CN115216431B - 一株来源玉米芯的多功能枯草芽孢杆菌及其应用 - Google Patents
一株来源玉米芯的多功能枯草芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株来源玉米芯的多功能枯草芽孢杆菌及其应用,其分类命名为枯草芽孢杆菌(Bacillus subtilis),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,保藏日期为2022年04月24日,保藏号为CGMCC NO:24759。本发明从玉米芯中发酵出能高效分泌淀粉酶、纤维酶和蛋白酶的菌株Bacillus subtilis LY12,并将该菌株再次接回玉米加工副产物中进行发酵,菌株筛选及产品发酵的目的针对性强,能较好的分解玉米加工副产物中粗纤维含量,提高其蛋白质、葡萄糖等营养物质,改善了饲料品质,再加上益生菌的大量生成,促进了玉米加工副产物发酵制剂产品的营养价值提升和推广应用,拓宽了玉米加工副产物生产产业链,具有良好的应用前景。
Description
技术领域
本发明属于食品生物技术领域,具体涉及一株来源玉米芯的多功能枯草芽孢杆菌及其应用。
背景技术
玉米芯是玉米果穗脱去子粒后的穗轴,主要成分包括多糖54.5%、粗蛋白质2.2%、粗脂肪0.4%、粗纤维29.7%、矿物质1.2%,以淀粉多糖和粗纤维为主,并含有一定量的粗蛋白。我国每年生产大量的玉米芯,达2千万吨以上,但目前大部分玉米芯仅被当做燃料烧掉,一方面对环境造成了较大的污染,另一方面也是一种资源浪费。
随着2020年7月开始中国饲料全面禁抗,微生物发酵饲料成为了饲料“禁抗替抗”研究的热点。研究表明,通过微生物发酵玉米芯、秸秆、米糠等农业副产物,不仅能提升这些副产物的经济效益,能防止环境污染,还能开发出一系列新型的饲料资源,拓宽农业副产物产业链,促进资源的多级转化和循环发展。
有鉴于此,本发明提供一株来源于玉米芯的多功能枯草芽孢杆菌及其应用。
发明内容
本发明的目的在于提供一株来源于玉米芯的多功能枯草芽孢杆菌及其应用。
为了实现上述目的,本发明采用的技术方案如下:
本发明从玉米芯中筛选获得能够同时高产淀粉酶、蛋白酶和纤维酶的枯草芽孢杆菌,其分类命名为枯草芽孢杆菌(Bacillus subtilis),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,保藏日期为2022年04月24日,保藏号为CGMCC NO:24759。
将所述枯草芽孢杆菌(Bacillus subtilis),接种至LB液体培养基中发酵扩培至总菌数不低于108CFU/mL后,再按2~3%接种量与经过干燥的玉米芯、玉米皮(包括玉米须)、玉米粉等载体混合,含水量为35~40%,然后装入带有单向呼吸阀的PE薄膜中进行发酵7~10d,制备成玉米加工副产物发酵制剂。
玉米加工副产物发酵制剂产品发酵前后的性能测定如表1所示。发酵后样品颜色加深,其产品色泽、手感、质地明显改善;与发酵前相比,玉米加工副产物发酵制剂产品的葡萄糖、粗蛋白含量明显增加,中性洗涤纤维、酸性洗涤纤维含量下降,其能量营养物质增加,提高了饲料消化率,改善了饲料品质,再加上益生菌的大量生成,促进了玉米加工副产物发酵制剂产品的营养价值提升和推广应用。
表1玉米加工副产物制剂发酵前后产品性能的测定
本发明具有以下有益效果:
本发明依次采用淀粉酶培养基、纤维素酶培养基和蛋白酶测定培养基从玉米芯粉中一株具有良好的能分泌淀粉酶、纤维酶和蛋白酶的菌株Bacillus subtilis LY12,并将该菌株接种至玉米芯、玉米皮、玉米粉等玉米加工副产物中进行发酵,制备发酵制剂,结果表明,所得的玉米加工副产物发酵制剂产品的葡萄糖、粗蛋白含量明显增加,中性洗涤纤维、酸性洗涤纤维含量下降,其能量营养物质增加,提高了饲料消化率,改善了饲料品质。
本发明筛选针对适合玉米芯、玉米皮、玉米粉等玉米加工副产物中的发酵菌株,从玉米芯中发酵出能高效分泌淀粉酶、纤维酶和蛋白酶的菌株Bacillus subtilis LY12,并将该菌株再次接回玉米加工副产物中进行发酵,菌株筛选及产品发酵的目的针对性强,能较好的分解玉米加工副产物中粗纤维含量,提高其蛋白质、葡萄糖等营养物质,改善了饲料品质,再加上益生菌的大量生成,促进了玉米加工副产物发酵制剂产品的营养价值提升和推广应用,拓宽了玉米加工副产物生产产业链,具有良好的应用前景。
附图说明
图1为菌株在不同培养基上的水解圈。
图2为菌株LY12的平板菌落形态。
图3为PCR电泳图。
图4为菌株LY12的测序序列结果。
图5为LY12菌株同源关系图。
具体实施方式
实施例1菌株的筛选、分离鉴定
依次采用淀粉酶培养基、纤维素酶培养基和蛋白酶测定培养基从玉米芯粉中分离发酵菌株,并通过培养菌落的形态观察、显微鉴定,确定所分离发酵菌株的基本生物学特性,并对该菌株进行16srDNA系列引物测序,在NCBI上与已知菌株进行序列比对,确定该菌株的种属类型和亲缘关系。
具体工艺流程如下:
(1)配置淀粉酶培养基:蛋白胨10g,酵母膏4~6g,NaCl 4~6g,可溶性淀粉9~10g,琼脂20~25g,加水至1000mL,pH 8.0~8.2。
配置纤维素酶培养基:磷酸二氢钾0.5~0.6g,硫酸镁0.20~0.30g,枯树叶4~6g,琼脂20~25g,加水至1000mL,pH值7.0~7.2
配置蛋白酶培养基(明胶培养基):牛肉膏2~4g,蛋白胨8~10g,氯化4~6g,明胶120~180g,加水至1000mL,pH值7.2~7.4。
(2)菌株的分离
取粉碎后玉米芯粉末10g,接入到90mL无菌水中,再依次梯度稀释到10-6。
吸取10-4、10-5、10-6稀释的菌悬液0.1mL涂布在淀粉酶初筛培养基中,挑选水解圈大的菌株再进行适当梯度稀释涂布于纤维素酶培养基,再次挑选水解圈较大菌株进行适当梯度稀释涂布于蛋白酶培养基中,最终挑选得到一株具有良好的能分泌淀粉酶、纤维酶和蛋白酶的菌株LY12(图1、表2)。
表2蛋白酶、淀粉酶、纤维素酶的水解圈直径(mm)
(3)菌株的鉴定
将选定平板菌株(命名LY12)进行LB培养皿平板菌落纯化,观察其菌落及显微形态鉴定,提取其DAN,送至北京奥维森基因科技有限公司进行测序,登录NCBI比对,确定其进化关系和属性。平板菌落形态见图2所示。结果表明:所挑选的菌符合细菌的基本形态及生物学特征,菌落形态为灰白色圆形,菌落边缘微突起,不透明,表面褶皱,边缘锯齿状。革兰氏染色阳性,显微镜下观察菌体形态菌体呈杆状,有芽孢、端生,不膨大,椭圆型,宽0.7~0.9μm,长2.2~3μm。PCR-测定菌种16srDNA序列,引物序列采用微生物通用引物27F和1492R,进行PCR扩增,反应体系为(25μL):PCR TaqMix(2*)12.5μL、PrimerF(10μM)0.2μL、PrimerR(10μM)0.2μL、DNA模板1.0μL、H2O 11.1μL。PCR扩增程序为:94℃,5min;94℃60S;60℃60S;72℃1min;30个Cycle;72℃10min,1%琼脂糖凝胶电泳检测。扩增片段长度740bp左右,PCR电泳图和测定的序列如图3、图4所示。
将该序列登录NCBI进行Blast比对,其同源关系如图5所示。结果表明,所分离的LY12菌株与枯草芽孢杆菌(Bacillus subtilis)的几个菌株的亲源关系最近,相似性均为1005,综合LY03菌株的形态特征以及l6S rDNA序列分析结果,将其鉴定为定为枯草芽孢杆菌(Bacillus subtilis),并将所保存的菌株送至中国普通微生物菌种保藏管理中心(China General Microbiological Culture Collection Center,CGMCC)进行专利保藏,保藏号:CGMCC No.24759。
实施例2玉米加工副产物发酵制剂的制备
将分离保藏的LY12菌株,接种至LB液体培养基中发酵扩培至总菌数不低于108CFU/mL后,再按2%的接种量与经过干燥的玉米芯、玉米皮、玉米粉混合,其中,各组分重量份如下,玉米芯35份,玉米皮25份,玉米粉5,蒸馏水35份,然后装入带有单向呼吸阀的PE薄膜中进行发酵10d,发酵温度为28℃,制备成玉米加工副产物发酵制剂。
发酵成品性能检测:外观颜色变深、气味柔和,不刺彝,手抓较松散,质地柔软而略带湿润。总菌数达到5.8×1010CFU/g;发酵后营养组成为(%,m/m):葡萄糖含量13.78%,中性洗涤纤维含量18.56%、酸性洗涤纤维含量6.38%,粗蛋白含量31.31%。
实施例3玉米加工副产物发酵制剂的制备
将分离保藏的LY12菌株,接种至LB液体培养基中发酵扩培至总菌数不低于108CFU/mL后,再按3%的接种量与经过干燥的玉米芯、玉米皮、玉米粉混合,其中,各组分重量份如下,玉米芯35份,玉米皮15份,玉米粉10,蒸馏水40份,然后装入带有单向呼吸阀的PE薄膜中进行发酵7d,发酵温度为25℃,制备成玉米加工副产物发酵制剂。
发酵成品性能检测:外观颜色变深、气味柔和,不刺彝,手抓较松散,质地柔软而略带湿润。总菌数达到6.4×1010CFU/g;发酵后营养组成为(%,m/m):葡萄糖含量14.23%,中性洗涤纤维含量17.25%、酸性洗涤纤维含量6.35%,粗蛋白含量31.40%。
Claims (1)
1.枯草芽孢杆菌在玉米加工副产物发酵制剂中的应用,其特征在于,所述枯草芽孢杆菌命名为LY12,分类命名为枯草芽孢杆菌(Bacillus subtilis),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,保藏日期为2022年04月24日,保藏号为CGMCC NO:24759;
所述应用是将所述枯草芽孢杆菌LY12接种至LB液体培养基中,发酵扩培至总菌数不低于108CFU/mL后,再按2~3%接种量与经过干燥的玉米芯、玉米皮或玉米粉混合,控制含水量为35~40%,然后装入带有单向呼吸阀的PE薄膜中进行发酵7~10d,制备成玉米加工副产物发酵制剂。
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