To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca2+, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca2+ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca2+ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca2+ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.

Sauce-flavor baijiu is one of the twelve flavor types of Chinese distilled fermented product. Microbial composition plays a key role in the stacked fermentation of Baijiu, which uses grains as raw materials and produces flavor compounds, however, the active microbial community and its relationship remain unclear. Here, we investigated the total and active microbial communities of stacked fermented grains of sauce-flavored Baijiu using flow cytometry and high-throughput sequencing technology, respectively. By using traditional high-throughput sequencing technology, a total of 24 bacterial and 14 fungal genera were identified as the core microbiota, the core bacteria were Lactobacillus (0.08-39.05%), Acetobacter (0.25-81.92%), Weissella (0.03-29.61%), etc. The core fungi were Issatchenkia (23.11-98.21%), Monascus (0.02-26.36%), Pichia (0.33-37.56%), etc. In contrast, using flow cytometry combined with high-throughput sequencing, the active dominant bacterial genera after cell sorting were found to be Herbaspirillum, Chitinophaga, Ralstonia, Phenylobacterium, Mucilaginibacter, and Bradyrhizobium, etc., whereas the active dominant fungal genera detected were Aspergillus, Pichia, Exophiala, Candelabrochaete, Italiomyces, and Papiliotrema, etc. These results indicate that although the abundance of Acetobacter, Monascus, and Issatchenkia was high after stacked fermentation, they may have little biological activity. Flow cytometry and cell sorting techniques have been used in the study of beer and wine, but exploring the microbiome in such a complex environment as Chinese baijiu has not been reported. The results also reveal that flow cytometry and cell sorting are convenient methods for rapidly monitoring complex microbial flora and can assist in exploring complex environmental samples.

Baijiu is a traditional fermented Chinese beverage with a history of hundreds of years. The notable characteristics of Baijiu include diverse raw materials, complex technology, and the co-fermentation of multiple strains. Complex brewing technology has created different aromas and contributes to complex trace component formation in Baijiu. Among the different components, the alcohol, ester, and acid contents are the highest in Baijiu, constituting its aroma skeleton. Nitrogen compounds significantly constitute the aroma compounds of Baijiu and affect human health. Although present in low concentration, sulfur compounds significantly contribute to the taste and positively impact human health. We provide a systematic review of domestic and international reports on the trace components in Baijiu. The review introduces their sources, types, and effects on Baijiu aroma, describes their content and existence in mainstream aromas of Baijiu, such as the strong-, jiang-, and light-aromas, and discusses their health effects. Furthermore, this article summarizes and proposes several feasible research ideas. The systematic review of these trace components will possibly facilitate studies on Baijiu, particularly on its relationship with health.

Background L-lysine is widely used for feed and special diet products. The transformation of fermentation strains plays a decisive role in the development of these industries. Based on the mutation breeding theory and metabolic engineering methods, this study aimed to improve the regeneration rate of high-lethality protoplasts by combining multiple mutagenesis and homologous cell fusion techniques to efficiently concentrate multiple dominant mutations and optimize the L-lysine production strain Escherichia coli QDW. Results In order to obtain the best protoplasts, the optimal enzymolysis time was selected as 4 h. The optimal lysozyme concentration was estimated at 0.8 mg/mL, because the protoplast formation rate and regeneration rate reached 90% and 30%, respectively, and their product reached the maximum. In this study, it was necessary that UV mutagenesis be excessive to obtain an expanded mutation library. For high lethality protoplasts, under the premise of minimal influence on its recovery, the optimal time for UV mutagenesis of protoplasts was 7 min, and the optimal time for thermal inactivation of protoplasts at 85 ℃ was 30 min. After homologous fusion, four fusion strains of E. coli were obtained, and their stability was analyzed by flow cytometry. The L-lysine yield of QDW-UH3 increased by 7.2% compared with that of QDW in a fermentation experiment, which promoted the expression of key enzymes in L-lysine synthesis, indicating that the combination of ultraviolet mutagenic breeding and protoplast fusion technology improved the acid-production level of the fusion strain. Conclusion This method provides a novel approach for the targeted construction of microbial cell factories.

Polyphenols are important functional substances produced in the acetic acid fermentation (AAF) of Shanxi aged vinegar (SAV). Previous studies have shown that the metabolic activity of microorganisms is closely related to polyphenol production and accumulation. In this study, microorganisms in the AAF of SAV were analyzed to explore how to increase the polyphenol yield by changing the microorganisms and reveal the potential mechanism of the microbial influence on the polyphenol yield. Macrotranscriptome analysis showed that acetic and lactic acid bacteria dominated the AAF fermentation process and initially increased and decreased. Spearman correlation analysis and verification experiments showed that the co-addition of Acetobacter pasteurianus and Lactobacillus helveticus promoted the accumulation of polyphenols, and the total polyphenol content increased by 72% after strengthening.

This study aimed at examining the structure-role modeling and antifatigue mechanism of polysaccharides, including M. androsaceus exopolysaccharide 3 (MEPS3), isolated from Marasmius androsaceus fermentation broth. The molecular weight of MEPS3 was 10.47 kDa. Furthermore, monosaccharide analysis showed the presence of mannose, glucose, and galactose in MEPS3 in a molar ratio of 0.08 : 0.34 : 1.46. Mannose, α -galactose, and α -d-glucose anomeric hydrogen signals were detected using nuclear magnetic resonance spectroscopy. MEPS3 was found to contain glyoxylic acid, forming rod-like chains that support high-purity polymerization. The weight-loaded swimming test results showed that MEPS3 treatment reduced lactic acid (LA) levels by 25.72% and increased the lactate dehydrogenase (LDH) activity by 5.67% in the plasma. Furthermore, it lowered malondialdehyde (MDA) levels by 47.09% and increased reactive oxygen species (ROS) and glutathione peroxidase (GSH-Px) levels by 52.42 and 97.03%, respectively, in the plasma. In addition, MEPS3 treatment reduced MDA and ROS levels in the liver by 28.85 and 18.64% while increasing superoxide dismutase (SOD) and GSH-Px levels by 17.41 and 38.13%, respectively. MEPS3 treatment increased the expression of nuclear factor-erythroid 2-related factor 2, glutamate-cysteine ligase, quinone oxidoreductase 1, and heme oxygenase 1 by 22.5, 24.8, 20.3, and 43.1%, respectively, in the liver. These findings demonstrate that MEPS3 effectively alleviates fatigue by removing harmful metabolites and indicate that the antifatigue mechanism is related to the Nrf-2 signaling pathway.

Aspergillus niger is the main industrial workhorse for global citric acid production. This fungus has complex sensing and signaling pathways to respond to environmental nutrient fluctuations. As the preferred primary carbon source, glucose also acts as a critical signal to trigger intracellular bioprocesses. Currently, however, there is still a knowledge gap in systems-level understanding of metabolic and cellular responses to this vital carbon source. In this study, we determined genome-wide transcriptional changes of citric acid-producing Aspergillus niger in response to external glucose gradient. It demonstrated that external glucose fluctuation led to transcriptional reprogramming of many genes encoding proteins involved in fundamental cellular process, including ribosomal biogenesis, carbon transport and catabolism, glucose sensing and signaling. The major glucose catabolism repressor creA maintained a stable expression independent of external glucose, while creB and creD showed significant downregulation and upregulation by the glucose increase. Notably, several high-affinity glucose transporters encoding genes, including mstA, were greatly upregulated when glucose was depleted, while the expression of low-affinity glucose transporter mstC was glucose-independent, which showed clear concordance with their protein levels detected by in situ fluorescence labeling assay. In addition, we also observed that the citric acid exporter cexA was observed to be transcriptionally regulated by glucose availability, which was correlated with extracellular citric acid secretion. These discoveries not only deepen our understanding of the transcriptional regulation of glucose but also shed new light on the adaptive evolutionary mechanism of citric acid production of A. niger.

Cellulosome is a highly efficient multi-enzyme self-assembly system and is found on the extracellular surface or in the free environment of microorganisms. However, with a lack of Ca2+ in vivo, cellulosome assembly is challenging. In this study, a novel design method was used to directionally modify the Ca2+-binding site, and four double-site dockerin A (DocA) mutants were obtained. At a Ca2+ concentration between 1.00 × 10−7 and 1.00 × 10−4 M, the mutant DocA-D3 had the strongest binding capacity to cohesion (Coh), which was 8.01 times that of DocA. The fluorescence signal intensity of the fusion proteins assembled using mutants was up to 1.26 × 107 in Escherichia coli, which indicated that these mutants could interact with Coh in vivo. The molecular dynamics simulation results showed that DocA-D3 could maintain a stable angle structure without Ca2+, and when applied to L-lysine fermentation, the yield was increased by 24.1%; when applied to β-alanine fermentation, the product accumulation was increased by 2.13–2.63 times. These findings lay the foundation for assembly design in cells.

In this work, a new strain of Bacillus amyloliquefaciens SY07 isolated from a traditional fermented soybean food was reported to possess remarkable α-glucosidase inhibitor-producing ability. Different culture media were applied for the proliferation of B. amyloliquefaciens SY07, and it was found that fermented okara broth presented the highest α-glucosidase inhibitory activity, while Luria-Bertani medium showed a negative effect. The extract from fermented okara broth acted in a dose-dependent manner to inhibit α-glucosidase activity, with an IC50 value of 0.454 mg/mL, and main inhibitors in the fermentation extract presented a reversible, uncompetitive pattern according to Lineweaver–Burk plots. Moreover, 1-deoxynojirimycin, a recognized α-glucosidase inhibitor, was found in the extract. Results indicated that B. amyloliquefaciens SY07 could utilize okara, a by-product from the soy processing industry, to generate α-glucosidase inhibitors effectively, and be regarded as a novel excellent microbial candidate for safe, economical production of potential functional foods or ingredients with hypoglycemic effect.

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.

Objective The docking and adhesion proteins present in cellulosomes are critical for the efficiency of their assembly. In this study, a in silico design method was used to directionally modify the calcium ion-binding site of a key component, namely, the type I docking protein DocA, of the cellulosomes of Clostridium thermocellum . Results The results indicated that a mutated DocA-D41 exhibited a highest binding capacity for the type I adhesion protein Coh at a calcium ion concentration of 5 × 10 −4 mol/L, which was 4.11 times the capacity of the original DocA. A molecular dynamics simulation showed that the high-frequency RMSD values for DocA-D40 and DocA-D41 were 0.232 and 0.228, lower than that of the original DocA, which implies that the mutants DocA-D40 and DocA-D41 were more stable than that of DocA. Conclusion The results of this study provide an efficient method for constructing efficient C. thermocellum -derived cellulosomes, and will lay the foundation for the design of other types of cellulosomes.

Xylanase, a glycoside hydrolase, is widely used in the food, papermaking, and textile industries; however, most xylanases are inactive at high temperatures. In this study, a xylanase gene, CFXyl3, was cloned from Cellulomonas flavigena and expressed in Escherichia coli BL21 (DE3). To improve the thermostability of xylanase, four hybrid xylanases with enhanced thermostability (designated EcsXyl1–4) were engineered from CFXyl3, guided by primary and 3D structure analyses. The optimal temperature of CFXyl3 was improved by replacing its N-terminus with the corresponding area of SyXyn11P, a xylanase that belongs to the hyperthermostable GH11 family. The optimal temperatures of the hybrid xylanases EcsXyl1–4 were 60, 60, 65, and 85°C, respectively. The optimal temperature of EcsXyl4 was 30 C higher than that of CFXyl3 (55°C) and its melting temperature was 34.5°C higher than that of CFXyl3. After the hydrolysis of beechwood xylan, the main hydrolysates were xylotetraose, xylotriose, and xylobiose; thus, these hybrid xylanases could be applied to prebiotic xylooligosaccharide manufacturing.

In this work, the changes of properties and microstructure of cellulose (bleached hardwood kraft pulp (BHKP)) subjected to different enzyme pretreatment times (0–10 h) were explored for further fibrillation. The various properties of the pretreated cellulose gradually decrease with the elapse of time relative to the pristine material, such as yield, water retention value, aspect ratio and degree of polymerization, etc. Enzyme pretreatment can promote the peeling of fibrils and loosen the amorphous areas of cellulose identified by Scanning Electron Microscope (SEM) and X-ray diffraction (XRD). A thorough investigation of the relation between pretreatment and evolution of inter-/intra-molecular H-bonds in cellulose was conducted including content and cleave sequence of H-bonds by Fourier transform infrared spectroscopy (FTIR), second derivative analysis and generalized two-dimensional correlation spectroscopy (2DCOS). The intermolecular H-bonds with the most significant decrease in content was cleaved first relative to the intramolecular H-bonds. These discoveries provide theoretical support to more effective pretreatment method for commercial production of fibrils from cellulosic fibers.

Nanofibrillated cellulose is obtained from cellulose resources (e.g., Natural cellulose, bacterial cellulose) and is deemed to be environmentally friendly, green, and inexhaustible. Accordingly, we reported an innovative method for fluorine-free water- and oil/grease-repellent surface based on porous cellulose-based materials. In order to improve oil/grease resistance, sulfonated nanofibrillated cellulose (SNFC) was coated on the surface to fill the porous, the obtained paper was soaked in chitosan aqueous solution as the second layer. The-SNFC-chitosan-coated paper was applied with modified polyvinyl alcohol (MPVA) as the third layer to increase water resistance and water vapour transmittance. The experimental results showed 5.2 g/m2 SNFC, 2.5 g/m2 chitosan and 7% MPVA demonstrated excellent kit rating value of 12/12 (maximum grease resistance) (oil resistance contact angle is 103.93°), good water resistance (water contact angle is 108.6°) and high water vapour transmission rate (4.8 × 10–14 g cm/cm2 s Pa) under the condition of 90% relative humidity. This non-fluorinated and practical method is an excellent alternative to existing fluorinated materials, which will further promote the development of cellulose-based packaging.

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.

Polyphenols extracted from Shanxi-aged vinegar (SAVEP) can alleviate oxidative stress and inflammatory stress caused by high-fat diets, improving intestinal microbial disorders. SAVEP may be a novel treatment of the metabolic syndrome.

食品中的多酚类化合物种类丰富,是天然的抗氧化剂和重要的功能因子.对食品中的多酚类化合物进行有效地提取和检测,有助于更全面地研究食品中多酚类化合物的种类和含量,能够更好地解析食品对人体的保健功能.因此,近年来受到国内外学者的广泛关注.文中对食品中多酚类化合物的种类结构、提取工艺和检测手段进行综述,旨在为食品中多酚类化合物的研究提供指导,为含多酚类化合物的功能性食品开发提供新思路.

Zhenjiang aromatic vinegar (ZAV) is one of the well-known fermented condiments in China, which is produced by solid-state fermentation. It can be classified into traditional Zhenjiang aromatic vinegar (TZAV) and industrial Zhenjiang aromatic vinegar (IZAV) because of different production methods. The purpose of the study was to evaluate the variations and differences on chemical compositions and antioxidant activities of TZAV and IZAV during the aging process. The proximate composition, organic acids content, total phenolic content (TPC), total flavonoid content (TFC), total antioxidant activity (TAA) and phenolic compounds composition of TZAV and IZAV were detected during the aging process. Organic acids contents, TPC, TFC, TAA and phenolic compounds contents in ZAV were increased during the aging process. Acetic acid, lactic acid and pyroglutamic acid in ZAV were major organic acids. With the extension of aging time, TZAV and IZAV had similar proximate compositions and organic acids content. The values of TPC, TFC and TAA were higher in TZAV than in IZAV when aging is more than 3 years. Rutin and p-coumaric acid were detected in TZAV but not in IZAV. In principal component analysis (PCA), TZAV and IZAV can be divided into two groups according to their phenolic compounds composition. These findings provide references for evaluating TZAV and IZAV on the basis of their characterizations.

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.