Correlation between the bacterial community succession and purine compound changes during Huangjiu fermentation.

Purine is mainly culprit of hyperuricemia (HUA) and gout, which is widely present in Huangjiu in the form of free bases. Bacterial succession plays an important role in quality control in Huangjiu. The correlation between the purine compound content and the bacterial communities during the fermentation process has not yet been evaluated. In this study, high-throughput sequencing (HTS) technology was used to monitor the bacterial community composition of Huangjiu at different fermentation stages. The correlation between the bacterial community and the contents of physicochemical properties and purine compounds were evaluated using the Spearman analysis method. The key enzymes of purine metabolism pathway in the microbial community were analyzed by bioinformatics using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). The results showed that the purine content in Huangjiu increased gradually in 0∼9d of fermentation (21.05-65.71 mg/L), and stabilized gradually in 12∼18d (65.63-69.55 mg/L), while the abundance of lactic acid bacteria (LAB) of bacterial microbial flora were increased (0∼9d) and then stabilized (12∼18d). Moreover, Lactobacillus acetotolerans and Lactobacillus helveticus were highly correlated positively with purine contents, while Limosilactobacillus fermentum and Lactiplantibacillus plantarum were correlated negatively. In addition, the dominant strains of bacteria were involved in the metabolism of purine, and the key enzymes for purine compound synthesis were more abundant than that for purine degradation. This study is helpful to scientifically understand the formation mechanism of purines, providing a basis for screening functional strains of purine degrading to accurately regulate purine level in Huangjiu.

Quality improvement of soybean meal by yeast fermentation based on the degradation of anti-nutritional factors and accumulation of beneficial metabolites.

BACKGROUND: Soybean meal (SBM) is the main protein source for animal diets but its anti-nutritional constituents affect animal growth and immunity. The yeast culture of soybean meal (SBM-YC) that fermented with yeast and hydrolyzed by protease simultaneously could reduce anti-nutritional factors effectively and accumulate beneficial metabolites. RESULTS: The crude protein and acid-soluble protein content of SBM-YC reached 542.5 g kg-1 and 117.2 g kg-1 , respectively, and the essential amino acid content increased by 17.9%. Raffinose and stachyose decreased over 95.0%, and the organic acid content such as acetic acid, butyric acid, citric acid, lactic acid, succinic acid, and propionic acid produced by fermentation reached 6.1, 3.8, 3.6, 2.5, 1.2, and 0.4 g kg-1 , respectively. As biomarkers of yeast culture, nucleosides and their precursors reached 1.7 g kg-1 ; in particular, the inosine content increased from 0 to 0.3 g kg-1 . The total antioxidant capacity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical activity, metal chelating ability, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability were increased by 50.3%, 46.1%, 43.9%, and 20.6%, respectively. CONCLUSION: This study established a diversified evaluation index, which could lay the foundations for the production and quality control of SBM-YC in the future. © 2023 Society of Chemical Industry.

Potential nutritional and functional matters in yeast culture prepared by soybean meal fermentation.

BACKGROUND: Yeast culture (YC) is a product fermented on a specific medium, which is a type of postbiotic of anaerobic solid-state fermentation. Although YC has positive effects on the animal growth and health, it contains a variety of beneficial metabolites as dark matter, which have not been quantified. In the present study, liquid chromatography-tandem mass spectrometry is employed to identify the unknown metabolites. Following their identification, the important chemicals are quantified using HPLC-diode array detection methods. RESULTS: Non-targeted metabolomics studies showed that 670 metabolites in total were identified in YC, of which 23 metabolites significantly increased, including organic acids, amino acids, nucleosides and purines, isoflavones, and other substances. The chemical quantitative analysis showed that the contents of succinic acid, aminobutyric acid, glutamine, purine and daidzein increased by 84.42%, 51.07%, 100%, 68.85% and 4.60%, respectively. CONCLUSION: Therefore, the use of non-targeted metabolomics combined with chemical quantitative analysis to reveal the nutritional and functional substances of YC could help to elucidate the postbiotic mechanism and provide theoretical support for the regulation of the directional accumulation of beneficial metabolites. © 2024 Society of Chemical Industry.

Development of a novel SRAP-SCAR marker for rapid identification of lager and ale types in brewer's yeast.

BACKGROUND: Beer is a globally consumed and universally popular beverage. According to the fermentation conditions of brewer's yeast, ale yeast and lager yeast are the two major varieties. Normal phenotypic and genotypic approaches are insufficient and time-consuming for identifying these two forms of yeast. Therefore, a method for the rapid and cost-effective identification of lager and ale-type brewer's yeasts is necessary. METHODS AND RESULTS: In this study, we analysed the genetic diversity of 23 industrial brewer's yeasts from around the world using sequence-related amplified polymorphism (SRAP) markers and produced stable sequence characteristic amplification region (SCAR) markers. The specific DNA fragments identified by the SRAP marker were sequenced and primers were constructed; the resultant SCAR marker (757 bp) was then confirmed against the indicated brewer's yeast type. CONCLUSION: The development of SRAP-SCAR marker is more economical, simple, and fast compared to morphological markers.

Evaluation of genetic diversity and development of core collections of industrial brewing yeast using ISSR markers.

Germplasm of industrial brewing yeast of the worldwide have a richer diversity, and various successes in improving the performance of brewing yeasts. However, they are limited in that they have relatively low odds of combining desirable traits in a correct manner. To improve germplasm resource preservation, management, and utilization efficiency. In this study, the genetic diversity of 35 industrial brewing yeasts were analyzed based upon inter simple sequence repeat (ISSR) markers, in which 151 out of 167 SSR loci (90.42%) were polymorphic between two or more strains. Three preliminary core collections were established using ISSR data, and based on three different strategies as follows: an advanced maximization (M) strategy, an allele preferred sampling (A) strategy, and a random sampling (R) strategy. Comparison of genetic parameters, including polymorphic information content, Nei's genetic diversity (H), effective allele number, observed allele number, Shannon's index (I), and principal coordinate analyses, confirmed that all the core collections accurately recapitulated the diversity of the initial germplasm. Considering the loci retention ratio and trait coverage efficiency, Core1 was considered the best core collection.

The production and application of enzymes related to the quality of fruit wine.

Grape wine is the most widely consumed fruit wine in the world. With the increasing diversification of consumers' needs, the variety of fruit wines in the market is becoming more and more abundant. Whether it is the production of grape wine or other fruit wines these processes are inseparable from the participation of enzymes. The quality of these wines is closely related to the application of enzymes in the winemaking process. Enzymes are involved in pretreatment, fermentation, filtration, flavoring, aging and storage of fruit wines. This review systematically illustrated the role of pectinase, ß-glucanase, ß-glucosidase, glucose oxidase, lysozyme, protease, tannase and urease in the production of wines and their current production status and also provided a theoretical basis for better application of various enzymes in the production of various fruit wines. This knowledge could be great significance to improve the quality of fruit wines and reduce the production costs in the fruit wine industry.

Development of a novel multi-strain wheat Qu with high enzyme activities for Huangjiu fermentation.

BACKGROUND: Wheat Qu has long been used as a fermentation starter to produce Huangjiu. Wheat Qu quality depends on its microbial community structure and the hydrolytic enzymes generated by the micro-organisms. RESULTS: Strain YF1 and YF2 were successfully screened as they exhibited high acidic protease (231.9 ± 1.4 U g-1 ) and cellulase (7.1 ± 0.6 U g-1 ) activities. Based on a morphological and sequence analysis of the internal transcribed spacer (ITS) gene, YF1 and YF2 were identified as Rhizopus oryzae and Aspergillus niger, respectively. Cooked wheat Qu was produced using mixed fungal starter fermentations with Aspergillus oryzae SU-16, YF1, and YF2. For Qu-making, the optimized conditions for fermentation time, water content, and inoculum size were 47.8 h, 69.4%, and 6.1%, respectively. Under these conditions, compared with single-strain cooked wheat Qu, enzyme activities of amylase, acidic protease, and cellulase increased by 27.4%, 657.1%, and 1276.2%, respectively. Short peptides and free amino acids contents increased by 19.6% and 131.8%, respectively. This wheat Qu was used for Huangjiu brewing, and the alcohol content increased by approximately 14.6% because of the increased starch hydrolysis efficiency mainly attributed to its high enzyme activity. CONCLUSION: Using mixed fungal strains as starter cultures may be an efficient strategy to improve wheat Qu quality, with great potential for application in industrial Huangjiu production. © 2021 Society of Chemical Industry.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Cloning and expression of ferulic acid esterase gene and its effect on wort filterability.

OBJECTIVES: To optimize the expression of type A ferulic acid esterase (FaeA) from Aspergillus niger in Pichia pastoris X-33 using codon optimization. RESULTS: Recombinant FaeA was purified from the fermentation broth, with the maximum specific activity of 48.4 ± 0.1 U mg-1. Adding it during mashing process for beer brewing raised the filtration rate by 14.5% while the turbidity and viscosity declined by 22 and 6.9%, respectively. Addition of FaeA increased the concentrations of free ferulic acid (FA) and arabinoxylan (AX) in the wort, while the polymeric arabinoxylans content declined significantly. CONCLUSIONS: Recombinant FaeA was capable to prevent the oxidative gelation of PAX formation by breaking the cross-linking of FA among AX chains and improve the filtration performance of wort.

Microbial community dynamics of Dan'er barley grain during the industrial malting process.

The microbes associated with barley grains are known to have either deleterious or beneficial effects on the malting process, thereby affecting the quality of malt and even beer. Dan'er barley is the major cultivar grown in Jiangsu Province in China, malt derived from this cultivar, however, invariably causes filtration problems. To understand the microbial community of Dan'er barley and its probable effects on malt quality and subsequent brewing process, the present study combined culture-dependent methods and culture-independent PCR denaturing gradient gel electrophoresis (PCR-DGGE) strategies. The results showed that as the dominant microbes, bacterial community structure varied considerably during the malting process, while the fungi load increased at the end of malting process, even in the kilning stage. The great abundance of gram-negative bacteria, which can produce exopolysaccharides and form biofilms, may be the main reason for the filtration defects of Dan'er malt. Pathogenic fungi may also have a negative impact on grain germination and malt nutritional value. Several beneficial lactic acid bacteria and fungi strains were also found. These results may contribute to our knowledge of barley grain-associated microbes, and help optimizing the malting process using the Dan'er barley cultivar to get malt product with better quality.

Characterization of the aroma compounds in crystal malt.

Crystal malt, the most popular type of specialty malt used in beer brewing, plays a vital role in forming complex flavor and color. Nevertheless, crystal malt is only defined based on the malting process, and there is not any standard to evaluate its quality. In the current study, the volatile aroma constituents of commercial crystal malt samples were analyzed with headspace solid-phase microextraction combined with gas chromatography-mass spectrometry, in order to explore the characteristic aroma compounds of crystal malt. The average concentration of volatile aroma compounds in 10 crystal malt samples is 587 µg L-1 , ranging from 347 to 1265 µg L-1 . A total of 38 aroma compounds were identified, 47% of which were existed in all the 10 samples. Based on principal component analysis and odor activity value, isobutyraldehyde, 2-methylbutanal, furfural, 2-acetyl-1H-pyrrole, oct-1-en-3-ol, 4-methyl-2-phenyl-2-pentenal, and (2E)-2-isopropyl-5-methyl-2-hexenal could be considered the characteristic aroma compounds of crystal malt. The results of this present study would help to establish a standard to assess the quality traits of crystal malt sample.

Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues.

There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.

The antifungal substance from Bacillus velezensis B-3 improved the malt quality against premature yeast flocculation induced by Fusarium graminearum infection.

Malt produced from barley contaminated with Fusarium graminearum can cause premature yeast flocculation (PYF) in beer fermentation, which ultimately affects the production efficiency and flavor quality of beer. In this study, a strain of Bacillus velezensis B-3 that was isolated from soil displayed strong antifungal activity against F. graminearum. The antifungal substances were extracted and separated by ammonium sulfate fractional precipitation and semipreparative high-performance liquid chromatography to obtain fraction VI with the strongest antifungal activity. Ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry was used to identify that the fraction contained lipopeptides such as iturin A, surfactant B, and surfactant C. The minimum inhibitory concentration of the fraction was 0.2 mg/mL, and confocal laser scanning microscopy showed that the morphology of F. graminearum hyphae was obviously abnormal, with most of them were dead. Antifungal fraction VI could inhibit the growth of F. graminearum and avoid the production of PYF factor during malting but did not affect other qualities of malt. Therefore, the development of antimicrobial agents against Fusarium provides an effective potential strategy to control the production of PYF factors for malt-manufacturing enterprises. PRACTICAL APPLICATION: Antifungal fraction VI could inhibit the growth of F. graminearum and the formation of DON during malting but did not affect other qualities of malt. The levels of soluble nitrogen, free amino nitrogen, and color experienced a substantial reduction compared to the malt infected by F. graminearum. The PYF phenomenon was significantly improved, the number of suspended yeast cells and the degree of fermentation were increased, and the level of residual extract in the wort was low, close to that of control malt.

Structure, antioxidant properties, and protective effects on DNA damage of exopolysaccharides from Clostridium butyricum.

Exopolysaccharides (EPSs) of probiotics are naturally nontoxic antioxidants with some interesting biological activities. This research aims to explore the structural and antioxidant properties of the EPS from Clostridium butyricum, a probiotics widely existed in human and animal intestines. EPS of C. butyricum RO-07 was purified through a combination of anion-exchange column chromatography and gel chromatography and determined to be composed of glucosamine, arabinose, galactosamine, galactose, glucose, and xylose in a molar ratio of 1:1:1:2:1:1 with a molecular weight 1.23 × 104  Da. It exhibited a stronger antioxidant activity than ascorbic acid, with scavenging activities up to 75.2% and 95.0% against hydroxyl radical (•OH) and superoxide radical (O2 - •), respectively. It also performed protective effects on DNA against radiation destruction by ultraviolet and reactive oxygen species generated oxidation stress. With these superior advantages in oxidants and radiation resistance, the EPS from C. butyricum RO-07 therefore has great potential to be applied in food and cosmetic industry.

Barley Husk Degraded by Fusarium graminearum MH1 Induced Premature Yeast Flocculation.

Premature yeast flocculation (PYF) is one of the pivotal problems affecting beer flavor and production. PYF is induced by certain non-starch polysaccharides produced by the degradation of malted barley husks upon the growth of contaminated microorganisms, such as Fusarium graminearum. In this research, the formation mechanism of PYF was uncovered by investigating the secretome of F. graminearum MH1 inoculated to the barley husk. The polysaccharide extract of degraded husk was ultrafiltrated into four fractions and characterized by the minimum PYF concentration, molecular mass distribution, monosaccharide composition, and zeta potential. Among the four fractions, the high-molecular-weight polysaccharide fraction had the highest content of uronic acid and the most negative zeta potential, which contributed to the most severe PYF phenomenon. In addition, the PYF yeast showed a more negative zeta potential than the control yeast during the small-scale brewing process. This is aligned to the negatively charged polysaccharides potentially bonded to the surface of yeast cells through the calcium cation in the same fermentation system, which results in rapid flocculation and precipitation. Approximately 12% of the 214 proteins identified in the Fusarium graminearum MH1 secretome were hemicellulases, which substantially interpreted the mechanism of polysaccharides inducing PYF yeast during beer brewing.

Enhancement Effect of Chitosan Coating on Inhibition of Deoxynivalenol Accumulation by Litsea cubeba Essential Oil Emulsion during Malting.

The purpose of this work was to study the enhancement effect of chitosan coating on inhibition of deoxynivalenol (DON) accumulation by Litsea cubeba essential oil emulsion during malting. Firstly, the primary emulsion suitable for malting process was screened and the improvement effect of chitosan coating on the properties of primary emulsion was studied. On this basis, chitosan-based Litsea cubeba essential oil emulsion was applied to malting processing. The results showed that the primary emulsion of Litsea cubeba essential oil had good antifungal properties and a minimal effect on the germinability of barley compared with other primary emulsions. The addition of chitosan can improve the physical stability and antifungal ability of the emulsion and reduce the effect of the emulsion on barley germination. When 100 g of chitosan-based Litsea cubeba essential oil emulsion (40 mg/g) was applied to the malting process, the germination rate of barley was 87.7% and the DON concentration of finished malt was reduced to 690 µg/kg, which was 20.9% lower than that of the control. Meanwhile, the other indexes of malt produced by secondary emulsion treatment (after adding chitosan) increased significantly compared with those of malt produced by primary emulsion. This study was of great significance for the application of emulsion to inhibit the accumulation of mycotoxin during malting.

Key Compounds and Metabolic Pathway Responsible for the Browning in Dangshan Pear (Pyrus spp.) Wine.

Based on hydrophilic interaction liquid chromatography (HILIC) liquid chromatography-mass spectrometry (LC-MS), untargeted differential metabolomics analysis was performed on the pear wine samples before and after browning to determine the key compounds that affect the browning of Dangshan pear wine. A total of 196 significantly differential metabolites were found, 22 of which might be related to the browning of Dangshan pear wine. d-(+)-glucose, l-phenylalanine, l-norleucine, methionine, d-(+)-proline, aloin, and rutin were the key differential metabolites in pear wine before and after browning. The Maillard reaction of d-(+)-glucose, l-norleucine, methionine, and the oxidation of aloin played critical roles in the browning of Dangshan pear wine. The reaction of aloin and glucose to form 5-hydroxyaloin A, 7-hydroxyaloin B, and elgonica-dimer A was one of the important metabolic pathways in which the phenolic compounds formed anthraquinone during the browning process of Dangshan pear wine.

Profiling the key metabolites produced during the modern brewing process of Chinese rice wine.

The study quantitatively profiled 83 low-molecular-weight metabolites in the categories of alcohols, aldehydes, amino acids, esters, fatty acids, organic acids, and reducing sugars produced during the advanced brewing process of Chinese rice wine, using multiply chromatography and mass spectrum. In the primary fermentation, vigorous metabolisms were demonstrated by the production of ethanol at the level of 14% by volume, and the consumption of reducing sugars from the maximum level of 100 g/L to 20 g/L. Meantime, more than 70% of the contents of organic acids, fatty acids, higher alcohols, and aldehydes were formed. The metabolisms slowed down in the secondary fermentation, whereas 60% of the contents of amino acids and esters were accumulated in this stage. The nutrients, such as amino acids, organic acids, and reducing sugars reached 10 g/L, 5 g/L, and 3 g/L at the end of brewing, respectively. In terms of flavor and taste attributes to the brewed rice wine, the organoleptic activity value (i.e. the ratio of content to threshold value) was above 1 for 17 compounds, including six organic acids, namely acetic acid, citric acid, lactic acid, malic acid, succinic acid, and tartaric acid, four amino acids, namely cysteine, aspartic acid, glutamic acid, and lysine, three higher alcohols namely isoamyl alcohol, isobutanol, and phenethyl alcohol, three esters, namely ethyl acetate, ethyl butyrate, and ethyl hexanoate, and an aldehyde, namely benzaldehyde.

Effect of surfactants on the production of polysaccharides from Schizophyllum commune through submerged fermentation.

Schizophyllum commune (S. commune) polysaccharides are biomacromolecules with multiple biological activities and wide applications. In this study, polysaccharide production through submerged fermentation of S. commune using different surfactants was investigated. The addition of 1 g/L of polyoxyethylene sorbitan monooleate (Tween 80) at the beginning of the fermentation showed the best promotional effects on collective exopolysaccharide (EPS) production (which increased by 37.17%) while shortening the production cycle by 2 days. The monosaccharide composition of the EPS produced when the added Tween 80 was similar to that of the control; however, the molecular weight (Mw) was lower. Notably, the addition of Tween 80 significantly increased the ATP levels and the transcription levels of phosphoglucomutase and ß-glucan synthase genes in the polysaccharide synthesis pathway. The addition of Tween 80 reduced the pellet size of the mycelium compared to that of the control, but did not significantly change the microstructure of the mycelial cells. This study proposes an efficient strategy for the production of polysaccharides through submerged fermentation of S. commune, and elucidates the detailed mechanism of using Tween 80 as a fermentation stimulatory reagent.

Levan from Bacillus amyloliquefaciens JN4 acts as a prebiotic for enhancing the intestinal adhesion capacity of Lactobacillus reuteri JN101.

Improving intrinsic adhesion performance of the known probiotics facilitates their residence and colonization, and therefore exerts more beneficial effects on the human or animal host. In this study, through adaptive culture with levan, Lactobacillus reuteri JN101 achieved the same biomass and exhibited 2.6 times higher adhesion capacity to HT-29 cells than those grown with glucose. The mechanism study related to this adhesion enhancement showed that the elevated proportion of unsaturated fatty acids facilitated the bacterial cells to overcome repulsive forces to approach the intestinal epithelial cell. At the same time, and the greater amounts of cell membrane proteins, such as S-layer protein (3.2 folds), elongation factor Tu (2.6 folds) and phosphoglycerate kinase (2.4 folds) probably enhanced the complementary interactions to the receptor on the epithelial cell. These results presented here indicated levan could be used as a potential prebiotic to regulate the adhesion capacity of probiotics, and provide ground for developing the specific-probiotics oriented functional food.