CN111549006B - 富含淀粉酶的复合酶的制备及其菌株和应用 - Google Patents

富含淀粉酶的复合酶的制备及其菌株和应用 Download PDF

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CN111549006B
CN111549006B CN202010511002.2A CN202010511002A CN111549006B CN 111549006 B CN111549006 B CN 111549006B CN 202010511002 A CN202010511002 A CN 202010511002A CN 111549006 B CN111549006 B CN 111549006B
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culture
fermentation
amylase
aspergillus oryzae
culture medium
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CN111549006A (zh
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吴勃
王云龙
徐永雷
王天珍
王云祥
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Hangzhou Biocom Biological Technology Co ltd
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Hangzhou Biocom Biological Technology Co ltd
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Abstract

本发明提出了一种富含淀粉酶的复合酶的制备及其菌株和应用，培养的发酵产物含有淀粉酶、中性蛋白酶类、木聚糖酶类、纤维素酶类、葡聚糖酶类以及果胶酶类。米曲霉(Aspergillus oryzae)，命名为米曲霉BAK200312，其保藏号为CGMCC No.19617。本发明提供一个丰富的复合酶系统；使用的米曲霉菌株是通过经严格驯化得到，其酶产酶能力较强，淀粉酶产量较高，酶活较高，是一株具有高效产淀粉酶能力的米曲霉菌株。

Description

富含淀粉酶的复合酶的制备及其菌株和应用
技术领域
本发明涉及复合酶领域，特别是指一种富含淀粉酶的复合酶的制备及其菌株和应用。
背景技术
饲料原料种类繁多，包括粮食原粮，谷物、大豆、玉米、豆粕、杂粕、鱼粉、肉骨粉、乳清粉、氨基酸、维生素等诸多品种，共同构成动物能量及营养来源。淀粉是饲料中最丰富的营养物质，也是畜禽的主要能量来源，占畜禽日粮表观代谢能的60％左右。淀粉是葡萄糖的多聚体，以白色固体淀粉微粒的形式贮藏于玉米、小麦、高粱等能量饲料中。天然淀粉一般含有两种成分：直链淀粉和支链淀粉。直链淀粉是由α-葡萄糖残基通过α-1-4-糖苷键连接成的不分支的长链分子；支链淀粉中连接相连的葡萄糖残基的糖苷键也是α-1-4-糖苷键，只是在分支点还存在α-1-6连接。
淀粉是能量的主要形式和来源，淀粉的利用率决定着动物能量的利用率。淀粉的消化利用与动物肠道中淀粉的结构和淀粉消化酶的活性密切相关。研究表明幼龄动物淀粉利用率较低，回肠末端淀粉消化率低。通过食糜检查可知大部分淀粉颗粒没有被消化进入后肠发酵，导致能量损失。而且早期畜禽肠道淀粉酶活性极低，导致畜禽能量利用率降低，制约早期生长。在幼畜日粮中添加适量的外源性淀粉酶，可以补充内源性淀粉酶的不足，帮助幼畜消化利用淀粉，促进生长。
每种酶类发挥作用都是由一系列单一酶组分协同发挥作用体现的。淀粉酶是动物机体分泌的一类帮助动物消化的活性物质，是作用于各种淀粉糖苷键的一类酶的总称。主要的淀粉分解酶包括α-淀粉酶、β-淀粉酶、糖化酶、支链淀粉酶和异淀粉酶。α-淀粉酶作用于α-1,4-糖苷键，将淀粉水解为双糖、寡糖和糊精，只能分解直链淀粉和支链淀粉的直链部分。β-淀粉酶作用于淀粉的β-1,4-糖苷键，将淀粉水解为双糖、寡糖和糊精。糖化酶水解线性的双糖、寡糖和糊精，产生葡萄糖和果糖，并从淀粉的非还原末端，依次水解α-1,4-糖苷键生成葡萄糖。异淀粉酶作用于α-1,6-糖苷键，生成直链淀粉和糊精。多种酶协同作用，共同水解淀粉源。
然而，除同一底物需要同种酶类中不同酶组分协同降解之外，同一物质也需要不同酶类共同发挥酶解功能。饲料原料通常同时含有多种组分(淀粉、蛋白质、纤维素、半纤维素、果胶等)，动物消化吸收这些组分需要多种酶共同发挥作用。李鹏等研究表明在饲料中添加消化酶为主的饲用酶制剂，可补充动物内源性酶分泌量的不足，提高淀粉、蛋白质等饲料消化利用，促进消化道的发育，使肠壁吸收功能大为增强。有研究表明多种酶制剂单一配比的效果往往较天然形成的复合酶体系差，以淀粉酶为主，以其它多种酶为辅的天然复合酶制剂具有巨大的潜能。
发明内容
本发明提出一种富含淀粉酶的复合酶的制备及其菌株和应用，解决了现有技术中酶制剂采用单一配比导致复配效果差的问题。
本发明的技术方案是这样实现的：
一种富含淀粉酶的复合酶的制备，其中，
所用菌株为米曲霉(Aspergilus oryzae)BAK200312，保藏号CGMCC No.19617；
培养的发酵产物含有淀粉酶、中性蛋白酶类、木聚糖酶类、纤维素酶类、葡聚糖酶类以及果胶酶类。
在一些实施例中，所述复合酶的制备包括固体发酵步骤；
将液体种子接种在固体培养基上，进行发酵培养；培养环境温度设置为30-35℃、湿度为40-55％，发酵培养5-7d；
所用固体发酵培养基为：麸皮70-85％、豆粕5-10％、玉米粉5-10％、玉米芯粉2-5％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％。
在一些实施例中，所述固体发酵培养基在121℃灭菌50-60min。
在一些实施例中，所述固体发酵培养基替换为：
麸皮80-85％、豆粕粉5-10％、玉米粉5-10％、硫酸铵1-2％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.2-0.3％。
在一些实施例中，所述液体种子的制备如下：
(1)斜面培养
取米曲霉BAK200312，在无菌环境下采用无菌操作在灭菌后的PDA试管斜面上进行划线培养；
所用PDA试管斜面培养基配方为：葡萄糖1-2％、土豆汁20-30％、琼脂粉1.2-1.8％；
(2)菌种液体培养
取出步骤(1)中培养结束的试管斜面菌种，全部转接至二级液体培养基中，所述培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％；
(3)菌种液体扩培
将步骤(2)中培养所得的种子接种至液体发酵罐中进行扩大培养，接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d；
所用液体发酵罐培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％。
在一些实施例中，所述步骤(1)的培养基替换为下述培养基：
蔗糖2-3％、硝酸钠0.2-0.3％、磷酸氢二钾0.05-0.1％、硫酸镁0.05-0.1％、氯化钾0.05-0.1％、硫酸亚铁0.001-0.002％、琼脂粉1.2-1.8％；
或，
动物组织胃蛋白酶水解物和胰酪胨等量混合物1％、葡萄糖4％、琼脂粉1.2-1.8％，pH 5.4-5.8。
本发明还提供一种米曲霉(Aspergilus oryzae)，命名为米曲霉BAK200312，其保藏号为CGMCC No.19617。
本发明还提供一种畜禽饲料，包括上述的米曲霉BAK200312的发酵产物。
本发明相比于现有技术具有以下有益效果：
(1)本发明提供的固体发酵产物经酶制剂酶活测定结果可知，除含有较高酶活的淀粉酶外，还含有较高酶活的中性蛋白酶、木聚糖酶、纤维素酶、葡聚糖酶、果胶酶类。
(2)本发明提供的固体发酵产物经蛋白质组学分析，除去固体发酵培养基原料所含蛋白以外，产物中还含有较多酶蛋白组分，是一个丰富的复合酶系统。
(3)本发明使用的米曲霉菌株是通过经严格驯化得到，其酶产酶能力较强，淀粉酶产量较高，酶活较高，是一株具有高效产淀粉酶能力的米曲霉菌株。
(4)本发明固体发酵原料及设备简单，成本较低，工艺操作简便，发酵产物较丰富，废弃物较少，更加环保。
(5)本发明提供的固体发酵产物能够显著提高动物的生产性能，降低料肉/蛋比，节约饲料成本。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
生物保藏：
米曲霉(Aspergilus oryzae)BAK200312，于2020年4月8号保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC；地址为北京市朝阳区北辰西路1号院3号中国微生物研究所，邮政编码100101)，保藏编号：CGMCC No.19617。
ITS鉴定：
该菌株ITSrDNA的区段序列(见序列表)比对结果：本发明使用的菌株与米曲霉属基因序列相似度最高。因此将其命名为米曲霉BAK200312。
本发明中提供的米曲霉BAK200312，是经过严格驯化培育得到的高产复合酶菌株。驯化前的菌株发酵最高酶活为9507U/g。
实施例1
一种富含淀粉酶的复合酶的制备，制备方法包括如下步骤：
1斜面培养
取米曲霉BAK200312菌株，在无菌环境下采用无菌操作在灭菌后的PDA试管斜面上进行划线培养，划线后的试管斜面置于30℃培养箱中培养5-7d。所用PDA试管斜面培养基配方为：葡萄糖1-2％、土豆汁20-30％、琼脂粉1.2-1.8％，121℃灭菌20min。
2菌种液体培养
取出步骤1中培养结束的试管斜面菌种，在超净工作台中采用无菌操作将试管斜面菌种全部转接至二级液体培养基中，于30℃摇床中振荡培养2-3d，摇床转速为160-200rpm。所用培养基采用半合成培养基(在天然有机物的基础上加入已知盐类)，培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
3菌种液体扩培
将步骤2中培养所得的三角瓶液体种子接种至液体发酵罐中进行扩大培养，接种过程中谨慎操作，防止染菌。接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d。所用液体发酵罐培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
4菌种固体发酵培养
将固体发酵培养基提前置于灭菌蒸球中进行灭菌处理，灭菌后的固体培养基冷却备用。将液体罐培养的液体扩培种子通过管道输送至装有冷却后的固体培养基的灭菌蒸球中进行搅拌混匀，使液体种子在固体培养基上均匀分布。将搅拌后的混合物分装于呼吸膜发酵袋中，装料量为4-7kg/袋，置于发酵车间进行发酵培养。发酵车间进行严格的控温控湿处理，期间进行观察。培养环境温度设置为30-35℃、湿度为40-55％，发酵培养5-7d。所用固体发酵培养基为：麸皮70-85％、豆粕5-10％、玉米粉5-10％、玉米芯粉2-5％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％。121℃灭菌50-60min。
5发酵产物主要酶类酶活测定
取出固体发酵产物，对其进行55-65℃烘干粉碎，过筛处理(40目)，取筛下物进行酶活测定。酶活检测方法严格依据国标酶制剂检测方法进行，得到的检测结果如表1所示，由结果可以看出，本发明固体发酵产物中淀粉酶为主要酶类，酶活最高，达21092U/g；其次为中性蛋白酶类，酶活达13784U/g；木聚糖酶类、纤维素酶类、葡聚糖酶类、果胶酶类酶活也较高。
表1主要酶类酶活汇总
Figure BDA0002528244740000071
6发酵产物酶蛋白组分测定
取出烘干后过40目筛的发酵产物，加入8M尿素溶液提取样品中的蛋白质组分，通过还原烷基化打开蛋白三维结构，酶解后抽提出肽段，再运用LC-MS质谱技术得到这些肽段的质谱图，使用蛋白质鉴定软件在相应数据库中进行鉴定，将比对结果进一步进行分析。
由检测结果可以看出，本发明提供的固体发酵产物中含有丰富的酶蛋白组分(近60种)，酶种繁多。除淀粉酶类、中性蛋白酶类、木聚糖酶类、纤维素酶类、葡聚糖酶类、果胶酶类之外，还有磷酸酶类、抗氧化物酶类、阿魏酸酯酶类。部分酶蛋白组分列举如下：
葡糖淀粉酶、酸性α-淀粉酶、葡聚糖内切1,3-β-葡糖苷酶eglC、果胶酯酶A、β-葡萄糖苷酶、木糖醛酸内切酶A、胞外菊粉酶、内聚半乳糖醛酸酶I、内聚半乳糖醛酸酶A、1,4-β-D-木聚糖木糖水解酶xlnD、β-葡萄糖醛酸酶、碱性磷酸酶、丝氨酸/苏氨酸蛋白磷酸酶、过氧化物酶、阿魏酸酯酶B、α-木糖苷酶A、葡聚糖酶、超氧化物歧化酶、1,4-β-木聚糖内切酶C、α-葡萄糖苷酶、内切葡聚糖酶A、β-葡萄糖苷酶A、1,4-β-D-葡聚糖纤维糖水解酶A、羧肽酶、1,4-β-D-葡聚糖纤维二糖水解酶B、内切葡聚糖酶B、肽水解酶、羧肽酶(丝氨酸型内肽酶活性)、肽酶S53前肽(丝氨酸型内肽酶活性)、中性蛋白酶2、精氨酸酶/胍丁胺酶/甲酰谷氨酰胺酶、二肽酶、肽酶M20、羧肽酶Y同源物A、氨基酰脯氨酸氨基肽酶、枯草杆菌蛋白酶样丝氨酸蛋白酶pepC、亮氨酸氨基肽酶2、过氧化氢酶A等。
6发酵产物动物试验
6.1发酵产物对仔猪生产性能的影响
选取200头28日龄的断奶仔猪，采取同窝分组的方式随机分成对照组与试验组。试验期间，试验组日粮在对照组基础上添加处理后的发酵产物300克/吨，两组饲养管理条件完全相同，于断奶后20天进行个体称重，记录每组耗料情况，统计试验期间日增重及料重比。试验结果如下：
表2发酵产物对仔猪生产性能的影响
项目 试验组 对照组
仔猪头数 100 100
进苗起始重(kg) 6.97±1.42 7.02±1.15
断奶20天平均体重(kg/头) 13.52±1.67 12.97±1.27
断奶20天平均日增重(g/头) 328±1.40 298±2.50
平均净增重(kg/头) 6.545 5.955
料肉比 1.46 1.60
由表2可以看出，在整个试验过程中，试验组在对仔猪的增重上优势较大，平均日增重比对照组增加10.07％，说明在仔猪饲料中添加一定量的本公司发酵产物有助于提高仔猪的生产性能。此外，本发明提供的发酵产物可以显著降低料肉比，提高仔猪的饲料转化与吸收能力。
6.2发酵产物对蛋鸡生产性能的影响
试验选取选取60周龄健康的蛋鸡200只，随机分成2个处理组。试验分为对照组(基础日粮)和试验组(基础日粮+200克/吨发酵产物)，试验期42天。按照蛋鸡常规饲养管理进行试验，期间进行观察记录。
表3发酵产物对蛋鸡生产性能的影响
项目 对照组 试验组
平均日采食量(g) 122.4 117.8
平均产蛋率(％) 84.9 86.1
平均蛋重(g) 63.6 64.4
料蛋比 2.24 2.12
由表3结果可以看出，在日粮中添加本发明固体发酵产物相比对照组，可以提高蛋鸡的产蛋率，增加蛋重，降低料蛋比。
实施例2
一种富含淀粉酶的复合酶的制备，制备方法包括如下步骤：
1斜面培养
取米曲霉BAK200312菌株，在无菌环境下采用无菌操作在灭菌后的试管斜面上进行划线培养，划线后的试管斜面置于30℃培养箱中培养7-10d。所用试管斜面培养基为高盐察氏培养基，其培养基配方为：蔗糖2-3％、硝酸钠0.2-0.3％、磷酸氢二钾0.05-0.1％、硫酸镁0.05-0.1％、氯化钾0.05-0.1％、硫酸亚铁0.001-0.002％、琼脂粉1.2-1.8％。121℃灭菌20min。
2菌种液体培养
取出步骤1中培养结束的试管斜面菌种，在超净工作台中采用无菌操作将试管斜面菌种全部转接至二级液体培养基中，于30℃摇床中振荡培养2-3d，摇床转速为160-200rpm。所用培养基采用半合成培养基(在天然有机物的基础上加入已知盐类)，培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
3菌种液体扩培
将步骤2中培养所得的三角瓶液体种子接种至液体发酵罐中进行扩大培养，接种过程中操作谨慎，防止染菌。接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d。所用液体发酵罐培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
4菌种固体发酵培养
将固体发酵培养基提前置于灭菌蒸球中进行灭菌处理，灭菌后的固体培养基冷却备用。将液体罐培养的液体扩培种子通过管道输送至装有冷却后的固体培养基的灭菌蒸球中进行搅拌混匀，使液体种子在固体培养基上均匀分布。将搅拌后的混合物分装于呼吸膜发酵袋中，置于发酵车间进行发酵培养。发酵车间进行严格的控温控湿处理，期间进行观察。培养环境温度设置为30-35℃、湿度为40-55％，发酵培养5-7d。所用固体发酵培养基为：麸皮70-85％、豆粕5-10％、玉米粉5-10％、玉米芯粉2-5％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌50-60min。
实施例3
一种富含淀粉酶的复合酶的制备，制备方法包括如下步骤：
1斜面培养
取米曲霉BAK200312菌株，在无菌环境下采用无菌操作在灭菌后的试管斜面上进行划线培养，划线后的试管斜面置于30℃培养箱中培养5-8d。所用培养基为SDA培养基，其培养基配方为：动物组织胃蛋白酶水解物和胰酪胨等量混合物1％、葡萄糖4％、琼脂粉1.2-1.8％，pH 5.4-5.8，121℃灭菌20min。
2菌种液体培养
取出步骤1中培养结束的试管斜面菌种，在超净工作台中采用无菌操作将试管斜面菌种全部转接至二级液体培养基中，于30℃摇床中振荡培养2-3d，摇床转速为160-200rpm。所用培养基采用半合成培养基(在天然有机物的基础上加入已知盐类)，培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
3菌种液体扩培
将步骤2中培养所得的三角瓶液体种子接种至液体发酵罐中进行扩大培养，接种过程中操作谨慎，防止染菌。接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d。所用液体发酵罐培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
4菌种固体发酵培养
将固体发酵培养基提前置于灭菌蒸球中进行灭菌处理，灭菌后的固体培养基冷却备用。将液体罐培养的液体扩培种子通过管道输送至装有冷却后的固体培养基的灭菌蒸球中进行搅拌混匀，使液体种子在固体培养基上均匀分布。将搅拌后的混合物分装于呼吸膜发酵袋中，置于发酵车间进行发酵培养。发酵车间进行严格的控温控湿处理，期间进行观察。培养环境温度设置为30-35℃、湿度为40-55％，发酵培养5-7d。所用固体发酵培养基为：麸皮70-85％、豆粕5-10％、玉米粉5-10％、玉米芯粉2-5％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌50-60min。
实施例4
一种富含淀粉酶的复合酶的制备，制备方法包括如下步骤：
1斜面培养
取米曲霉BAK200312菌株，在无菌环境下采用无菌操作在灭菌后的PDA试管斜面上进行划线培养，划线后的试管斜面置于30℃培养箱中培养5-7d。所用PDA试管斜面培养基配方为：葡萄糖1-2％、土豆汁20-30％、琼脂粉1.2-1.8％，121℃灭菌20min。
2菌种液体培养
取出步骤1中培养结束的试管斜面菌种，在超净工作台中采用无菌操作将试管斜面菌种全部转接至二级液体培养基中，于30℃摇床中振荡培养2-3d，摇床转速为160-200rpm。所用培养基采用半合成培养基(在天然有机物的基础上加入已知盐类)，培养基配方为：细麸皮1-2％、豆粕粉2-5％、麦芽糊精1.5-2％、土豆汁0.3-0.6％、氯化钙0.2-0.5％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
3菌种液体扩培
将步骤2中培养所得的三角瓶液体种子接种至液体发酵罐中进行扩大培养，接种过程中操作谨慎，防止染菌。接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d。所用液体发酵罐培养基配方为：细麸皮1-2％、豆粕粉2-5％、麦芽糊精1.5-2％、土豆汁0.3-0.6％、氯化钙0.2-0.5％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
4菌种固体发酵培养
将固体发酵培养基提前置于灭菌蒸球中进行灭菌处理，灭菌后的固体培养基冷却备用。将液体罐培养的液体扩培种子通过管道输送至装有冷却后的固体培养基的灭菌蒸球中进行搅拌混匀，使液体种子在固体培养基上均匀分布。将搅拌后的混合物分装于呼吸膜发酵袋中，置于发酵车间进行发酵培养。发酵车间进行严格的控温控湿处理，期间进行观察。培养环境温度设置为30-35℃、湿度为40-55％，发酵培养5-7d。所用固体发酵培养基为：麸皮70-85％、豆粕5-10％、玉米粉5-10％、玉米芯粉2-5％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌50-60min。
实施例5
一种富含淀粉酶的复合酶的制备，制备方法包括如下步骤：
1斜面培养
取米曲霉BAK200312菌株，在无菌环境下采用无菌操作在灭菌后的PDA试管斜面上进行划线培养，划线后的试管斜面置于30℃培养箱中培养5-7d。所用PDA试管斜面培养基配方为：葡萄糖1-2％、土豆汁20-30％、琼脂粉1.2-1.8％，121℃灭菌20min。
2菌种液体培养
取出步骤1中培养结束的试管斜面菌种，在超净工作台中采用无菌操作将试管斜面菌种全部转接至二级液体培养基中，于30℃摇床中振荡培养2-3d，摇床转速为160-200rpm。所用培养基采用半合成培养基(在天然有机物的基础上加入已知盐类)，培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
3菌种液体扩培
将步骤2中培养所得的三角瓶液体种子接种至液体发酵罐中进行扩大培养，接种过程中操作谨慎，防止染菌。接种量为2-8％，接种完成后发酵罐温度设置为30℃，转速为120-180rpm，发酵培养2-3d。所用液体发酵罐培养基配方为：玉米粉2-5％、豆粕粉2-5％、土豆汁0.3-0.6％、氯化钙0.5-1％、硫酸镁0.05-0.1％、磷酸氢二钾0.1-0.2％，121℃灭菌30-40min。
4菌种固体发酵培养
将固体发酵培养基提前置于灭菌蒸球中进行灭菌处理，灭菌后的固体培养基冷却备用。将液体罐培养的液体扩培种子通过管道输送至装有冷却后的固体培养基的灭菌蒸球中进行搅拌混匀，使液体种子在固体培养基上均匀分布。将搅拌后的混合物分装于呼吸膜发酵袋中，置于发酵车间进行发酵培养。发酵车间进行严格的控温控湿处理，期间进行观察。培养环境温度设置为30-35℃、湿度为40-55％，发酵培养4-6d。所用固体发酵培养基为：麸皮80-85％、豆粕粉5-10％、玉米粉5-10％、硫酸铵1-2％、氯化钙1-2％、硫酸镁0.05-0.1％、磷酸氢二钾0.2-0.3％。121℃灭菌50-60min。
序列表
<110> 杭州保安康生物技术有限公司
<120> 富含淀粉酶的复合酶的制备及其菌株和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 569
<212> DNA
<213> 米曲霉BAK200312(Aspergillus oryzae)
<400> 1
tgcggaagga tcattaccga gtgtagggtt cctagcgagc ccaacctccc cacccgtgtt 60
tactgtacct tagttgcttc ggcgggcccg ccattcatgg ccgccggggg ctctcagccc 120
cgggcccgcg cccgccggag acaccacgaa ctctgtctga tctagtgaag tctgagttga 180
ttgtatcgca atcagttaaa actttcaaca atggatctct tggttccggc atcgatgaag 240
aacgcagcga aatgcgataa ctagtgtgaa ttgcagaatt ccgtgaatca tcgagtcttt 300
gaacgcacat tgcgccccct ggtattccgg ggggcatgcc tgtccgagcg tcattgctgc 360
ccatcaagca cggcttgtgt gttgggtcgt cgtcccctct ccggggggga cgggccccaa 420
aggcagcggc ggcaccgcgt ccgatcctcg agcgtatggg gctttgtcac ccgctctgta 480
ggcccggccg gcgcttgccg aacgcaaatc aatcttttcc aggttgacct cggatcaggt 540
agggataccc gctgaactta agcatatca 569

Claims (8)

1.一种富含淀粉酶的复合酶的制备方法，其特征在于，
所用菌株为米曲霉（Aspergillus oryzae）BAK200312，保藏号CGMCC No.19617；
培养的发酵产物含有淀粉酶、中性蛋白酶类、木聚糖酶类、纤维素酶类、葡聚糖酶类以及果胶酶类。
2.根据权利要求1所述的一种富含淀粉酶的复合酶的制备方法，其特征在于，所述复合酶的制备方法包括固体发酵步骤；
将液体种子接种在固体培养基上，进行发酵培养；培养环境温度设置为30-35℃、湿度为40-55%，发酵培养5-7 d；
所用固体发酵培养基为：麸皮70-85%、豆粕5-10%、玉米粉5-10%、玉米芯粉2-5%、氯化钙1-2%、硫酸镁0.05-0.1%、磷酸氢二钾0.1-0.2%。
3.根据权利要求2所述的一种富含淀粉酶的复合酶的制备方法，其特征在于，所述固体发酵培养基在121℃灭菌50-60 min。
4.根据权利要求2所述的一种富含淀粉酶的复合酶的制备方法，其特征在于，所述固体发酵培养基替换为：
麸皮80-85%、豆粕粉5-10%、玉米粉5-10%、硫酸铵1-2%、氯化钙1-2%、硫酸镁0.05-0.1%、磷酸氢二钾0.2-0.3%。
5.根据权利要求2所述的一种富含淀粉酶的复合酶的制备方法，其特征在于，所述液体种子的制备如下：
（1）斜面培养
取米曲霉BAK200312，在无菌环境下采用无菌操作在灭菌后的PDA试管斜面上进行划线培养；
所用PDA试管斜面培养基配方为：葡萄糖1-2%、土豆汁20-30%、琼脂粉1.2-1.8%；
（2）菌种液体培养
取出步骤（1）中培养结束的试管斜面菌种，全部转接至二级液体培养基中，所述培养基配方为：玉米粉2-5%、豆粕粉2-5%、土豆汁0.3-0.6%、氯化钙0.5-1%、硫酸镁0.05-0.1%、磷酸氢二钾0.1-0.2%；
（3）菌种液体扩培
将步骤（2）中培养所得的种子接种至液体发酵罐中进行扩大培养，接种量为2-8%，接种完成后发酵罐温度设置为30℃，转速为120-180 rpm，发酵培养2-3 d；
所用液体发酵罐培养基配方为：玉米粉2-5%、豆粕粉2-5%、土豆汁0.3-0.6%、氯化钙0.5-1%、硫酸镁0.05-0.1%、磷酸氢二钾0.1-0.2%。
6.根据权利要求5所述的一种富含淀粉酶的复合酶的制备方法，其特征在于，所述步骤（1）的培养基替换为下述培养基：
蔗糖2-3%、硝酸钠0.2-0.3%、磷酸氢二钾0.05-0.1%、硫酸镁0.05-0.1%、氯化钾0.05-0.1%、硫酸亚铁0.001-0.002%、琼脂粉1.2-1.8%；
或，
动物组织胃蛋白酶水解物和胰酪胨等量混合物1%、葡萄糖4%、琼脂粉1.2-1.8%，pH5.4-5.8。
7.一种米曲霉（Aspergillus oryzae），命名为米曲霉BAK200312，其保藏号为CGMCCNo.19617。
8.一种畜禽饲料，包括权利要求7所述的米曲霉BAK200312的发酵产物。
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