CN110714037A - 一种木聚糖酶的制备方法及其在啤酒生产中的应用 - Google Patents
一种木聚糖酶的制备方法及其在啤酒生产中的应用 Download PDFInfo
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- CN110714037A CN110714037A CN201911199189.0A CN201911199189A CN110714037A CN 110714037 A CN110714037 A CN 110714037A CN 201911199189 A CN201911199189 A CN 201911199189A CN 110714037 A CN110714037 A CN 110714037A
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- Prior art keywords
- xylanase
- beer
- malt
- wort
- mash
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Abstract
本发明公开了一种木聚糖酶的制备方法及其在啤酒生产中的应用，属于啤酒生产技术领域。本发明将木聚糖酶添加到大麦麦芽的糖化醪液中，能够使麦汁中的聚合态***木聚糖含量最高下降80％，麦汁的粘度降低7.2％，过滤速度提高80％。对于改善啤酒品质、提高啤酒产量具有重要意义。
Description
技术领域
本发明涉及一种木聚糖酶的制备方法及其在啤酒生产中的应用，属于啤酒生产技术领域。
背景技术
啤酒糖化生产中，糖化醪液经过滤槽分离而得到透明澄清的麦汁，糖化醪液中麦汁的分离速度即过滤速度。大麦麦芽的过滤速度对啤酒生产效率和成品啤酒的品质均具有重要的影响：过滤速度慢，糖化醪液的粘度高，不利于酶与底物的接触和降解，导致非淀粉多糖、蛋白和淀粉等大分子物质不能充分降解，浸出物收得率较低，延长了啤酒单批次的生产时间，增加了生产成本。
***木聚糖是大麦胚乳细胞壁中最主要的组成成分，属于非淀粉多糖，其约占胚乳细胞壁的干重的20％，占大麦种子总重量的4％～10％。研究表明，麦汁和成品啤酒中仍然含有较高浓度的***木聚糖：36种国内外啤酒中，***木聚糖的最高浓度达到了849mg/L，严重影响了麦汁粘度和过滤速度。添加能够降解***木聚糖的外源微生物酶是解决这一问题的有效方法。
***木聚糖的完全降解需要一系列酶共同完成，该酶系主要包括：内切-β-1,4-木聚糖酶(EC 3.2.1.8)，β-1,4-木糖苷酶(EC 3.2.1.37)，α-L-***呋喃糖苷酶(EC3.2.1.55)及阿魏酸酯酶(EC 3.1.1.6)，这些酶的作用位点见图1。A位点为β-1,4-木聚糖酶，其以内切方式作用于***木聚糖主链中未被***呋喃糖基团取代的β-1,4-木糖苷键，生成聚合度不同的低聚木糖和少量的木糖；其中B位点为α-L-***呋喃糖苷酶。α-L-***呋喃糖苷酶能够将***木聚糖侧链基团切除。C位点为阿魏酸酯酶，其作用于O-5位上与***呋喃糖基团以酯键相连的阿魏酸，释放阿魏酸；D位点为β-木糖苷酶，作用于木聚糖酶的水解产物——低聚木糖，从非还原端进一步降解低聚木糖生成β-木糖，在将***木聚糖彻底降解为木糖的过程中起重要作用。
内切木聚糖酶是降解***木聚糖的关键酶，也是目前被研究最多、最透彻的***木聚糖降解酶。由于不同来源的木聚糖酶的底物特异性，***木聚糖的分子结构造成的空间位阻效应、木聚糖酶对***木聚糖的亲和性及是否具有侧链水解活性等因素，造成微生物木聚糖酶对不同***木聚糖的降解能力存在差异，目前还没有找到一种能够适用于降解啤酒大麦麦芽中的高分子量***木聚糖降解的木聚糖酶，从而影响了部分高***木聚糖含量的大麦麦芽在啤酒工业中的应用。
发明内容
为解决上述问题，本发明提供一种木聚糖酶在啤酒生产中应用。
本发明的技术方案如下：
本发明提供了一种降解麦汁中高分子量***木聚糖的方法，所述方法为在大麦麦芽糖化过程中添加氨基酸序列如SEQ ID NO.1所示的木聚糖酶。
在本发明的一种实施方式中，将所述木聚糖酶以15～30U/g麦芽的添加量在糖化开始时与麦芽一同加入。
在本发明的一种实施方式中，所述方法包含如下步骤：①将麦芽与所述木聚糖酶一起投入水中，于40～50℃下保温60～120min，制备得到醪液；②将醪液以0.5～1.5℃/min的速率升温至50～60℃，于50～60℃下保温35～45min；③将醪液以0.5～1.5℃/min的速率升温至70～75℃，直至淀粉完全分解。
本发明提供了一种降解麦汁中高分子量***木聚糖的方法在提高麦芽过滤速度中的应用。
本发明提供了一种木聚糖酶的微生物制备方法，以里氏木霉CICC41495为发酵菌种，以玉米芯和麸皮为碳源进行产酶发酵。
在本发明的一种实施方式中，所述里氏木霉为CICC41495，购买于中国工业微生物菌种保藏管理中心，保藏地址为北京市朝阳区酒仙桥中路24号院6号楼，保藏编号为CICC41495。
在本发明的一种实施方式中，所述玉米芯和麸皮的比例为1～5:1。
在本发明的一种实施方式中，所述发酵为在28～35℃下培养150～200h。
在本发明的一种实施方式中，对发酵后的发酵液进行分离，分离包含如下步骤：(1)用硫酸铵沉淀得到粗酶液；(2)将步骤(1)得到的粗酶液过脱盐柱后收集的溶液过离子交换柱，收集具有木聚糖活性的组分，将其进行包埋浓缩；(3)浓缩后的酶液利用凝胶过滤色谱柱进一步纯化，收集具有木聚糖酶活性的组分。
在本发明的一种实施方式中，分离的步骤具体为：
(1)采用70～80％饱和度的硫酸铵沉淀粗酶溶液中的蛋白，10,000～12,000×g离心10～20min，弃上清，沉淀用15～25mmol/L Tris-HCl缓冲溶液(pH7.5～8.0)溶解，得到酶液；
(2)上述酶液用SephadexG-25柱脱盐后，上样于DEAE-Sepharose Fast Flow离子交换柱，用350～410mL含0～0.50mol/L NaCl的15～25mmol/L Tris-HCl缓冲溶液(pH8.0)梯度洗脱，流速为80～120mL/h。收集具有木聚糖酶活性的组分，用PEG20000包埋浓缩；
(3)取浓缩后的酶液，采用Sephacryl S-100凝胶过滤色谱柱进一步纯化，流动相为pH5.0～5.5、80～150mmol/L的乙酸-乙酸钠缓冲溶液，流速18～25mL/h，收集具有木聚糖酶活性的组分
本发明提供了一种降解麦汁中高分子量***木聚糖的方法在饮品中应用。
本发明的一种实施方式中，所述饮品包含熟啤酒、生啤酒、鲜啤酒、干啤酒、冰啤酒、低醇啤酒、无醇啤酒、小麦啤酒、浑浊啤酒、果蔬汁型啤酒、果蔬味型啤酒。
有益效果：本发明在糖化投料时，将本发明制备的木聚糖酶Ⅲ添加于大麦麦芽的糖化醪液中，促进了麦汁中的***木聚糖的降解，使高分子量***木聚糖含量从从301mg/L降至38mg/L，降低了87.4％；降低了麦汁的粘度，提高了过滤速度，使过滤速度从5.0mL/min提高至9.0mL/min，提高了80％；改善了产品品质，对于麦芽和啤酒的生产都具有重要的意义。
附图说明
图1为***木聚糖降解酶系的酶切位点，A表示β-1,4-木聚糖酶，B表示α-L-***呋喃糖苷酶，C表示阿魏酸酯酶，D表示β-木糖苷酶。
图2为木聚糖酶Ⅲ纯化过程的SDS-PAGE图谱。泳道1：Sephacryl S-100纯化后的木聚糖酶Ⅲ；泳道2：DEAE Sepharose Fast Flow分离后的木聚糖酶Ⅲ；泳道3：发酵液；M，marker。
图3为木聚糖酶Ⅲ的质谱图。
图4为添加木聚糖酶Ⅲ对麦汁中***木聚糖含量、粘度和过滤速度的影响。
具体实施方式
内切木聚糖酶活力的测定以浓度10mg/mL燕麦木聚糖为底物。取2mL的底物溶液与2mL适当稀释的酶液混合，在37℃下反应30min，加入5.0mL DNS试剂终止反应，沸水浴加热5min，于540nm处测定OD值，根据标准曲线计算木聚糖酶活力。一个酶活力单位(U)是指在测定条件下每分钟释放1μmol的木糖所需要的酶量。
实施例1里氏木霉CICC41495发酵液的制备
配制种子培养基为：硫酸铵1.4g/L，葡萄糖10g/L，磷酸二氢钾2.0g/L，酵母粉1.0g/L，氯化钙0.3g/L，硫酸镁0.3g/L，氯化钴2.0mg/L，硫酸亚铁5.0mg/L，硫酸锌1.4mg/L，硫酸锰1.6mg/L，pH为自然。
配制产酶培养基：将种子培养基中的碳源---葡萄糖替换为玉米和麸皮(玉米30g/L和麸皮10g/L)，其余成分不变。
里氏木霉CICC41495保存在马铃薯---葡萄糖---琼脂斜面上，使用时用0.9％的NaCl溶液收集孢子用于接种。250mL三角瓶装25mL上述种子培养基，接入孢子液，培养温度为30℃，摇床转速为200r/min，培养36～48h。
在250mL三角瓶中装25mL产酶培养基，接入2.5mL种子液，培养温度为30℃，摇床转速为200r/min，培养168h。发酵液经10000×g离心15min，冷冻干燥后，获得里氏木霉CICC41495全培养液中的蛋白并于4℃保存备用。
实施例2内切木聚糖酶Ⅲ的纯化
内切木聚糖酶Ⅲ的纯化步骤为：
(1)采用75％饱和度的硫酸铵沉淀粗酶溶液中的蛋白，10,000×g离心15min，弃上清，沉淀用20mmol/L Tris-HCl缓冲溶液(pH8.0)溶解；
(2)上述酶液用SephadexG-25柱脱盐后，上样于DEAE-Sepharose Fast Flow离子交换柱，用400mL含0～0.50mol/L NaCl的20mmol/L Tris-HCl缓冲溶液(pH8.0)梯度洗脱，流速为100mL/h。收集具有木聚糖酶活性的组分，用PEG20000包埋浓缩；
(3)取浓缩后的酶液，采用Sephacryl S-100凝胶过滤色谱柱进一步纯化，流动相为pH5.5、100mmol/L的乙酸-乙酸钠缓冲溶液，流速20mL/h，收集具有木聚糖酶活性的组分，SDS-PAGE的结果表明，纯化后的木聚糖酶达到了电泳纯，结果见图2。
在纯化的过程中，采用考马斯亮蓝法测定样品的蛋白浓度，采用SDS-PAGE对抑制蛋白的纯度进行分析。具体方法为：
(1)将样品与4倍体积的上样缓冲溶液(2％SDS,0.1％溴酚蓝10％甘油)混合，沸水浴5min；
(2)取30μL上样(SDS-PAGE的分离胶浓度为12.5％，浓缩胶浓度为5％)，采用60V电压直至溴酚蓝指示带到达浓缩胶底部成一条直线→80V电压直到溴酚蓝指示带到达分离胶底部；
(3)经固定液(甲醇：乙酸：水比例为5：1：4)固定30min，用0.25％的考马斯亮蓝R-250溶液染色1h；
(4)用脱色液(甲醇：乙酸：水比例为1：1：8)脱至背景清晰。
纯化得到的木聚糖酶，经基质辅助激光解析电离串联飞行时间质谱鉴定为内切-β-1,4-木聚糖酶Ⅲ(EC 3.2.1.8)(图3)。该酶的理论分子量值为38.0kDa，理论pI为6.97，属于糖苷水解酶GHF10家族；经SDS-PAGE测定的木聚糖酶的分子量为32.0kDa，pI值约为9.0。
表1木聚糖酶的质谱鉴定结果
实施例3纯化后的内切木聚糖酶Ⅲ在大麦麦芽糖化工艺中的应用
(1)糖化工艺为：
模拟工业化生产的糖化麦汁的制备方法为：
①25.0kg细粉碎麦芽与100L 46℃的自来水一起投入到糖化锅中(糖化锅容积为200L)，于45℃下保温90min，使木聚糖酶对大麦麦芽高分子量***木聚糖的降解更加充分；
②将醪液以1℃/min的速率升温至65℃，于65℃下保温40min；
③将醪液以1℃/min的速率升温至72℃，隔5min进行碘试，直到显色完全；
④将麦芽醪液泵入到过滤槽中，静置30min；
⑤采用过滤槽底部的筛板过滤麦汁，收集清亮的麦汁，计算单位时间内收集到的麦汁体积。过滤速度(V)以单位时间内收集到的麦汁体积表示(单位为mL/min)。
糖化结束后，测定麦汁中的***木聚糖含量、粘度和过滤速度等指标，对照为未加酶的糖化麦汁，以反映木聚糖酶对糖化过滤指标的改善效果。
(2)间苯三酚法测定***木聚糖含量
配制显色剂：0.5g间苯三酚用1mL无水乙醇助溶，再分别加入1mL浓盐酸、0.5mL17.5g/L的葡萄糖溶液和55mL冰醋酸，混匀，贮存于棕色瓶中。
配制标准曲线：分别配制20，40，60，80和100mg/L的系列木糖工作溶液。分别取2mL各浓度的工作溶液，向各试管分别加入10mL显色剂，对照用2mL蒸馏水代替，混匀后，于沸水浴中准确反应25min，冷却至室温，测定552nm下的吸光度值，绘制标准曲线。
高分子量***木聚糖(high molecular weight arabinoxylan，HMW-AX)含量的测定
取2mL麦汁与3mL无水乙醇混合均匀在4℃下过夜沉淀，10000×g离心15min，弃上清，沉淀用2mL蒸馏水复溶，取复溶后的溶液0.1mL，加入1.9mL蒸馏水，按照制备标准曲线的方法采用间苯三酚法测定***木聚糖的含量。
(3)过滤速度(V)：在32cm漏斗中，以中性滤纸为介质，单位时间内收集的麦汁体积来表示；粘度采用HAAKE落球式粘度计进行测定。
外加不同活力的纯化后的木聚糖酶Ⅲ对协定糖化麦汁中***木聚糖的降解、粘度和过滤速率的影响如图4示。
图4的结果表明，木聚糖酶Ⅲ的添加大大提高了糖化醪液的过滤性能。在糖化醪液中添加25U/g麦芽的木聚糖酶Ⅲ时，协定麦汁的聚合态***木聚糖含量从301mg/L降至38mg/L，降解率为80％；粘度由1.508mPa·s降低至1.4mPa·s，降低了7.2％。过滤速度由5.0mL/min提高至9.0mL/min，过滤速度提高了80％。
实施例4木聚糖酶Ⅲ水解大麦麦芽高分子量***木聚糖的产物分析
为研究木聚糖酶Ⅲ能显著提高糖化过程中大麦麦芽醪液过滤速度的原因，采用高效液相色谱法对其降解大麦麦芽高分子量***木聚糖的产物进行了分析。
表2木聚糖酶Ⅲ水解高分子量***木聚糖的产物分析
表2的结果表明，木聚糖酶Ⅲ在37℃下与高分子量***木聚糖反应30min后，高分子量***木聚糖水解产物主要为木糖和***糖，其组成为***糖41.4％，木糖44.0％，没有木二糖和木三糖等典型内切木聚糖酶的水解产物。高效液相色谱法的分析结果表明，木聚糖酶Ⅲ作用于麦芽高分子量***木聚糖时具有水解侧链***糖的功能，这可能是木聚糖酶Ⅲ能够显著降解糖化醪液中高分子量***木聚糖，提高过滤速度的原因。
对比例1
具体实施方式同实施例3，区别在于添加黑曲霉木聚糖酶、枯草芽孢杆菌木聚糖酶，各25U/g麦芽，测定麦汁中高分子量***木聚糖的含量、过滤速度及粘度。
表3添加不同木聚糖酶的麦汁过滤指标
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种木聚糖酶的制备方法及其在啤酒生产中的应用
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 347
<212> PRT
<213> Trichoderma reesei
<400> 1
Met Lys Ala Asn Val Ile Leu Cys Leu Leu Ala Pro Leu Val Ala Ala
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Leu Pro Thr Glu Thr Ile His Leu Asp Pro Glu Leu Ala Ala Leu Arg
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Ala Asn Leu Thr Glu Arg Thr Ala Asp Leu Trp Asp Arg Gln Ala Ser
35 40 45
Gln Ser Ile Asp Gln Leu Ile Lys Arg Lys Gly Lys Leu Tyr Phe Gly
50 55 60
Thr Ala Thr Asp Arg Gly Leu Leu Gln Arg Glu Lys Asn Ala Ala Ile
65 70 75 80
Ile Gln Ala Asp Leu Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp
85 90 95
Gln Ser Leu Glu Asn Asn Gln Gly Gln Leu Asn Trp Gly Asp Ala Asp
100 105 110
Tyr Leu Val Asn Phe Ala Gln Gln Asn Gly Lys Ser Ile Arg Gly His
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Thr Leu Ile Trp His Ser Gln Leu Pro Ala Trp Val Asn Asn Ile Asn
130 135 140
Asn Ala Asp Thr Leu Arg Gln Val Ile Arg Thr His Val Ser Thr Val
145 150 155 160
Val Gly Arg Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu
165 170 175
Ile Phe Asn Glu Asp Gly Thr Leu Arg Ser Ser Val Phe Ser Arg Leu
180 185 190
Leu Gly Glu Glu Phe Val Ser Ile Ala Phe Arg Ala Ala Arg Asp Ala
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Asp Pro Ser Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Arg Ala
210 215 220
Asn Tyr Gly Lys Val Asn Gly Leu Lys Thr Tyr Val Ser Lys Trp Ile
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Ser Gln Gly Val Pro Ile Asp Gly Ile Gly Ser Gln Ser His Leu Ser
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Gly Gly Gly Gly Ser Gly Thr Leu Gly Ala Leu Gln Gln Leu Ala Thr
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Val Pro Val Thr Glu Leu Ala Ile Thr Glu Leu Asp Ile Gln Gly Ala
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Pro Thr Thr Asp Tyr Thr Gln Val Val Gln Ala Cys Leu Ser Val Ser
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Lys Cys Val Gly Ile Thr Val Trp Gly Ile Ser Asp Lys Asp Ser Trp
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Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp Ala Asn Phe Asn Pro Lys
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Pro Ala Tyr Asn Ser Ile Val Gly Ile Leu Gln
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Claims (10)
1.一种降解麦汁中高分子量***木聚糖的方法，其特征在于，在麦芽处理过程中添加氨基酸序列如SEQ ID NO.1所示的木聚糖酶。
2.根据权利要求1所述的方法，其特征在于，将所述木聚糖酶以15～30U/g麦芽的添加量在糖化开始时与麦芽一同加入。
3.根据权利要求1所述的方法，其特征在于，所述方法包含如下步骤：①将麦芽与所述木聚糖酶一起投入水中，于40～50℃下保温60～120min，制备得到醪液；②将醪液以0.5～1.5℃/min的速率升温至50～60℃，于50～60℃下保温35～45min；③将醪液以0.5～1.5℃/min的速率升温至70～75℃，直至淀粉完全分解。
4.权利要求1～3所述的方法在提高麦汁过滤速度中的应用。
5.一种木聚糖酶的微生物制备方法，其特征在于，以里氏木霉CICC41495为发酵菌种，以玉米芯和麸皮为碳源进行产酶发酵。
6.根据权利要求5所述的制备方法，其特征在于，玉米芯和麸皮的比例为1～5:1。
7.根据权利要求5所述的制备方法，其特征在于，在28～35℃下培养150～200h。
8.根据权利要求5所述的制备方法，其特征在于，对发酵后的发酵液进行分离，分离包含如下步骤：(1)用脱色液脱色后，用硫酸铵沉淀得到粗酶液；(2)将步骤(1)的粗酶液过脱盐柱后收集的溶液过离子交换柱，收集具有木聚糖活性的组分，将其进行包埋浓缩；(3)浓缩后的酶液利用凝胶过滤色谱柱进一步纯化，收集具有木聚糖酶活性的组分。
9.权利要求1～3任一所述方法在饮品制备中的应用。
10.根据权利要求9所述的应用，其特征在于，所述饮品包含熟啤酒、生啤酒、鲜啤酒、干啤酒、冰啤酒、低醇啤酒、无醇啤酒、小麦啤酒、浑浊啤酒、果蔬汁型啤酒、果蔬味型啤酒。
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