Discovery and mechanism explanation of a novel green biocatalyst esterase Bur01 from Burkholderia ambifaria for ester synthesis under aqueous phase.

Biocatalyst catalyzing the synthesis of esters under aqueous phase is an alternative with green and sustainable characteristics. Here, a biocatalyst esterase Bur01 was identified through genome sequencing and gene library construction from a Burkholderia ambifaria BJQ0010 with efficient ester synthesis property under aqueous phase for the first time. Bur01 was soluble expressed and the purified enzyme showed the highest activity at pH 4.0 and 40 °C. It had a broad substrate spectrum, especially for ethyl esters. The structure of Bur01 was categorized as a member of α/ß fold hydrolase superfamily. The easier opening of lid under aqueous phase and the hydrophobicity of substrate channel contribute to easier access to the active center for substrate. Molecular docking and site-directed mutation demonstrated that the oxyanion hole Ala22, Met112 and π-bond stacking between His24 and Phe217 played essential roles in catalytic function. The mutants V149A, V149I, L159I and F137I enhanced enzyme activity to 1.42, 1.14, 1.32 and 2.19 folds due to reduced spatial resistance and increased hydrophobicity of channel and ethyl octanoate with the highest conversion ratio of 68.28 % was obtained for F137I. These results provided new ideas for developing green catalysts and catalytic basis of mechanistic studies for ester synthetase under aqueous phase.

Caproicibacter sp. BJN0012, a potential new species isolated from cellar mud for caproic acid production from glucose.

Acids play a crucial role in enhancing the flavor of strong-aroma Baijiu, and among them, caproic acid holds significant importance in determining the flavor of the final product. However, the metabolic synthesis of caproic acid during the production process of Baijiu has received limited attention, resulting in fluctuations in caproic acid content among fermentation batches and generating production instability. Acid-producing bacteria found in the cellar mud are the primary microorganisms responsible for caproic acid synthesis, but there is a lack of research on the related microbial resources and their metabolic properties. Therefore, it is essential to identify and investigate these acid-producing microorganisms from cellar mud to ensure stable caproic acid synthesis. In this study, a unique strain was isolated from the cellar mud, exhibiting a 98.12 % similarity in its 16 S rRNA sequence and an average nucleotide identity of 79.57 % with the reference specie, together with the DNA-DNA hybridization of 23.20 % similarity, confirming the distinct species boundaries. The strain was able to produce 1.22 ± 0.55 g/L caproic acid from glucose. Through genome sequencing, annotation, and bioinformatics analysis, the complete pathway of caproic acid synthesis from glucose was elucidated, and the catalytic mechanism of the key thiolase for caproic acid synthesis was investigated. These findings provide useful fundamental data for revealing the metabolic properties of caproic acid-producing bacteria found in cellar mud.

Functional activity of endophytic bacteria G9H01 with high salt tolerance and anti-Magnaporthe oryzae that isolated from saline-alkali-tolerant rice.

It holds significant practical importance to screen and investigate endophytic bacteria with salt-tolerant activity in rice for the development of relevant microbial agents. A total of 179 strains of endophytic bacteria were isolated from 24 samples of salt-tolerant rice seeds, with almost 95 % of these bacteria exhibiting tolerance to a salt content of 2 % (0.34 mol/L). Following the screening process, a bacterium named G9H01 was identified, which demonstrated a salt tolerance of up to 15 % (2.57 mol/L) and resistance to Magnaporthe oryzae, the causal agent of rice blast disease. Phylogenetic analysis confirmed G9H01 as a strain of Bacillus paralicheniformis. The complete genome of G9H01 was sequenced and assembled, revealing a considerable number of genes encoding proteins associated with salt tolerance. Further analysis indicated that G9H01 may alleviate salt stress in a high-salt environment through various mechanisms. These mechanisms include the utilization of proteins such as K+ transporters, antiporters, and Na+/H+ antiporters, which are involved in K+ absorption and Na+ excretion. G9H01 also demonstrated the ability to uptake and accumulate betaine, as well as secrete extracellular polysaccharides. Collectively, these findings suggest that Bacillus paralicheniformis G9H01 has potential as a biocontrol agent, capable of promoting rice growth under saline-alkali-tolerant conditions.

Complete genome sequence of Acinetobacter indicus and identification of the hydrolases provides direct insights into phthalate ester degradation.

A strain designated Acinetobacter indicus WMB-7 with the ability to hydrolyze phthalate esters (PAEs) was isolated from the fermented grains of Baijiu. The genome of the strain was sequenced with a length of 3,256,420 bp and annotated with 3183 genes, of which 36 hydrolases encoding genes were identified. The hydrolases were analyzed by protein structure modeling and molecular docking, and 14 enzymes were docked to the ligand di-butyl phthalate with the catalytic active regions, and showed binding affinity. The 14 enzymes were expressed in E. coli and 5 of them showed the ability for PAEs hydrolysis. Enzyme GK020_RS15665 showed high efficiency for PAEs hydrolysis and could efficiently hydrolyze di-butyl phthalate under an initial concentration of 1000 mg/L with a half-life of 4.24 h. This work combined a series of methods for identifying PAEs hydrolases and offered a molecular basis for PAEs degradation of A. indicus strains from Baijiu. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01334-w.

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.

Isolation and Characterization of Yeast with Benzenemethanethiol Synthesis Ability Isolated from Baijiu Daqu.

Baijiu, a prevalent alcoholic beverage, boasts over 2000 aroma compounds, with sulfur-containing compounds being the most influential in shaping its flavor. Benzenemethanethiol, a distinctive odorant in baijiu, is known to enhance the holistic flavor profile of baijiu. Despite its importance, there is very little literature on the biotransformation mechanism of benzenemethanethiol. Thus, extensive research efforts have been made to elucidate the formation mechanism of this compound in order to improve baijiu production. In this study, 12 yeast strains capable of generating benzenemethanethiol were isolated from baijiu daqu, and the Saccharomyces cerevisiae strain J14 was selected for further investigation. The fermentation conditions were optimized, and it was found that the optimal conditions for producing benzenemethanethiol were at 28 °C for 24 h with a 4% (v/v) inoculum of 3.025 g/L L-cysteine. This is the first time that yeast has been shown to produce benzenemethanethiol isolated from the baijiu fermentation system. These findings also suggest that benzenemethanethiol can be metabolized by yeast using L-cysteine and benzaldehyde as precursor substrates.

Roles of sulfur-containing compounds in fermented beverages with 2-furfurylthiol as a case example.

Aroma is a critical component of the flavor and quality of beverages. Among the volatile chemicals responsible for fragrance perception, sulfur compounds are unique odorants due to their extremely low odor threshold. Although trace amounts of sulfur compounds can enhance the flavor profile of beverages, they can lead to off-odors. Sulfur compounds can be formed via Maillard reaction and microbial metabolism, imparting coffee aroma and altering the flavor of beverages. In order to increase the understanding of sulfur compounds in the field of food flavor, 2-furfurylthiol (FFT) was chosen as a representative to discuss the current status of their generation, sensory impact, enrichment, analytical methods, formation mechanisms, aroma deterioration, and aroma regulation. FFT is comprehensively reviewed, and the main beverages of interest are typically baijiu, beer, wine, and coffee. Challenges and recommendations for FFT are also discussed, including analytical methods and mechanisms of formation, interactions between FFT and other compounds, and the development of specific materials to extend the duration of aroma after release.

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.

Characteristics and Correlation of the Microbial Communities and Flavor Compounds during the First Three Rounds of Fermentation in Chinese Sauce-Flavor Baijiu.

Sauce-flavor Baijiu is representative of solid-state fermented Baijiu. It is significant to deeply reveal the dynamic changes of microorganisms in the manufacturing process and their impact on the formation of flavor chemicals correlated with the quality of Baijiu. Sauce-flavor Baijiu manufacturing process can be divided into seven rounds, from which seven kinds of base Baijius are produced. The quality of base Baijiu in the third round is significantly better than that in the first and second rounds, but the mystery behind the phenomenon has not yet been revealed. Based on high-throughput sequencing and flavor analysis of fermented grains, and correlation analysis, the concentrations of flavor chemicals in the third round of fermented grains were enhanced, including esters hexanoic acid, ethyl ester; octanoic acid, ethyl ester; decanoic acid, ethyl ester; dodecanoic acid, ethyl ester; phenylacetic acid, ethyl ester; 3-(methylthio)-propionic acid ethyl ester; acetic acid, phenylethyl ester; hexanoic acid, butyl ester, and other flavor chemicals closely related to the flavor of sauce-flavor Baijiu, such as tetramethylpyrazine. The changes in flavor chemicals should be an important reason for the quality improvement of the third round of base Baijiu. Correlation analysis showed that ester synthesis was promoted by the bacteria genus Lactobacillus and many low abundances of fungal genera, and these low abundances of fungal genera also had important contributions to the production of tetramethylpyrazine. Meanwhile, the degrading metabolic pathway of tetramethylpyrazine was investigated, and the possible microorganisms were correlated. These results clarified the base Baijiu quality improvement of the third round and helped to provide a theoretical basis for improving base Baijiu quality.

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.

Reducing the background interference of liquid-liquid extraction method during Baijiu aroma analysis.

Liquid-liquid extraction coupled with gas chromatography-mass spectrometry (LLE-GC/MS) is a useful methodology for Baijiu aroma analysis. However, the background problems caused by LLE have rarely been investigated. In the present study, synthetic Baijiu was analyzed by traditional LLE-GC/MS to assess background problems, and a series of ghost peaks of some compounds were detected within the retention time of 47.0-60.0 min, including 2,4-di-tert-butylphenol (2,4-DTBP), palmitic acid and oleanitrile. 2,4-DTBP, which has a phenolic odor, is an important component in both strong and light-flavor Baijiu. Single-factor experiments confirmed that 2,4-DTBP is not a constituent of Baijiu but migrates from sodium sulphate and sodium chloride, and, high-temperature baking was an effective approach to eliminate these compounds. A combined strategy using standard cleaning coupled with high-temperature baking was proposed to reduce the background problems during LLE. Furthermore, more trace compounds could be identified through this process in the future.

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.

Recent trends in aroma release and perception during food oral processing: A review.

The dynamic and complex peculiarities of the oral environment present several challenges for controlling the aroma release during food consumption. They also pose higher requirements for designing food with better sensory quality. This requires a comprehensive understanding of the basic rules of aroma transmission and aroma perception during food oral processing and its behind mechanism. This review summarized the latest developments in aroma release from food to retronasal cavity, aroma release and delivery influencing factors, aroma perception mechanisms. The individual variance is the most important factor affecting aroma release and perception. Therefore, the intelligent chewing simulator is the key to establish a standard analytical method. The key odorants perceived from the retronasal cavity should be given more attention during food oral processing. Identification of the olfactory receptor activated by specific odorants and its binding mechanisms are still the bottleneck. Electrophysiology and image technology are the new noninvasive technologies in elucidating the brain signals among multisensory, which can fill the gap between aroma perception and other senses. Moreover, it is necessary to develop a new approach to integrate the relationship among aroma binding parameters, aroma concentration, aroma attributes and cross-modal reactions to make the aroma prediction model more accurate.

Insights into a new alternative method with graphene oxide/polyacrylamide/Fe3O4 nanocomposite for the extraction of six odor-active esters from Strong-aroma types of Baijiu.

Liquid-liquid extraction (LLE) is the most commonly utilized technique for the extraction of odor-active esters (OAEs) in strong-aroma types of Baijiu (SAB). However, since the contents of different OAEs in SAB vary widely, it is still a puzzle to ensure that all OAEs to be thoroughly extracted by LLE without the problem of saturated adsorption. Herein, a novel approach of magnetic solid phase extraction (MSPE), based on the magnetic graphene oxide nanocomposite modified with polyacrylamide (GO/PAM/Fe3O4), was employed for the efficient extraction of six OAEs from SAB. Compared with LLE, GO/PAM/Fe3O4 exhibited highly selective recognition properties and larger adsorption capacities for OAEs (ranging from 13.68 to 39.06 mg/g), resulting in better extraction performances for OAEs. Coupled with GC-MS, six OAEs in real SAB were successfully determined, with recoveries ranged from 70.1 âˆ¼ 90.0% and LODs at 0.08 âˆ¼ 1.35 µg/L. Overall, the MSPE-GC/MS is a promising alternative for accurate determination of OAEs in SAB.

Clostridium btbubcensis BJN0001, a potentially new species isolated from the cellar mud of Chinese strong-flavor baijiu, produces ethanol, acetic acid and butyric acid from glucose.

A novel strain, designated BJN0001, was isolated from the cellar mud of Chinese strong-flavor baijiu. The complete genome of strain BJN0001 was 2,688,791 bp and annotated with 2610 genes. Whole-genome similarity metrics such as average nucleotide identity (ANI) of BJN0001 with reference genomes reveals clear species boundaries of < 95% ANI value for species. The DNA-DNA hybridization (DDH) values of BJN0001 with the type species were all lower than 70% DDH value for species. Based on these results, the strain BJN0001 was considered a potentially new species of the genus Clostridium. Meanwhile, the fermentation characteristics indicated that the strain had the capability to convert glucose to ethanol, acetic acid and butyric acid, which could provide basic data for revealing its function in baijiu fermentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03271-7.

Synthetic methods of lipid-coated mesoporous silica nanoparticles as drug carriers.

The lipid-coated mesoporous silica nanoparticles (LMSNs) that can synergistically harness the advantages and mitigate the disadvantages of the liposomes and MSNs are considered potential drug carriers. So far, several methods have been developed to prepare LMSNs, including vesicle fusion, thin-film hydration, and solvent exchange. Despite their wide application in LMSN preparation, these methods are short of detailed elaboration and comparison, which hinders their further development. In this review, for the first time, the three methods are systematically summarized, including their mechanisms, influence factors, advantages, and limitations. Although these methods are all based on lipid self-assembly, there is still a difference between them. In order to efficiently prepare LMSNs, we proposed that a suitable method should be selected based on the actual situation. It is conceivable that the elaboration and comparison in this review will make these methods easy to be understood and provide guidance for the design of LMSNs as drug carriers.

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.