CN107937305B - 一株耐丁酸、抗逆性强的丁酸梭菌菌剂及其应用 - Google Patents
一株耐丁酸、抗逆性强的丁酸梭菌菌剂及其应用 Download PDFInfo
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Abstract
本发明属于微生物发酵技术领域,具体涉及一株耐丁酸、抗逆性强的丁酸梭菌菌剂及其应用。所述丁酸梭菌具体为丁酸梭菌(Clostridium butyricum)LXKJYB‑1,保藏编号为:CCTCC NO:M 2017485。菌株LXKJYB‑1具有发酵水平高、有机酸产量高、可抑制多种肠道病原菌、对胃肠液、胆盐及湿热耐受能力高等特点。该菌株及由其制备的菌剂适合应用于畜禽类养殖,提高养殖效益,生产无抗畜禽产品。
Description
技术领域:
本发明属于微生物发酵技术领域,具体涉及一株耐丁酸、抗逆性强的丁酸梭菌菌剂及其应用。
背景技术:
丁酸梭菌是一种完全厌氧的革兰氏阳性芽孢杆菌,是人和动物肠道的正常菌群,也存在于土壤、奶酪和天然酸奶中;周身鞭毛,具运动性;芽孢偏心或次端生(内生),呈圆形或椭圆形,无孢外壁和附属丝。丁酸梭菌芽孢结构能够抵抗动物胃肠道恶劣环境,同时能够耐受饲料加工过程中的高温、高湿过程;丁酸梭菌只对新生霉素、万古霉素和四环素等少数抗生素敏感,对其他多种抗生素具有很强的耐药性,具有良好的应用前景。
丁酸梭菌能够防止病原菌及腐败菌在肠道内的异常增殖,同时还可以促进肠道有益菌群增殖、繁育,从而纠正肠道菌群紊乱,减少肠毒素的产生,其代谢产生的活性物质能增强动物的免疫功能,促进动物的生长,提高动物的生产性能,其代谢产生的酶类可降解饲料,提高饲料营养,代谢产生的丁酸、乙酸等有机酸不仅可以改善肠道环境、促进有益菌繁殖,还可以抑制有害菌增殖,提高饲料的适口性等;其还可以在肠道内代谢产生多种酶类、氨基酸、B族维生素和维生素K等,为宿主补充营养物质兼具其他保健功能;丁酸梭菌本身还能够分解胺类等有害物质,改善养殖环境;其重要代谢产生的氢气可以修复肝脏、肾脏的损伤。
目前丁酸梭菌已经做为饲料添加剂被允许在饲料中添加,而且做为抗腹泻类抗生素的替代产品已经在无抗养殖中起到了替抗效果,甚至作用比许多促生长抗生素效果还要明显。但是由于其发酵水平较低严重影响着丁酸梭菌的应用,如何降低丁酸梭菌生产成本迫在眉睫,通过诱变育种筛选高产并且抗逆性强的丁酸梭菌菌株,是丁酸梭菌产品推广的主要途径。
丁酸梭菌的发酵过程代谢产生大量的丁酸、乙酸等有机酸,丁酸、乙酸作为主要的益生物质决定了丁酸梭菌的益生效果,但是丁酸、乙酸的大量产生对丁酸梭菌发酵发酵水平及芽孢率影响很大,筛选耐丁酸、抗逆性强的丁酸梭菌是解决目前丁酸梭菌发酵水平及其应用的一种重要途径,因此筛选能够抵御酸胁迫丁酸梭菌菌种具有重要意义。在丁酸梭菌应用过程的高温处理以及进入动物胃肠道后的极端环境,是否能够耐热,耐胃肠道极端环境是丁酸梭菌筛选的一个重要指标,也是其应用价值高低的衡量指标。因此筛选抗逆性强且发酵水平高的丁酸梭菌菌种是丁酸梭菌广泛推广应用的重要途径。
发明内容:
为了解决上述技术问题,本发明筛选出了一株耐丁酸、抗逆性强的丁酸梭菌突变株,该菌株具体为丁酸梭菌(Clostridium butyricum)LXKJYB-1,已于2017年9月8日保藏于中国典型培养物保藏中心,地址:中国,武汉,武汉大学,邮编430072,保藏编号为:CCTCCNO:M 2017485。
所述LXKJYB-1是以丁酸梭菌(Clostridium butyricum)LXKJ-1为出发菌株,通过ARTP常温室压等离子诱变所得,该菌株菌落呈圆形,乳白色,菌落突起;芽孢呈椭圆形、芽孢壁厚。经诱变后,LXKJYB-1较LXKJ-1具有更高的抗逆性和抗菌活性,并且发酵水平较初始菌种提高很多。
具体如下:
(1)耐丁酸能力:较起始菌LXKJ-1耐丁酸能力提高了三倍,能够耐受40g/L丁酸钠;
(2)发酵能力:菌株LXKJYB-1发酵后,体系中的活菌数达到7.19×109cfu/mL,芽孢率达到98.79%;
(3)抗菌活性:该菌对大肠杆菌等病原菌的抑制率较初始菌提高了35-52%;对动物体内双歧杆菌、乳酸菌等益生菌促进增生的作用较原菌株也明显增强。
(4)抗逆性方面:耐高温能力、抗胃肠道极端环境能力较出发菌种增强。
本发明还提供一种采用上述耐丁酸的丁酸梭菌制备的益生菌菌剂,制备方法如下:
(1)将丁酸梭菌LXKJYB-1进行发酵培养,得到丁酸梭菌LXKJYB-1发酵液;
(2)发酵液离心后,冻干粉碎,制得冻干菌粉,由于其具有高芽孢率,耐冷、热性能强,所有不需要添加任何保护剂直接可以冷冻干燥。
应用效果:日粮中添加0.01%~0.08%丁酸梭菌LXKJYB-1冻干菌粉能够提高樱桃谷肉鸭成活率,促进生长,提高日增重;同时降低了料肉比、腹泻率和死亡率;增加樱桃谷肉鸭盲肠中乳酸菌和双歧杆菌的数量,降低大肠杆菌数量;该菌株在促生长,尤其降低腹泻率方面,较出发菌株更为突出,因此具有更强的应用价值。
有益效果:
1、本发明提供的丁酸梭菌突变株解决了传统菌株耐受丁酸能力差而导致的发酵水平相对较低的情况,本发明筛选的丁酸梭菌其发酵水平比起始对照菌株提高了3-4倍,厌氧发酵24h后活菌数达到7.19×109cfu/mL,芽孢率为98.79%,极大的提高了发酵水平,提高了经济效益。
2、本发明所制得的益生菌制剂,耐丁酸能力提高。丁酸梭菌在代谢过程中,丁酸类的代谢会降低生长环境的pH,在较低的酸性环境下,不利于有害菌的生长,而其本生的生长也会受到抑制,活菌数和芽孢数会降低。本发明,将诱变后丁酸梭菌单菌落接种于含有一定浓度丁酸钠的种子培养液中,培养12-15h,发现,诱变后丁酸梭菌的耐丁酸钠能力较诱变前提高了三倍多,由原来的18g/L丁酸纳最大承受浓度,提高到40g/L,并且在含有40g/L丁酸钠的培养基中培养24h后,OD600仍能达到1.432。
3、本发明所筛选的丁酸梭菌抗逆性较起始菌株有明显的提高,90℃处理15min后存活率保持在89±2.2%,且在人工胃液、肠液中的生存能力更强,在人工胃液中处理1h后,存活率保持在98.4±2.4%,在人工肠液处理2h后,存活率在98.0±2.3%,该菌株能够直接应用于畜禽养殖,生产无抗畜禽产品。
4、本发明提供的丁酸梭菌在实际应用过程中,对病原菌的抑制能力得到提高,对大肠杆菌、沙门氏杆菌、单增李斯特菌、志贺氏菌和金黄色葡萄球菌的抑制率分别达到84.34%、75.53%、73.07%、69.25%和80.03%,具有更强的应用价值。
5、本发明提供的丁酸梭菌生产有机酸的能力得到提高,经过20-24h的发酵培养,丁酸和乙酸产量分别达到12.19g/L、3.06g/L,从而提高了丁酸梭菌的益生效果。
6、本发明中,诱变后菌株所用发酵培养基价格低廉,更适于工厂规模化生产。
附图说明:
图1丁酸梭菌LXKJ-1生长曲线;
图2丁酸梭菌LXKJ-1致死曲线
图3丁酸梭菌LXKJYB-1与菌株LXKJ-1传代稳定性。
具体实施方式:
为了使本专利的目的、技术方案及优点更加清楚明白,以下结合具体实施例,对本专利进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本专利,并不用于限定本发明。
本发明涉及的培养基如下:
RCM液体培养基:酵母浸膏3g,牛肉浸膏10g,胰蛋白胨10g,葡萄糖5g,可溶性淀粉1g,氯化钠5g,三水合乙酸钠3g,半胱氨酸盐酸盐0.15g,蒸馏水1000ml,pH:7.1
TPY培养基:胰蛋白胨10g,大豆胨5g,酵母粉3g,月示蛋白胨10g,葡萄糖10g,L-半胱氨酸盐酸盐0.3g,磷酸氢二钾2.5g,硫代乙醇酸钠0.3g,氯化钠3g,琼脂20g,蒸馏水1000mL,pH6.5
活化及种子液培养基:RCM培养基。
斜面培养基为:酵母膏1%,葡萄糖1%,琼脂2%,pH 6.5~7.0。
一级种子培养基为:硫酸铵2-5%,玉米粉2-5%,酵母浸膏2-5%,尿素1-3%,磷酸氢二钾0.5-3%,pH 6.5~7.0;
二级种子培养基为:硫酸铵2-5%,玉米粉2-5%,酵母浸膏2-5%,尿素1-3%,磷酸氢二钾0.5-3%,pH 6.5~7.0,碳酸钙0.5-1%;
三级发酵罐培养基为:硫酸铵2-5%,玉米粉2-5%,酵母浸膏2-5%,尿素1-3%,磷酸氢二钾0.5-3%,pH 6.5~7.0,碳酸钙0.5-1%;
以上所有的培养基在使用前都需要蒸汽灭菌(121℃,30min),冷却待用。
实施例1、LXKJYB-1的诱变培育
(1)悬浮液制备:
将甘油管保藏的丁酸梭菌LXKJ-1菌株进行活化,挑取单菌落接种在RCM液体培养基中,根据生长曲线培养到8-10h,即在对数期时收集菌液进行离心,收集菌体,用生理盐水洗涤两到三次,制得诱变悬浮液后进行诱变,诱变液菌体浓度为2×106cfu/ml。生长曲线见图1。
(2)制作致死曲线
诱变处理剂量或时间不仅影响致死率,同时能显著影响突变效率,在一个合适的突变效率区间进行有效的筛选是菌株选育的关键之处。本研究首先对出发菌株进行不同时间的等离子体射流处理:诱变时间为:15s、30s、45s、60s、90s、120s、150s、180s,设置:射频功率(W):115;等离子体发射源与载样台之间的距离(mm):2;气体流速(L/min):10。
将诱变后的菌悬液,用生理盐水稀释至10-3,10-4,加样量100μL,涂布至计数TPY培养基上,37℃厌氧培养24h,待平板上长出成熟菌落体后,记录数据,同时根据诱变时间测得致死率,根据诱变致死率绘制致死曲线。
使用ARTP常温室压等离子诱变技术对菌悬液进行诱变,常温室压等离子体对出发菌株的损伤作用效果明显,随着等离子体射流处理时间的增加,其致死率逐渐增大。处理时间90s杀死约90%的菌体;处理时间达到120s以上时,菌株致死率接近100%。据文献报道,当致死率在90%左右时,诱变处理对细胞的诱变效应较强,所以诱变时间采用90s处理时间(致死率约为90%)对菌株进行诱变。致死曲线见附图2。
(3)耐丁酸菌株的初筛:
将90s诱变后长出的菌落,分别编号,进行甘油管保藏。后将保藏菌株活化至RCM液体培养基中,再接种到含有不同浓度丁酸钠的初筛培养基37℃厌氧,24h培养。通过紫外分光光度计在OD600处分别测得最大吸光值,记录,选出高出原菌株丁酸钠耐性的菌株,再转接到斜面培养基上进行保存。
初筛培养基:蛋白胨2%,酵母浸粉2%,磷酸氢二钾0.05%,氯化钠0.05%;
丁酸钠添加量:添加1-40g/L的丁酸钠。
(4)复筛:
将初筛所得耐丁酸能力强的菌株转接五代后,进行摇瓶发酵复筛:以起始菌株活菌数作为对照,通过发酵水平比较,培养条件为(37±1)℃,厌氧,培养20h,测定发酵液活菌数及芽孢数。挑选较高活菌数的菌株,复筛结果见表2。
复筛培养基:硫酸铵2-5%,玉米粉2-5%,酵母浸膏2-5%,尿素1-3%,磷酸氢二钾0.5-3%,pH 6.5~7.0。
(5)结果与分析:
在最佳诱变时间90s的条件处理下,共得到长势较好的菌株共计228株,通过丁酸钠筛选,后得到丁酸钠耐性较高的菌株,共11株,正突变率达到0.04%(正突变率(%)=正突变株株数÷所有筛选的总株数×100%)。初筛结果见表1
表1.丁酸梭菌LXKJ-1诱变处理后初筛结果
表2.丁酸梭菌LXKJ-1诱变处理后复结筛果
由以上结果可以看初筛得到的11株耐丁酸菌株,复筛结果发现这11株菌较原始菌株LXKJ-1发酵水平都有不同程度的提高其中一株菌发酵水平突出,其编号为LXKJYB-1,菌株LXKJYB-1发酵水平比起始菌株LXKJ-1高3-4倍,同时传代稳定性比起始菌株更稳当。传代稳定性结果见图3。
实施例2、不同发酵培养基对耐丁酸高产菌株LXKJYB-1发酵水平的影响
将甘油管冻藏的LXKJYB-1接到活化培养基中,37℃静置厌氧培养18h;活化后的丁酸梭菌LXKJYB-1接种到种子培养基,接种量为5%,37℃厌氧培养24h得种子液。
发酵培养基1:硫酸铵2%,玉米粉2%,酵母浸膏2%,尿素1%,磷酸氢二钾0.5%,pH 6.5~7.0,碳酸钙0.5%;去离子水溶解,玻璃棒搅拌混合均匀,分装后灭菌,121℃灭菌20min;
发酵培养基2:硫酸铵4%,玉米粉4%,酵母浸膏4%,尿素2%,磷酸氢二钾1.5%,pH 6.5~7.0,碳酸钙0.7%;去离子水溶解,玻璃棒搅拌混合均匀,分装后灭菌,121℃灭菌20min;
发酵培养基3:硫酸铵5%,玉米粉5%,酵母浸膏5%,尿素5%,磷酸氢二钾3%,pH6.5~7.0,碳酸钙1%;去离子水溶解,玻璃棒搅拌混合均匀,分装后灭菌,121℃灭菌20min;
按照7%的接种量,将LXKJYB-1种子液分别接种至上述3在发酵培养基中,纱布塞加牛皮纸封口,放入厌氧培养箱中,利用厌氧设备抽放气三次,用N2置换三角瓶中的空气,37℃条件下培养24h,后对丁酸梭活菌数及芽孢数进行检测。比较结果见表3。
表3.不同发酵培养基对菌株LXKJYB-1发酵水平影响
由结果可以看出发酵培养基2更适合菌株LXKJYB-1发酵水平的提高。
实施例3、丁酸梭菌(Clostridium butyricum)LXKJYB-1的培养
(1)活化及种子培养
将甘油管冻藏的LXKJYB-1以及LXKJ-1菌株分别接到活化培养基中,37℃静置厌氧培养18h;活化后的丁酸梭菌LXKJYB-1以及LXKJ-1分别接种到种子培养基,接种量为5%,37℃厌氧培养24h得种子液。
(2)一级种子培养
分别将步骤(1)活化后的LXKJYB-1以及LXKJ-1种子液按5%接种量接种至一级种子培养基中,厌氧,37℃,培养12h进行扩增得到一级种子液,培养结束进行活菌数和芽孢数比较,见表4。
(3)二级种子培养
分别将步骤(2)获得的丁酸梭菌LXKJ-1、LXKJYB-1的一级种子液按5%接种量接入二级种子培养基中,厌氧,37℃,培养12h作为二级种子液,培养结束进行活菌数和芽孢数比较,见下表4。
(4)三级发酵罐培养
分别将步骤(3)获得的丁酸梭菌LXKJ-1、LXKJYB-1的二级种子液按5%接种量接入三级发酵罐培养基中,氮气环境下,37℃,培养24h,培养结束后即得到丁酸梭菌LXKJ-1,与诱变后菌株LXKJYB-1的培养物,同时进行活菌数、芽孢数及产酸能力比较结果见下表4、表5。
表4.菌株LXKJ-1与菌株LXKJYB-1发酵水平比较
表5.菌株LXKJ-1与菌株LXKJYB-1产酸水平比较
(5)冻干菌粉制备
离心收集菌体:将上述所得的LXKJ-1与LXKJYB-1的三级发酵培养液12000rmp离心后,冻干时间24h后粉碎,制得冻干菌粉。
由于其具有高芽孢率,耐冷、热性能强,所有不需要添加任何保护剂直接可以冷冻干燥。
冻干粉中丁酸梭菌LXKJ-1的活菌数及芽孢数为:5.32×1011cfu/g,芽孢率为:95.79%。
冻干粉中丁酸梭菌LXKJYB-1的活菌数及芽孢数为:1.41×1012cfu/g,芽孢率为:99.23%。
实施例4、菌株LXKJYB-1与LXKJ-1抗逆性比较试验
(1)耐热性(湿热)
分别取下酸梭菌生长末期已完全形成芽孢的培养物1mL于4℃、3000rpm离心、15min,PBS洗涂两次,重悬后分别置于60℃、80℃、90℃水浴中处理15min,后置于0℃冰水混合物中冷却,梯度稀释平板法测定其存活率,3个重复/样。测定结果见表6.
表6菌株LXKJ-1与菌株LXKJYB-1耐热性比较
从以上结果可以看出菌株LXKJ-1与菌株LXKJYB-1能够100%耐受80℃15min处理,但是在90℃处理15分钟,菌株LXKJYB-1耐热性略高于起始菌株LXKJ-1。
(2)人工胃液和人工肠液耐受性(人工胃液、肠液按照中国药典进行配制)
人工胃液耐受性实验:取生长末期培养物1ml于4℃、3000rpm离心15min,PBS洗涤两次后重悬,以107cfu/mL接种于人工胃液中,震荡混匀,37℃水浴孵育,1h后取样,利用梯度稀释平板法迸行活菌计数,3个重复/样。
人工肠液耐受性:取生长末期培养物1ml于4℃、3000rpm离心15min,PBS洗涤两次后重悬,以l07cfu/mL接种于人工肠液中处理2h后取样,利用梯度稀释平板法,进行活菌计数,3个重复/样。测定结果见表7。
表7菌株LXKJ-1与菌株LXKJYB-1耐人工胃液、人工肠液比较
从以上结果可以看出菌株LXKJ-1与菌株LXKJYB-1能够都能够耐受人工胃液、人工肠液环境,但是菌株LXKJYB-1在人工胃液、肠液中的耐受性更强。
(3)胆盐耐受性
在RCM液体培养基加入猪胆盐,质量分数分别为0.20%、0.40%、0.60%、0.80%、1.0%,121℃、15min灭菌。按1%接种,置厌氧培养箱内培养,观察菌体生长情兄,3个重复/样品。(“+++”表示生长良好,“++”表示次之,“+”表示略有生长,“-”表示不生长)测定结果见表8。
表8菌株LXKJ-1与菌株LXKJYB-1耐猪胆盐比较
从以上结果可以看出菌株LXKJ-1与菌株LXKJYB-1能够都能够耐受一定浓度的胆盐,在高浓度胆盐环境中菌株LXKJYB-1耐受性更强。
实施例5、菌株LXKJYB-1与LXKJ-1体外抑菌活性比较
采用浊度法测定大肠杆菌、沙门氏菌、单增李斯特菌、志贺氏菌、金黄色葡萄球菌,抑菌效果对比,方法如下:
(1)将丁酸梭菌LXKJ-1与诱变后菌株LXKJYB-1的发酵液,离心后收集清液备用。
(2)将靶标菌菌梯度稀释至108cfu/mL,取0.5ml接种到9mL LB液体培养基中。
(3)将菌株LXKJ-1与菌株LXKJYB-1的离心清液各取0.5ml,分别加入到接种靶标菌的LB液体培养基中,37℃,培养箱培养18h。以LB培养基作为空白培养基,测定各培养管OD600。依次计算抑制率,结果见表9。
表9菌株LXKJ-1与菌株LXKJYB-1体外抑菌能力比较
从结果可以看出,菌株LXKJ-1与菌株LXKJYB-1发酵液对大肠杆菌、金黄色葡萄球菌等靶标菌都具有一定的抑菌活性,菌株LXKJYB-1其发酵液抑菌能力更强,尤其对大肠杆菌及金黄色葡萄球菌均有很强的抑制活性,适宜作为由大肠杆菌引起的畜禽腹泻的预防和治疗菌株,应用于养殖业。
实施例6、丁酸梭菌LXKJYB-1和LXKJ-1对樱桃谷肉鸭生产影响的比较
(1)本次实验采用丁酸梭菌诱变后菌株LXKJYB-1与出发菌株LXKJ-1菌剂(每克制剂含丁酸梭菌活菌≥2.0×108cfu/g)。
(2)选取1日龄樱桃谷肉鸭进行试验,平均分成对照组和试验组。对照组喂基础日粮,试验组在基础日粮中添加丁酸梭菌制剂,添加量为100g/t,200g/t,400g/t,800g/t,试验周期42天。
(3)生长性能测定:在试验开始第一天、1周,2周、3周、4周、5周、6周末时,每组随机选50只樱桃谷肉鸭进行称重,取平均值,计算各组樱桃谷肉鸭重量。记录试验前期和后期的耗料量,计算樱桃谷肉鸭平均日增重、平均日采食量和料肉比。
(4)腹泻率和死亡率:记录饲喂过程中各组腹泻只数和死亡只数,计算腹泻率和死亡率。
(5)粪便中菌群数量的测定:各组采集1.0g新鲜粪样,采用10倍梯度稀释法稀释至10-8,选择连续三个稀释度,分别涂布于EMB、MRS、BBL等鉴别培养基平板上,接种量为100μL/9cm平板,每个稀释度做3个平行,37℃恒温培养24-48h。主要检测粪便中大肠杆菌、沙门氏菌、乳酸菌、双歧杆菌的含量。试验结果见表10、表11。
表10丁酸梭菌LXKJ-1和LXKJYB-1对樱桃谷肉鸭生长性能的影响
表11丁酸梭菌LXKJ-1和LXKJYB-1对樱桃谷肉鸭盲肠微生物的影响
日粮中添加0.01%~0.08%丁酸梭菌LXKJ-1制剂与LXKJYB-1制剂都具有提高樱桃谷肉鸭成活率、促进生长及提高日增重,降低了料肉比、腹泻率和死亡率,同时增加樱桃谷肉鸭盲肠中乳酸菌和双歧杆菌的数量,降低大肠杆菌数量的作用,但是丁酸梭菌LXKJYB-1对乳酸菌的促进作用较LXKJ-1提高11-55%,对动物病原菌大肠杆菌、沙门氏菌抑制能力提高了20-50%,同时在降低腹泻率和死亡率方面的效果明显优于菌株LXKJ-1,因此菌株LXKJYB-1更适合做为促生长,抗腹泻,提高养殖生产水平的微生态制剂应用于樱桃谷肉鸭的养殖。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本专利构思的前提下,上述各实施方式还可以做出若干变形、组合和改进,这些都属于本专利的保护范围。因此,本专利的保护范围应以所附权利要求为准。
Claims (5)
1. 一种丁酸梭菌菌剂,其特征在于,所述菌剂由丁酸梭菌(Clostridium butyricum)LXKJYB-1制备,丁酸梭菌(Clostridium butyricum)LXKJYB-1保藏编号为:CCTCC NO:M2017485。
2.权利要求1所述丁酸梭菌菌剂的制备方法,其特征在于,具体如下:
(1)将丁酸梭菌LXKJYB-1进行发酵培养,得到丁酸梭菌LXKJYB-1发酵液;
(2)发酵液离心后,冻干粉碎,制得冻干菌粉,无需添加保护剂。
3.权利要求2所述的丁酸梭菌菌剂的制备方法,其特征在于,发酵培养所用的培养基质量百分比组成为:硫酸铵4%,玉米粉4%,酵母浸膏4%,尿素2%,磷酸氢二钾1.5%,碳酸钙0.7%,其余为去离子水,pH 6.5~7.0。
4.权利要求1所述丁酸梭菌菌剂在制备动物饲料中的应用。
5.权利要求4所述丁酸梭菌菌剂在制备动物饲料中的应用,其特征在于,在动物日粮中添加0.01%~0.08%丁酸梭菌LXKJYB-1。
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