【作者】 金昭；

【导师】 陆健；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 过滤性能（包括麦汁的过滤速度、粘度和浊度）是啤酒大麦麦芽品质的一项重要指标，严重影响啤酒的生产效率和成品啤酒的质量。针对国内啤酒大麦主产区之一江苏地区的大麦（单二和港啤1号为主产品种）所生产麦芽过滤性能不良的缺点，本文以过滤性能优良且普遍进口的加拿大品种Metcalfe和澳大利亚品种Baudin的大麦麦芽为参照，在分析单二和港啤1号大麦麦芽过滤性能缺陷及直接影响因素的基础上，通过比较蛋白质组学的策略，解析了单二和港啤1号麦芽与Metcalfe和Baudin麦芽之间差异的活性蛋白质，并就差异蛋白质对过滤性能的影响及其作用途径展开深入研究，以揭示造成江苏啤酒大麦麦芽过滤性能缺陷的内源性因素。主要研究结果如下：（1）通过对麦芽协定麦汁常规指标的统计分析表征了江苏啤酒大麦麦芽过滤性能的缺陷：四个年份的24个单二和港啤1号麦芽比20个Baudin和Metcalfe麦芽协定麦汁的过滤速度、粘度和浊度分别平均慢44.2%，高11.4%和260.5%；造成这种缺陷的直接原因是制麦和糖化过程中大分子物质的降解程度较低：协定麦汁中高分子的β-葡聚糖、阿拉伯木聚糖和蛋白质的含量与过滤性能显著相关且均在单二和港啤1号中较高，而原料大麦之间β-葡聚糖的含量差异并不显著，Metcalfe和Baudin大麦中阿拉伯木聚糖的含量反而比单二和港啤1号的平均高11.2%。（2）建立了基于双向电泳的啤酒大麦麦芽中活性蛋白质组的研究方法。确定采用低盐溶液从麦芽中提取的蛋白质可获得理想的双向电泳效果，并通过MALDI-TOF-TOF分析单二、港啤1号、Baudin和Metcalfe啤酒大麦麦芽的双向电泳凝胶中共同存在的347个蛋白质斑点，其中314个斑点鉴定为具有参与代谢功能的活性蛋白质，即主要是酶和病程相关蛋白质（包括酶的抑制剂）。（3）通过比较蛋白质组学策略，从定性和定量差异的角度研究筛选了江苏啤酒大麦麦芽过滤性能缺陷相关的活性蛋白质。在港啤1号和单二中表达量较低且根据其功能分析对过滤性能有利的蛋白质主要有β-淀粉酶、α-淀粉酶1、极限糊精酶、阿拉伯呋喃糖苷酶I（Arabinoxylan arabinofuranohydrolase I, AXAH-I）、β-D-葡聚糖外切酶I和预测的酯酶、Cu-Zn超氧化物歧化酶、抗坏血酸氧化还原酶和热激蛋白；在港啤1号和单二中表达量较高且根据功能分析对过滤性能不利的蛋白质主要有过氧化物酶、丝氨酸蛋白酶抑制剂Z7、α-淀粉酶/胰蛋白酶抑制剂CMb、非特异性脂肪酸转移酶1和B3-醇溶蛋白质。其中，β-淀粉酶、AXAH-I、丝氨酸蛋白酶抑制剂Z7和过氧化物酶在过滤性能差异的麦芽中表达量差别明显。（4）通过将单二和港啤1号中表达量不足较为严重的β-淀粉酶和AXAH-I从单二麦芽中分离纯化，并补充到单二麦芽的协定糖化中，研究它们对过滤性能的影响和途径，结果表明：β-淀粉酶通过降解可形成浑浊的糊精而明显改善麦汁浊度，AXAH-I通过促进阿拉伯木聚糖和蛋白质的溶解和水解而显著改善麦汁过滤速度，且两种水解酶具有协同作用。糖化起始时，当β-淀粉酶添加量达到80.0U g-1时，单二协定麦汁浊度和粘度分别降低了66.3%和7.2%，过滤速度加快了14.4%；当AXAH-I添加量达到6.0mU g-1时，麦汁过滤速度加快了34.2%，粘度降低了5.5%，而浊度增加约32.5%，阿拉伯木聚糖和总蛋白质的含量分别增加了5.7%和1.7%，说明添加AXAH-I促进了阿拉伯木聚糖和蛋白质的溶解，进而有利于水解，从而使多聚阿拉伯木聚糖含量、阿拉伯糖和木糖的比值、高分子蛋白质和β-葡聚糖含量分别降低了6.7%、31.0%、3.7%和12.4%。同时补充β-淀粉酶（80.0U g-1）和AXAH-I（6.0mU g-1），麦汁的过滤速度加快了58.5%，粘度和浊度分别降低了4.3%和30.7%。（5）丝氨酸蛋白酶抑制剂Z7主要通过与β-葡聚糖和多聚阿拉伯木聚糖的结合对单二协定麦汁过滤速度有较大的负面影响，而且过氧化物酶通过促进丝氨酸蛋白酶抑制剂与多酚的氧化结合对过滤性能具有协同的负面作用。糖化起始时，当丝氨酸蛋白酶抑制剂Z7添加量达到600μg g-1麦芽时，麦汁的过滤速度减慢了48.0%，粘度升高了2.7%，浊度降低了11.4%，β-葡聚糖和多聚阿拉伯木聚糖含量分别降低了12.3%和7.8%。单独添加400U g-1辣根过氧化物酶到单二麦芽的协定糖化中，麦汁过滤速度相对于空白对照减慢了8.9%；粘度增加了5.4%，浊度增加了9.2%，还原性多酚含量降低了35.5%，而同时添加600μg g-1丝氨酸蛋白酶抑制剂Z7和400U g-1辣根过氧化物酶使麦汁的过滤速度、粘度、浊度和还原性多酚含量相对于空白对照分别降低了62.2%、增加了6.8%、增加了10.0%和降低了47.8%。（6）改良制麦和糖化工艺发现，提高麦芽中β-淀粉酶、AXAH-I、α-淀粉酶1、β-D-葡聚糖外切酶I的表达量，降低非特异性脂肪酸转移酶1、过氧化物酶、丝氨酸蛋白酶抑制剂Z7的表达量，延长糖化阶段β-淀粉酶和阿拉伯呋喃糖苷水解酶I的作用时间有利于单二大麦麦芽过滤性能的改善。将β-淀粉酶和阿拉伯呋喃糖苷酶I初步补充到商品酶制剂中对过滤性能的改善效果更显著，当β-淀粉酶、AXAH-I和商品酶制剂（主要含有β-葡聚糖酶内切酶、阿拉伯木聚糖内切酶和蛋白酶活力）在单二麦芽协定糖化起始时的添加量分别为80.0U g-1、6.0mU g-1和0.4mg g-1时，相对于空白对照和单独添加0.4mg g-1的酶制剂，麦汁的过滤速度分别加快了81.3%和20.9%，粘度分别降低了7.3%和2.6%，浊度分别降低了48.5%和59.7%。更多还原

【Abstract】 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 β-glucan, arabinoxylan (AX) and protein hadsignificant correlation with filterability parameters, and were higher in Dan’er and Gangpi1Congress wort. While, the difference of β-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 β-amylase,α-amylase1, limit dextrinase, arabinoxylan arabinofuranohydrolase I (AXAH-I), β-D-glucanexohydrolase I, predicted esterase, Cu-Zn superoxide dismutase, ascorbic acid redox enzymeand heat shock protein. In contrast, peroxidase, serpin Z7, α-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 β-amylase significantly decreased the wort turbidity by degrading starch contributing to turbidity. Adding AXAH-Iincreased the filtrating rate by promoting dissolution of AX, β-glucan and protein andfacilitating their hydrolysis. Moreover, the filterability was synergistically improved byβ-amylase and AXAH-I. When80.0U g-1of β-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, β-glucan and protein, and ratio of arabinose to xylosedecreased by6.7%,12.4%,3.7%and31.0%, respectively. When80.0U g-1of β-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 β-glucan and AX. And peroxidase had synergistic effect with serpin Z7by promoting the cross-link of polyphenol and serpin Z7. When600μg g-1of serpin Z7wasadded, the filtering rate and turbidity decreased by48.0%and11.4%, viscosity increased by2.7%, and contents of β-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. When600μg 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 β-amylase, AXAH-I, α-amylase1and β-D-glucan exohydrolase I, anddecreasing the abundance of LTP1, peroxidase and serpin Z7. Extending the action time ofβ-amylase and AXAH-I during mashing can improve filterability. Compounding β-amylaseand AXAH-I to an commercial enzyme preparation (mainly consist of endo-β-(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 β-amylase,6.0mU g-1ofAXAH-I and0.4mg g-1of the enzyme preparation.更多还原