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CN111549020B - 富含酸性果胶酶的复合酶的制备及其菌株和应用 - Google Patents

富含酸性果胶酶的复合酶的制备及其菌株和应用 Download PDF

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CN111549020B
CN111549020B CN202010511065.8A CN202010511065A CN111549020B CN 111549020 B CN111549020 B CN 111549020B CN 202010511065 A CN202010511065 A CN 202010511065A CN 111549020 B CN111549020 B CN 111549020B
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pectinase
aspergillus niger
complex enzyme
rich
acidic
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CN111549020A (zh
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吴勃
王云龙
徐永雷
王天珍
刘广晓
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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酸性果胶酶的复合酶的制备及其菌株和应用；培养的发酵产物中含有果胶酶、木聚糖酶、甘露聚糖酶、酸性蛋白酶、纤维素酶、淀粉酶以及β‑葡聚糖酶。黑曲霉(Aspergillus niger)，命名为黑曲霉BAK200345，其保藏号为CGMCC No.19614。本发明发酵产物提供的富含酸性果胶酶的复合酶中果胶酶种类丰富，果胶酯酶、果胶水解酶、果胶裂解酶、聚半乳糖醛酸酶、鼠李糖半乳糖醛酸酶均存在，是一个完整的果胶酶体系。酸性果胶酶酶活较高，最高可达132960U/g；该复合酶能够降低料蛋比，提高蛋鸡产蛋率，减少粘粪现象，改善肠道健康，应用效果好。

Description

富含酸性果胶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，特别是指一种富含酸性果胶酶的复合酶的制备及其菌株和应用。
背景技术
果胶是以一种高分子多糖化合物，广泛存在于果蔬、玉米、大豆等植物中，是植物细胞壁的主要组成成分，夹杂于植物初生细胞壁和细胞间隙中。目前，饲料原料组成大部分为植物原料，而果胶作为抗营养因子的一种存在于植物饲料原料中，进入消化道后提高食糜粘度，影响动物对营养物质的消化吸收。
果胶主要分为果胶、果胶酸、原果胶三类。果胶是半乳糖醛酸酯及少量半乳糖醛酸通过α-1,4-糖苷键连接而成的长链高分子化合物；果胶酸是由约100个半乳糖醛酸通过α-1,4-键连接而成的直链，主要存在于中层中；原果胶的分子量比果胶酸和果胶高，甲酯化程度介于二者之间，主要存在于初生壁中，不溶于水，在稀酸和原果胶酶的作用下转变为可溶性的果胶。在植物组织中果胶可与纤维素、半纤维素、木质素、蛋白质等物质结合，表现出各部位组织的固有形态。
裂解果胶需要一系列的酶参与。果胶酶是分解果胶类物质的一类多酶复合体系，它可瓦解植物细胞壁，使高分子的半乳糖醛酸降解为半乳糖醛酸和果胶酸小分子物质，从而快速彻底的脱除果胶。根据对底物作用方式的不同可将其分为多聚半乳糖醛酸酶、果胶裂解酶、果胶酯酶。聚半乳糖醛酸酶可进一步分为外切聚半乳糖醛酸酶、内切聚半乳糖醛酸酶；果胶酸裂解酶分为内切聚半乳糖醛酸裂解酶、外切聚半乳糖醛酸裂解酶、内切聚甲基半乳糖醛酸裂解酶、外切聚甲基半乳糖醛酸裂解。按其最适作用pH又可分为酸性果胶酶和碱性果胶酶。酸性果胶酶是目前工业酶制剂的主要来源，主要由真菌产生；碱性果胶酶主要由细菌产生，适用于碱性环境中。
由于动物体细胞无细胞壁结构，不能分泌产生果胶酶，因此在畜禽生产中一般为外源添加。许多研究试验结果表明，在饲粮中添加适宜水平果胶酶可改善动物生产性能，提高畜禽产品品质。姜晓霞等研究发现，日粮中添加果胶酶能降解植物细胞壁的果胶，引起细胞壁裂解，有利于日粮中CF、NDF、AD的吸收和利用，对提高肉鸡生产性能、生长速度、腿肌率及降低腹脂率、促进营养物质的消化吸收有显著作用；张倩等研究发现，日粮中添加适量的果胶酶制剂能显著提高鹅对纤维素的消化率，并能促进鹅对钙、磷的消化吸收。此外，有研究发现，植物细胞壁的彻底裂解需要果胶酶、纤维素酶、半纤维素酶、蛋白酶等的协同作用，形成多酶复合体系，使消化道内的消化作用得以顺利进行。Tahir等研究表明果胶酶对纤维素酶和半纤维素酶在提高蛋白质和干物质消化率方面的协同作用显著；对单糖的释放也有类似作用；Zyla等研究表明，果胶酶和蛋白酶配合使用可提高火鸡骨骼矿物质和钙磷存留量。
目前果胶酶的应用大部分集中于食品等行业，且以液体发酵居多。液体发酵产物较单一，对于成分复杂的植物饲料原料而言，需要多种酶协同发挥作用。因此，有必要进一步研究复合酶体系。
发明内容
本发明提出一种富含酸性果胶酶的复合酶的制备及其菌株和应用，解决了现有技术中发酵产物中酶系单一的问题。
本发明的技术方案是这样实现的：
一种富含酸性果胶酶的复合酶的制备，其中，
所用菌株为黑曲霉(Aspergillus niger)BAK200345，保藏号CGMCC No.19614；
培养的发酵产物中含有果胶酶、木聚糖酶、甘露聚糖酶、酸性蛋白酶、纤维素酶、淀粉酶以及β-葡聚糖酶。
在一些实施例中，所述复合酶的制备包括固体发酵步骤；
将种子液接种于固体发酵培养基上，搅拌混匀，在曲房中发酵培养；
所述固体发酵培养基按下述比例配制：麸皮80-90％、玉米粉5％-10％、豆粕粉2％-5％、硫酸铵1％-2％、硫酸镁0.05％-0.1％、磷酸氢二钾0.2％-0.3％、初始水分60％-70％，121℃灭菌40min。
进一步的，所述固体发酵培养基采用下述配方替换：
麸皮80-85％、豆粕5-10％、玉米粉5-10％、氯化铵1-2％、硫酸镁0.05％-0.1％、碳酸钙0.5％-1％、磷酸氢二钾0.2％-0.3％，初始水分60％-65％，121℃灭菌40min；
或，
麸皮80-90％、豆粕5-10％、棕榈粕粉5-10％、硫酸铵1-2％、硫酸镁0.05％-0.1％、硫酸锰0.01-0.02％、碳酸钙1％-2％、磷酸氢二钾0.2％-0.3％，初始水分60％-65％，121℃灭菌40min。
在一些实施例中，对所述曲房进行控温控湿处理，控制曲房相对湿度50％以上，料温28℃-32℃；发酵培养4-8d至菌丝明显、厚实、酶活增长缓慢为止。
进一步的，所述种子液的制备如下：
(1)斜面培养
将黑曲霉BAK200345划线接种至经灭菌后的斜面固体培养基上，25-35℃培养4-8d；其培养基为察氏培养基：蔗糖3％、硝酸钠0.3％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％、琼脂粉1.2-1.8％，121℃灭菌20min；
(2)摇瓶培养
刮取1-2环步骤(1)中得到的斜面种子至二级摇瓶液体培养基中培养，摇瓶装液量为100-150mL/瓶；温度25-35℃、摇床转速160-200rpm培养2-6d，得到二级摇瓶种子液；其培养基配方为：麸皮2-6％、蛋白胨1-5％、葡萄糖2-6％、麦芽糊精4-8％，121℃灭菌40min；
(3)液体罐培养
将上述经步骤(2)得到的二级摇瓶种子液接种至三级液体罐种子液中，接种量为2-10％，温度25-35℃、转速100-180rpm培养2-6d得到三级种子液；其培养基配方为：麸皮2-6％、蛋白胨1-5％、葡萄糖2-6％、麦芽糊精4-8％，121℃灭菌40min。
进一步的，步骤(1)中的培养基可用下述培养基替换：
葡萄糖2％、土豆汁20％、琼脂粉1.2-1.8％，121℃灭菌20min；或
麸皮汁2-5％、牛肉膏1-3％、蛋白胨0.5％-2％、磷酸氢二钾0.1-0.6％、硫酸镁0.2-0.4％、琼脂粉1.2-1.8％，121℃灭菌20min。
在一些实施例中，还包括发酵产物的烘干步骤和粉碎步骤；其中，烘干的温度为55-65℃。
本发明还提供一种黑曲霉(Aspergillus niger)，命名为黑曲霉BAK200345，其保藏号为CGMCC No.19614。
本发明还提供一种畜禽饲料，包括上述的黑曲霉BAK200345的发酵产物。
本发明相比于现有技术具有以下有益效果：
(1)本发明发酵产物提供的富含酸性果胶酶的复合酶中果胶酶种类丰富，果胶酯酶、果胶水解酶、果胶裂解酶、聚半乳糖醛酸酶、鼠李糖半乳糖醛酸酶均存在，是一个完整的果胶酶体系。
(2)本发明发酵得到的酸性果胶酶酶活较高，最高可达132960U/g；
(3)本发明提供的复合酶能够降低料蛋比，提高蛋鸡产蛋率，减少粘粪现象，改善肠道健康，应用效果好。
附图说明
为了更清楚地说明本发明实施方案或现有技术中的技术方案，下面将对实施方案或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施方案，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1发酵产物LC-MS谱图结果。
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：
黑曲霉(Aspergillus niger)BAK200345，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.19614。
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黑曲霉属基因序列相似度最高。因此将其命名为黑曲霉BAK200345。
本发明中提供的黑曲霉BAK200345，是经过严格驯化培育得到的高产复合酶菌株。
实施例1
一种富含酸性果胶酶的复合酶的制备，具体制备方法如下：
1斜面培养
将黑曲霉BAK200345划线接种至经灭菌后的一级试管斜面固体培养基上，25-35℃培养4-8d。其培养基为察氏培养基：蔗糖3％、硝酸钠0.3％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％、琼脂粉1.2-1.8％，121℃灭菌20min。
2摇瓶培养
刮取1-2环步骤1中得到的一级试管斜面种子至二级摇瓶液体培养基中培养，摇瓶装液量为100-150mL/瓶。温度25-35℃、摇床转速160-200rpm培养2-6d，得到二级摇瓶种子液。其培养基配方为：麸皮2-6％、蛋白胨1-5％、葡萄糖2-6％、麦芽糊精4-8％，121℃灭菌40min。
3液体罐培养
将上述经步骤2得到的二级摇瓶种子液接种至三级液体罐种子液中，接种量为2-10％，温度25-35℃、转速100-180rpm培养2-6d得到三级种子液。其培养基配方为：麸皮2-6％、蛋白胨1-5％、葡萄糖2-6％、麦芽糊精4-8％，121℃灭菌40min。
4固体发酵工艺
将步骤3中的三级种子液接种于固体发酵培养基上，搅拌混匀，灌装于无菌发酵透气袋中，装料量为5-8kg/袋，平铺放置于浅盘上，在曲房中进行发酵培养。期间进行控温控湿处理，控制曲房相对湿度50％以上，料温28℃-32℃。发酵培养4-8d至菌丝明显、厚实、酶活增长缓慢为止。
固体发酵培养基按下述比例配制：麸皮80-90％、玉米粉5％-10％、豆粕粉2％-5％、硫酸铵1％-2％、硫酸镁0.05％-0.1％、磷酸氢二钾0.2％-0.3％、初始水分60％-70％，121℃灭菌40min。
5酶活检测结果
将由本公司提供的固体发酵工艺发酵得到的产物进行55-65℃烘干粉碎并过筛处理(40目)。依据国标酶制剂检测方法，对其进行酶活检测。由检测结果可知，该发酵产物中不仅含有较高酶活的果胶酶，还含有木聚糖酶、甘露聚糖酶、酸性蛋白酶、纤维素酶、淀粉酶、β-葡聚糖酶等。其中果胶酶酶活达132960U/g、木聚糖酶9024U/g、甘露聚糖酶5246U/g、酸性蛋白酶857U/g、纤维素酶2620U/g、β-葡聚糖酶1264U/g、淀粉酶2203U/g，结果如表1。
表1主要酶类酶活汇总表
Figure BDA0002528293860000071
实施例2
实施例1中的斜面培养基使用下述培养基替换；
葡萄糖2％、土豆汁20％、琼脂粉1.2-1.8％，121℃灭菌20min。
或，
麸皮汁2-5％、牛肉膏1-3％、蛋白胨0.5％-2％、磷酸氢二钾0.1-0.6％、硫酸镁0.2-0.4％、琼脂粉1.2-1.8％，121℃灭菌20min。
其余条件不变。
实施例1中的固体培养基使用下述培养基替换；
麸皮80-85％、豆粕5-10％、玉米粉5-10％、氯化铵1-2％、硫酸镁0.05％-0.1％、碳酸钙0.5％-1％、磷酸氢二钾0.2％-0.3％，初始水分60％-65％，121℃灭菌40min。
或，
麸皮80-90％、豆粕5-10％、棕榈粕粉5-10％、硫酸铵1-2％、硫酸镁0.05％-0.1％、硫酸锰0.01-0.02％、碳酸钙1％-2％、磷酸氢二钾0.2％-0.3％，初始水分60％-65％，121℃灭菌40min。
其余条件不变。
上述替换，均能实现本发明目的。
实施例3
一种富含酸性果胶酶的复合酶的制备，具体制备方法如下：
1斜面培养
将黑曲霉菌株CGMCC No.19614划线接种至经灭菌后的一级试管斜面固体培养基上，25-35℃培养6-10d。其培养基为察氏培养基：蔗糖3％、硝酸钠0.3％、磷酸氢二钾0.1％、硫酸镁0.05％、氯化钾0.05％、硫酸亚铁0.001％、琼脂粉1.2-1.8％，121℃灭菌20min。
2三角瓶培养
在超净工作台中刮取经步骤1培养得到的一级试管斜面孢子至装有150-200mL无菌水(内含100-500μL吐温-80)的玻璃珠瓶中，震荡混匀，使孢子充分悬浮于无菌水中。再按1-3mL/瓶固体种子量接种至二级三角瓶固体培养基上培养。28-35℃培养2-4d。培养基配方为：麸皮80-90％、豆粕10-15％、氯化铵1％-5％，初始水分55％-70％，121℃灭菌40min。每瓶培养基装量为15-20g。
3固体发酵工艺
向经步骤2发酵培养得到的二级三角瓶固体培养基种子中加入无菌水，搅拌使其成为孢子悬浮液，加水量为300-500mL/瓶。按照15-20瓶/吨固体料的量接种至固体发酵料上，搅拌混匀。灌装于无菌发酵透气袋中，装料量为5-8kg/袋，平铺放置于浅盘上，在曲房中进行发酵培养。期间进行控温控湿处理，控制曲房相对湿度50％以上，料温28℃-32℃，发酵培养4-8d至菌丝明显、厚实、酶活增长缓慢为止。
固体发酵培养基按下述比例配制：麸皮80-90％、玉米粉5％-10％、豆粕粉2％-5％、硫酸铵1％-2％、硫酸镁0.05％-0.1％、磷酸氢二钾0.2％-0.3％、初始水分60％-70％，121℃灭菌40min。
实施例4
酶蛋白检测分析结果
将由本公司提供的固体发酵工艺发酵得到的产物进行55-65℃烘干粉碎并过筛处理(40目)，向样品中加入8M尿素溶液提取样品中蛋白组分，利用LC-MS对其进行蛋白组分鉴定。先对蛋白进行定量，通过还原烷基化打开蛋白三维结构，酶解后抽提出肽段，运用质谱技术得到这些肽段的质谱图，最后运用蛋白质鉴定软件，鉴定出样品中的蛋白质。将所得结果在蛋白质库(黑曲霉库)中进行搜索比对，对比对结果进一步进行分析。
经检测分析结果可知，由本公司提供的固体发酵工艺发酵得到的发酵产物中含有丰富的酶蛋白组分(近60种)。是一个以果胶酶为主，其它多种酶为辅的复合酶系统。其中果胶酶种类多达18种，涉及果胶水解全过程，木聚糖酶类11种、纤维素酶类11种以及其它各酶类较多种。LC-MS谱图及部分酶蛋白组分如下：
果胶酯酶、果胶酯酶A、果胶裂解酶A、果胶裂解酶B、果胶裂解酶D、果胶裂解酶F、果胶酶I、果胶酶A、果胶酶B、果胶酶D、果胶酶E、半乳糖醛酸酶B、外聚半乳糖醛酸酶X、外聚半乳糖醛酸酶C、酸性α-淀粉酶、鼠李糖半乳糖醛酸裂合酶A、内聚半乳糖醛酸酶C、β-半乳糖苷酶C、阿拉伯半乳聚糖内酯β-1,4-半乳糖苷酶A、内切-1,4-β-木聚糖酶B、外-1,4-β-木糖苷酶xlnD、内切1,4-β-半乳聚糖酶A、α-L-阿拉伯呋喃糖苷酶axhA、α-L-阿拉伯呋喃糖苷酶B、NAD(P)H依赖性D-木糖还原酶xyl1、α-半乳糖苷酶B、α-岩藻糖苷酶A、β-葡萄糖醛酸酶、木聚糖酶A、阿魏酰基酯酶A、α-淀粉酶A型1/2、β-葡萄糖苷酶O、β-甘露糖苷酶A、葡糖淀粉酶I、α-葡萄糖苷酶、纤维素酶A、β-葡萄糖苷酶I、纤维素酶B、葡聚糖内切1,3-β-葡糖苷酶eglC、β-葡萄糖苷酶A、外切葡聚糖酶B、β-1,4-内切葡聚糖酶内切酶D、1,4-β-D-葡聚糖纤维二糖水解酶A、β-葡萄糖苷酶A、α-木糖苷酶A、葡聚糖内酯-1,3-β-葡萄糖苷酶EGLC、内切-β-1,4-甘露聚糖酶A、β-半乳糖苷酶A、阿魏酰基酯酶B、甘露糖基寡糖α-1,2-甘露糖苷酶1B、甘露醇-1-磷酸5-脱氢酶、β-半乳糖苷酶B、胞外菊粉酶、羧肽酶、枯草杆菌蛋白酶样丝氨酸蛋白酶pepC、天冬氨酸肽链内切酶、过氧化氢酶B等。
固体发酵产物应用效果评估实例(选用实施例1的发酵产物)
试验选在浙江某大型养鸡场进行。选用4000只体重相近、采食产蛋正常的47周龄的蛋鸡进行试验。随机分配为对照组与试验组，各2000只，两组试验同时进行。试验鸡自由采食及饮水，笼养，每日光照时间为16h，严格按照饲养管理进行试验，每日观察鸡群状况，并记录数据。
表2日粮中添加酸性果胶酶制剂对蛋鸡生产性能的影响
Figure BDA0002528293860000101
Figure BDA0002528293860000111
结果发现，在日粮中添加本发明提供的固体发酵产物(试验组)相比对照组，能够降低料蛋比，提高产蛋率。还可以减少粘粪现象，改善肠道健康。
序列表
<110> 杭州保安康生物技术有限公司
<120> 富含酸性果胶酶的复合酶的制备及其菌株和应用
<141> 2020-06-05
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> 黑曲霉BAK200345(Aspergillus niger)
<400> 1
tgcggaagga tcattaccga gtgcgggtcc tttgggccca acctcccatc cgtgtctatt 60
ataccctgtt gcttcggcgg gcccgccgct tgtcggccgc cgggggggcg cctttgcccc 120
ccgggcccgt gcccgccgga gaccccaaca cgaacactgt ctgaaagcgt gcagtctgag 180
ttgattgaat gcaatcagtt aaaactttca acaatggatc tcttggttcc ggcatcgatg 240
aagaacgcag cgaaatgcga taactaatgt gaattgcaga attcagtgaa tcatcgagtc 300
tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360
tgccctcaag cccggcttgt gtgttgggtc gccgtccccc tctccggggg gacgggcccg 420
aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc acatgctctg 480
taggattggc cggcgcctgc cgacgttttc caaccatttt ttccaggttg acctcggatc 540
aggtagggat acccgctgaa cttaagcata tcaata 576

Claims (7)

1.一种富含酸性果胶酶的复合酶的制备方法，其特征在于，
所用菌株为黑曲霉（Aspergillus niger）BAK200345，保藏号CGMCC No.19614；
培养的发酵产物中含有果胶酶、木聚糖酶、甘露聚糖酶、酸性蛋白酶、纤维素酶、淀粉酶以及β-葡聚糖酶。
2.根据权利要求1所述的一种富含酸性果胶酶的复合酶的制备方法，其特征在于，所述复合酶的制备包括固体发酵步骤；
将种子液接种于固体发酵培养基上，搅拌混匀，在曲房中发酵培养；
所述固体发酵培养基按下述比例配制：麸皮80-90%、玉米粉5%-10%、豆粕粉2%-5%、硫酸铵1%-2%、硫酸镁0.05%-0.1%、磷酸氢二钾0.2%-0.3%、初始水分60%-70%，121℃灭菌40 min。
3.根据权利要求2所述的一种富含酸性果胶酶的复合酶的制备方法，其特征在于，所述固体发酵培养基采用下述配方替换：
麸皮80-85%、豆粕5-10%、玉米粉5-10%、氯化铵1-2%、硫酸镁0.05%-0.1%、碳酸钙0.5%-1%、磷酸氢二钾0.2%-0.3%，初始水分60%-65%，121℃灭菌40 min；
或，
麸皮80-90%、豆粕5-10%、棕榈粕粉5-10%、硫酸铵1-2%、硫酸镁0.05%-0.1%、硫酸锰0.01-0.02%、碳酸钙1%-2%、磷酸氢二钾0.2%-0.3%，初始水分60%-65%，121℃灭菌40 min。
4.根据权利要求2所述的一种富含酸性果胶酶的复合酶的制备方法，其特征在于，对所述曲房进行控温控湿处理，控制曲房相对湿度50%以上，料温28℃-32℃；发酵培养4-8 d至菌丝明显、厚实、酶活增长缓慢为止。
5.根据权利要求2所述的一种富含酸性果胶酶的复合酶的制备方法，其特征在于，还包括发酵产物的烘干步骤和粉碎步骤；其中，烘干的温度为55-65℃。
6.一种黑曲霉（Aspergillus niger），命名为黑曲霉BAK200345，其保藏号为CGMCCNo.19614。
7.一种畜禽饲料，包括权利要求1中所述的黑曲霉BAK200345的发酵产物。
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