Yongxian Li

Circular dichroism and infrared spectroscopic characterization of secondary structure components of protein Z during mashing and boiling processes

In beer brewing, protein Z is hypothesized to stabilize beer foam. However, few investigations have revealed the relationship between conformational alterations to protein Z during the brewing process and beer foam. In this report, protein Z from sweet wort was isolated during mashing and boiling processes. Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) were used to monitor the structural characteristics of protein Z. The results showed that the α-helix and β-sheet content decreased, whereas the content of β-turn and random coil increased. The complex environment rich in polysaccharides may facilitate conformational alterations and modifications to protein Z. Additionally, the formation of extended structural features to protein Z provides access to reactive amino acid side chains that can undergo modifications and the exposure of hydrophobic core regions of the protein. Analyzing structural transformations should provide a deeper understanding of the mechanism of protein Z on maintaining beer foam.

Evaluation of emerging factors blocking filtration of high-adjunct-ratio wort.

Corn starch has become a common adjunct for beer brewing in Chinese breweries. However, with increasing ratio of corn starch, problems like poor wort filtration performance arise, which will decrease production capacity of breweries. To solve this problem, factors affecting wort filtration were evaluated, such as the size of corn starch particle, special yellow floats formed during liquefaction of corn starch, and residual substance after liquefaction. The effects of different enzyme preparations including β-amylase and β-glucanase on filtration rate were also evaluated. The results indicate that the emerging yellow floats do not severely block filtration, while the fine and uniform-shape corn starch particle and its incompletely hydrolyzed residue after liquefaction are responsible for filtration blocking. Application of β-amylase preparation increased the filtration rate of liquefied corn starch. This study is useful for our insight into the filtration blocking problem arising in the process of high-adjunct-ratio beer brewing and also provides a feasible solution using enzyme preparations.

Production of a thermostable 1,3-1,4-β-glucanase mutant in Bacillus subtilis WB600 at a high fermentation capacity and its potential application in the brewing industry

1,3-1,4-β-glucanase was an important biotechnological aid in the brewing industry. In a previous research, a Bacillus BglTO mutant (BglTO) with high tolerance towards high temperature and low-pH conditions was constructed and expressed in Escherichia coli. However, E. coli was not a suitable host for enzyme production in food industry. Therefore, the present work aimed to achieve the high-level expression of BglTO in Bacillus subtilis WB600 and to test its effect in Congress mashing. The β-glucanase mutant was successfully expressed in B. subtilis WB600 and favorable plasmid segregation and structural stability were observed. The maximal extracellular activity of β-glucanase in recombinant B. subtilis WB600 reached 4840.4UmL-1 after cultivation condition optimization, which was 1.94-fold higher than that before optimization. The fermentation capacity of recombinant B. subtilis reached 242.02UmL-1h-1, which was the highest among all reported β-glucanases. The addition of BglTO in Congress mashing significantly reduced the filtration time and viscosity of mash by 29.7% and 12.3%, respectively, which was superior to two commercial enzymes. These favorable properties indicated that B. subtilis WB600 was a suitable host for production of BglTO, which was promising for application in the brewing industry.

The FKS family genes cause changes in cell wall morphology resulted in regulation of anti-autolytic ability in Saccharomyces cerevisiae

The aim of this study was to discuss the functions of FKS family genes which encode β-1, 3-glucan synthase regarding the viability and autolysis of yeast strain. Loss of FKS1 gene severely influences the viability and anti-autolytic ability of yeast. Mutation of FKS1 and FKS2 genes led to cell reconstruction, resulting in a sharp shrinkage of cell volume and decreased stress resistance, viability, and anti-autolytic ability. Deletion of FKS3 gene did not clearly influence the synthesis of β-1, 3-glucan of yeast but increased the strains stress resistance, viability, and anti-autolytic ability. It is suggested that FKS3 would be the potential target for improving the stress resistance of yeast. The results revealed the relationship among FKS family genes and demonstrated their functions on yeast cell wall construction and anti-autolytic ability.

Monitoring the Microbial Conditions in Breweries in Yangtze River Delta Region, China

ABSTRACT This study aimed to investigate the contamination degree and quality control of seven breweries from the Yangtze River Delta region, China. A total of 205 spoilage bacteria strains were isolated and no wild yeast was observed. The vast majority of the spoilage bacteria belonged to Lactobacillus, Lactococcus, and Leuconostoc species and 28.8% of them could grow in beer. Among the seven sampling points, fermented beer, finished beer, and equipment contained larger amounts of spoilage bacteria. The most abundant of the spoilage bacteria that could grow in beer existed in fermented beer and the number of spoilage bacteria was significantly reduced in the finished beer. The equipment in fermentation and packaging workshops showed high percentages of spoilage bacteria that could grow in beer. This indicated that more attention should be paid in controlling the spoilage bacteria in the fermentation and packaging processes. The hop tolerance related gene analysis showed that the majority of spoilage bacteria contained at least one of the HorA, HorC and HitA genes. None of these genes were observed in the two spoilage bacteria strains that could grow in the beer. This information could be useful for breweries in the Yangtze River Delta region for removing risks in order to produce high-quality products in good microbial condition.

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.

Comparative analysis of the effect of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability: Role of N-glycosylation and glycation

This study aimed to elaborate the effect of N-glycosylation and glycation of protein Z4 from barley malt and recombinant Pichia pastoris on beer foam stability. The malt protein Z4 and recombinant protein Z4 showed similar N-glycosylation patterns while recombinant protein Z4 was glycosylated at a higher degree. In the simulated mashing and boiling, malt protein Z4 and deglycosylated malt protein Z4 preferred to glycate with glucose and maltose while recombinant protein Z4 and deglycosylated recombinant protein Z4 showed preference towards fructose. The addition of protein Z4 and protein Z4-saccharide complexes in finished beer showed that the addition of glycosylated protein Z4 only slightly enhanced the beer foam stability while the addition of glycated protein Z4 and protein Z4 with both glycation and glycosylation could significantly increase the beer foam stability. Therefore, glycation instead of N-glycosylation of protein Z4 played important roles in maintaining beer foam stability.

Process optimization of the extraction condition of β‐amylase from brewer's malt and its application in the maltose syrup production

β‐Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of β‐amylase from brewers malt and the optimal dosage of β‐amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of β‐amylase from brewers 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 β‐amylase activity of 1,290.99 U g−1, which was close to the experimental β‐amylase activity of 1,230.22 U g−1. The optimal dosages of β‐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 β‐amylases from soybean and microbe (P < 0.01). Thus, β‐amylase from brewers malt was beneficial for production of high maltose syrup.