CN110760399A - 一种阿拉伯呋喃糖苷酶在啤酒生产中的应用 - Google Patents
一种阿拉伯呋喃糖苷酶在啤酒生产中的应用 Download PDFInfo
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- CN110760399A CN110760399A CN201911203088.6A CN201911203088A CN110760399A CN 110760399 A CN110760399 A CN 110760399A CN 201911203088 A CN201911203088 A CN 201911203088A CN 110760399 A CN110760399 A CN 110760399A
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- Prior art keywords
- arabinofuranosidase
- beer
- mash
- malt
- wort
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- 238000001914 filtration Methods 0.000 claims abstract description 19
- 235000007340 Hordeum vulgare Nutrition 0.000 claims abstract description 13
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- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
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Abstract
本发明公开了一种阿拉伯呋喃糖苷酶在啤酒生产中的应用。本发明将糖苷水解酶第62家族的α‑L‑阿拉伯呋喃糖苷水解酶添加到大麦麦芽的糖化醪液中,可以使高分子量阿拉伯木聚糖含量从305mg/L降至190mg/L,降低了36.7%,使麦汁的使过滤速度提高了32%。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
本发明涉及一种阿拉伯呋喃糖苷酶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。大麦麦芽麦汁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。
阿拉伯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的阿拉伯木聚糖含量,36种国内外啤酒中,阿拉伯木聚糖的最高浓度达到了849mg/L。醪液中高含量的阿拉伯木聚糖影响麦汁的粘度和过滤速度。添加能够降解阿拉伯木聚糖的外源微生物酶是解决这一问题的有效方法。
阿拉伯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-阿拉伯呋喃糖苷酶(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阿拉伯木聚糖主链中未被阿拉伯呋喃糖基团取代的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-阿拉伯呋喃糖苷酶。α-L-阿拉伯呋喃糖苷酶能够将阿拉伯木聚糖侧链基团切除。C位点为阿魏酸酯酶,其作用于O-5位上与阿拉伯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,在将阿拉伯木聚糖彻底降解为木糖的过程中起重要作用。
大麦麦芽阿拉伯木聚糖的结构中的侧链基团,会在空间上阻碍酶-底物诱导构象的形成,使木聚糖酶无法打开木聚糖的糖苷键,降低到内切木聚糖酶的降解效率;部分木聚糖酶不能降解侧链被取代的木糖基团之间的糖苷键,只有通过α-L-阿拉伯呋喃糖苷酶将支链切除,水解才能进行。因而具有切支作用的阿拉伯呋喃糖苷酶对阿拉伯木聚糖的降解非常关键。
根据活性中心的结构以及氨基酸序列,α-L-阿拉伯呋喃糖苷酶可划分为糖苷水解酶的第3、43、51、54、62和127家族,里氏木霉的基因组中分别有两种第54家族和一种第62家族的控制基因,在不同的诱导培养基和培养条件下可分别分泌不同家族的α-L-阿拉伯呋喃糖苷酶。
发明内容
本发明提供了一种阿拉伯呋喃糖苷酶及其在啤酒糖化中的应用,本发明的技术方案如下:
本发明提供了一种提高大麦麦芽麦汁过滤速度的方法,将阿拉伯呋喃糖苷酶添加入麦芽糖化过程中,所述的阿拉伯呋喃糖苷酶为α-L-阿拉伯呋喃糖苷酶,属于糖苷水解酶第62家族,氨基酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中,所述阿拉伯呋喃糖苷酶以6~12mU/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将麦芽与所述阿拉伯呋喃糖苷酶一起投入纯水,于40~50℃下保温25~35min,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60~120min;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。
本发明提供了一种将所述提高大麦麦芽麦汁过滤速度的方法在糖化工艺中的应用。
在本发明的一种实施方式中,所述糖化工艺包含如下步骤:(1)将麦芽与所述阿拉伯呋喃糖苷酶一起投入纯水,于40~50℃下保温25~35min,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60~120min;(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SEQ ID NO.1所示的阿拉伯呋喃糖苷酶微生物制备方法。
在本发明的一种实施方式中,以里氏木霉CICC41495为发酵菌种,以玉米芯和麸皮为碳源进行发酵,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,所述方法包含如下步骤:(1)将里氏木霉接种于种子培养基,在25~35℃培养36~48h;(2)将种子液接入含有玉米芯和麸皮的培养基在25~35℃培养150~200h。
在本发明的一种实施方式中,所述种子培养基含有:硫酸铵1.0~2.0g/L,葡萄糖6.0~12.0g/L,磷酸二氢钾1.5~3.0g/L,酵母粉0.5~2.0g/L,氯化钙0.2~0.6g/L,硫酸镁0.2~0.6g/L,氯化钴1.5~3.0mg/L,硫酸亚铁4.5~5.5mg/L,硫酸锌1.0~2.0mg/L,硫酸锰1.0~2.0mg/L,pH为自然。
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。
本发明提供了一种将所述提高大麦麦芽麦汁过滤速度的方法在以麦汁为原料的饮品中应用。
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:本发明在大麦麦芽投料时,添加糖苷水解酶第62家族的α-L-阿拉伯呋喃糖苷酶于大麦麦芽的糖化醪液中,促进了麦汁中的高分子量阿拉伯木聚糖的降解,使高分子量阿拉伯木聚糖含量从305mg/L降至190mg/L,降低了36.7%;麦汁粘度从1.51mPa·s降低至1.44mPa·s,过滤速度从5.0mL/min提高至6.6mL/min,提高了32%,提高了麦汁的生产效率和品质,有助于啤酒工业的生产。
附图说明
图1为阿拉伯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-阿拉伯呋喃糖苷酶,C表示阿魏酸酯酶,D表示β-木糖苷酶。
图2为纯化得到的阿拉伯呋喃糖苷酶的SDS-PAGE图谱。
图3为添加阿拉伯呋喃糖苷酶对麦汁中高分子量阿拉伯木聚糖含量的影响。
图4为添加阿拉伯呋喃糖苷酶对过滤速度的影响。
图5为添加阿拉伯呋喃糖苷酶对粘度的影响。
具体实施方式
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,在30℃、200r/min的条件下,培养36~48h。
在250mL三角瓶中装25mL产酶培养基,接入2.5mL种子液,在30℃、200r/min的条件下,培养168h。发酵液经10000×g离心15min,冷冻干燥后,获得里氏木霉CICC41495分泌的胞外酶蛋白并于4℃保存备用。
实施例2阿拉伯呋喃糖苷酶的纯化
α-L-阿拉伯呋喃糖苷酶活力的测定以4-硝基苯基-α-L-阿拉伯糖醛酸苷为底物,取0.5mL的底物溶液与适当稀释的酶液混合,在50℃下反应30min,加入0.5mol/L的碳酸钠1.0mL终止反应,于410nm处测定OD值,根据标准曲线计算α-L-阿拉伯糖呋喃糖苷酶活力。一个酶活力单位(U)是指在测定条件下每分钟水解1μmol的4-硝基苯基-α-L-阿拉伯糖醛酸苷所需的酶量。
阿拉伯呋喃糖苷酶的纯化步骤为:
(1)采用75%饱和度的硫酸铵沉淀粗酶溶液中的蛋白,10000×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。收集具有阿拉伯呋喃糖苷酶活力的组分,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,收集含阿拉伯呋喃糖苷酶活力的组分,SDS-PAGE显示纯化后的阿拉伯呋喃糖苷酶达到了电泳纯(图1)。如图1所示,M为标准分子量蛋白,泳道1为纯化后得到的阿拉伯呋喃糖苷酶。
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(60mmol/L pH6.8Tris-HCl,2%SDS,0.1%溴酚兰,25%甘油,14.4mmol/Lβ-巯基乙醇)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。
本发明纯化得到的阿拉伯呋喃糖苷酶经质谱鉴定为糖苷水解酶第62家族的α-L-阿拉伯呋喃糖苷酶,结果见表1。该阿拉伯呋喃糖苷酶的分子量理论值为22163Da,理论pI为7.77。SDS-PAGE测定,其在本发明中的分子量为32000Da,pI值约为7.4。
表1纯化得到的阿拉伯呋喃糖苷酶的质谱鉴定结果
实施例3纯化后的α-L-阿拉伯呋喃糖苷酶在大麦麦芽糖化工艺中的应用
(1)糖化工艺为:
添加阿拉伯呋喃糖苷酶的麦汁的制备方法为:
①25.0kg细粉碎麦芽与不同单位的阿拉伯呋喃糖苷酶及100L 46℃的自来水一起投入到200L糖化锅中,于45℃下保温30min;
②将醪液以1℃/min的速率升温至55℃,于55℃下保温90min,以保证α-L-阿拉伯呋喃糖苷酶充分发挥作用;
③将醪液以1℃/min的速率升温至72℃,隔5min进行碘试,直到淀粉完全降解(加入碘液不变色);
④将糖化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)。
(2)间苯三酚法测定阿拉伯木聚糖含量
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。
高分子量阿拉伯木聚糖(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,按照间苯三酚法测定阿拉伯木聚糖的含量。
(3)粘度采用HAAKE落球式粘度计进行测定。
糖化结束后,测定麦汁中的阿拉伯木聚糖含量、粘度和过滤速度等指标,对照为未加酶的糖化麦汁,以反映阿拉伯呋喃糖苷酶对糖化过滤指标的改善效果。糖化过程中外加阿拉伯呋喃糖苷酶对麦芽中阿拉伯木聚糖的降解、麦汁过滤速度和粘度的影响,结果分别见图2、3、4。
图2的结果表明,随着第62家族的α-L-阿拉伯呋喃糖苷酶的添加量的增加,高分子量阿拉伯木聚糖被逐渐降解,在添加量为0-10mU/g麦芽之间有一个显著的降低过程,麦汁的高分子量阿拉伯木聚糖含量从305mg/L降至190mg/L,当添加量为10mU/g麦芽时,高分子量阿拉伯木聚糖的降解率为36.7%,继续加大阿拉伯呋喃糖苷酶的量,高分子阿拉伯木聚糖的含量变化不大。
图3的结果表明,第62家族的α-L-阿拉伯呋喃糖苷酶的添加提高了糖化醪液的过滤速度。在糖化醪液中添加10mU/g麦芽的阿拉伯呋喃糖苷酶时,麦汁的过滤速度从5.0mL/min提高至6.6mL/min,过滤速度提高了32%。
图4的结果表明,阿拉伯呋喃糖苷酶的添加降低了麦汁的粘度,在添加量为7.5~10mU/g麦芽时,麦汁粘度可达到最低,为1.44mPa·s,下降了4.6%。
对比例1
具体实施方式同实施例3,区别在于添加α-L-阿拉伯呋喃糖苷酶(第54家族),各10mU/g’麦芽。测定麦汁中HMW-AX的含量、过滤速度及粘度。
表2添加不同家族α-L-阿拉伯呋喃糖苷酶的协定麦汁指标
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> 一种阿拉伯呋喃糖苷酶在啤酒生产中的应用
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 322
<212> PRT
<213> Trichoderma reesei
<400> 1
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Ser Ser Asn Pro Ser Ser Val Asn Gly Trp Ser Ser Pro Gln Pro Leu
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Claims (10)
1.一种提高大麦麦芽麦汁过滤速度的方法,其特征在于,将阿拉伯呋喃糖苷酶添加入麦芽糖化过程中,所述阿拉伯呋喃糖苷酶为α-L-阿拉伯呋喃糖苷酶,氨基酸序列如SEQ IDNO.1所示,所述阿拉伯呋喃糖苷酶的添加量为6~12mU/g麦芽。
2.根据权利要求1所述的方法,其特征在于,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)将麦芽与所述阿拉伯呋喃糖苷酶一起投入酿造水,于40~50℃下保温25~35min,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60~120min;(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所述的方法在糖化工艺中的应用。
5.根据权利要求4所述的应用,其特征在于,所述糖化工艺包含如下步骤:(1)将麦芽与所述阿拉伯呋喃糖苷酶一起投入酿造水,于40~50℃下保温25~35min,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60~120min;(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。
6.权利要求1所述的SEQ ID NO.1所示的阿拉伯呋喃糖苷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发酵。
7.根据权利要求6所述的制备方法,其特征在于,所述玉米芯和麸皮比例为1~5:1。
8.根据权利要求6所述的制备方法,其特征在于,所述方法包含如下步骤:(1)将里氏木霉接种于种子培养基,在25~35℃培养36~48h;(2)将种子液接入含有玉米芯和麸皮的培养基在25~35℃培养150~200h。
9.权利要求1~3所述的方法在以麦汁为原料的饮品制备中的应用。
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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