Revealing the Succession of Spatial Heterogeneity of the Microbial Community during Broad Bean Paste Fermentation.

This study aimed to elaborate the assembly processes and metabolic regulation of the microbial community under the conditions of environmental factors and artificial intervention using broad bean paste (BBP) fermentation as a tractable research object. Spatial heterogenicity of amino acid nitrogen, titratable acidity, and volatile metabolites were observed between upper and lower layers after fermentation for 2 weeks. Amino nitrogen contents in the upper fermented mash reached 0.86, 0.93, and 1.06 g/100 g at 2, 4, and 6 weeks, respectively, which were significantly higher than those of mash located at the lower layer (0.61, 0.79, and 0.78 g/100 g). Moreover, higher concentrations of titratable acidity were accumulated in upper layers (2.05, 2.25 and 2.56 g/100g) than those in lower layers, and the differentiation of volatile metabolites was the greatest (R = 0.543) at 36 days, after which the BBP flavor profiles converged with the fermentation progress. The successive heterogenicity of the microbial community in the mid-late stage was also found during fermentation, and Zygosaccharomyces, Staphylococcus, and Bacillus had heterogeneous characteristics driven by sunlight, water activity, and microbial interactions. This study provided new insights into the mechanisms underlying the succession and assembly of the microbial community of BBP fermentation, which also laid new clues for researches of the microbial communities in complex ecosystems. IMPORTANCE Gaining insights into the community assembly processes is essential and valuable for the elaboration of underlying ecological patterns. However, current studies about microbial community succession in multispecies fermented food usually treat the research object as a whole, are focused exclusively on temporal dimensions, and have ignored the changes of community structure in spatial dimensions. Therefore, dissecting the community assembly process from the view of spatiotemporal dimensions will be a more comprehensive and detailed perspective. Here, we found the heterogenicity of the BBP microbial community under the traditional production technology from spatial and temporal scales, systematically analyzed the relationship between the spatiotemporal succession of community and the difference of BBP quality, and elucidated the roles of environmental factors and microbial interactions to drive the heterogeneous succession of the microbial community. Our findings provide a new insight into understanding the association between microbial community assembly and the quality of BBP.

Transcriptomic and metabolomic analysis reveals genes related to stress tolerance in high gravity brewing.

The fermentation performance of yeast is the key of beer production. High gravity brewing is a commonly used technique in industrial lager beer production and it is environmentally friendly. Therefore, there has been extensive effort toward improving high gravity brewing. In this study, through transcriptomic and metabolomic analysis of two homologous lager yeasts, genes that relate to stress tolerance in high gravity brewing were screened. The results showed EMP pathway and multiple amino acid metabolism pathway were the most enriched pathways, and pyruvate might be the core metabolite. Overexpression and knockdown strains were constructed to verify the genes' functions. The overexpression of MAN2, PCL1 and PFK26 genes were beneficial to fermentation without significantly changes in flavor profiles. The relative intracellular ATP levels can help us understand the change of metabolic flux such as enhancement of sugar consumption. This work is helpful to reveal the stress tolerance mechanism of high gravity brewing and breed yeast strains with improved performance.

Genome comparison of three lager yeasts reveals key genes affecting yeast flocculation during beer fermentation.

Yeast flocculation plays an essential role in industrial application. Appropriate flocculation of yeast cells at the end of fermentation benefits the cell separation in production, which is an important characteristic of lager yeast for beer production. Due to the complex fermentation environment and diverse genetic background of yeast strains, it is difficult to explain the flocculation mechanism and find key genes that affect yeast flocculation during beer brewing. By analyzing the genomic mutation of two natural mutant yeasts with stronger flocculation ability compared to the parental strain, it was found that the mutated genes common in both mutants were enriched in protein processing in endoplasmic reticulum, membrane lipid metabolism and other pathways or biological processes involved in stress responses. Further functional verification of genes revealed that regulation of RIM101 and VPS36 played a role in lager yeast flocculation under the brewing condition. This work provided new clues for improving yeast flocculation in beer brewing.

Screening lager yeast with higher ethyl-acetate production by adaptive laboratory evolution in high concentration of acetic acid.

Ethyl-acetate is important for the flavor and aroma of the alcoholic beverages, therefore, there have been extensive efforts toward increasing its production by engineering yeast strains. In this study, we reported a new approach to breed non-genetic modified producing yeast strain with higher ethyl-acetate production for beer brewing. First, we demonstrated the positive effect of higher acetic acid concentration on inducing the expression of acetyl-CoA synthetase (ACS). Then, we applied adaptive laboratory evolution method to evolve strain with higher expression level of ACS. As a result, we obtained several evolved strains with increased ACS expression level as well as ethyl-acetate production. In 3 L scale fermentation, the optimal strain EA60 synthesized more ethyl-acetate than M14 at the same time point. At the end of fermentation, the ethyl-acetate production in EA60 was 21.4% higher than M14, while the other flavor components except for acetic acid were changed in a moderate degree, indicating this strain had a bright prospect in industrial application. Moreover, this study also indicated that ACS1 played a more important role in increasing the acetic acid tolerance of yeast, while ACS2 contributed to the synthesis of cytosol acetyl-CoA, thereby facilitating the production of ethyl-acetate during fermentation.

Evaluation and prediction of the biogenic amines in Chinese traditional broad bean paste.

Biogenic amines (BAs) are a threat to the safety of broad bean paste, and biosynthetic mechanism of BA and its regulation are unknown. This study aimed to assess microbial BA synthesis in Chinese traditional broad bean paste and determine favorable fermentation conditions for BA regulation. The BAs content in 27 pastes was within the safe range. 64 strains with potential decarboxylation were screened in Luria-Bertani Glycerol medium and identified as Bacillus spp. Although Bacillus amyloliquefaciens produced highest levels of BAs (70.14 ± 2.69 mg/L) in LBAA, Bacillus subtilis produced 6% more BAs than B. amyloliquefaciens. Meanwhile, temperature was the most remarkable factor affecting BAs production by B. amyloliquefaciens 1-13. Furthermore, the fermented broad bean paste model revealed that BA content increased by 61.2 mg/kg every 10 days at 45 °C, which was approximately threefold of that at 25 °C. An ARIMA prediction model of BAs content was constructed, and the total BAs content of 40 mg/100 g was set as the critical value. This study not only contributed to understanding the BAs formation mechanism, but also provided potential measures to control the BAs in fermented soybean products.

A Bottom-Up Approach To Develop a Synthetic Microbial Community Model: Application for Efficient Reduced-Salt Broad Bean Paste Fermentation.

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function.IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

Screening of thermosensitive autolytic mutant brewer's yeast and transcriptomic analysis of heat stress response.

Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast autolysis is most affected by medium composition and temperature. In this study, a thermosensitive autolytic brewer's yeast P-510 was obtained with atmospheric and room temperature plasma mutagenesis plus 5-bromo-chloro-3-indolyl phosphate screening. The mutant rapidly autolyzed at 37 °C and the autolysates contained more active components and showed higher antioxidant activities compared with that of the parental strain, which indicated that the mutant's autolysates can potentially be used as functional food and nutritional ingredients. Transcriptomic analysis of the mutant and parental strains at 28 and 37 °C suggested that thermosensitive autolysis of P-510 was probably caused by mitochondrial disfunction, glycogen metabolic flux of glycolysis and pentose phosphate pathway disorder, as well as hexose transport inhibition. The results revealed the important role of mitochondrial metabolism and glycogen utilization regulation in heat stress response of yeast.

Residue analysis of gibberellic acid isomer (iso-GA3) in brewing process and its toxicity evaluation in mice.

Gibberellic acid (GA3) is an efficient plant growth regulator, which could speed up barley germination in the brewing industry. However, the residue of GA3 in malt gets denatured into an isomer, termed iso-GA3. In this study, the concentration of iso-GA3 and the conversion rate of GA3 to iso-GA3 during the brewing process was studied by high performance liquid chromatography and the potential toxicity of iso-GA3 was evaluated in ICR mice. The concentration of iso-GA3 increased in the saccharification and wort boiling processes while its concentration was stable during fermentation. The maximum conversion rates of GA3 to iso-GA3 in Canadian malt, Australian malt SCO and Australian malt FAQ were 88%, 87% and 87%, respectively. In the acute oral toxicity study, the median lethal dose (LD50) of iso-GA3 was 2.82 g/kg body weight (BW). In the 28-day repeated dose toxicity study, the iso-GA3 could cause weight loss in mice. And the mice of high-dose group showed a slight decrease in food consumption. Moreover, inflammation and cell necrosis were found in kidney and liver tissue, which were alleviated during the recovery phase. These results establish a practical reference for food safety in products, in which GA3 is added as a food additive.

Simultaneous enhancement of barley ß-amylase thermostability and catalytic activity by R115 and T387 residue sites mutation.

OBJECTIVE: To simultaneously increase the thermostability and catalytic activity of barley ß-amylase. METHODS: The amino acid sequences of various barley ß-amylases with different enzyme properties were aligned, two amino acid residues R115 and T387 were identified to be important for barley ß-amylase properties. R115C and T387V were then generated using site-directed and saturation mutagenesis. RESULTS: R115C and T387V mutants increased the enzyme catalytic activity and thermostability, respectively. After combinational mutagenesis, the T50 value and t(1/2,60oC) value of R115C/T387V mutant reached 59.4 °C and 48.8 min, which were 3.6 °C higher and 29.5 min longer than those of wild-type. The kcat/Km value of mutant R115C/T387V were 59.82/s·mM, which were 54.7% higher than that of wild-type. The increased surface hydrophobicity and newly formed strong hydrogen bonds and salt bridges might be responsible for the enzyme thermostability improvement while the two additional hydrogen bonds formed in the active center may lead to the catalytic property enhancement. CONCLUSIONS: The mutant R115C/T387V showed high catalytic activity and thermostability indicating great potential for application in industry.

Reverse metabolic engineering in lager yeast: impact of the NADH/NAD+ ratio on acetaldehyde production during the brewing process.

Acetaldehyde is synthesized by yeast during the main fermentation period of beer production, which causes an unpleasant off-flavor. Therefore, there has been extensive effort toward reducing acetaldehyde to obtain a beer product with better flavor and anti-staling ability. In this study, we discovered that acetaldehyde production in beer brewing is closely related with the intracellular NADH equivalent regulated by the citric acid cycle. However, there was no significant relationship between acetaldehyde production and amino acid metabolism. A reverse engineering strategy to increase the intracellular NADH/NAD+ ratio reduced the final acetaldehyde production level, and vice versa. This work offers new insight into acetaldehyde metabolism and further provides efficient strategies for reducing acetaldehyde production by the regulating the intracellular NADH/NAD+ ratio through cofactor engineering.

Engineering the cytosolic NADH availability in lager yeast to improve the aroma profile of beer.

OBJECTIVE: To improve the aroma profile of beer by using metabolic engineering to increase the availability of cytosolic NADH in lager yeast. RESULTS: To alter NADH levels in lager yeast, the native FDH1 (YOR388C) encoding NAD+-dependent formate dehydrogenase was overexpressed in the yeast strain M14, yielding strain M-FDH1. This led to a simultaneous increase of NADH availability and NADH/NAD+ ratio in the M-FDH1 strain during fermentation. At the end of the main fermentation period, ethanol production by strain M-FDH1 was decreased by 13.2%, while glycerol production was enhanced by 129.4%, compared to the parental strain respectively. The production of esters and fusel alcohols by strains M14 and M-FDH1 was similar. By contrast, strain M-FDH1 generally produced less organic acids and off-flavor components than strain M14, improving the beer aroma. CONCLUSIONS: Increased NADH availability led to rerouting of the carbon flux toward NADH-consuming pathways and accelerated the NADH-dependent reducing reactions in yeast, greatly impacting the formation of aroma compounds and improving the beer aroma.

Process optimization of the extraction condition of ß-amylase from brewer's malt and its application in the maltose syrup production.

ß-Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of ß-amylase from brewer's malt and the optimal dosage of ß-amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of ß-amylase from brewer's malt was composed of 1:17 (g/v) material/liquid ratio, 44°C extraction temperature, pH 6.4 buffer pH, 2.3 H extraction time, and 1.64 g L-1 NaSO3 dosage with a predicted ß-amylase activity of 1,290.99 U g-1 , which was close to the experimental ß-amylase activity of 1,230.22 U g-1 . The optimal dosages of ß-amylase used in maltose syrup production were 455.67 U g-1 starch and its application in maltose syrup production led to a 68.37% maltose content in maltose syrup, which was 11.2% and 28.9% higher than those using ß-amylases from soybean and microbe (P < 0.01). Thus, ß-amylase from brewer's malt was beneficial for production of high maltose syrup.

Rationally designed perturbation factor drives evolution in Saccharomyces cerevisiae for industrial application.

Saccharomyces cerevisiae strains with favorable characteristics are preferred for application in industries. However, the current ability to reprogram a yeast cell on the genome scale is limited due to the complexity of yeast ploids. In this study, a method named genome replication engineering-assisted continuous evolution (GREACE) was proved efficient in engineering S. cerevisiae with different ploids. Through iterative cycles of culture coupled with selection, GREACE could continuously improve the target traits of yeast by accumulating beneficial genetic modification in genome. The application of GREACE greatly improved the tolerance of yeast against acetic acid compared with their parent strain. This method could also be employed to improve yeast aroma profile and the phenotype could be stably inherited to the offspring. Therefore, GREACE method was efficient in S. cerevisiae engineering and it could be further used to evolve yeast with other specific characteristics.

Simultaneous determination of diethylacetal and acetaldehyde during beer fermentation and storage process.

BACKGROUND: Acetaldehyde is an important flavor component in beer which is possibly carcinogenic to humans. Owing to the limitations of present detection methods, only free-state acetaldehyde in beers has been focused on, while acetal in beers has hardly been reported so far. RESULTS: A sensitive headspace gas chromatography method was developed for the determination of diethylacetal and acetaldehyde in beer. The column DB-23 was chosen with a total run time of 22.5 min. The optimal addition amount of NaCl, equilibrium temperature and equilibrium time were 2.0 g, 70 °C and 30 min respectively. For both diethylacetal and acetaldehyde analyses, the limit of detection was 0.005 mg L-1 with relative standard deviation < 5.5%. The recoveries of acetaldehyde and diethylacetal were 95-110 and 95-115% respectively. The diethylacetal and acetaldehyde average contents in 24 beer products were 11.83 and 4.36 mg L-1 respectively. The Pearson correlation coefficient between diethylacetal and acetaldehyde was the highest (0.963). Both diethylacetal and acetaldehyde contents increased to a peak value after fermentation for 3 days and then decreased to a lower value. During both normal and forced aging storage, the diethylacetal content decreased and the acetaldehyde content increased gradually over time. When beers were forced aged for 4 days, the increased ratio of acetaldehyde could be above 40.00%. CONCLUSION: The newly established method can be used to assess acetaldehyde level and flavor quality in beer more scientifically. © 2018 Society of Chemical Industry.

Cell wall polysaccharides: before and after autolysis of brewer's yeast.

Brewer's yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of ß-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.

Rational design of thermostability in bacterial 1,3-1,4-ß-glucanases through spatial compartmentalization of mutational hotspots.

Higher thermostability is required for 1,3-1,4-ß-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial ß-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous ß-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures. The calcium region was predicted to be the crucial region for thermostability of bacterial 1,3-1,4-ß-glucanases, and optimization of four residue sites in this region by iterative saturation mutagenesis greatly increased the thermostability of a mesophilic ß-glucanase (BglT) from Bacillus terquilensis. The E46P/S43E/H205P/S40E mutant showed a 20 °C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature (T m) compared to wild-type BglT. Its half-life values at 60 and 70 °C were 3.86-fold and 7.13-fold higher than those of wild-type BglT. The specific activity of E46P/S43E/H205P/S40E mutant was increased by 64.4 %, while its stability under acidic environment was improved. The rational design strategy used in this study might be applied to improve the thermostability of other industrial enzymes.

Construction of a highly thermostable 1,3-1,4-ß-glucanase by combinational mutagenesis and its potential application in the brewing industry.

OBJECTIVES: To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-ß-glucanase by combinational mutagenesis and to test its effect in congress mashing. RESULTS: A mutant ß-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T 50 value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k cat value were 42,734 U mg-1 and 189 s-1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml-1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively. CONCLUSIONS: The mutant ß-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

Simultaneous enhanced catalytic activity and thermostability of a 1,3-1,4-ß-glucanase from Bacillus amyloliqueformis by chemical modification of lysine residues.

The thermostablility and enzymatic activity of 1,3-1,4-ß-glucanase (BglA) from Bacillus amyloliquefaciens was improved by modifying five (out of 12) ε-amino groups in lysine residues with nitrous acid. The optimal modification condition for BglA was determined as 30 mM nitrous acid at, 40 °C for 30 min. The optimally-modified BglA had higher specific activity and T 50 value, which were 3,370 U/mg and 70 °C, respectively. Its half-life values at 50 and 60 °C were extended and reached 58.5 and 49.5 min, respectively. Circular dichroism analysis showed that the secondary structures in modified BglA were almost the same with that of wild-type BglA. Thus, modification of lysine residues can simultaneously improve the activity and thermostability of ß-glucanase which are ideal targets for further protein engineering.

Enhanced expression of a novel trypsin from Streptomyces fradiae in Komagataella phaffii GS115 through combinational strategies of propeptide engineering and self-degredation sites modification.

This study aimed to enhance the expression level of a novel trypsin gene from Streptomyces fradiae ATCC14544 in Komagataella phaffii GS115 through the combinational use of propeptide engineering and self-degradation residues modification strategies. An artificial propeptide consisted of thioredoxin TrxA, the bovine propeptide DDDDK and the hydrophobic peptide FVEF was introduced to replace the original propeptide while the self-degradation residue sites were predicted and analyzed through alanine screening. The results showed that the quantity and enzymatic activity of asft with engineered propeptide reached 47.02 mg/mL and 33.9 U/mL, which were 9.6 % and 59.29 % higher than those of wild-type (42.9 mg/mL and 13.8 U/mL). Moreover, the introduction of R295A/R315A mutation further enhanced the enzymatic activity (58.86 U/mL) and obviously alleviated the phenomena of self-degradation. The tolerance of trypsin towards alkaline environment was also improved since the optimal pH was shifted from pH 9.0 to pH 9.5 and the half-life value at pH 10 was significantly extended. Finally, the fermentation media composition and condition were optimized and trypsin activity in optimal condition reached 160.58 U/mL, which was 2.73-fold and 11.64-fold of that before optimization or before engineering. The results obtained in this study indicated that the combinational use of propeptide engineering and self-degradation sites modification might have great potential application in production of active trypsins.

Improving ribonucleic acid production in Saccharomyces pastorianus via in silico genome-scale metabolic network model.

Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we used a genome-scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03). Yeast 8.5.0 was used as the base model, which accurately predicted G03's growth. Using OptForce, we found that overexpression of ARO8 and ATP1 among six different strategies increased the RNA content of G03 by 58.0% and 74.8%, respectively. We also identified new metabolic targets for improved RNA production using a modified GSMM called TissueModel, constructed using the GIMME transcriptome constraint tool to remove low-expressed reactions in the model. After running OptKnock, the RNA content of G03-△BNA1 and G03-△PMA1 increased by 44.6% and 39.8%, respectively, compared to G03. We suggest that ATP1, ARO8, BNA1, and PMA1 regulate cell fitness, which affects RNA content. This study is the first to identify strategies for RNA overproduction using GSMM and to report that regulation of ATP1, ARO8, BNA1, and PMA1 can increase RNA content in S. pastorianus. These findings also provide valuable knowledge on model reconstruction for S. pastorianus.