Exercise Combined with a Low-Calorie Diet Improves Body Composition, Attenuates Muscle Mass Loss, and Regulates Appetite in Adult Women with High Body Fat Percentage but Normal BMI.

BACKGROUND: The present study aimed to examine the effects of a 500 kcal reduction in daily energy intake alone and in combination with 90 min of moderate-to-vigorous aerobic exercise per week on body weight, body composition, and appetite sensations in young women with normal BMI and abnormal body fat percentage. METHODS: sixty-six young women with normal BMI and abnormal body fat percentage (21.33 ± 1.20 kg/m2 and 34.32 ± 2.94%) were randomly assigned into three groups: (1) caloric restriction (CR; n = 22), (2) caloric restriction with exercise (CR-EX; n = 22), and (3) control (C; n = 22). Data on anthropometry, blood samples, and subjective appetite sensations pre- and post-intervention were collected. RESULTS: After 4 weeks of intervention, CR and CR-EX groups both reduced body weight, fat percentage, and waist and hip circumferences compared to the C group (p < 0.05). Muscle mass of the CR group was significantly lower than that of the C group (-1.21 ± 0.86 kg vs. -0.27 ± 0.82 kg, p < 0.05), and no significant difference between CR-EX and C groups was observed. For appetite sensations, the subjects of the CR group showed significant increases in change of scores in desire to eat and prospective consumption than that of the C group (p < 0.05), while no significant difference between CR-EX and C groups was observed. CONCLUSION: A 500 kcal reduction in daily energy intake alone and in combination with 90 min of moderate-to-vigorous aerobic exercise per week could both reduce weight and improve body composition in young adult women with normal BMI and abnormal body fat percentage. More importantly, calorie restriction combined with exercise intervention was superior to calorie restriction alone in improving muscle mass loss and regulating appetite sensations.

Improvement of thermostability and catalytic efficiency of xylanase from Myceliophthora thermophilar by N-terminal and C-terminal truncation.

Introduction: Extracting xylanase from thermophilic filamentous fungi is a feasible way to obtain xylanase with good thermal stability. Methods: The transcriptomic data of Myceliophthora thermophilic destructive ATCC42464 were differentially expressed and enriched. By comparing the sequences of Mtxylan2 and more than 10 xylanases, the N-terminal and C-terminal of Mtxylan2 were truncated, and three mutants 28N, 28C and 28NC were constructed. Results and discussion: GH11 xylan Mtxylan2 was identified by transcriptomic analysis, the specific enzyme activity of Mtxylan2 was 104.67 U/mg, and the optimal temperature was 65°C. Molecular modification of Mtxylan2 showed that the catalytic activity of the mutants was enhanced. Among them, the catalytic activity of 28C was increased by 9.3 times, the optimal temperature was increased by 5°C, and the residual enzyme activity remained above 80% after 30 min at 50-65°C, indicating that redundant C-terminal truncation can improve the thermal stability and catalytic performance of GH11 xylanase.

Temporal Profile of the Microbial Community and Volatile Compounds in the Third-Round Fermentation of Sauce-Flavor baijiu in the Beijing Region.

Sauce-flavor baijiu produced in the Beijing and Guizhou regions has regional characteristic flavors, but the differences in flavor compounds and reasons for their formation remain unclear. The sauce-flavor baijiu brewing process involves several rounds of fermentation. In this study, we investigated the temporal distribution of microbial communities and flavor substances during the third round of sauce-flavor baijiu fermentation in the Beijing region, and we then compared and analyzed the differences of flavor substances and microorganisms in the fermented grains of sauce-flavor baijiu in the Beijing and Guizhou regions. It was found that 10 bacterial genera and 10 fungal genera were dominant in the fermented grains. The acidity of the fermented grains had a significant driving effect on the microbial community succession. A total of 81 volatile compounds were identified and quantified in the fermented grains, of which esters and alcohols were relatively abundant. The differences in 30 microbial community compositions and their resulting differences in terms of the fermentation parameters of fermented grains are responsible for the differences in the profiles of flavor compounds between sauce-flavor baijiu produced in the Beijing and Guizhou regions.

Spatial heterogeneity of microbiota and flavor across different rounds of sauce-flavor baijiu in Northern China.

Sauce-flavor baijiu (SFB) is a traditional Chinese distilled liquor crafted through a distinctive brewing process, involving seven rounds of stack fermentation (SF) and pit fermentation (PF). To date, there remains a knowledge gap regarding the microbial succession and flavor throughout all rounds of SFB with distinctive northern characteristics. Through LEfSe analysis, Saccharopolyspora, Virgibacillus, Thermoascus and Thermomyces, and Lactobacillus and Issatchenkia were found to be the most differentially representative genera in SF and PF, respectively. A total of 93 volatile flavor compounds were found in base baijius through the gas-chromatography mass spectrometry. Moreover, 29 volatile flavor substances with significant difference in base baijius of different rounds were revealed using the OPLS-DA model and VIP values and Spearman correlation analysis shows that bacteria have a greater impact on differential flavor compounds than fungi. This study provides a new perspective and insight into the brewing of northern SFB.

Correlation of preoperative CT imaging shift parameters of the lateral plateau with lateral meniscal injury in Schatzker IV-C tibial plateau fractures.

BACKGROUND: Schatzker IV-C is a high-energy tibial plateau fracture often accompanied by lateral meniscus injuries. While imaging examinations are routine preoperative measurements, the correlation between CT imaging shift parameters of the lateral plateau and lateral meniscal injury in Schatzker IV-C fractures remains uncovered. METHODS: This retrospective study enrolled a total of 60 patients with Schatzker IV-C tibial plateau fractures at the First People's Hospital of Hefei. Prior to surgery, CT imaging was used to measure the numerical values of lateral plateau depression (LPD) and lateral plateau widening (LPW). The degree of lateral meniscus injury was confirmed based on intraoperative direct vision, with patients being classified into meniscus injury and non-meniscus injury groups. Dichotomous logistic regression was employed to evaluate the correlation between LPD, LPW, and lateral meniscus injury, while the optimal cut-off points for predicting lateral meniscal injury with LPD and LPW were determined using receiver operator characteristic (ROC) curves. RESULTS: The meniscus injury group exhibited a mean LPD of 15.3 ± 3.5 mm, which was significantly higher than the non-meniscus injury group's mean LPD of 8.4 ± 3.4 mm (P < 0.05). Similarly, the meniscus injury group had a larger mean LPW of 9.4 ± 1.8 mm compared to the non-meniscus injury group's mean LPW of 6.9 ± 0.9 mm (P < 0.05). The optimal cut-off points for predicting lateral meniscal injury were determined to be 8.40 mm for LPD (with a sensitivity of 95%, specificity of 85%, and AUC of 0.898) and 7.90 mm for LPW (with a sensitivity of 75%, specificity of 90%, and AUC of 0.897). CONCLUSIONS: Patients with Schatzker IV-C tibial plateau fractures are at a significantly higher risk of lateral meniscal injury when the LPD exceeds 8.40 mm and/or the LPW exceeds 7.90 mm. Our results may provide novel reference metrics for the early diagnosis of lateral meniscal injury in Schatzker IV-C tibial plateau fracture patients when the MRI examination is not available.

Molecular Modeling Insights into Metal-Organic Frameworks (MOFs) as a Potential Matrix for Immobilization of Lipase: An In Silico Study.

CRL is a highly versatile enzyme that finds extensive utility in numerous industries, which is attributed to its selectivity and catalytic efficiency, which have been impeded by the impracticality of its implementation, leading to a loss of native catalytic activity and non-reusability. Enzyme immobilization is a necessary step for enabling its reuse, and it provides methods for regulating the biocatalyst's functional efficacy in a synthetic setting. MOFs represent a novel category of porous materials possessing distinct superlative features that make MOFs an optimal host matrix for developing enzyme-MOF composites. In this study, we employed molecular modeling approaches, for instance, molecular docking and MD simulation, to explore the interactions between CRL and a specific MOF, ZIF-8. The present study involved conducting secondary structural analysis and homology modeling of CRL, followed by docking ZIF-8 with CRL. The results of the molecular docking analysis indicate that ZIF-8 was situated within the active site pocket of CRL, where it formed hydrogen bonds with Val-81, Phe-87, Ser-91, Asp-231, Thr-132, Lue-297, Phe-296, Phe-344, Thr-347, and Ser-450. The MD simulation analysis revealed that the CRL and ZIF-8 docked complex exhibited stability over the entire simulation period, and all interactions presented in the initial docked complex were maintained throughout the simulation. The findings derived from this investigation could promote comprehension of the molecular mechanisms underlying the interaction between CRL and ZIF-8 as well as the development of immobilized CRL for diverse industrial purposes.

Comparison of fungal communities and flavour substances in surface and inner layers of fermented grains during stacking fermentation of sauce-flavour baijiu.

The stacking fermentation process plays a vital role in the production of sauce-flavour baijiu. The aim of this paper is to elucidate the effects of environmental variables on the fungal communities of different layers of fermented grains (zaopei) during the sixth round of stacking and the changes in volatile flavour substances during this process. The composition of the fungal communities in different layers during the stacking fermentation process was analysed. Principal coordinate analysis (pCoA) showed that the dominant fungal communities in different layers differed significantly with the stacking fermentation process. The dominant fungal genera were Thermoascus, Thermomyces and Issatchenkia. The total content of flavouring substances in the surface layer of zaopei were higher, but the types of flavouring substances were less than in the inner layer. The relationship between temperature, moisture content, acidity, starch content and reducing sugar content and microbial community was analysed by Redundancy analysis. The results showed that the correlation between microbial communities and physicochemical indexes in different layers of zaopei varied. The core fungal genera in the surface layer were mainly influenced by acidity, and the microorganisms in the inner layer were more strongly correlated with temperature. Spearman correlation coefficient revealed the correlation between fungal community and volatile flavour substances, and the results showed that microorganisms in different layers of zaopei have different correlations with flavour substances. This study contributes to the understanding of the evolution of different layers fungal communities during the stacking of sauce-flavour baijiu and their relationship with volatile aroma substances.

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus.

The GH11 xylanase XynA from Streptomyces rameus L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations 11YHDGYF16, 23AP24/23SP24, and 32GP33. The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the "thumb" of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency.

Marine Chitosan-Oligosaccharide Ameliorated Plasma Cholesterol in Hypercholesterolemic Hamsters by Modifying the Gut Microflora, Bile Acids, and Short-Chain Fatty Acids.

This study evaluated the cholesterol-alleviating effect and underlying mechanisms of chitosan-oligosaccharide (COS) in hypercholesterolemic hamsters. Male hamsters (n = 24) were divided into three groups in a random fashion, and each group was fed one particular diet, namely a non-cholesterol diet (NCD), a high-cholesterol diet (HCD), and an HCD diet substituting 5% of the COS diet for six weeks. Subsequently, alterations in fecal bile acids (BAs), short-chain fatty acids (SCFAs), and gut microflora (GM) were investigated. COS intervention significantly reduced and increased the plasma total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels in hypercholesteremic hamsters. Furthermore, Non-HDL-C and total triacylglycerols (TG) levels were also reduced by COS supplementation. Additionally, COS could reduce and increase food intake and fecal SCFAs (acetate), respectively. Moreover, COS had beneficial effects on levels of BAs and GM related to cholesterol metabolism. This study provides novel evidence for the cholesterol-lowering activity of COS.

Butyriproducens baijiuensis BJN0003: a potential new member of the family Oscillospiraceae isolated from Chinese Baijiu.

Acid-producing bacteria are one kind of crucial species for Baijiu fermentation. The strain BJN0003 with the ability of producing butyric acid was isolated from the cellar mud of Baijiu, and the 16S rRNA gene sequence similarity was 94.2% to its most closely related type species Caproicibacterium lactiferaments JNU-WLY1368T, less than the threshold value of 94.5% for distinguishing genera. Furthermore, the genome of BJN0003 showed a length of 2,458,513 bp and a DNA G + C content of 43.3% through high throughput sequence. BJN0003 exhibited whole-genome average nucleotide identity value of 68.9% to the most closely related species, while the whole-genome digital DNA-DNA hybridization value was only 23.1%, which were both below the delineation thresholds of species. These results indicated BJN0003 could represent a potential novel species of a new genus of the family Oscillospiraceae, and was proposed the name as Butyriproducens baijiuensis. In addition, gene annotation and metabolic analysis showed that BJN0003 harbored the metabolic pathway of converting glucose to butyric acid. The discovery of the new species provided bacterial resource for Baijiu production and the revealing of genetic characteristics would promote the investigation of acid synthesis during Baijiu manufacturing process. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03624-w.

Simulated Fermentation of Strong-Flavor Baijiu through Functional Microbial Combination to Realize the Stable Synthesis of Important Flavor Chemicals.

The solid-state fermentation of Baijiu is complicated by the co-fermentation of many microorganisms. The instability of the composition and abundance of the microorganisms in the fermentation process leads to fluctuations of product quality, which is one of the bottleneck problems faced by the Strong-flavor Baijiu industry. In this study, we established a combination of functional microorganisms for the stable fermentation of the main flavor compounds of Baijiu, including medium and long-chain fatty acid ethyl esters such as hexanoic acid, ethyl ester; butanoic acid, ethyl ester; octanoic acid, ethyl ester; acetic acid, ethyl ester; 9,12-octadecadienoic acid, ethyl ester; and decanoic acid, ethyl ester in the fermented grains. Our study investigated the effects of microbial combinations on the fermentation from three aspects: microbial composition, microbial interactions, and microbial association with flavor compounds. The results showed that the added functional microorganisms (Lactobacillus, Clostridium, Caproiciproducens, Saccharomyces, and Aspergillus) became the dominant species in the fermentation system and formed positive interactions with other microorganisms, while the negative interactions between microorganisms were significantly reduced in the fermentation systems that contained both Daqu and functional microorganisms. The redundancy analysis showed that the functional microorganisms (Lactobacillus, Saccharomyces, Clostridium, Cloacibacterium, Chaenothecopsis, Anaerosporobacter, and Sporolactobacillus) showed strong positive correlations with the main flavor compounds (hexanoic acid, ethyl ester; lactic acid, ethyl ester; butanoic acid, ethyl ester; acetic acid, ethyl ester; and octanoic acid, ethyl ester). These results indicated that it was feasible to produce Baijiu with a functional microbial combination, and that this could promote stable Baijiu production.

Three Molecular Modification Strategies to Improve the Thermostability of Xylanase XynA from Streptomyces rameus L2001.

Glycoside hydrolase family 11 (GH11) xylanases are the preferred candidates for the production of functional oligosaccharides. However, the low thermostability of natural GH11 xylanases limits their industrial applications. In this study, we investigated the following three strategies to modify the thermostability of xylanase XynA from Streptomyces rameus L2001 mutation to reduce surface entropy, intramolecular disulfide bond construction, and molecular cyclization. Changes in the thermostability of XynA mutants were analyzed using molecular simulations. All mutants showed improved thermostability and catalytic efficiency compared with XynA, except for molecular cyclization. The residual activities of high-entropy amino acid-replacement mutants Q24A and K104A increased from 18.70% to more than 41.23% when kept at 65 °C for 30 min. The catalytic efficiencies of Q24A and K143A increased to 129.99 and 92.26 mL/s/mg, respectively, compared with XynA (62.97 mL/s/mg) when using beechwood xylan as the substrate. The mutant enzyme with disulfide bonds formed between Val3 and Thr30 increased the t1/260 °C by 13.33-fold and the catalytic efficiency by 1.80-fold compared with the wild-type XynA. The high thermostabilities and hydrolytic activities of XynA mutants will be useful for enzymatic production of functional xylo-oligosaccharides.

Correlational Analysis of the Physicochemical Indexes, Volatile Flavor Components, and Microbial Communities of High-Temperature Daqu in the Northern Region of China.

Daqu is a microbial-rich baijiu fermentation starter. The high-temperature Daqu plays an essential role in the manufacturing of sauce-flavored baijiu. However, few studies have focused on three kinds of high-temperature Daqu (white, yellow, and black Daqu) in northern China. In this study, the physicochemical indexes, volatile flavor compounds, and microbial characteristics of the three different colors of high-temperature Daqu in northern China were comparatively analyzed to reveal their potential functions. White Daqu (WQ) exhibited the highest liquefying power and starch, and black Daqu (BQ) showed the highest saccharifying and esterifying powers. A total of 96 volatile components were identified in the three types of Daqu, and the contents of the volatile components of yellow Daqu (YQ) were the highest. The microbial community structure analysis showed that Bacillus and Byssochlamys were dominant in BQ, Kroppenstedtia and Thermoascus were dominant in WQ, and Virgibacillus and Thermomyces dominated the YQ. The RDA analysis revealed the correlation between the dominant microorganisms and different physicochemical indexes. The Spearman correlation analysis indicated that Oceanobacillus, Saccharopolyspora, Staphylococcus, Pseudogracilibacillus, Byssochlamys, and Thermomyces showed positive correlations with part of the majority of the key volatile flavor compounds. This work provides a scientific basis for the actual production of different colors of high-temperature Daqu in the northern region of China for sauce-flavored baijiu.

Improved thermostability, acid tolerance as well as catalytic efficiency of Streptomyces rameus L2001 GH11 xylanase by N-terminal replacement.

The N-terminal of xylanase from 11 family of glycoside hydrolases (GH11 xylanase) has an important effect on its thermostability and catalytic properties. Previous studies have unearthed five important residues located in the N-terminal and successfully improved the thermostability of several GH11 xylanases using amino acid substitutions. In the present study, we applied this tactic to construct a mutant of XynA from Streptomyces rameus L2001, XynAR, and studied its biochemical, catalytic and hydrolytic properties. The results showed that thermostability, acid tolerance as well as catalytic efficiency of XynAR significantly improved compared to those of XynA, while the hydrolytic characteristics changed. Computer simulation analysis showed that this tactic created new hydrogen bonds and electrostatic interactions in the N-terminal, resulting in decrease in the flexibility of N-terminal and surface electrostatic potential as well as a change in the profile of hydrogen bonds between the subsites and substrate in the cleft region of xylanase. This study showed that amino acid substitutions at the key sites of the N-terminal of GH11 xylanase can improve its thermostability and catalytic properties.

Dynamic Transcriptome Analysis Reveals Transcription Factors Involved in the Synthesis of Ethyl Acetate in Aroma-Producing Yeast.

Ethyl acetate is an important flavor element that is a vital component of baijiu. To date, the transcription factors that can help identify the molecular mechanisms involved in the synthesis of ethyl acetate have not been studied. In the present study, we sequenced and assembled the Wickerhamomyces anomalus strain YF1503 transcriptomes to identify transcription factors. We identified 307 transcription factors in YF1503 using high-throughput RNA sequencing. Some transcription factors, such as C2H2, bHLH, MYB, and bZIP, were up-regulated, and these might play a role in ethyl acetate synthesis. According to the trend of ethyl acetate content, heat map results and STEM, twelve genes were selected for verification of expression levels using quantitative real-time PCR. This dynamic transcriptome analysis presents fundamental information on the transcription factors and pathways that are involved in the synthesis of ethyl acetate in aroma-producing yeast. Of significant interest is the discovery of the roles of various transcription factor genes in the synthesis of ethyl acetate.

Exploring bioactive peptides as potential therapeutic and biotechnology treasures: A contemporary perspective.

In preceding years, bioactive peptides (BAPs) have piqued escalating attention owing to their multitudinous biological features. To date, many potential BAPs exhibiting anti-cancer activities have been documented; yet, obstacles such as their safety profiles and consumer acceptance continue to exist. Moreover, BAPs have been discovered to facilitate the suppression of Coronavirus Disease 2019 (CoVID-19) and maybe ideal for treating the CoVID-19 infection, as stated by published experimental findings, but their widespread knowledge is scarce. Likewise, there is a cornucopia of BAPs possessing neuroprotective effects that mend neurodegenerative diseases (NDs) by regulating gut microbiota, but they remain a subject of research interest. Additionally, a plethora of researchers have attempted next-generation approaches based on BAPs, but they need scientific attention. The text format of this critical review is organized around an overview of BAPs' versatility and diverse bio functionalities with emphasis on recent developments and novelties. The review is alienated into independent sections, which are related to either BAPs based disease management strategies or next-generation BAPs based approaches. BAPs based anti-cancer, anti-CoVID-19, and neuroprotective strategies have been explored, which may offer insights that could help the researchers and industries to find an alternate regimen against the three aforementioned fatal diseases. To the best of our knowledge, this is the first review that has systematically discussed the next-generation approaches in BAP research. Furthermore, it can be concluded that the BAPs may be optimal for the management of cancer, CoVID-19, and NDs; nevertheless, experimental and preclinical studies are crucial to validate their therapeutic benefits.

An updated review on food-derived bioactive peptides: Focus on the regulatory requirements, safety, and bioavailability.

Food-derived bioactive peptides (BAPs) are recently utilized as functional food raw materials owing to their potential health benefits. Although there is a huge amount of scientific research about BAPs' identification, purification, characterization, and physiological functions, and subsequently, many BAPs have been marketed, there is a paucity of review on the regulatory requirements, bioavailability, and safety of BAPs. Thus, this review focuses on the toxic peptides that could arise from their primary proteins throughout protein extraction, protein pretreatment, and BAPs' formulation. Also, the influences of BAPs' length and administration dosage on safety are summarized. Lastly, the challenges and possibilities in BAPs' bioavailability and regulatory requirements in different countries were also presented. Results revealed that the human studies of BAPs are essential for approvals as healthy food and to prevent the consumers from misinformation and false promises. The BAPs that escape the gastrointestinal tract epithelium and move to the stomach are considered good peptides and get circulated into the blood using different pathways. In addition, the hydrophobicity, net charge, molecular size, length, amino acids composition/sequences, and structural characteristics of BAPs are critical for bioavailability, and appropriate food-grade carriers can enhance it. The abovementioned features are also vital to optimize the solubility, water holding capacity, emulsifying ability, and foaming property of BAPs in food products. In the case of safety, the possible allergenic and toxic peptides often exhibit physiological functions and could be produced during the hydrolysis of food proteins. It was also noted that the production of iso-peptides bonds and undesirable Maillard reaction might occur during protein extraction, sample pretreatments, and peptide synthesis.

Effect of Environmental Microorganisms on Fermentation Microbial Community of Sauce-Flavor baijiu.

The compositions of the microbial community in fermented grains of Sauce-flavor baijiu produced in different regions have diverse characteristics; however, the reasons for this remain unclear. The present study investigated the contributions of environmental microorganisms to the microbial community as well as the volatile compounds in the fermented grains of Sauce-flavor baijiu produced in the Beijing region using high-throughput sequencing combined with sourcetracker analysis, and compared the differences of environmental microorganism and their roles in the production process of Sauce-flavor baijiu from different regions.The results showed that the environmental microorganisms in the tools were the main contributors of the bacterial and fungal communities in fermented grains during heap fermentation and at the beginning of pit fermentation. At the end of pit fermentation, pit mud was the main environmental source of bacterial community in fermented grains, while tools and Daqu were the main environmental sources of fungal community in fermented grains.Environmental microorganisms thrived on the functional microorganisms in the fermented grains of Sauce-flavor baijiu produced in the Beijing region and thus shaped the profiles of volatile compounds. Environmental microorganisms of Sauce-flavor baijiu in the Guizhou province and the Beijing region differed significantly, which is partially responsible for the distinctive characteristics in the microbial community structure of Sauce-flavor baijiu-fermented grains from different regions.

Molecular mechanism of LIP05 derived from Monascus purpureus YJX-8 for synthesizing fatty acid ethyl esters under aqueous phase.

Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu. Monascus purpureus YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to α/ß fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The π-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu.

Correlation between microbial communities and flavor compounds during the fifth and sixth rounds of sauce-flavor baijiu fermentation.

Sauce-flavor baijiu is a representative of Chinese traditional fermented baijiu using grains as the raw materials through the co-fermentation of microorganisms. The whole manufacturing process includes 7 times of distillation and generates 7 kinds of base baijius. The final product is a mixture of the 7 kinds of base baijius. Thus the base baijius greatly affect the quality of the final product. The quality of the base baijiu obtained by the sixth distillation is obviously poorer than that of the fifth one. However, the reason is still unclear and limits the quality control of baijiu fermentation. In this study, the flavor substances and microbiota in the up, middle and bottom layers of fermented grains in the fifth and sixth rounds were compared. Some flavor esters showed obviously decreased concentrations in the sixth round, including ethyl benzoneacetic acid, ethyl hexanoic acid, ethyl dodecanoic acid, diethyl butanedioic acid, and ethyl 2-hydroxyl-propanoic acid. Meanwhile, an off-flavor p-cresol was detected in the sixth round. Correlation analysis of flavor chemicals and microbiota indicated that fungi in the fifth round played an important role for ester synthesis. Some bacterial and fungal species were both positively correlated with p-cresol synthesis, and the related p-cresol metabolic pathways were proposed for the first time. These results revealed flavor divergences of fermented grains between the fifth and sixth rounds, and will ultimately help to improve baijiu quality.