CN117106676B - 一株枯草芽孢杆菌及其在饲料生产中的应用 - Google Patents
一株枯草芽孢杆菌及其在饲料生产中的应用 Download PDFInfo
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- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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Abstract
本发明从猪场分离得到一株新的枯草芽孢杆菌BS‑22株,具有较高的产纤维素酶能力,对于金黄色葡萄球菌、大肠杆菌K88和K99株均具有较强的抑制作用,在亚硝酸盐降解方面也表现优异,具有优越的耐热性能。将所述的BS‑22株用于豆粕的发酵后,能够充分分解蛋白质,将其降解为吸收和利用效果更好的小肽类成分,发酵豆粕中小肽含量≥22.4%;在基础日粮的基础上添加本发明的枯草芽孢杆菌BS‑22株菌粉,可以提高保育猪的日均增重,降低料肉比。
Description
技术领域
本发明属于生物技术领域,涉及一株枯草芽孢杆菌(Bacillus subtilis),还涉及其在饲料生产中的具体应用,例如作为发酵剂、微生态制剂等。
背景技术
枯草芽孢杆菌(Bacillus subtilis)也称为枯草杆菌,是一类需氧型、内生抗逆孢子的杆状革兰氏阳性菌,自身没有致病性,可以分泌淀粉酶、蛋白酶、脂肪酶和纤维素酶等酶类。枯草芽孢杆菌在土壤及植物体表广泛存在,可形成孢子,且抗逆性好,在芽孢状态具有良好的稳定性,通常在120℃的高温处理下能够存活20min左右,酸性环境也可保持存活,因此,其可以在畜禽养殖生产和应用当中保持较高的活力,属于一种高效微生态制剂菌种。另外,有研究表明,枯草芽孢杆菌产生溶菌物质因而具有抑菌活性;还能够在畜禽肠道内生营养细胞,与肠道粘膜位点结合、定植,形成生物屏障后可以有效阻止病原微生物的吸附与生长;通过产生各种消化酶,例如蛋白酶、脂肪酶和淀粉酶等,能够提高饲料的转化率,促进动物的生长。近年来,也有研究表明微生态制剂可以迅速降解、吸附和转化水体中的有机污染物、氮和磷等,其中枯草芽孢杆菌能将动物***物、残存饲料、硝酸盐、磷酸盐等分解为小分子有机酸、氨基酸,为水体中的藻类提供营养,防止水体的富营养化,维持水体生态平衡;还可以有效降低粪便中的氨气、吲哚等有害气体的浓度,减轻粪便臭味,其代谢产物也能抑制蚊蝇的生长繁殖,改善养殖环境,有利于粪便的资源化。
目前,枯草芽孢杆菌在饲料生产中应用广泛,我公司也着力于分离获得性能更好的新菌株,并积极拓展枯草芽孢杆菌在饲料生产、养殖业中的应用范围,先后分离得到了枯草芽孢杆菌C-1株(CGMCC No:4628)、枯草芽孢杆菌1-c-7株(CGMCC No:17348),并申请了发明专利CN201110116641.X和CN20201161705.6,前者具有极强的抑菌作用和产纤维素酶的能力,可以用于畜禽的养殖,后者主要集中在水产领域应用,可以提高水产动物肝脏组织的健康水平,增强鳜鱼的抗病能力。也有大量其他公司或高校研究单位对枯草芽孢杆菌进行了分离和鉴定,探索了其在饲料生产中的应用,且随着饲料“无抗”概念的提出和推广,枯草芽孢杆菌的分离和应用被提到了前所未有的新高度。目前枯草芽孢杆菌在饲料行业、养殖产业大规模应用的瓶颈问题仍然是优良菌株的分离和筛选,因此,如何筛选得到可以有效突破胃酸、胆汁屏障,真正进入肠道内繁殖并发挥益生作用、产酶能力强、抑菌活性高、应用范围广的菌株是目前亟需解决的技术问题。
发明内容
为了解决现有技术中存在的问题,本发明旨在提供一株抗逆性强、益生功能优秀,且具有抑菌作用和较强亚硝酸盐降解能力的枯草芽孢杆菌,并探索其在饲料生产中的应用。
为解决以上技术问题,本发明采用如下技术方案:
本发明提供一株枯草芽孢杆菌BS-22株,其特征在于,所述枯草芽孢杆菌BS-22株于2023年05月18日在中国微生物菌种保藏管理委员会普通微生物中心进行保存,命名为BS-22株,分类命名为:枯草芽孢杆菌(Bacillus subtilis),保藏号为:CGMCC No:27370,保藏地址为: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。
本发明还请求保护所述枯草芽孢杆菌BS-22株菌粉的制备方法,其特征在于,包括如下步骤:
步骤一,枯草芽孢杆菌BS-22株的发酵:
(1)发酵培养基的配制:玉米面0.5%,黄豆饼粉1%,蔗糖0.4%,鱼粉0.6%,KH2PO40.1%,FeSO4.7H2O 0.025%,MgSO4.7H2O 0.05%,MnSO4 0.024%,CaCO3 0.1%,消泡剂0.05%,pH约7.2;
(2)发酵罐培养:121℃灭菌30min,降温至37℃,向其中接入菌龄为12h的种子液,接种量为1%,发酵期间保持转速220rpm,通气量1:0.3vvm,罐压0.05MPa,培养至24h为发酵终点,检测枯草芽孢杆菌BS-22株的活菌数≥1.0×1011CFU/mL;
步骤二,喷雾干燥制备菌粉:在枯草芽孢杆菌BS-22株发酵菌液中加入10%w/v的玉米淀粉作为保护剂,搅拌均匀后进行喷雾干燥,喷雾干燥条件为:进风口温度160℃,出风口温度85℃,进料速度3L/h。收集得到枯草芽孢杆菌干粉制剂,经检测,菌粉中枯草芽孢杆菌BS-22株的活菌数≥5.0×1010CFU/g。
本发明还请求保护一种枯草芽孢杆菌BS-22株菌粉,所述菌粉采用上述方法制备得到。
本发明还请求保护所述的枯草芽孢杆菌BS-22株在制备发酵豆粕中的应用。
进一步地,所述发酵豆粕采用如下方法制备得到:将豆粕、麸皮粉与水按照1:0.5:1的质量比例混合均匀,经121℃灭菌15分钟,冷却后接种枯草芽孢杆菌BS-22株菌粉混合均匀得到发酵组合物,其中枯草芽孢杆菌的添加量为108CFU/kg,32℃下发酵3天,发酵结束后,将发酵豆粕烘干后,测定其中的小肽含量,小肽含量≥22.4%。
本发明还请求保护枯草芽孢杆菌BS-22株作为饲料添加剂的应用,其特征在于,在普通日粮中添加0.1‰的枯草芽孢杆菌BS-22株菌粉(活菌数≥5.0×1010CFU/g)后用于动物的饲喂。
进一步,优选地,所述动物为畜、禽、反刍动物,优选为保育猪。
基于以上技术方案,本发明具有如下优点和有益效果:
本发明从猪场分离得到一株新的枯草芽孢杆菌BS-22株,相对而言,该菌株具有较高的产纤维素酶能力,对于金黄色葡萄球菌、大肠杆菌K88和K99株均具有较强的抑制作用,在亚硝酸盐降解方面也表现优异,且经溶血试验表明其也安全无害,具有优越的耐热性能,能避免高温加工对于菌株活性的影响,更加适合饲料添加剂和饲料加工过程。将所述的BS-22株用于豆粕的发酵后,具有相对较强的降解能力,能够充分分解蛋白质,将其降解为吸收和利用效果更好的小肽类成分,发酵豆粕中小肽含量≥22.4%;在基础日粮的基础上添加本发明的枯草芽孢杆菌BS-22株菌粉,在没有大幅改变平均日采食量的情况下,可以提高保育猪的日均增重,降低料肉比。
实施方式
实施例1:枯草芽孢杆菌的分离与筛选
1.1 菌株的初步分离:
取本集团公司旗下黑龙江地区、辽宁地区的猪场猪粪、猪粪废水暂存池、两级AO工段污水和沼液还田土壤等环境样品共30份,每个样品取样10 g,取样后分别置于100 ml猪粪模拟培养基(猪粪模拟培养基配方为:蛋白胨10 g/L、酵母粉10 g/L、乙酸钠10 g/L、柠檬酸钠10 g/L、植物油5 g/L、甘油5 g/L、羧甲基纤维素钠5 g/L、氯化铵2 g/L、硝酸钠2 g/L、磷酸氢二钾2 g/L、硫酸亚铁0.1 g/L、硫酸镁0.5 g/L、氯化钙0.1 g/L,pH 6.8-7.3)中,在25℃、200 rpm的条件下培养5-6天,然后取10 ml培养液接种至新的猪粪模拟培养基中进行传代,连续传代3次后即得到富集培养液,而后送由北京大北农饲用微生物工程国家重点实验室进行进一步的分离、筛选与鉴定。
采用无菌PBS液将富集培养液进行10倍稀释,振荡混匀制成菌悬液,放入水浴锅中,90℃加热15分钟,之后取上清液,8000rpm离心10分钟,弃去上清,菌体沉淀加入10mL人工胃液(1%胃蛋白酶,0.85%氯化钠,采用HCl溶液调节pH至2.0,过滤除菌后制备得到人工胃液)中,37℃静置2h,而后6000rpm离心10分钟,弃去上清,菌体沉淀加入10mL人工胆盐(在液体肉汤培养基中添加0.3%的猪胆盐,高压灭菌后制备得到人工胆盐)中,37℃静置4h。震荡混匀后,采用无菌PBS梯度稀释后,涂布到LB培养基上,30℃培养24h后,纯化菌落。共分离获得了5株疑似枯草芽孢杆菌,而后将初步分离得到的5株菌株继续在LB培养基上进行分离纯化,分别命名为BS-05,BS-10,BS-17,BS-22,BS-28。
1.2 菌株的复筛
1.2.1 枯草芽孢杆菌的溶血性测试
将分离得到的5株枯草芽孢杆菌BS-05,BS-10,BS-17,BS-22,BS-28分别划线接种至新鲜的血液培养基上,37℃培养24h,经观察,5株菌株均未出现溶血环,表明分离得到的5株枯草芽孢杆菌没有溶血活性,对于动物而言是安全的。
1.2.2 产纤维素酶能力的筛选
采用接种环将分离纯化出来的菌株在0.5%羧甲基纤维素的LB-CMC培养基上进行点菌,30℃培养24h,用刚果红染色1小时,弃去染料后再用1M的NaCl溶液洗涤1h,根据菌落周围有无出现水解透明圈,测定透明圈与菌落直径,计算透明圈与菌落直径的比例(H/C),同时以本公司早期分离得到的枯草芽孢杆菌C-1株(CGMCC No:4628)、枯草芽孢杆菌1-c-7株(CGMCC No:17348)作为对照菌株。具体检测结果如下表1所示:
表1 枯草芽孢杆菌产纤维素酶能力测试-透明圈与菌落直径的比例(H/C)
基于以上试验结果可知,本发明所分离得到的BS-17株、BS-22株均表现出较高的产纤维素酶能力,其与本公司先前分离得到的高产纤维素酶的C-1株的产酶能力相当,其中BS-22株的产酶能力相对而言最高。
1.2.3 抑菌能力测定
以大肠杆菌K88、大肠杆菌K99和金黄色葡萄球菌为指示菌,检测分离得到的枯草芽孢杆菌的抑菌活性。具体将LB固体培养基融化后,冷却至45℃左右,加入过夜培养的指示菌培养液(每100毫升培养基加入100微升菌液),震荡混匀,倒入无菌平板,每皿倒入15-20mL。每个平板上预先放置2只经灭菌的牛津杯,琼脂凝固后将牛津杯取出,平板上形成琼脂孔。向琼脂孔中加入150μL待测菌液,设置3个重复,盖好皿盖,小心移到37℃培养箱中进行培养,平皿正放,静置培养。培养20小时后,打开皿盖,用卡尺测量抑菌圈的直径。具体抑菌效果如下表2所示:
表2 枯草芽孢杆菌的抑菌能力测定结果
基于以上抑菌性能检测结果可知,本发明分离得到的BS-17和BS-22株具有较好的抑菌能力,其中BS-22的抑菌能力最为突出,其对于金黄色葡萄球菌、大肠杆菌K88和大肠杆菌K99株均具有很强的抑菌性能,其对于大肠杆菌的抑菌能力接近C-1株的2倍。
1.2.4 枯草芽孢杆菌的降解亚硝酸盐能力测试
张峰峰(北农学报,2009,24(4):218-221)等报道了枯草芽孢杆菌可以迅速降低养殖水体中的硝酸盐和亚硝酸盐,具有净化水体的作用。而本发明的枯草芽孢杆菌即分离自猪场猪粪、猪粪废水暂存池、两级AO工段污水和沼液还田土壤等环境样品,其分离环境中含有大量的亚硝酸盐,因此,本发明进一步对枯草芽孢杆菌BS-17和BS-22株降解亚硝酸盐的能力进行了测试。具体测试方法如下:
(1)标准曲线绘制:称取分析纯亚硝酸钠配置浓度梯度标准溶液,各取200μL至96孔板中,每孔先后加入格里斯试剂A、B各 20μL。用酶标仪测定550nm波长下的吸光值。以吸光值为横坐标,亚硝酸钠浓度为纵坐标绘制标准曲线。
(2)亚硝酸盐降解培养基配制:LB液体培养基80mL,亚硝酸钠2.5mg,加蒸馏水定容至1000mL,分装到250mL三角瓶中,每瓶100mL,表明覆盖5mL石蜡油,以隔绝空气。121℃高压灭菌 20min。
(3)菌株降解亚硝酸盐能力测定:将过夜培养的菌液按1%接种量转接到亚硝酸盐降解培养基中,在第0h、12h、24h和48h分别取发酵液1ml,离心。吸取200μL上清液至96孔板中,先后加入格里斯试剂A、B各20μL,于波长550nm测定吸光值。根据标准曲线计算发酵液中亚硝酸钠的浓度。具体结果如下表3所示:
表3 枯草芽孢杆菌降解亚硝酸盐的能力测定
基于以上表3的结果可知,本发明所分离得到的枯草芽孢杆菌BS-22株降解亚硝酸盐的能力最强,其在12h内能够降解培养基95.2%的亚硝酸盐,在24h内能够降解98.4%的亚硝酸盐,在48h内降解率达到99.6%,其相比BS-17株以及现有的C-1株和1-c-7株具有相对更强的亚硝酸盐降解能力。
基于以上试验可知,本次分离得到的BS-22株相对其他分离株而言具有更强的纤维素降解能力、抑菌性能,在亚硝酸盐降解方面也表现优异,且经溶血试验表明其也安全无害,因此,将其确定为本次分离得到的最优菌株。
实施例2:枯草芽孢杆菌BS-22株的鉴定与保藏(CGMCC No:27370)
2.1形态学及生化鉴定:
经观察,枯草芽孢杆菌BS-22株细胞呈杆状,0.9-1.1μm×1.7-1.9μm,着色均匀,无荚膜,鞭毛侧生,运动,革兰氏染色呈现阳性。芽孢呈椭圆柱状,0.6-0.7μm×0.8-1.2μm。菌落为圆形,不闪光不透明,培养48h以后,菌落变厚变干,边缘出现不整齐的情况,菌落表面呈现微黄色。
根据《伯杰细菌鉴定手册》和《常见细菌鉴定手册》,对枯草芽孢杆菌BS-22株进行生理生化鉴定试验。具体生理生化特征见下表4。
表4 枯草芽孢杆菌BS-22株生理生化特征
注释:“+”表示阳性;“—”表示阴性。
2.2 16S rDNA序列测定
利用细菌16s rDNA通用引物(上游引物为5´-AGAGTTTGATCCTGGCTCAG-3´、下游引物:5´-GGTTACCTTGTTACGACTT-3´)扩增细菌的16s rDNA片段,测序后得到序列片段,所得序列与Genbank 中的16s rDNA序列进行分析比对,结果显示该菌与枯草芽孢杆菌C-1株的同源性高达99.5%,结合生理生化试验结果,进一步将该分离菌鉴定为枯草芽孢杆菌。
2.3 枯草芽孢杆菌BS-22株的保藏
对于分离得到的菌株于2023年05月18日在中国微生物菌种保藏管理委员会普通微生物中心进行保存,命名为BS-22株,分类命名为:枯草芽孢杆菌(Bacillus subtilis),保藏号为:CGMCC No:27370,保藏地址为: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。
实施例3:枯草芽孢杆菌BS-22株的抗逆性检测
(1)菌种活化:将保存的枯草芽孢杆菌BS-22株划线接种到LB培养基斜面,37℃培养24h。
(2)种子液制备:挑取活化后的单菌落2环接种到LB培养基,37℃,150rpm摇床培养24h。
(3)发酵液的制备:将种子液按照2%的接种量接种到LB培养基,37℃,150rpm摇床培养24h,制备得到枯草芽孢杆菌BS-22株发酵液。
3.1温度敏感性试验:
取发酵液分别在25℃、80℃、85℃、90℃、95℃和100℃条件下保温处理10min,以25℃处理组作为对照组,对各组菌液进行梯度稀释,均匀涂布在LB固体培养基平板,37℃培养48h后进行菌落计数,测定各处理组的活菌数和存活率,各组重复3次试验。具体结果见下表5。
表5. 枯草芽孢杆菌BS-22株耐热性能的测定
基于以上表的结果可知,本发明分离得到的枯草芽孢杆菌BS-22株具有优越的耐热性能,其经过90℃的温度处理10min后,存活率仍高达97.44%,高于C-1株报道的95.4%;经过95℃的温度处理后,存活率仍高达95.51%,在100℃处理10min,其残存活菌数仍高达89.26%,以上试验结果表明,本发明分离得到的枯草芽孢杆菌BS-22株具有优越的耐热性能,能避免高温加工对于菌株活性的影响,更加适合饲料添加剂和饲料加工过程。
3.2 耐人工胃液、人工胆盐试验:
人工胃液的配制方法:1%胃蛋白酶,0.85%氯化钠,用HCl调节pH至2.0,细菌过滤器过滤除菌备用。
人工胆盐的配制方法:在LB培养基中添加0.3%或0.6%的猪胆盐(分析纯)制备得到不同浓度的人工胆盐培养基,高压灭菌后备用。
取发酵液1mL,加入9mL pH=2的人工胃液作为试验组,同时取发酵液1mL,加入9mL生理盐水作为对照组,37℃静置培养5h,对两组发酵液分布进行梯度稀释,而后均匀涂布到LB培养基,37℃厌氧培养48h后计数。
存活率(%)=人工胃液处理组的活菌数/对照组活菌数×100%。
取发酵液1mL,加入9mL含有0.3%或0.6%胆盐溶液中,同时取发酵液1mL,加入9mL生理盐水作为对照组,37℃静置培养4h,对两组发酵液分布进行梯度稀释,而后均匀涂布到MRS培养基,37℃厌氧培养48h后计数。
存活率(%)=胆盐处理组的活菌数/对照组活菌数×100%。
表6 耐人工胃、胆盐试验结果
基于以上试验可知,本次新分离得到的枯草芽孢杆菌BS-22株对于人工胃酸和胆盐都具有很好的耐受能力,人工胃肠液处理后的存活率均高达94.14%,0.3%的胆盐处理对于枯草芽孢杆菌BS-22株的活力影响不大,高达0.6%的胆盐处理后,其存活率仍高达90%以上,相比C-1株具有更好的耐受性。
实施例4:枯草芽孢杆菌BS-22株菌粉的制备
4.1 枯草芽孢杆菌BS-22株的发酵:
(1)发酵培养基的配制:玉米面0.5%,黄豆饼粉1%,蔗糖0.4%,鱼粉0.6%,KH2PO40.1%,FeSO4.7H2O 0.025%,MgSO4.7H2O 0.05%,MnSO4 0.024%,CaCO3 0.1%,消泡剂0.05%,pH约7.2。
(2)发酵罐培养:121℃灭菌30min,降温至37℃,向其中接入菌龄为12h的种子液,接种量为1%,发酵期间保持转速220rpm,通气量1:0.3vvm,罐压0.05MPa,培养至24h为发酵终点,检测枯草芽孢杆菌BS-22株的活菌数≥1.0×1011CFU/mL。
4.2 喷雾干燥制备菌粉:
在枯草芽孢杆菌BS-22株发酵菌液中加入10%w/v的玉米淀粉作为保护剂,搅拌均匀后进行喷雾干燥,喷雾干燥条件为:进风口温度160℃,出风口温度85℃,进料速度3L/h。收集得到枯草芽孢杆菌干粉制剂,经检测,菌粉中枯草芽孢杆菌BS-22株的活菌数≥5.0×1010CFU/g。
实施例5:枯草芽孢杆菌BS-22株在制备发酵豆粕中的应用
将豆粕、麸皮粉与水按照1:0.5:1的质量比例混合均匀,经121℃灭菌15分钟,冷却后接种枯草芽孢杆菌菌粉混合均匀得到发酵组合物,其中枯草芽孢杆菌的添加量约为108CFU/kg,32℃下发酵3天。空白组为不接种枯草芽孢杆菌的豆粕和水的混合物,对照组采用的菌种为枯草芽孢杆菌C-1株,试验组采用的是枯草芽孢杆菌BS-22株。
发酵结束后,将发酵豆粕烘干后,测定其中的小肽含量。经检测,空白组中小肽含量为3.5%,对照组的小肽含量约为17.4%,而试验组的小肽含量≥22.4%。由此可见,本发明分离得到的BS-22株具有相对较强的降解能力,能够充分分解蛋白质,将其降解为吸收和利用效果更好的小肽类成分。
实施例6:枯草芽孢杆菌BS-22株作为饲料添加剂的应用
本试验在本公司黑龙江地区的猪场进行,在保育舍中随机选取初重相当、公母各半的2栏保育猪进行试验,每栏数量为20头,设其中1栏为对照组,1栏为试验组。试验组在普通日粮中添加0.1‰的枯草芽孢杆菌BS-22株菌粉(活菌数≥5.0×1010CFU/g)。对照组不添加菌种,饲喂普通日粮。试验周期为31天,普通日粮的组成相同,饲喂过程均由同一名饲养员完成,记录每只猪饲喂前后的重量。具体结果如下表7所示:
表7 保育猪饲喂结果
基于以上试验可知,在基础日粮的基础上添加本发明的枯草芽孢杆菌BS-22株菌粉,在没有大幅改变平均日采食量的情况下,可以提高保育猪的日均增重,降低料肉比。由此可见,本发明的枯草芽孢杆菌BS-22株菌粉可提高保育猪的消化吸收能力,增强动物对饲料的消化、转化能力,促进动物生长,显著地提高饲料利用率,提高养殖的经济效益。
Claims (4)
1.枯草芽孢杆菌(Bacillus subtilis)BS-22在制备饲料原料中的应用,所述枯草芽孢杆菌BS-22的保藏编号为:CGMCC No:27370,所述饲料原料为发酵豆粕。
2.根据权利要求1所述的应用,其特征在于,所述发酵豆粕采用如下方法制备得到:将豆粕、麸皮粉与水按照1:0.5:1的质量比例混合均匀,经121℃灭菌15分钟,冷却后接种所述枯草芽孢杆菌BS-22菌粉混合均匀得到发酵组合物,其中所述枯草芽孢杆菌BS-22的添加量为108CFU/kg,32℃发酵3天,发酵结束后,将发酵豆粕烘干后,测定其中的小肽含量,小肽含量≥22.4%。
3.枯草芽孢杆菌BS-22在制备饲料添加剂中的应用,其特征在于,在普通日粮中添加0.1‰的所述枯草芽孢杆菌BS-22菌粉后用于动物的饲喂,所述枯草芽孢杆菌BS-22菌粉中的活菌数≥5.0×1010CFU/g,所述枯草芽孢杆菌BS-22的保藏编号为:CGMCC No:27370,所述动物为保育猪。
4.根据权利要求3所述的应用,其特征在于,所述枯草芽孢杆菌BS-22菌粉的制备方法包括如下步骤:
步骤一,枯草芽孢杆菌BS-22发酵:
(1)发酵培养基的配制:玉米面 0.5%,黄豆饼粉 1%,蔗糖 0.4%,鱼粉 0.6%,KH2PO40.1%,FeSO4 .7H2O 0.025%,MgSO4.7H2O 0.05%,MnSO4 0.024%,CaCO3 0.1%,消泡剂0.05%,pH 7.2;
(2)发酵罐培养:121℃灭菌30min,降温至37℃,向其中接入菌龄为12h的种子液,接种量为1%,发酵期间保持转速220rpm,通气量1:0.3vvm,罐压0.05MPa,培养至24h为发酵终点,发酵活菌数≥1.0×1011CFU/mL;
步骤二,喷雾干燥制备菌粉:在枯草芽孢杆菌BS-22发酵菌液中加入10%w/v的玉米淀粉作为保护剂,搅拌均匀后进行喷雾干燥,喷雾干燥条件为:进风口温度160℃,出风口温度85℃,进料速度3L/h,收集得到枯草芽孢杆菌BS-22干粉制剂,经检测,菌粉中枯草芽孢杆菌BS-22的活菌数≥5.0×1010CFU/g。
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