•Salt significantly affected microbial dynamics during doubanjiang fermentation.•Salt reduction increased the concentration of amino acid nitrogen in doubanjiang.•Low salt led to more kinds and ...higher amount of volatile flavor compounds in doubanjiang.•More total acids and biogenic amines were observed in lower salt doubanjiang.•Conditional pathogens were observed in lower salt doubanjiang fermentation.
This study aimed to elaborate the roles of salt concentration on doubanjiang (broad bean paste) fermentation. Three sets of doubanjiang samples which had lower salt concentration than commercial doubanjiang were prepared and the physicochemical parameters, biogenic amines, flavor, microbial dynamics were analyzed during fermentation. The salt reduction showed significant effect on the dynamics of bacteria and fungi, thus leading to doubanjiang samples with different properties. Salt reduction during fermentation relieved the osmotic pressure towards microbes, which favored the accumulation of amino acid nitrogen, amino acids, and volatile flavor compounds. However, higher concentrations of total acids and biogenic amines and the existence of conditional pathogens, such as Klebsiella, Cronobacter and Acinetobacter genera, were observed in salt reduced doubanjiang samples, which was undesirable for doubanjiang quality. This study would deep our understanding of the roles of salt on doubanjiang fermentation.
1,3-1,4-β-glucanase is an important biocatalyst in brewing industry and animal feed industry, while its low thermostability often reduces its application performance. In this study, the ...thermostability of a mesophilic β-glucanase from Bacillus terquilensis was enhanced by rational design and engineering of disulfide bonds in the protein structure. Protein spatial configuration was analyzed to pre-exclude the residues pairs which negatively conflicted with the protein structure and ensure the contact of catalytic center. The changes in protein overall and local flexibility among the wild-type enzyme and the designated mutants were predicted to select the potential disulfide bonds for enhancement of thermostability. Two residue pairs (N31C-T187C and P102C-N125C) were chosen as engineering targets and both of them were proved to significantly enhance the protein thermostability. After combinational mutagenesis, the double mutant N31C-T187C/P102C-N125C showed a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature (Tm) compared to wild-type enzyme. The catalytic property of N31C-T187C/P102C-N125C mutant was similar to that of wild-type enzyme. Interestingly, the optimal pH of double mutant was shifted from pH6.5 to pH6.0, which could also increase its industrial application. By comparison with mutants with single-Cys substitutions, the introduction of disulfide bonds and the induced new hydrogen bonds were proved to result in both local and overall rigidification and should be responsible for the improved thermostability. Therefore, the introduction of disulfide bonds for thermostability improvement could be rationally and highly-effectively designed by combination with spatial configuration analysis and molecular dynamics simulation.
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.
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.
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.
(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main ...source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8′s dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus.
Acetaldehyde is regarded as an important flavor compound in alcoholic beverages. With the advantages of rapidity, low cost and high sensitivity, fluorescent probe could be used as a new tool for the ...detection of acetaldehyde. Here, an effective fluorescence sensing method based on fluorescent probe N1 (FPN1) was established in this study. The function of FPN1 relies on the nucleophile substitution reaction and photoinduced electron transfer (PET), resulting in a fluorescence increase. Remarkably, the pretreatment background removal method (BRM) was successfully applied for removal of the interference of pyruvate and acetal. The linearity range (LR), limit of detection (LOD) and recovery of the fluorescence sensing method with BRM were 0.0053–200 mg/L, 0.0016 mg/L and 94.02–108.12%, respectively, which showed a broader detection range and better performance on sensitivity compared with the traditional quantitation using gas chromatography (GC). Furthermore, successful application of the method in real samples indicated the advantages of low-cost and rapidity for small-scale detection while assuring the accuracy, which provides a new strategy for the detection of acetaldehyde concentration in alcoholic beverages.
Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with ...increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry.
1,3-1,4-β-Glucanase received great interest due to its application in brewing and feed industries. Application of 1,3-1,4-β-glucanase in brewing industry helps make up for the defect that ...plant-derived β-glucanases are heat-sensitive. A new strain, CGX5-1, exhibited remarkable 1,3-1,4-β-glucanase, was isolated from Asian giant hornet nest and identified
Bacillus tequilensis
. Moreover, a new 1,3-1,4-β-glucanase gene from
B. tequilensis
was cloned and measured to be 720 bp encoding 239 amino acids, with a predicted molecular weight of 26.9 kDa. After expressed in
Escherichia coli
BL21, active recombinant enzyme of 24 kDa was detected in the supernatant of cell culture, with the activity of 2,978.2 U/mL. The new enzyme was stable in the pH 5.0–7.5 with the highest activity measured at pH 6.0. Moreover, it is thermostable within 45 to 60 °C. The property of the new recombinant enzyme makes this enzyme a broad prospect in brewing industry. Moreover, this is the first report on 1,3-1,4-β-glucanase produced by
B. tequilensis
.
Lager-brewing yeasts are mainly used for the production of lager beers. Illumina and PacBio-based sequence analyses revealed an approximate genome size of 22.8 Mb, with a GC content of 38.98%, for ...the Chinese lager-brewing yeast
sp. strain M14. Based on
prediction, 9,970 coding genes were annotated.
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