vinegar and its analytical characterization by aroma wheel.

A solid-phase microextraction followed by gas chromatography-mass spectrometry method was developed to determine the volatile compounds in Shanxi aged vinegar. The optimal extraction conditions were: 50 °C for 20 min with a PDMS/DVB fiber. This analytical method was validated and showed satisfactory repeatability (0.5 %

Metabolomic studies on special processing of Asari Radix et Rhizoma in Guilingji / 中草药

Objective: To explore the underlying reasons why Asari Radix et Rhizoma (ARR) should be processed by Shanxi aged vinegar when used in Guilingji. Methods: 1H-NMR-based metabolomic approach combined with multivariate statistical analysis was used to investigate the differential chemical components among the raw and two vinegar-baked ARR. Results: More than 30 metabolites were identified in the 1H-NMR spectra of ARR. Raw and two vinegar-baked ARR could be separated obviously, and the rice vinegar-baked ARR and the Shanxi aged vinegar-baked ARR also differed to each other, in which the priamary metabolites showed bigger difference than that of secondary metabolites. Conclusion: This study laid a basis for the modern research of the special processing technology for Guilingji.