CN107254424A - 一种畜禽用新型液态复合微生态制剂及其制备方法 - Google Patents
一种畜禽用新型液态复合微生态制剂及其制备方法 Download PDFInfo
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- CN107254424A CN107254424A CN201710569185.1A CN201710569185A CN107254424A CN 107254424 A CN107254424 A CN 107254424A CN 201710569185 A CN201710569185 A CN 201710569185A CN 107254424 A CN107254424 A CN 107254424A
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Abstract
本发明属于微生态制剂生产制备技术领域,具体涉及一种畜禽用新型液态复合微生态制剂及其制备方法。包括步骤:(1)两种发酵菌株活化;(2)发酵种子液制备;(3)两株菌混和共发酵;(4)液固混合;(5)装瓶。本发明提供的方法能够加速液态发酵速率,利于高浓度菌液制备,所得微生态制剂可以有效防治金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌所引起的疾病,补充畜禽肠道益生菌,可辅助防治畜禽热应激综合征;同样,本发明所提供的方法还能有效与中草药浸膏体外复配构建新的液体制剂,利于多样化操作,工艺简单;最后是本发明方法不需要严格灭菌操作,显著降低工作难度,降低能源浪费,节约成本。
Description
技术领域
本发明属于微生态制剂生产制备技术领域,具体涉及一种畜禽用新型液态复合微生态制剂及其制备方法。
背景技术
我国是农业大国,畜牧业发展占据重要比重,根据国家近年来供给侧改革,全国畜牧规模及其发展将极大带动产业发展,因此,全局来看,我国畜牧业的产业化、规模化、标准化的发展将进一步推动,我国畜禽养殖业在未来“十三五”期间的发展潜力巨大,空间巨大,意义重大。然而,我国养殖业存在抗生素滥用现象十分严重的现象,近些年国家已加大国内畜禽养殖业抗生素滥用的管理工作,国家农业部分别于2016年和2017年出台部分抗生素禁用的公告。同时,国内乃至全球对无抗养殖的呼声越来越大,不同的抗生素替代物应运而生,目前科学家发现益生菌发酵制备的微生态制剂是其中最具有潜力的一类,固态和液态微生态制剂的发展迅速。现在科学研究证明微生态制剂不仅能提高药效、无毒无残留,还能积极补充肠道益生菌,利于肠道菌群的修复和再平衡。
当前,微生态制剂剂型中以发酵型微生态制剂产品效果更佳,发酵型微生态制剂按照剂型可分为液态微生态制剂、半固态微生态制剂和固态微生态制剂三类。液态微生态制剂是利用微生物间的协同作用,发挥了群体的联合作用优势,很好地提高了菌体的稳定性,保持了生物活性,为调节动物生理活性发挥重要作用,而且制备工艺简单,有效地降低了成本,能够产生巨大的经济效益。综合现有资料可归结液态微生态制剂可进行饮水用、口服用、拌料用和喷洒用四类使用方式,但以饮水用、口服用和拌料用稍多。然而,目前液态微生态制剂产品任然具有急需解决的问题:①液态发酵前需要严格灭菌,操作复杂且耗时;②瓶装液态微生态制剂产品部分出现胀瓶现象,缩减产品货架期;③液态发酵缺少优秀的协同发酵混合菌株;④液态发酵中发酵促进剂种类少,更缺少药食同源性发酵促进剂。
发明内容
为克服现有技术中液态微生态制剂制备时所存在的难题,本发明的目的在于提供一种畜禽用新型液态复合微生态制剂及其制备方法。
为实现上述目的,本发明采取的技术方案如下:
一种畜禽用新型液态复合微生态制剂的制备方法,其特征在于,步骤如下:
(1)、两种发酵菌株的活化:
粪肠球菌(Enterococcus faecalis)BD-1的活化:将粪肠球菌BD-1试管斜面菌种在无菌环境下用无菌生理盐水冲洗,然后接种到含太子参须提取物0.2~0.5%的MRS液体培养基中,35~37℃静止培养42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计(即100mL MRS液体培养基含太子参须提取物0.2~0.5g);粪肠球菌BD-1为新菌株,申请人已将该菌株保藏于中国典型培养物保藏中心,地址:武汉市武昌珞珈山,保藏日期:2017年7月7日;保藏编号:CCTCC NO:M2017416;
丁酸梭菌(Clostridium butyricum)RCM-2的活化:将丁酸梭菌RCM-2试管斜面菌种在无菌环境下用无菌生理盐水冲洗,然后接种到含太子参须提取物0.2~0.5%的MRS液体培养基中,35~37℃静止培养42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计(即100mL MRS液体培养基含太子参须提取物0.2~0.5g);丁酸梭菌RCM-2为新菌株,申请人已将该菌株保藏于中国典型培养物保藏中心,地址:武汉市武昌珞珈山,保藏日期:2017年6月29日;保藏编号:CCTCC NO M2017396;
(2)、分别制备粪肠球菌BD-1和丁酸梭菌RCM-2的发酵种子液:
粪肠球菌BD-1的发酵种子液:无菌环境下,将步骤(1)所得粪肠球菌BD-1的活化液按体积百分比3~5%接种到含质量百分比为0.2~0.5%的太子参须提取物的MRS液体培养基中,35~37℃下静止发酵42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计(即100mL MRS液体培养基含太子参须提取物0.2~0.5g);
丁酸梭菌RCM-2的发酵种子液:无菌环境下,将步骤(1)所得丁酸梭菌RCM-2的活化液按体积百分比3~5%接种到含质量百分比为0.2~0.5%的太子参须提取物的MRS液体培养基中,35~37℃下静止发酵42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计(即100mL MRS液体培养基含太子参须提取物0.2~0.5g);
(3)、两株菌混和共发酵:
首先向顶部带有排气阀的发酵罐内注入50~60℃的热水,此时排气阀呈完全关闭状态,然后加入营养成分,搅拌均匀,密封罐体,待发酵罐内水温降至40℃以下时再加入茶多酚粉、大蒜素粉,搅拌均匀,密封罐体保存30min~1h,再接入粪肠球菌BD-1的发酵种子液与丁酸梭菌RCM-2的发酵种子液按体积比1∶1~5∶1组成的混合种子液,盖盖密封,32~37℃静止发酵6~8h时,半开排气阀,然后继续发酵36~40h时关闭排气阀,终止发酵;
所述营养成分为太子参须提取物、红糖、酵母浸膏、维生素C、CMC和麦芽汁;以占热水的百分比计,太子参须提取物占0.2~0.5%、红糖占10~15%、酵母浸膏占5~8%、维生素C占1~2%、CMC占0.5~1.5%、麦芽汁占1~5%、茶多酚粉占0.1~0.25%、大蒜素粉占0.01~0.025%、混合种子液占5~10%,其中麦芽汁和混合种子液的百分比为体积比,其余均为质量体积比g/mL;
(4)、液固混合:
所述“液”是指步骤(3)所得发酵液,所述“固”是指下述的固体①或固体②:
固体①的组成为:茶多酚粉、大蒜素粉和益生元,其中益生元为低聚异麦芽糖、低聚果糖的一种或两种,以占液固混合物总量的质量百分比计,茶多酚粉占0.25~1.5%、大蒜素粉占0.05~0.1%、益生元占1~5%;
固体②的组成为:茶多酚粉、大蒜素粉和能提高畜禽免疫力的中草药浸膏;以占液固混合物总量的质量百分比计,茶多酚粉占0.1~0.8%、大蒜素粉占0.02~0.05%、中草药浸膏占10~20%;
(5)、待步骤(4)结束后,再次密封罐体保存1~3h,即得畜禽用新型液态复合微生态制剂。
作为优选,以质量份数计,所述中草药浸膏由下述配比的各原料药经醇提或/和水提而得:太子参须20~60份、陈皮10~30份、山楂10~30份和六神曲10~30份。
进一步,所述中草药浸膏的提取步骤为:
(i)按配方称取各原料药,加75%乙醇回流提取1~3小时,冷却后,渣液分离得药渣和乙醇提取液,药渣加水煎煮提取1~2次,合并乙醇提取液和水提取液;
(ii)将步骤(i)合并的提取液减压浓缩到28℃相对密度为1.2~1.35的稠膏,即得中草药浸膏。
利用所述制备方法制备的畜禽用新型液态复合微生态制剂。
本发明中,太子参须提取物可按现有技术获得,比如但不限于申请人于2016年8月30日申请的名称为“一种太子参须提取物的制备方法”的专利(申请号201610758305.8)中公开的方法。
有益效果:
1、本发明中粪肠球菌BD-1分离于柘荣县下村农户(坐标:119。51′59.7〞E,27。13′34.0〞N)自养健康成年猪盲肠黏膜层,所得粪肠球菌BD-1安全无毒,37℃ MRS培养基中培养48h菌落表面光滑凸起,周边整齐,菌落颜色灰白色,菌落直径0.5~1.2mm,镜检G+,无芽孢,菌体长度约0.5~1μm;本发明丁酸梭菌RCM-2分离于柘荣县下村农户(坐标:119。52′04.1〞E,27。13′32.7〞N)自圈山林地养殖的健康成年公鸡的盲肠黏膜段,所得丁酸梭菌RCM-2安全无毒,37℃ RCM培养基中培养48h菌落表面光滑凸起,周边整齐,菌落颜色乳黄色不透明,略有酸臭味,菌落直径0.5~1.5cm,镜检G+,有芽孢,芽孢卵圆,芽孢端生或次端生,生芽孢的菌体呈梭状,菌体长度约3.5~7.0μm;
2、本发明的发酵菌株粪肠球菌BD-1和丁酸梭菌RCM-2为具有优良的耐茶多酚和大蒜素能力的新菌株,另外,具有较好的协同发酵能力,所述的太子参须提取物更是提供了一种新型的药食同源性发酵促进剂,可显著促进该协同发酵速率,节省发酵时间;同时,发酵菌株粪肠球菌BD-1和丁酸梭菌RCM-2能有效防治金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌所引起的疾病,并能补充畜禽肠道益生菌,利于修复受损的肠道微生态环境,可辅助防治畜禽热应激综合征;
3、本发明制备方法的发酵前段工作是采用一般性热水常态降温并添加百分比为0.1~0.25%的茶多酚粉和0.01~0.025%的大蒜素粉用来抑制杂菌生长,而非选择传统性的高温高压灭菌(121℃,20~30min),不需要严格灭菌操作,显著降低工作难度,节省能耗,节约时间,简化操作难度。同时,发酵后再次添加茶多酚、大蒜素,酸性的发酵液环境能够极显著促进茶多酚、大蒜素的稳定性,不仅利于茶多酚、大蒜素的有效期延长,还能协同增进发酵液抑菌能力和降低该类液态微生态制剂货架期内胀瓶的现象;另外,茶多酚、大蒜素也广泛用于畜禽养殖中的抗氧化剂和诱食剂,能够辅助防治畜禽热应激综合征;同样,本发明所提供的方法还能有效与中草药浸膏体外复配构建新的液态微生态制剂,利于多样化操作,工艺简单。
附图说明
图1:粪肠球菌BD-1的***进化树。
图2:丁酸梭菌RCM-2的***进化树。
具体实施方式
以下实施例中,太子参提取物为按专利申请号为CN201610758305.8中实施例1公开的方法制备而得;各培养基的配方为:
MRS液体培养基(1L):蛋白胨10g,牛肉粉5g,葡萄糖20g,酵母粉4g,乙酸钠5g,磷酸氢二钾2g,硫酸镁0.2g,柠檬酸三铵2g,硫酸锰0.05g,吐温80 1mL,纯化水1L,pH值6.2±0.2;
MRS琼脂平板或MRS斜面培养基(1L):蛋白胨10g,牛肉粉5g,葡萄糖20g,酵母粉4g,乙酸钠5g,磷酸氢二钾2g,硫酸镁0.2g,柠檬酸三铵2g,硫酸锰0.05g,吐温80 1mL,琼脂15g,纯化水1L,pH值6.2±0.2;
RCM琼脂平板或RCM斜面培养基(1L):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、琼脂15g,纯化水1L,pH值6.8±0.2,灭菌备用;
淀粉培养基(1L):牛肉膏5g、蛋白胨5g、氯化钠0.5g、可溶性淀粉20g、琼脂18g,纯化水1L,pH=7.2±0.1,灭菌备用;
其中,含太子参提取物、茶多酚或大蒜素的MRS液体培养基均是指在上述MRS液体培养基配方基础上,再另外对应添加太子参提取物、茶多酚或大蒜素;含溴甲酚紫指示剂、茶多酚或大蒜素的MRS/RCM琼脂平板内均是指在上述MRS/RCM琼脂平板配方基础上,再另外对应添加溴甲酚紫指示剂、茶多酚或大蒜素。
实施例1--粪肠球菌BD-1的分离与鉴定
1、粪肠球菌BD-1的分离、富集、纯化
无菌操作条件下,取福建省宁德市柘荣县下村农户(坐标:119。51′59.7〞E,27。13′34.0〞N)自养健康成年猪盲肠黏膜段,用灭菌的载玻片刮取黏膜黏液于EP管,用无菌生理盐水稀释102倍,取0.1mL涂布于含溴甲酚紫指示剂 (0.01%,g/mL )的MRS琼脂平板上,涂布后将MRS琼脂平板放置厌氧培养箱中,37℃培养48h,挑选培养基变黄的菌落划线纯化,选取镜检革兰氏染色阳性、无芽孢的菌株于含溴甲酚紫 (0.01%,g/mL )、茶多酚(0.01%,g/mL)和大蒜素(0.01%,g/mL)的MRS琼脂平板上复筛,最终选取菌落黄色颜色明显、菌落表面光滑凸起,周边整齐的作为对象菌株,菌株命名为BD-1,并以MRS斜面培养基对菌种进行斜面保藏。
2、形态学观察
观察菌株BD-1的菌落形态特征,可知:MRS琼脂平板上菌落表面光滑凸起,周边整齐,菌落颜色灰白色,菌落直径0.5~1.2mm,镜检G+,无芽孢,菌体长度约0.5~1μm。
3、生理生化特征
按乳酸菌的生化鉴定试验对菌株BD-1进行鉴定,分别进行了运动性、氧化酶试验等生化特性鉴定,试验结果对照《伯杰氏细菌鉴定手册》进行综合判定。结果见表1。
分离菌株的生理系列化指标测定结果表明与产乳酸肠球菌株结果相符。
4、BD-1菌株的16S rDNA测序比对
利用细菌基因组DNA抽提试剂盒(DP302-02,TIANGEN)提取菌株BD-1的总DNA,以菌株BD-1的总DNA为模板,在引物P0(5’- AGAGTTTGATCCTGGCTCAG -3’)和PC3(5’-GGTTACCTTGTTACGACTT -3’)的引导下PCR扩增该菌株的16S rDNA序列,PCR反应体系为:10×Buffer 5.0μL,dNTP(10mmol/L)2.0μL,Primer 1(20μmol/L)1.0μL,Primer2(20μmol/L)1.0μL,模板DNA 2.5μL,Taq酶(5.0U/μL)0.5μL,超纯水38μL,总50μL。PCR反应条件为:先94℃5min;然后94℃1min,55℃1min,72℃1min,共30个循环;最后72℃延伸8min。反应结束后,对PCR扩增产物进行1%琼脂糖凝胶电泳检测,比对marker获得目的条带。用购自Axygen公司的琼脂糖凝胶回收试剂盒回收并纯化该目的条带,送生工生物工程(上海)股份有限公司进行DNA序列测定,测序结果见SEQ ID No.1。
将所测得的序列与Genbank中的16S rDNA序列进行Blastn相似性分析比较,结果菌株BD-1与Enterococcus faecium strain KCI1、Enterococcus lactis strain K12419C的16S rDNA序列的同源性达到了100%,因此BD-1鉴定为粪肠球菌,其***进化树如图1。
结合上述形态学、生理生化特征和测序及进化树结果,将本发明菌株BD-1鉴定并分类命名为粪肠球菌(Enterococcus faecalis)BD-1。
5、茶多酚对粪肠球菌BD-1生长、pH的影响
(1)粪肠球菌BD-1的活化:将粪肠球菌BD-1试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到MRS液体培养基中,37℃静止培养48h;
(2)按接种量3%(体积比)将步骤(1)所得粪肠球菌BD-1的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内茶多酚的梯度依次设置为(g/mL):0%、0.10%、0.25%、0.50%、1.00%,待培养终点时测定发酵液pH值和活菌数,结果见表2。
由表2结果可知:本发明粪肠球菌BD-1可耐受茶多酚的浓度为1.0%,该浓度下菌体存活率为70.26%。
6、大蒜素对粪肠球菌BD-1生长、pH的影响
(1)粪肠球菌BD-1的活化:同本实施例第5项下的步骤(1);
(2)按接种量3%(体积比)将步骤(1)所得粪肠球菌BD-1的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内大蒜素的梯度依次设置为(g/mL):0%、0.010%、0.025%、0.050%、0.10%,待培养终点时测定发酵液pH值和活菌数,结果见表3;
由表3结果可知:本发明粪肠球菌BD-1可耐受大蒜素的浓度为0.1%,该浓度下菌体存活率为70.43%;
(3)粪肠球菌耐受茶多酚及大蒜素生长对比:
按接种量3%(体积比)分别将步骤(1)所得粪肠球菌BD-1(计为A)的活化液和对照菌B、C、D的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内①茶多酚的梯度依次设置为(g/mL):0%、1.00%、2.00%,②大蒜素的梯度依次设置为(g/mL):0%、0.05%、0.1%,连续培养48h,由0%+0%作为对照(活菌数定义为X0),通过公式:菌体损失率=(X0-X)/ X0*100%来计算粪肠球菌菌体损失率,结果见表4。
由表4结果可知:在茶多酚(2%)和大蒜素(0.1%)联合作用下本发明粪肠球菌BD-1存活率可达53.7%,超出50%,远高于市场同类菌株。
7、粪肠球菌抑菌实验
(1)粪肠球菌BD-1的活化:同本实施例第5项下的步骤(1);
(2)粪肠球菌BD-1在MRS液体培养基中厌氧静止发酵:
按接种量3%(体积比)将步骤(1)所得粪肠球菌BD-1的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,37℃静止培养48h,待发酵终点时取发酵液利用牛津杯法检测对金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌的抑菌效果,以未接种的MRS液体培养基为对照;
(3)粪肠球菌BD-1在MRS液体培养基中有氧发酵:
按接种量(体积比)3%将步骤(1)所得粪肠球菌BD-1的活化液接入装有100 mL MRS液体培养基的500 mL三角瓶中,37℃,150rpm下培养48h,待培养终点时取发酵液利用牛津杯法检测对金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌的抑菌效果,以未接种的MRS液体培养基为对照。
抑菌圈直径大小见表5。
由表5结果可知:本发明粪肠球菌BD-1可显著抑制致病菌金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌的生长,且厌氧培养物较强于有氧培养物。
实施例2--丁酸梭菌RCM-2的分离与鉴定
1、丁酸梭菌RCM-2的分离、富集、纯化
无菌操作条件下,取福建省宁德市柘荣县下村农户(坐标:119。52′04.1〞E,27。13′32.7〞N)自圈山林地养殖的健康成年公鸡的盲肠黏膜段,用灭菌的载玻片刮取黏膜黏液于EP管,用无菌生理盐水稀释102倍,然后加热至60℃、30min,取0.1mL涂布于含溴甲酚紫指示剂 (0.01%,g/mL )的RCM琼脂平板上,涂布后将RCM琼脂平板放置厌氧培养箱中,37℃培养48h,挑选培养基变黄的菌落划线纯化,选取镜检革兰氏染色阳性、有芽孢的杆状菌株于含溴甲酚紫 (0.01%,g/mL)、茶多酚(0.01%,g/mL)和大蒜素(0.01%,g/mL)的RCM琼脂平板上复筛,最后选取菌落周围黄色颜色明显、菌落表面光滑凸起,乳黄色不透明,有酸臭味的作为预备菌株,然后再将预备菌株涂布于淀粉培养基,37℃培养48h后挑选透明圈最大的作为对象菌株,菌株命名为RCM-2,并以RCM斜面培养基对菌种进行斜面保藏。
2、形态学观察
观察菌株RCM-2的菌落形态特征,可知:RCM琼脂平板上菌落表面光滑凸起,菌落颜乳黄色不透明,略有酸臭味,菌落直径0.5~1.5cm,镜检G+,有芽孢,芽孢卵圆,芽孢端生或次端生,生芽孢的菌体呈梭状,菌体长度约3.5~7.0μm。
3、生理生化特征
参照《伯杰细菌鉴定手册》对菌株进行生理生化鉴定,分别以接触酶、淀粉酶、硝酸盐还原和糖等进行生理生化试验,结果见表6。
将其与《伯杰细菌鉴定手册》和常见细菌***鉴定手册中丁酸梭菌的生理生化特性进行比较,结果发现菌株RCM-2与丁酸梭菌的生理生化特性基本相符,因此初步鉴定菌株RCM-2为丁酸梭菌。
4、测序及进化树
利用细菌基因组DNA抽提试剂盒(DP302-02,TIANGEN)提取菌株RCM-2的总DNA,以菌株RCM-2的总DNA为模板,在引物P0(5’- AGAGTTTGATCCTGGCTCAG -3’)和PC3(5’-GGTTACCTTGTTACGACTT -3’)的引导下PCR扩增该菌株的16S rDNA序列,PCR反应体系为:10×Buffer 5.0μL,dNTP(10mmol/L)2.0μL,Primer 1(20μmol/L)1.0μL,Primer2(20μmol/L)1.0μL,模板DNA 2.5μL,Taq酶(5.0U/μL)0.5μL,超纯水38μL,总50μL。PCR反应条件为:先94℃5min;然后94℃1min,55℃1min,72℃1min,共30个循环;最后72℃延伸8min。反应结束后,对PCR扩增产物进行1%琼脂糖凝胶电泳检测,比对marker获得目的条带。用购自Axygen公司的琼脂糖凝胶回收试剂盒回收并纯化该目的条带,送生工生物工程(上海)股份有限公司进行DNA序列测定,测序结果见SEQ ID No.2。
将所测得的序列与Genbank中的16S rDNA序列进行Blastn相似性分析比较,结果菌株RCM-2与Clostridium butyricum strain 1005-15098的16S rDNA序列的同源性达到了98%,因此RCM-2鉴定为丁酸梭菌,其***进化树如图2。
结合上述形态学、生理生化特征和测序及进化树结果,将本发明菌株RCM-2鉴定并分类命名为丁酸梭菌(Clostridium butyricum)RCM-2。
5、茶多酚对丁酸梭菌RCM-2生长、pH的影响
(1)丁酸梭菌RCM-2的活化:将丁酸梭菌RCM-2试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到MRS液体培养基中,37℃静止培养48h;
(2)按接种量3%(体积比)将步骤(1)所得丁酸梭菌RCM-2的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内茶多酚的梯度依次设置为(g/mL):0%、0.10%、0.25%、0.50%、1.00%,待培养终点时测定发酵液pH值和活菌数,结果见表7。
由表7结果可知:本发明丁酸梭菌RCM-2可耐受茶多酚的浓度为1.0%,该浓度下菌体存活率为87.3%。
6、大蒜素对丁酸梭菌RCM-2生长、pH的影响
(1)丁酸梭菌RCM-2的活化:同本实施例第5项下的步骤(1);
(2)按接种量3%(体积比)将步骤(1)所得丁酸梭菌RCM-2的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内大蒜素的梯度依次设置为(g/mL):0%、0.010%、0.025%、0.050%、0.10%,待培养终点时测定发酵液pH值和活菌数,结果见表8;
由表8结果可知:本发明丁酸梭菌RCM-2可耐受大蒜素的浓度为0.1%,该浓度下菌体存活率为85.5%;
(3)丁酸梭菌耐受茶多酚及大蒜素生长对比:
按接种量3%(体积比)将步骤(1)所得丁酸梭菌RCM-2(计为A)的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,其中厌氧血清瓶内的MRS液体培养基内①茶多酚的梯度依次设置为(g/mL):0%、1.00%、2.00%,②大蒜素的梯度依次设置为(g/mL):0%、0.05%、0.1%,连续培养48h,由0%+0%作为对照(活菌数定义为X0),通过公式:菌体损失率=(X0-X)/X0*100%来计算丁酸梭菌菌体损失率,结果见表9。
由表9结果可知:在茶多酚(1%)和大蒜素(0.05%)联合作用下本发明丁酸梭菌RCM-2存活率可达88.5%,超出50%,远高于市场同类菌株。
7、丁酸梭菌抑菌实验
(1)丁酸梭菌RCM-2的活化:同本实施例第5项下的步骤(1);
(2)按接种量3%(体积比)将步骤(1)所得丁酸梭菌RCM-2的活化液于厌氧箱中接种至含MRS液体培养基的厌氧血清瓶中,37℃静止培养48h,待发酵终点时取发酵液利用牛津杯法检测对金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌的抑菌效果,以未接种的MRS液体培养基为对照;抑菌圈直径大小见表10。
由表10结果可知:本发明丁酸梭菌RCM-2可显著抑制致病菌金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌的生长。
实施例3--粪肠球菌BD-1和丁酸梭菌RCM-2共生长
(1)菌株的活化
粪肠球菌BD-1的活化:将粪肠球菌BD-1的试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到MRS液体培养基中,37℃静止培养48h;
丁酸梭菌RCM-2的活化:将丁酸梭菌RCM-2的试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到MRS液体培养基中,37℃静止培养48h;
(2)然后将粪肠球菌BD-1的活化液、丁酸梭菌RCM-2的活化液以及两者按体积比1:1复合的活化液分别按照接种量3%(体积比)接种于MRS液体培养基的200 mL厌氧血清瓶内,其中厌氧血清瓶内的MRS液体培养基内太子参须提取物的梯度依次设置为(质量比):0%、0.25%,37℃静止培养48h,待培养终点时测定发酵液活菌数,另外测定丁酸梭菌RCM-2的芽孢率;结果见表11。
由表11可知:太子参须提取物可显著促进粪肠球菌BD-1和丁酸梭菌RCM-2的共生长,且在同等浓度下对两株菌共生促进程度大于两株单菌株数量之和;另外,0.25%浓度下可分别促进RCM-2的芽孢率,共生培养的芽孢率有所进一步提高。
实施例4--微生态制剂的制备
制备步骤如下:
(1)、两种发酵菌株的活化:
将粪肠球菌BD-1试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到装有含太子参须提取物0.25%的MRS液体培养基的500 mL厌氧血清瓶内,37℃静止培养48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
将丁酸梭菌RCM-2试管斜面菌种在无菌环境下用无菌生理盐水冲洗,洗下培养基表面菌苔,然后接种到装有含太子参须提取物0.25%的MRS液体培养基的500 mL厌氧血清瓶内,37℃静止培养48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
(2)、分别制备粪肠球菌BD-1和丁酸梭菌RCM-2的发酵种子液:
粪肠球菌BD-1的发酵种子液:无菌环境下,将步骤(1)所得粪肠球菌BD-1活化液按体积百分比3%接种到装有含太子参须提取物0.25%的MRS液体培养基的1L厌氧血清瓶内,37℃下静止发酵36h,制得粪肠球菌BD-1发酵种子液;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
丁酸梭菌RCM-2的发酵种子液:无菌环境下,将步骤(1)所得丁酸梭菌RCM-2活化液按体积百分比3%接种到装有含太子参须提取物0.25%的MRS液体培养基的1L厌氧血清瓶内,37℃下静止发酵36h,制得丁酸梭菌RCM-2发酵种子液;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
(3)、两株菌混和共发酵:
首先向顶部带有排气阀的发酵罐内注入55℃的热水,此时排气阀呈完全关闭状态,然后加入营养成分,90rpm搅拌5min,密封罐体,待发酵罐内水温降至37℃时再加入茶多酚粉、大蒜素粉,搅拌均匀,密封罐体保存45min,再接入粪肠球菌BD-1的发酵种子液与丁酸梭菌RCM-2的发酵种子液按体积比1∶1组成的混合种子液,盖盖密封,37℃静止发酵6h时,半开排气阀,然后继续发酵38h时关闭排气阀,终止发酵;
所述营养成分为太子参须提取物、红糖、酵母浸膏、维生素C、CMC和麦芽汁;以占热水的百分比计,太子参须提取物占0.25%、红糖占15%、酵母浸膏占8%、维生素C占2%、CMC占1.0%、麦芽汁占3%、茶多酚粉占0.15%、大蒜素粉占0.02%、混合种子液占7%,其中麦芽汁和混合种子液的百分比为体积比,其余均为质量体积比g/mL;
(4)、液固混合,混匀搅拌转速为100rpm,时间为20min:
所述“液”是指步骤(3)所得发酵液,所述“固”是指茶多酚粉、大蒜素粉和益生元,其中益生元为低聚异麦芽糖,以占液固混合物总量的质量百分比计,茶多酚粉占0.5%、大蒜素粉占0.1%、益生元占3%;
(5)、待步骤(4)结束后,再次密封罐体保存2h,即得畜禽用新型液态复合微生态制剂,可称量和瓶装分装,瓶子采用250mL棕色塑料瓶,每瓶装液量为200mL。
分别取10瓶,等量分为2组A和B,A组现场开盖取样分别检测杂菌率、致病菌(金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌)抑菌圈直径;B组则在温度35℃空调控温房内存放6个月,再分别检测杂菌率、致病菌(金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌)抑菌圈直径和胀瓶率。结果见表12。
由表12可知:本实施例制备的液态微生态制剂无胀瓶现象,延长其货架期。
以本实施例制备的产品为实验药剂,以金黄色葡萄球菌∶藤黄微球菌∶大肠埃希氏菌=1∶1∶1致病菌复合液(金黄色葡萄球菌、藤黄微球菌、大肠埃希氏菌三种致病菌分别单独进行营养液体培养基活化,活化后的菌液按体积比3%添加至含100mL营养液体培养基的三角药瓶进行100rpm的药瓶培养,24h获得菌液,最后按照体积比1∶1∶1制成致病菌复合液,其中1L营养液体培养基成分为:蛋白胨10g、牛肉膏粉3g、氯化钠5g、最终pH 7.3±0.2)连续灌喂健康鸡2d使试验鸡人为感染致病来建造模型动物,其中模型鸡的总数为200只,分为两组,一组采用该实施例产品治疗,一组不进行治疗(对照组)。采用该实施例产品治疗的利用采用灌喂方式治疗,一日三次,每次2mL/只,饮水、采食和活动自由,治疗3d。三天后检查治愈率(治愈是指鸡的临床症状完全消失,精神状况也完全恢复正常;治愈率是治愈的鸡只数占该组鸡只总数的百分比)、死亡率(死亡率是死亡的鸡只数占该组鸡只总数的百分比)、鸡新鲜粪便情况,结果见下表13。
实施例5--微生态制剂的制备
与实施例4的不同之处在于:步骤(4)中,所述“固”是指茶多酚粉、大蒜素粉和能提高畜禽免疫力的中草药浸膏,以占液固混合物总量的质量百分比计,茶多酚粉占0.20%、大蒜素粉占0.04%、中草药浸膏占15%;所述中草药浸膏由下述配比的各原料药提取而得:太子参须40份、陈皮20份、山楂20份和六神曲20份;提取步骤为:
(i)按配方称取各原料药,加75%乙醇回流提取2小时,冷却后,渣液分离得药渣和乙醇提取液,药渣加水煎煮提取2次,合并乙醇提取液和水提取液;
(ii)将步骤(i)合并的提取液减压浓缩到28℃相对密度为1.2的稠膏,即得中草药浸膏。
分别取10瓶,等量分为2组A和B,A组现场开盖取样分别检测杂菌率、致病菌(金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌)抑菌圈直径;B组则在温度35℃空调控温房内存放6个月,再分别检测杂菌率、致病菌(金黄色葡萄球菌、藤黄微球菌和大肠埃希氏菌)抑菌圈直径和胀瓶率。结果见表14。
由表14可知:本实施例制备的液态微生态制剂无胀瓶现象,延长其货架期。
以本实施例制备的产品为实验药剂,以金黄色葡萄球菌∶藤黄微球菌∶大肠埃希氏菌=1∶1∶1致病菌复合液灌喂健康鸡建造模型动物(方法同上实施例)连续灌喂健康鸡2d使试验鸡人为感染致病来建造模型动物,其中模型鸡的总数为200只,分为两组,一组采用该实施例产品治疗,一组以中草药浸膏为对照。采用该实施例产品和中草药浸膏治疗的利用采用灌喂方式治疗,一日三次,两者每次用药量分别为2mL/只、0.3g/只,饮水、采食和活动自由,治疗7d。试验3天后检查治愈率(治愈是指鸡的临床症状完全消失,精神状况也完全恢复正常;治愈率是治愈的鸡只数占该组鸡只总数的百分比)、死亡率(死亡率是死亡的鸡只数占该组鸡只总数的百分比)、鸡新鲜粪便情况。另外记录第1天和第7天每只存活试验鸡的体重再由第1天和第7天的鸡平均体重计算鸡平均增重量(g/只),结果见下表15。
SEQUENCE LISTING
<110> 福建贝迪药业有限公司
<120> 一种畜禽用新型液态复合微生态制剂及其制备方法
<130>
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 1461
<212> DNA
<213> 未知
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cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 900
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Claims (4)
1.一种畜禽用新型液态复合微生态制剂的制备方法,其特征在于,步骤如下:
(1)、两种发酵菌株的活化:
粪肠球菌(Enterococcus faecalis)BD-1的活化:将粪肠球菌BD-1试管斜面菌种在无菌环境下用无菌生理盐水冲洗,然后接种到含太子参须提取物0.2~0.5%的MRS液体培养基中,35~37℃静止培养42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;粪肠球菌BD-1为新菌株,保藏编号为CCTCC NO:M2017416;
丁酸梭菌(Clostridium butyricum)RCM-2的活化:将丁酸梭菌RCM-2试管斜面菌种在无菌环境下用无菌生理盐水冲洗,然后接种到含太子参须提取物0.2~0.5%的MRS液体培养基中,35~37℃静止培养42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;丁酸梭菌RCM-2为新菌株,保藏编号为CCTCC NO M2017396;
(2)、分别制备粪肠球菌BD-1和丁酸梭菌RCM-2的发酵种子液:
粪肠球菌BD-1的发酵种子液:无菌环境下,将步骤(1)所得粪肠球菌BD-1的活化液按体积百分比3~5%接种到含质量百分比为0.2~0.5%的太子参须提取物的MRS液体培养基中,35~37℃下静止发酵42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
丁酸梭菌RCM-2的发酵种子液:无菌环境下,将步骤(1)所得丁酸梭菌RCM-2的活化液按体积百分比3~5%接种到含质量百分比为0.2~0.5%的太子参须提取物的MRS液体培养基中,35~37℃下静止发酵42~48h;其中,所述太子参须提取物的百分比以质量体积百分比g/mL计;
(3)、两株菌混和共发酵:
首先向顶部带有排气阀的发酵罐内注入50~60℃的热水,此时排气阀呈完全关闭状态,然后加入营养成分,搅拌均匀,密封罐体,待发酵罐内水温降至40℃以下时再加入茶多酚粉、大蒜素粉,搅拌均匀,密封罐体保存30min~1h,再接入粪肠球菌BD-1的发酵种子液与丁酸梭菌RCM-2的发酵种子液按体积比1∶1~5∶1组成的混合种子液,盖盖密封,32~37℃静止发酵6~8h时,半开排气阀,然后继续发酵36~40h时关闭排气阀,终止发酵;
所述营养成分为太子参须提取物、红糖、酵母浸膏、维生素C、CMC和麦芽汁;以占热水的百分比计,太子参须提取物占0.2~0.5%、红糖占10~15%、酵母浸膏占5~8%、维生素C占1~2%、CMC占0.5~1.5%、麦芽汁占1~5%、茶多酚粉占0.1~0.25%、大蒜素粉占0.01~0.025%、混合种子液占5~10%,其中麦芽汁和混合种子液的百分比为体积比,其余均为质量体积比g/mL;
(4)、液固混合:
所述“液”是指步骤(3)所得发酵液,所述“固”是指下述的固体①或固体②:
固体①的组成为:茶多酚粉、大蒜素粉和益生元,其中益生元为低聚异麦芽糖、低聚果糖的一种或两种,以占液固混合物总量的质量百分比计,茶多酚粉占0.25~1.5%、大蒜素粉占0.05~0.1%、益生元占1~5%;
固体②的组成为:茶多酚粉、大蒜素粉和能提高畜禽免疫力的中草药浸膏;以占液固混合物总量的质量百分比计,茶多酚粉占0.1~0.8%、大蒜素粉占0.02~0.05%、中草药浸膏占10~20%;
(5)、待步骤(4)结束后,再次密封罐体保存1~3h,即得畜禽用新型液态复合微生态制剂。
2.如权利要求1所述的制备方法,其特征在于:以质量份数计,所述中草药浸膏由下述配比的各原料药经醇提和/或水提而得:太子参须20~60份、陈皮10~30份、山楂10~30份和六神曲10~30份。
3.如权利要求2所述的制备方法,其特征在于:所述中草药浸膏的提取步骤为:
(i)按配方称取各原料药,加75%乙醇回流提取1~3小时,冷却后,渣液分离得药渣和乙醇提取液,药渣加水煎煮提取1~2次,合并乙醇提取液和水提取液;
(ii)将步骤(i)合并的提取液减压浓缩到28℃相对密度为1.2~1.35的稠膏,即得中草药浸膏。
4.一种利用如权利要求1~3任一所述制备方法制备的畜禽用新型液态复合微生态制剂。
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