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.

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.

Enhancing citric acid tolerance of Acetobacter tropicalis using chemical and physical mutagenesis and adaptive evolution to improve the quality of lemon fruit vinegar.

The high concentration of citric acid in lemons limits the production of lemon fruit vinegar because it inhibits the metabolism of acetic acid bacteria and reduces the utilization of raw materials. This study aimed to enhance the citric acid tolerance of Acetobacter tropicalis by using complex mutagenesis and adaptive laboratory evolution (ALE) and improving the quality of lemon fruit vinegar. After mutagenesis and ALE, A. tropicalis JY-135 grew well under 40 g/L citric acid, and it showed high physiological activity and excellent fermentation performance under high concentrations of citric acid. The survival rate and ATP content of JY-135 were 15.27 and 9.30 times higher than that of the original strain J-2736. In the fermentation of lemon fruit vinegar, the acid production and the number of aroma-active compounds were 1.61-fold and 2.17-fold than J-2736. In addition, we found that citric acid tolerance of JY-135 is related to the respiratory electron-transport chain and the tricarboxylic acid (TCA) cycle. This work is of great significance for the production of high-quality lemon fruit vinegar and the enrichment of seed resources of acetic acid bacteria.

Inhibition mechanism of fusarium graminearum growth by g-C3N4 homojunction and its application in barley malting.

The increase of deoxynivalenol (DON) caused by Fusarium graminearum (F. graminearum) during the malting process is a serious safety problem. In our work, the inhibition mechanism of F. graminearum growth by g-C3N4 homojunction and its application in barley malting were studied. The reason why the growth activity of F. graminearum decreased after photocatalysis by g-C3N4 homojunction was that under visible light irradiation, a large amount of •O2- elicited by g-C3N4 homojunction destroyed the cell structure of F. graminearum, leading to the deficiency of cell membrane selective permeability and serious disorder of intracellular metabolism. The application of photocatalysis technology in malting can effectively inhibit the growth of F. graminearum and the accumulation of ergosterol was reduced by 30.55 %, thus reducing the DON content in finished malt by 31.82 %. Meanwhile, the physicochemical indexes of barley malt after photocatalytic treatment still met the requirements of second class barley malt in Chinese light industry standard QB/T 1686-2008. Our work provides a new idea for the control of fungal contamination in barley malt.

Evaluation of the differences between low-salt solid-state fermented soy sauce and high-salt diluted-state fermented soy sauce in China: from taste-active compounds and aroma-active compounds to sensory characteristics.

BACKGROUND: The present study aimed to determine the components related to sensory properties in soy sauce and to characterize the differences between low-salt solid-state fermented soy sauce (LSFSS) and high-salt diluted-state fermented soy sauce (HDFSS). The taste and aroma active components of 18 commercially available soy sauces (eight types of LSFSS and 10 types of HDFSS) were characterized. The relationship between these compounds, soy sauce samples, and sensory properties was modeled by partial least squares regression. RESULTS: The analysis showed that the 11 taste-active components, including glutamic acid, glycine, alanine, threonine, malic acid, citric acid, tartaric acid, acetic acid, lactic acid, reducing sugar and salt, contributed greatly to the taste of soy sauce. In addition, umami, saltiness and sweetness are the characteristic tastes of HDFSS, whereas sourness and bitterness were the characteristic tastes of LSFSS. At the same time, seven aroma-active compounds, namely 4-ethyl-2-methoxyphenol, ethanol, 3-methyl-1-butanol, ethyl acetate, 2-phenethyl alcohol, 3-methyl thiopropanol and 2-ethyl-4-hydroxy-5-methylfuran-3-one, played a decisive role in the flavor of soy sauce. In addition, HDFSS presented the aroma attributes of smoky, alcoholic, floral, fruity and caramel-like, whereas LSFSS mainly presented sour and malty aroma attributes. CONCLUSION: The present study reveals new insight into the relationship between the chemical composition and sensory characteristics of soy sauce, which is of great significance for developing an objective measurement system and providing a theoretical basis to improve the sensory quality of soy sauce. © 2023 Society of Chemical Industry.

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.

Screening and application of purine degrading Limosilactobacillus fermentum LF-1 from Huangjiu fermentation broth.

BACKGROUND: As the important building blocks of nucleic acids, purines are alkaloids and responsible for hyperuricemia and gout. The purine content in Huangjiu is higher, and mainly exists in the form of free bases, which is easier to be absorbed by human body. However, the currently available reports on purine in Huangjiu mainly focus on detection methods and content survey. No studies on the regulation of the purine content in Huangjiu have been reported. RESULTS: Eighty-four strains, with the degradation capacity of purine, were screened from the fermentation broth of Huangjiu. In detail, the isolated lactic acid bacteria (LAB) strain 75 # showed the strongest degradation ability of guanosine, inosine and four purines, which reduce their levels by 83.4% (guanosine), 97.4% (inosine), 95.1% (adenine), 95.0% (guanine), 94.9% (hypoxanthine) and 65.9% (xanthine), respectively. Subsequently, the LAB strain 75# was identified to be Limosilactobacillus fermentum by 16S rRNA gene sequencing, which was named as Limosilactobacillus fermentum LF-1 and applied to the fermentation of Huangjiu in the laboratory. Compared with the fermentation broth of Huangjiu without adding L. fermentum LF-1, the content of purine compounds in the fermentation broth inoculated with L. fermentum LF-1 was reduced by 64.7%. In addition, the fermented Huangjiu had richer flavor compounds, and the physicochemical indices were in accordance with the national standard of Chinese Huangjiu. CONCLUSION: The screened strain L. fermentum LF-1 may be a promising probiotic for the development of a novel that can efficiently degrade purine in Huangjiu. © 2023 Society of Chemical Industry.

A highly-sensitive sensor based on carbon nanohorns@reduced graphene oxide coated by gold platinum core-shell nanoparticles for electrochemical detection of carbendazim in fruit and vegetable juice.

Carbendazim (CBZ) is beneficial to fruit and vegetable cultivation, but its residue will cause fruit and vegetable juice pollution. In this work, an electrochemical sensor based on carbon nanohorns@reduced graphene oxide coated by gold platinum core-shell nanoparticles (Au@Pt/CNHs@RGO/GCE) was prepared for CBZ detection. The results showed that the assembly of CNHs and RGO assisted by ultrasound improved the electron transfer ability and electrochemical active surface area of CNHs@RGO. Moreover, the coating of Au@Pt nanoparticles further enhanced the sensitivity of the sensor. With the synergistic effect of the three materials, the sensor had a wider linear range (0.05 µmol/L-50 µmol/L), a lower limit of detection (1.64 nmol/L), and satisfactory recovery rates (90.60 % ∼ 97.60 %, carrot juice; 94.00-114.43 %, orange juice). Additionally, the sensor presented good anti-interference and repeatability. This work provides a simple, rapid, economical, sensitive, and accurate sensor for CBZ quantification in fruit and vegetable juice.

Evaluation of the antioxidant characteristics of craft beer with green tea.

The addition of green tea as antioxidants to beer can improve the beer's flavor stability by protecting against staling during storage. To analyze the effect of different green teas on the flavor stability of beer, we developed an approach to rapidly evaluate their antioxidant activity. Ten types of craft beer were produced by adding different kinds of green tea during brewing, and their antioxidant activity and phenolic profiles were evaluated. The results showed remarkable variations in antioxidant activity and antioxidative compound contents, which were considerably higher in green tea beers than in non-tea beer (p < 0.05). A comprehensive evaluation function was developed to evaluate the total antioxidant activity of beers using principal component analysis. The highest total antioxidant activity was observed in Taiping Houkui beer, with a comprehensive evaluation score of 2.53. Pearson correlation analysis suggested that (-)-epigallocatechin gallate, (-)-epicatechin gallate, and (-)-epigallocatechin were strongly correlated with the total antioxidant activity of green tea beers (p < 0.01). The summation of their contents represented more than 60% of the total phenolic content of the teas, which can be used to predict the total antioxidant activity of green tea beers. PRACTICAL APPLICATION: Flavor stability is of prime concern for brewers, and flavor aging is increasingly becoming the limiting factor in beer shelf life. The application of green tea as antioxidants in beer can improve the flavor stability by protecting against beer staling during storage. The analytical method developed in this study will contribute to the rapid comparison of the effect of different green teas on the flavor stability of beer. Furthermore, the research findings demonstrate the potential benefits of green teas to beer flavor stability, which is of considerable importance in promoting the development and consumption of green teas.

Inhibition of Fusarium graminearum growth and deoxynivalenol accumulation in barley malt by protonated g-C3N4/oxygen-doped g-C3N4 homojunction.

Barley malt, the main raw material for beer production, is at risk of Fusarium graminearum (F. graminearum) infection, leading to the possible production of large amounts of deoxynivalenol (DON) in malt. DON in malt can migrate into the final beer product, posing a food safety risk to consumers. In our work, a protonated g-C3N4/oxygen-doped g-C3N4 (CNH/OCN) composite was prepared and used for the inhibition of F. graminearum growth and DON accumulation in barley malt under visible light irradiation. The results showed that the inhibition rate of F. graminearum reached 100 % after 2.5 h of visible light irradiation, and the inhibition effect was still stable after 3 rounds of reuse. The possible pathway of inhibiting F. graminearum spores was that the photogenerated carriers in the CNH/OCN composite transferred in the form of a type II homojunction under visible light and stimulated O2 in the catalytic system to produce a large amount of O2- to kill spores. Compared with the untreated malt, the photocatalytic inhibition rates of the CNH/OCN composite material for ergosterol and DON in malt reached 73.33 % and 67.25 %, respectively. Although photocatalysis had a certain effect on the physicochemical indices of malt, the malt after photocatalysis still met the first-grade standard of Chinese industry standard QB/T 1686-2008.

Highly sensitive electrochemical detection of carbendazim residues in water by synergistic enhancement of nitrogen-doped carbon nanohorns and polyethyleneimine modified carbon nanotubes.

Carbendazim (CBZ) can protect crops from pathogens, but it is also easy to cause pesticide residues, threatening human health. In our work, an electrochemical sensor based on nitrogen-doped carbon nanohorns (N-CNHs) and polyethyleneimine-modified carbon nanotubes (PEI-CNTs) was developed for the detection of CBZ content in water. The results showed that N-doping provided the CN bonds for CNHs and improved the electrochemical reaction performance of N-CNHs surface. With the participation of PEI, the surface of CNTs was positively charged and contained a large number of NH bonds, which not only promoted the electrostatic assembly of N-CNHs and PEI-CNTs but also was beneficial to further enriching CBZ. After further ultrasound-assisted assembly of N-CNHs and PEI-CNTs, the electron transfer capacity, electrochemical active surface area, and catalytic activity of N-CNHs/PEI-CNTs were significantly improved. The sensor performed a wider linear range (15 nmol/L ~ 70 µmol/L), low detection limit (4 nmol/L) and satisfactory recovery (87.33 % ~ 117.67 %) under the optimal conditions. In addition, the sensor had good anti-interference, reproducibility, and stability. Our work provided a new strategy for quantification of CBZ in environment.

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.

Isolation and identification of the bitter compound from Huangjiu.

The strategy of taste-guided assisted by solvent extraction, solid-phase extraction and semipreparative HPLC were applied to isolate the main nonvolatile bitter components from mechanized Huangjiu. The potential fraction was identified by amino acid analysis and ultra-performance liquid chromatography-quadrupole-time-of-flight-MS/MS. Bitter pyroglutamate peptide Pyr-LFNPSTNPWHSP (PGP) was successfully identified from Huangjiu for the first time. Quantitative analysis showed that PGP contents ranged from below the limit of quantitation to 32.97 mg/L, among mechanized Huangjiu had higher contents than manual and commercial Huangjiu. The formation of PGP mainly occurred in the primary fermentation and it was stable in Huangjiu. Moreover, the PGP content of the Huangjiu brewed using raw wheat Qu was 112.6% higher than that using cooked wheat Qu, but presented subtle change with the increase of raw wheat Qu. The results revealed that PGP contributed the bitterness to Huangjiu, which may offer a possibility to reduce the bitterness of Huangjiu.

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.

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.

Objective measures of greengage wine quality: From taste-active compound and aroma-active compound to sensory profiles.

This study is sought to identify the components in greengage wine that predict the sensory properties. Taste-active compounds and aroma-active compounds of 20 commercially available greengage wines from different regions were characterized. The relationship between these compounds, wine samples and sensory attributes was modeled by partial least squares regression. The regression analysis indicated the taste-active compounds, alanine, leucine, proline, glutamic acid, lysine, malic acid, citric acid, sucrose, glucose, gallic acid, caffeic acid and tannin made a great contribution to the characteristic taste or mouthfeel of greengage wine. Meanwhile, the aroma-active compounds, including ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, 3-methylbutanol, 5-hydroxymethylfurfural, octanoic acid and benzaldehyde, modeled well with the flavor characteristic of greengage wine. The study revealed new insights into the relationship between chemistry and wine sensory characters, which has implications for developing an objective measurement system for determining greengage wine quality.

Uncovering mechanisms of greengage wine fermentation against acidic stress via genomic, transcriptomic, and metabolic analyses of Saccharomyces cerevisiae.

Acid stress is one of the most common adverse conditions during fermentation of fruit wines, and the acid tolerance of yeasts is, therefore, critical for fruit wine production. However, the biological mechanism underlying the acquired tolerance of yeasts against acid stress is poorly understood. We have previously obtained an evolved Saccharomyces cerevisiae strain ET008-c54 with increased tolerance against acid stress, and potentially, it serves as a promising yeast strain for greengage wine fermentation. In the current study, we further revealed the alterations responsible for the adaptation of ET008-c54 to low pH by whole-genome re-sequencing, transcriptomic, and metabolic analyses. Results confirmed the outstanding fermenting performance of ET008-c54 at low pH as compared with the parental ET008. More specifically, the growth rate of ET008-c54 at low pH was increased by 6.24 times and the fermentation time was shortened by 70%. Differences were also observed in the physiology of the strains through ergosterol, H+-ATPase activity, and aroma determinations. By integrating both RNA-seq and whole-genome re-sequencing data, we demonstrated some metabolic pathways in ET008-c54, namely ergosterol synthesis and ferrous iron uptake, in which several acid-responsive genes were involved being upregulated. Also, upregulation of the pathways responsible for aroma compound formation, including fatty acid ethyl ester synthesis and aromatic amino acid biosynthesis, was identified. Thus, the enhanced fermentation ability of ET008-c54 at low pH should be, at least partly, contributed by the altered gene expressions associated with the aforementioned pathways. By elucidating the biological mechanism of yeasts against acid stress, this current study allows better-defined targets for future studies of genetic improvement of wine yeasts and enhancement of the fermentation processes. KEY POINTS: • Metabolic analysis confirmed the excellent fermentation performance of ET008-c54. • Acid tolerance genes for ergosterol synthesis and ferrous iron uptake were upregulated. • Aroma genes for fatty acid ethyl ester and aromatic amino acid synthesis were upregulated.

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.

A multiple-step strategy for screening Saccharomyces cerevisiae strains with improved acid tolerance and aroma profiles.

Acid tolerance and aroma profile are crucial factors for wine production in Saccharomyces cerevisiae. However, most wine yeasts to date fail to endure low-pH environments, therefore resulting in problems such as lengthened fermentation and poor flavor during acidic fruit wine production. In the present study, we established a multiple-step screening strategy, which was composed of atmospheric and room temperature plasma (ARTP), high-throughput screening (HTS), and laboratory adaptive evolution (ALE), to screen yeast strains for potential wine-producing with enhanced performances during low pH conditions. Importantly, we obtained the S. cerevisiae strain from the mutant library, ET008-c54, which displayed exhibited excellent performances in survival rate, fermentation time, aroma profile, and genetic stability. More specifically, the survival rate of ET008-c54 at low pH was increased by 10-fold, the fermentation time of greengage plum wine was shortened by about 70%, and the content of main aroma compounds was significantly increased by 52%. Collectively, we demonstrate the practical application of the screening platform designed for discovering mutant strains in winemaking technology.