Study On The Filterability Defection Of Malts Produced From Barleys Grown In Jiangsu Province Based On Proteomics

The filterability (including the wort filtering rate, viscosity and turbidity) is an essentialquality parameter of barley malt and significantly impacts productive efficiency and quality ofbeer. Jiangsu province is an major grown area of malting barleys in China, with Dan'er andGangpi1as the dominant cultivars. However, the filterability defect of these barley maltsseverely impeded their application in beer production. ACanadian cultivar of Metcalfe and anAustralian cultivar of Baudin with superior malt filterability are widely imported in Chinese.In this dissertation, endogenous reasons for the filterability defect of Dan'er and Gangpi1malts were studied. Firstly, the filterability defect of Dan'er and Gangpi1and the direct factorwere analyzed by conventional parameters. Then, metabolic proteins related to filterabilitywere screened by comparative proteomics. At last, the impacting paths of several keyfilterability-related proteins were researched. The main results were as follows:(1) The specific filterability defects of Dan'er and Gangpi1malts were characterized byCongress wort and the direct factors were analyzed. The average filtrating rate of Congresswort of24Dan'er and Gangpi1malt samples was slower44.2%than that of20Metcalfe andBaudin, and viscosity and turbidity were higher11.4%and260.5%, respectively. The directreason was the lower degradation rate of some macromolecules in Dan'er and Gangpi1during malting and mashing, rather than the higher content of these macromolecules in barleymaterials. The contents of macromolecular ?-glucan, arabinoxylan (AX) and protein hadsignificant correlation with filterability parameters, and were higher in Dan'er and Gangpi1Congress wort. While, the difference of ?-glucan contents between the barleys wasn'tsignificant. The average content of arabinoxylan in Metcalfe and Baudin barleys was higher11.2%than that in Dan'er and Gangpi1barleys.(2) A research method based on two-dimensional electrophoresis (2DE) was constructedto study metabolic proteins in barley malts. Firstly, proteins in2DE can be clearly separatedby extracting for1h at4?with low-salt solution (5mmol L-1Tris/HCl pH7.5,1mmol L-1CaCl2) and blending for2min every15min. The shared347spots in2DE gels among Dan'er,Gangpi1, Metcalfe and Baudin malts were identified by MALDI-TOF-TOF. The resultsproved that about90%of protein spots were metabolic proteins, mainly including enzymesand pathogen-related proteins (some enzyme inhibitors included).(3) Filterability-related proteins were qualitatively and quantitatively screened usingcomparative proteomics. Metabolic proteins in Dan'er and Gangpi1of lower expressionlevels and benefits for filterability according to their functions, mainly included ?-amylase,?-amylase1, limit dextrinase, arabinoxylan arabinofuranohydrolase I (AXAH-I), ?-D-glucanexohydrolase I, predicted esterase, Cu-Zn superoxide dismutase, ascorbic acid redox enzymeand heat shock protein. In contrast, peroxidase, serpin Z7, ?-amylase/trypsin inhibitor CMb,non-special lipid transfer protein1(LTP1) and prolamine B3had higher expression levels andmight be harmful for filterability. Beta-amylase, AXAH-I, serpin Z7and peroxidase expressedremarkable differently between malts with distinct filtrability.(4) Beta-amylase and AXAH-I were purified from the Dan'er malt and then replenishedinto its Congress mashing process. The results showed that adding ?-amylase significantly decreased the wort turbidity by degrading starch contributing to turbidity. Adding AXAH-Iincreased the filtrating rate by promoting dissolution of AX, ?-glucan and protein andfacilitating their hydrolysis. Moreover, the filterability was synergistically improved by?-amylase and AXAH-I. When80.0U g-1of ?-amylase was added at the beginning ofmashing, the wort turbidity and viscosity decreased by66.3%and7.2%, and filtering rateincreased by14.4%. When6.0mU g-1of AXAH-I was added at the beginning of Congressmashing, the filtrating rate increased by34.2%, viscosity decreased by5.5%, turbidityincreased by32.5%, and contents of AX and protein increased by5.7%and1.7%, respectively.The contents of macromolecular AX, ?-glucan and protein, and ratio of arabinose to xylosedecreased by6.7%,12.4%,3.7%and31.0%, respectively. When80.0U g-1of ?-amylase and6.0mU g-1were added at the beginning of mashing, the wort turbidity and viscosity decreasedby30.7%and4.3%, and filterability rate increased by58.5%.(5) Serpin Z7had a great negative effect on the filtrating rate of Dan'er Congress wortby cross-linking with ?-glucan and AX. And peroxidase had synergistic effect with serpin Z7by promoting the cross-link of polyphenol and serpin Z7. When600?g g-1of serpin Z7wasadded, the filtering rate and turbidity decreased by48.0%and11.4%, viscosity increased by2.7%, and contents of ?-glucan and AX decreased by12.3%and7.8%, respectively. When400U g-1of horseradish peroxidase was added, the wort filtrating rate decreased by8.9%,viscosity increased by5.4%, turbidity increased by9.2%, and the reducing polyphenolcontent decreased by35.5%, respectively. When600?g g-1of serpin Z7and400U g-1of thehorseradish peroxidase were added together, the filtering rate and the reducing polyphenolcontent decreased by62.2%and47.8%, but viscosity and turbidity increased by6.8%and10.0%, respectively.(6) Modifying malting condition can improve filterability of Dan'er malt by increasingthe abundance of ?-amylase, AXAH-I, ?-amylase1and ?-D-glucan exohydrolase I, anddecreasing the abundance of LTP1, peroxidase and serpin Z7. Extending the action time of?-amylase and AXAH-I during mashing can improve filterability. Compounding ?-amylaseand AXAH-I to an commercial enzyme preparation (mainly consist of endo-?-(1,3;1,4)-glucanase, endoxylanase and protease) could improve filterability better than individualapplication of the enzyme preparation. Compared with control and only adding0.4mg g-1ofthe enzyme preparation at the beginning of Dan'er Congress mashing, the filtrating rate ofwort increased by81.3%and20.9%, viscosity decreased by7.3%and2.6%, turbiditydecreased48.5%and59.7%, respectively, by adding80.0U g-1of ?-amylase,6.0mU g-1ofAXAH-I and0.4mg g-1of the enzyme preparation.