CN117603889B - 饲用产酸性蛋白酶的枯草芽孢杆菌及其应用 - Google Patents
饲用产酸性蛋白酶的枯草芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域,提供一株饲用产酸性蛋白酶的枯草芽孢杆菌及其应用。本发明利用海带酶解粉含有丰富氨基酸、寡糖、维生素和微量元素等营养成分的优点,将其配制成高产酸性蛋白酶培养基,同时结合使用分离筛选得到的枯草芽孢杆菌SDHY‑2023001,可有效促进酸性蛋白酶的分泌表达,使得蛋白酶摇瓶发酵活力大幅度提高,最高可达7820 U/mL。该蛋白酶能够高效降解豆粕、棉粕和菜粕蛋白,使酸溶蛋白含量大幅度提高,该酶在猪、鸡等单胃动物饲料中具有很好的应用前景。
Description
技术领域
本发明属于微生物技术领域,具体地说,涉及一株饲用产酸性蛋白酶的枯草芽孢杆菌及其应用。
背景技术
饲料中蛋白质的消化是由消化道内的内源性蛋白水解酶系完成,通常包括胃蛋白酶、胰蛋白酶、糜蛋白酶、弹性蛋白酶、氨肽酶和羧肽酶等。幼龄动物由于消化道发育不完善,不能完全消化和吸收日粮中的蛋白质和氨基酸。未消化的日粮组分可能作为动物后肠微生物的发酵底物而发酵,产生胀气、腹泻等不良作用,进而导致单胃动物消化紊乱。饲料中添加外源性饲用蛋白酶可以通过提高日粮蛋白质的溶解性和水解作用而提高原料的蛋白质消化率,减少后肠不良发酵所需的底物。
猪、鸡等单胃动物的消化道前段呈酸性,更适合酸性蛋白酶发挥作用。因此,开发高效酸性蛋白酶的生产技术受到高度重视。除了饲料工业和食品工业以外,蛋白酶在酿造、皮革、纺织、医药等领域也都有着重要的应用。
发明内容
本发明的目的是提供一株饲用产酸性蛋白酶的枯草芽孢杆菌及其应用。
为了实现本发明目的,第一方面,本发明提供从山东威海荣成海边的土壤中分离并纯化得到的一株饲用酸性蛋白酶的专用菌株SDHY-2023001,该菌株在含有脱脂奶粉的固体筛选平板中可以产生透明圈。通过形态学观察、生理生化鉴定以及16S rDNA分子鉴定,最终确定菌株SDHY-2023001为枯草芽孢杆菌,分类命名为枯草芽孢杆菌Bacillus subtilis,现已保藏于中国微生物菌种保藏管理委员会普通微生物中心,地址北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编100101,保藏编号CGMCC No.29189,保藏日期2023年11月30日。
第二方面,本发明提供一种含有所述枯草芽孢杆菌SDHY-2023001的菌剂。
第三方面,本发明提供由所述枯草芽孢杆菌SDHY-2023001产生的酸性蛋白酶。
第四方面,本发明提供一种微生物发酵培养基(高产酸性蛋白酶培养基),50 mL发酵培养基中含有:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉7.5-15 g和海带粉(海带酶解粉)5.0-10.0 g。
优选地,50 mL发酵培养基中含有:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉15 g和海带粉5.0 g。
第五方面,本发明提供所述枯草芽孢杆菌SDHY-2023001、含有所述枯草芽孢杆菌SDHY-2023001的菌剂或由其产生的酸性蛋白酶在饲料领域中的应用。
第六方面,本发明提供所述枯草芽孢杆菌SDHY-2023001、含有所述枯草芽孢杆菌SDHY-2023001的菌剂或由其产生的酸性蛋白酶在制备饲料添加剂中的应用。
第七方面,本发明提供所述枯草芽孢杆菌SDHY-2023001、含有所述枯草芽孢杆菌SDHY-2023001的菌剂或由其产生的酸性蛋白酶在饲料原料的预消化中的应用。
所述饲料原料包括但不限于豆粕、棉粕、菜粕。
优选地,饲料原料的预消化条件为:pH3.0,温度40℃。
第八方面,本发明提供一种微生物发酵产酸性蛋白酶的方法,在所述发酵培养基中接种所述枯草芽孢杆菌SDHY-2023001,进行发酵培养,从获得的培养物中收集所产生的酸性蛋白酶。
进一步地,发酵条件为:接种量0.1% v/v,35℃、220 r/min发酵30 h。
50 mL发酵培养基中含有:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉15 g和海带粉5.0g。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明利用海带酶解粉含有丰富氨基酸、寡糖、维生素和微量元素等营养成分的优点,将其配制成高产酸性蛋白酶培养基,同时结合使用分离筛选得到的枯草芽孢杆菌SDHY-2023001,可有效促进酸性蛋白酶的分泌表达,使得蛋白酶摇瓶发酵活力大幅度提高,最高可达7820 U/mL。该蛋白酶能够高效降解豆粕、棉粕和菜粕蛋白,使酸溶蛋白含量大幅度提高,该酶在猪、鸡等单胃动物饲料中具有很好的应用前景。
附图说明
图1为本发明实施例1中菌株SDHY-2023001培养物在含有脱脂奶粉的固体筛选平板上的透明圈;其中,1为菌株SDHY-2023001。
图2为本发明菌株SDHY-2023001 16s rDNA基因PCR扩增结果。
图3为本发明较佳实施例中不同培养基中SDHY-2023001发酵上清液的酸性蛋白酶的相对催化活性。
图4为本发明较佳实施例中SDHY-2023001发酵上清液在不同温度下酸性蛋白酶的相对催化活性。
图5为本发明较佳实施例中SDHY-2023001发酵上清液在不同pH值条件下酸性蛋白酶的相对催化活性。
图6为本发明较佳实施例中酸性蛋白酶的耐酸性。
图7为本发明较佳实施例中酸性蛋白酶的耐热性。
图8为本发明较佳实施例中发酵温度对酸性蛋白酶活性的影响。
图9为本发明较佳实施例中发酵时间对酸性蛋白酶活性的影响。
图10为本发明较佳实施例中酸性蛋白酶水解豆粕、棉粕和菜粕的时间曲线。
具体实施方式
本发明提供一株产酸性蛋白酶的枯草芽孢杆菌及其应用。
发明人从山东威海荣成海边的土壤中分离并纯化得到了菌株SDHY-2023001。该菌株在含有脱脂奶粉的固体筛选平板中可以产生透明圈。采用芽孢杆菌16S rDNA基因特异引物对该菌株特异性片段进行扩增,将扩增产物测序,进行序列比对分析,结果发现与菌株SDHY-2023001高度同源的菌株为枯草芽孢杆菌(Bacillus subtilis),鉴定其为枯草芽孢杆菌。
本发明还提供一种微生物发酵培养基,枯草芽孢杆菌SDHY-2023001接种该培养基,发酵产生高活性酸性蛋白酶,该蛋白酶在pH3.0和40℃具有最佳催化活性;该菌株培养上清液可以高效降解豆粕、棉粕和菜粕蛋白。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
以下实施例中使用的海带粉购自威海市世代海洋生物科技有限公司。
实施例1 产酸性蛋白酶菌株的分离和筛选
称取5 g山东威海荣成海边有腐烂海带的土壤样品,溶解于100 mL LB液体培养基(胰蛋白胨10 g/L、酵母提取物5 g/L、氯化钠10 g/L,121℃高压灭菌15 min)中,37℃、220r/min振荡培养2~3 h,10000 r/min离心10 min收集菌体,用灭菌的生理盐水洗涤2~3次,用10 mL灭菌生理盐水悬浮,进行梯度稀释,直到10-8为止。涂布于LB平板(胰蛋白胨10 g/L、酵母提取物5 g/L、氯化钠10 g/L、琼脂10 g/L)上,37℃培养24 h,待菌落生长清晰为止。挑取单菌落划线接种LB平板培养基,37℃培养,待菌落生长清晰后,4℃冰箱保存。
挑取保存的菌种,接种于5 mL液体LB培养基中,37℃、220 r/min振荡培养24 h,10000 r/min离心5 min收集上清液。制备含有脱脂奶粉的固体筛选平板(琼脂2%、脱脂奶粉1%,pH3.0),在平板上打孔,在孔内加入100 μL培养上清液,37℃保温2 h,观察透明圈的大小,透明圈越大,蛋白酶活性越强。如图1所示。
实施例2 筛选菌株的分子鉴定
将实施例1中筛选出的菌株SDHY-2023001接种于LB液体培养基,在37℃摇床220r/min摇菌16 h以上,取1.5 mL培养液,于室温10000 r/min离心5 min,收集菌体,弃上清,用基因组DNA提取试剂盒(北京擎科生物科技股份有限公司产品)提取基因组DNA,Nanodrop测定DNA浓度。
PCR扩增体系包括:总基因组DNA 1.0 μL(约0.5 μg)、2×PCR mix 25.0 μL、引物27F和1492R各1.0 μL、灭菌水22.0 μL,总反应体积50.0 μL,混匀。引物和PCR mix均购自北京擎科生物科技股份有限公司。
引物27F:5’-AGAGTTTGATCCTGGCTCAG-3’;
引物1492R:5’-GGTTACCTTGTTACGACTT-3’。
PCR扩增条件为:94℃变性1 min,56℃退火30 s,72℃延伸2 min,扩增30个循环,1%琼脂糖凝胶电泳检测扩增产物,获得1条约1500 bp扩增条带,结果如图2所示。然后直接送公司测序,测序得到菌株SDHY-2023001的16s rDNA序列如SEQ ID NO:1所示。经过NCBI数据库在线比对(https://blast.ncbi.nlm.nih.gov/Blast.cgi),该菌与多种枯草芽孢杆菌的序列一致性都达到99%以上,因此,确定此菌种为枯草芽孢杆菌(Bacillus subtilis)。
实施例3 培养基组分对菌株SDHY-2023001发酵产酸性蛋白酶活力的影响
按照表1配制培养基,溶解于50 mL蒸馏水中,置于250 mL三角瓶中,调节pH值为7.0,121℃高温灭菌15~20 min。每个编号培养基做3个平行。菌种活化:挑取单菌落,接种于20 mL LB培养基,37℃、220 r/min振荡培养过夜,所得种子液的菌含量约为1.5×108CFU/mL,按0.1%的体积比转接发酵培养基,继续37℃、220 r/min振荡培养24 h,于4℃、10000 r/min离心5 min,收集上清液测定蛋白酶的活性(图3)。
表1 摇瓶发酵培养基组分构成
最适催化温度:在pH3.0条件下,测定20~70℃范围内测定上清液蛋白酶的相对催化活性,以最高酶活为100%,其他温度酶活与之相比较,结果如图4所示。
最适催化pH值:在40℃,测定pH2.0~7.0范围内上清液蛋白酶的相对催化活性,以最高酶活为100%,其他pH值的酶活与之相比较,结果如图5所示。
耐酸性测试:配制pH2.5的Na2HPO4-柠檬酸缓冲液。发酵上清液用该缓冲液按照1:10的体积比稀释,室温条件下(25℃)处理3 h,每隔30 min取样,在40℃和pH3.0的条件下测残余酶活。以未经过酸处理的酶活为100%,结果如图6所示。
耐热性测试:将发酵上清液分别在60℃、70℃、80℃和90℃处理30 min和60 min,然后在40℃和pH3.0的条件下测残余酶活,以未经处理的酶活为100%,结果如图7所示。
酸性蛋白酶活性测定参考行业标准(DB22/T 1819-2013)的方法进行,溶解酪蛋白为Na2HPO4-柠檬酸缓冲液(pH3.0)。计算每种培养基发酵的酶活平均值,以最高酶活为100%,其他酶活与之相比较,作图。
根据检测结果,3号培养基(50 mL培养基:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉15g、海带粉5.0 g),发酵产生酸性蛋白酶活性最高。
实施例4 SDHY-2023001的摇瓶发酵产酸性蛋白酶条件研究
培养基:选择实施例3中3号培养基。
菌株的活化:用接种环挑取SDHY-2023001单菌落接种于10 mL LB液体培养基中,于37℃摇床220 r/min培养过夜,备用。
发酵温度对酸性蛋白酶活性的影响:按照0.1% v/v接种量(种子液的菌含量约为1.5×108CFU/mL)接种发酵培养基,分别在31℃、33℃、35℃、37℃和39℃进行培养,摇床转速220 r/min,发酵时间24 h,每个温度同时做3个平行,计算酶活的平均值。以最高酶活为100%,其他温度的酶活与之相比较,计算相对酶活,结果如图8所示。
发酵时间对酶活的影响:按照0.1%接种量接种发酵培养基,在35℃、220 r/min,发酵时间48 h,每隔6 h取样测酶活。同时做3个平行,计算平均酶活。以最高酶活为100%,其他时间的酶活与之相比较,计算相对酶活,结果如图9所示。
菌株SDHY-2023001在35℃条件下,在3号培养基中发酵30 h,酸性蛋白酶的活性达到最高为7820 U/mL。
实施例5 SDHY-2023001发酵上清液对饲料原料的预消化效果评价
(1)酸性蛋白酶样品制备
配制3号培养基500 mL,置于10个250 mL三角瓶中,每瓶50 mL,高压灭菌。SDHY-2023001种子液制备同实施例3。35℃、220 r/min振荡培养30 h,10000 r/min离心10 min收集上清液。将酶液冻干,得酶粉,测定酶活为213300 U/g。
(2)豆粕、棉粕和菜粕的酶解效果评价
按照表2配制反应体系,缓冲液为Na2HPO4-柠檬酸缓冲液(pH3.0),置于250 mL三角瓶中。混匀后,封口膜封口,置于40℃水浴中连续酶解8 h,期间每隔30 min左右,摇匀30 s。每个样品同时做3个平行。每小时取样测定酸溶蛋白含量,做酶解时间曲线。按照农业行业标准“饲料原料中酸溶蛋白的测定(NY/T 3801-2020)”的方法测定酶解样品中酸溶蛋白的含量。从酸溶蛋白产量曲线(图10)可以看出,6 h酶解反应达到平衡,酸溶蛋白生成不再增加。
表2 不同饲料原料的酶解反应体系
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (8)
1.枯草芽孢杆菌(Bacillus subtilis)SDHY-2023001,保藏编号为CGMCC No.29189。
2.含有权利要求1所述枯草芽孢杆菌的菌剂。
3.权利要求1所述枯草芽孢杆菌或权利要求2所述菌剂在饲料领域中的应用。
4.权利要求1所述枯草芽孢杆菌或权利要求2所述菌剂在制备饲料添加剂中的应用。
5.权利要求1所述枯草芽孢杆菌或权利要求2所述菌剂在饲料原料的预消化中的应用;
所述饲料原料选自豆粕、棉粕、菜粕。
6.根据权利要求5所述的应用,其特征在于,饲料原料的预消化条件为:pH3.0,温度40℃。
7.微生物发酵产酸性蛋白酶的方法,其特征在于,在发酵培养基中接种权利要求1所述枯草芽孢杆菌,进行发酵培养,从获得的培养物中收集所产生的酸性蛋白酶;
50 mL发酵培养基中含有:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉7.5-15 g和海带粉5.0-10.0 g。
8.根据权利要求7所述的方法,其特征在于,发酵条件为:接种量0.1% v/v,35℃、220r/min发酵30 h;
50 mL发酵培养基中含有:葡萄糖2.5 g、玉米粉5.0 g、豆粕粉15 g和海带粉5.0 g。
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