Degradation Of Arabinoxylan In Barley Malt During The Mashing Of Beer Brewing

The main components of the barley endosperm cell wall areβ-glucan and arabinoxylan, of whichβ-glucan has always been regarded as the main substance that clogs the filter medium during beer brewing.At present,theβ-glucan content in barley malt,wort and beer is low,and its influence on viscosity and filtration speed has been eliminated.The latest research shows that whenβ-glucan content is very low,arabinoxylan has the same negative effect on filterability.In this study,a size exclusion chromatography method for the determination of the molecular weight of arabinoxylan was established.The Pearson method was used to analyze the correlation between the molecular weight of barley malt arabinoxylan and the filtration speed and viscosity.A new specification for evaluating the arabinoxylan content,which was responsible for filtration speed and viscosity,PWEAX₅₀,was established.The effects of mashing process parameters,endogenous xylanase and exogenous microbial xylanases on PWEAX₅₀ content during the mashing process were studied;2-DE was used to analyze the protein composition of the microbial xylanase with the best performance of PWEAX₅₀ degradation.The key enzyme in the crude xylanase was purified and its properties was studied.Finally,the fermentation process factors and medium composition of the key enzyme secreted by the microorganism were optimized.This study has a significant guidance for explaining the mechanism of arabinoxylan degradation during beer mashing process,improving beer production efficiency and strategy of compound enzymes for mashing.The main findings are as follows:（1）Precipitateβ-glucan with 75%saturated ammonium sulfate and precipitate polymer water-extractable arabinoxylan in wort（PWEAX）with 80%(v×v^-1)ethanol,using size exclusion chromatography to analyze the ofβ-glucan and arabinoxylan concentration with molecular weights>1000 kDa,500～1000 kDa,50～500 kDa and<50 kDa in barley malt.Pearson correlation analysis showed that the arabinoxylan concentration of molecular weight>50 kDa(PWEAX₅₀)was significantly negatively correlated with filtration rate and extremely positively correlated with viscosity;the concentration ofβ-glucan with molecular weight>1000 kDa was extremely significantly positively correlated with viscosity.The significant level of PWEAX₅₀ and the correlation coefficient were the highest compared with the content of arabinoxylan determined by other methods,and the PWEAX₅₀ content could more accurately reflect the high molecular weight arabinoxylan content,which affected the viscosity and filtration rate of the Congress wort.The higher molecular weight and higher concentration of PWEAX₅₀0 as well as the higher side chain substitution degree（A/X value）in the molecular structure were the reasons why PWEAX₅₀ caused high viscosity and slow filtration rate of wort.A linear equation reflecting the relationship between filtration rate and PWEAX₅₀ content of the Congress wort was constructed using SPSS19.0 linear regression method:V₃₀=485-0.852×PWEAX₅₀ content.The PWEAX₅₀ content in the agreement wort is controlled within≤334 mg·L^-1 according to the equation calculation,and the filtration speed of barley wort agreement wort can be controlled≥200 mL to avoid negative effects on the viscosity and filtration rate of wort.（2）During mashing,the PWEAX₅₀ concentration in mash is slightly different,but the difference is not large.When the temperature was 45°C and 55°C,the PWEAX₅₀concentration remained unchanged with time prolonged.When the temperature was kept at65°C and 75°C,the PWEAX₅₀ concentration increased slightly with time prolonged.The results showed that the mashing temperature and time had little effect on PWEAX₅₀degradation.When PWEAX₅₀0 was used as the substrate,in the range of pH4.5～6.0 and temperature 45～75°C,the endogenous xylanase of barley malt—X-Ⅰwas active,which indicated that under the conditions of mash,the X-Ⅰactivity was inhibited.（3）Malt soluble protein was extracted using 100 mmol·L^-1 of acetic acid-sodium acetate buffer solution（pH5.5）and an endogenous xylanase inhibitor was isolated with ion exchange and size exclusion chromatography.The inhibitory protein against X-Ⅰwas identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as barleyα-amylase/subtilisin inhibitor（BASI）.When the molar ratio was 2.75:1,the reaction time was20 min,the pH value was 6.0,and the temperature was 50°C,the inhibition activities were higher.BASI has strong inhibitory activity towards X-Ⅰunder the mashing temperature and pH,which was the main reason why PWEAX₅₀ was not degraded during the mashing process.With the addition of BASI in the reaction system between X-Ⅰand substrate,the K_m value increases and the V_max value remains unchanged.The result showed that BASI was a competitive inhibitor of X-Ⅰ.（4）The substrate specificity of WUAX and WEAX and the resistance to BASI inhibitory activity are the main factors affecting the degradation of PWEAX₅₀ by microbial xylanases during mashing.Fourteen kinds of microbial xylanases were supplemented to the mash of barley malt.The content of SAX in wort increased after adding 2^#,4^#,6^#,11^#and 12^#xylanases,and the content of PWEAX₅₀ in wort showed the same change trend.The results showed that the PWEAX₅₀ and SAX contents,viscosity and filtration rate of wort with 3^#,7^#and 14^#xylanases were little changed,and their catalytic capacities were inhibited by BASI.Both 1^#,5^#,8^#,9^#,10^#and 13^#xylanases had the ability to degrade PWEAX₅₀,which could reduce wort viscosity and increase filtration rate.When 8#xylanase（from Trichoderma reesei CICC41495）was supplemented to the mash,PWEAX₅₀ was completely degraded,the wort viscosity was reduced by 11.8%,and the filtration speed was increased by 93%,the synergistic effect reached 162%after TrAbf62A treatment for 2 h and then XYNⅢtreatment for 2 h.（5）The experiment on fermentation process conditions showed that,when the initial pH of the culture medium was 5.0,the culture temperature was 30°C,the inoculation amount was10%,the Tween 80 was 0.2%,the filling volume was 50 mL×250 mL^-1 Erlenmeyer flask,shaker speed was 180 r·min^-1,and the culture time was 168 h,the XYNⅢactivity in the fermentation broth was higher.Among the medium components,carbon and nitrogen sources have the greatest influence on the secretion of XYNⅢby T.reesei CICC41495.The Box-Benhnken response surface method was used to optimize the concentration of the components in the medium.The components that have a greater impact on enzyme production were corncob,bran,yeast powder and ammonium sulfate.The results showed that when the corncob concentration was 2.17 g·L^-1,the concentration of bran was 30.50 g·L^-1,the yeast powder concentration was 3.75 g·L^-1,and the XYNⅢactivity was the highest.After optimization,the XYNⅢactivity in the fermentation broth was XYNⅢactivity was 281U·mL^-1,increased by 406%.