CN114680251A - 一种菌株及其微生态制剂和应用 - Google Patents
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Abstract
本发明公开了一种菌株及其微生态制剂和应用,所述的菌株B1的分类命名为胶红酵母,其菌落呈圆形,凸起,边缘整齐,不透明,表面光滑或较光滑,颜色呈粉红色,大约为3mm,扫描电镜下,细胞长度为2‑5um,球形,出牙生殖.本申请的胶红酵母是从广西北部湾石角红树林根际微生物群落种中的红酵母进行筛选、分离和鉴定获得,是一类抗逆性较强的腐生菌,具有促进水生动物生长、显著提高抗氧化能力、提高成活率和增强机体的非特异性免疫能力的作用。
Description
技术领域
本发明涉及微生物技术领域,具体为一种菌株及其微生态制剂和应用。
背景技术
近年来养殖环境的污染问题、抗生素在养殖中的滥用等问题日益突出,导致养殖动物品质和食用安全性降低,影响人类健康。如今迫切需要寻找研发出绿色安全,对环境无污染,容易被动物吸收利用,营养健康的天然饲料。
红酵母富含有多种氨基酸和脂肪酸,具有丰富的营养,同时还含有多种生物活性成分,如亚麻酸、亚油酸和β-胡萝卜素等,并且,还可改善养殖环境。红树林蕴藏着大量的微生物种植资源,是陆地向海洋过渡的特殊生态系统。目前,从红树林根际微生物中筛选和挖掘了一批有潜在利用价值的微生物菌株资源。红酵母作为一类单细胞真核微生物,通过对红酵母的研究,发现其富含丰富的营养成分和不饱和脂肪酸、类胡萝卜素、虾青素等多种生物活性物质,应用于饲料产品的前景广阔,是非常重要的真菌资源。
但是现有的菌株大多数研究侧重于促进海洋红酵母的类胡萝卜素合成、发酵工艺优化等方面,而在水产养殖应用方面则鲜少研究,因此我们选择红树林地区采集的微生物样品,对海洋红酵母进行初步分离、筛选、鉴定,得到纯化菌株,分析和评价红酵母菌株的营养成分,并进行发酵培养条件优化,最终将红酵母及衍生物制成水产饲料产品应用于水产养殖业中。
发明内容
本发明的目的在于提供一种菌株及其微生态制剂和应用,以解决上述背景技术提出的现有的菌株大多数研究侧重于促进海洋红酵母的类胡萝卜素合成、发酵工艺优化等方面,而在水产养殖应用方面则鲜少研究的问题。
为实现上述目的,本发明采用以下技术方案予以实现:
本发明提供一种菌株,其分类命名为胶红酵母B1。
首先,提取菌株B1的DNA并扩增菌株的核糖体基因转录间隔区序列,经测定序列长度为588bp,在NCBI网站上进行网站下载与菌株B1密切相关的ITS序列,通过MEGA软件构建系统发育树。从利用近邻相接法构建的系统发育树可知,菌株B1和胶红酵母聚为一支,且具有高度的拓扑稳定性,自举支持率为99%,在利用最大似然法构建的系统发育树中,菌株B1和胶红酵母的关系同样被证实。
本发明的菌株B1在PDA培养基上的菌落形态一致,呈不透明的圆形,直径大约为3mm,菌落为粉红色,表面隆起、有光泽,边缘完整,扫描电镜观察下,菌株B1细胞长度2-5um,球形,出牙生殖。
综上所述,通过菌落形态和ITS序列分析,菌株B1属于胶红酵母。
进一步的,所述改善生态养殖优良海洋红酵母添加剂的制备方法,包括如下步骤:
(1)将所述胶红酵母B1接种于种子培养基中,30℃摇瓶培养3d,得到胶红酵母B1种子液;
(2)在50L发酵罐中加入20L所述的胶红酵母B1的最佳发酵培养基并灭菌40min,按摇瓶最适接种量1%将胶红酵母B1种子液接种于发酵罐中进行发酵,得到发酵菌液;
(3)将所述发酵菌液于4℃、8000rpm条件下冷冻离心15min后,去除上清液,保留沉淀,静置沉淀10min后于4℃、8000rpm冷冻离心15min后,去除上清液,保留沉淀,静置沉淀10min后,于4℃、800rpm冷冻离心15min后,去除上清液,保留沉淀,所得沉淀即为红酵母菌剂;
(4)将所述的红酵母菌剂于-10℃~-50℃条件下冷冻干燥3d,3d后取出,碾磨后即得到红酵母冻干粉;
(5)将红酵母冻干粉添加于基础饲料中,制成红酵母添加剂。
进一步的,所述步骤2发酵培养基组分为去皮马铃薯200g/L,葡萄糖20g/L,pH值自然。
进一步的,所述步骤(2)中的发酵条件为:温度设定为30℃,罐压设定为0.5公斤,pH值自然,发酵8h以上。
更近一步的,本发明提供了上述的红酵母添加剂的益生菌微生态制剂在罗非鱼饲料添加剂制剂中的应用。
进一步的,是应用于促进水产动物生长、提高水产动物生产性能方面。
进一步的,可以应用于提高鱼类的生产性能以及鱼类品质方面。
具体地,本发明中提高动物的生产性能指的是提高水产动物罗非鱼的增重率;改善水产动物罗非鱼品质值的是加深鱼肉色泽,并同时对谷丙转氨酶和谷草转氨酶以及超氧化物歧化酶活性有促进作用。
进一步的,所述红酵母饲料添加剂的使用添加量为0.5-2%。
本发明公开的红酵母微生态制剂作为鱼类饲料添加剂,制备方法简单,没有特殊设备要求,添加量较少,可以直接加入动物的食品、药品、饲料或饮水中,添加过程方便快捷,具有很好的推广应用前景。
本发明经过罗非鱼饲养试验发现:
用红酵母菌剂制备的生长促进剂可以使罗非鱼的平均体重显著提高了;可以改善罗非鱼品质,显著加深罗非鱼色泽;
与现有技术相比,本发明具有以下有益效果:
本发明的红酵母菌株产生的氨基酸、脂肪酸、β-胡萝卜素的含量也比出发野生菌株明显提高。此外,该菌株还具有遗传性稳定、无致病性、无毒副作用、容易培养、繁殖速度快、菌株生物量多等有点,明显优于未添加红酵母菌株的市售基础饲料。
本发明的红酵母菌株显著提高水产动物幼苗的存活率、提高饲料效果和增强动物体的免疫功能、减少抗生素用量、皮肤和肌肉色泽鲜艳、口味好等特点。
附图说明
图1为胶红酵母B1在PDA培养基28℃培养3d的菌落形态(A)及菌株B1的扫描电镜观察(B);
图2为胶红酵母B1的系统发育树;
图3为胶红酵母B1的全基因组图谱;
图4为胶红酵母的色素薄层色谱分析图,其中斑点1为菌株B1的色素提取液;斑点2为β-胡萝卜素标准品;斑点a和c,β-胡萝卜素,斑点b,圆酵母素;
图5为胶红酵母B1的β-胡萝卜素提取液高效液相色谱分析(A)及β-胡萝卜素标准品高效液相色谱分析(B);
图6为胶红酵母B1的氨基酸组成;
图7为胶红酵母B1的脂肪酸组成;
图8为添加不同红酵母添加量对罗非鱼生长性能的影响;
图9为试验4周后罗非鱼的免疫指标。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下列实施例中未注明具体条件的试验方法,通常按照常规条件,或按照制造厂商所建议的条件;未注明的材料、试剂等,均未市售产品。除非另外说明,否则百分比和分数按重量计算。
请参阅图1-9,本发明中所需要的培养基的配方如下:
(1)筛选培养基:YEPD固体培养基:酵母粉10g,蛋白胨20g,葡萄糖20g,琼脂15g,蒸馏水1000mL,氯霉素终浓度100mg/L;PDA培养基:马铃薯200g,葡萄糖20g,琼脂15g,蒸馏水1000mL,氯霉素终浓度为100mg/L.
(2)纯化培养基:PDA固体培养基(不含氯霉素)。
(3)扩大培养基(PDB液体培养基):马铃薯200g,葡萄糖20g,蒸馏水1000mL。
实施例一,胶红酵母B1的筛选和鉴定:
1、菌株B1在PDA培养基上的菌落形态一致,呈不透明的原型,直径大约为3mm,菌落为粉红色,表面隆起、有光泽,边缘完整(如图1A),扫描电镜观察下,菌株B1细胞长度为2-5um,球形,出牙生殖(如图1B)。利用NCBI网站下载与该菌株密切相关ITS序列,通过MEGA软件构建系统发育树(如图2)。
2、16SrRNA基因的扩增与序列分析:
(1)真菌基因组DNA提取参照试剂盒操作说明进行;(2)以上步的基因组为模板,利用ITS引物选择通用ITS和ITS4;(3)进行PCR扩增:利用50uL体系进行PCR扩增,体系成分包括:PCRMix(25μL)、ddH2O(22μL)、ITS1(1μL)、ITS2(1μL)、DNA模板(1μL)。扩增程序为:95℃预变性4min,95℃变性40s,55℃退火50s,72℃延伸1min,共进行32个循环。取产物5μL,利用1%琼脂糖凝胶进行凝胶电泳且电压120V,电泳25min,紫外成像检测结果,并将扩增产物测序。将测序结果在NCBI网站上进行BLAST比对,结合菌落形态,初步确定菌株的分类位置将测序结果在NCBI网站上进行BLAST比对,结合菌落形态,初步确定菌株的分类位置。用MEGA软件进行系统发育进化树的构建,从利用近邻相接法构建的系统发育树可知,菌株B1具有高度的拓扑稳定性,自举支持率99%,在利用最大似然法构建的系统发育树中,菌株B1和胶红酵母的关系同样被证实。
3.全基因组测序及数据分析:
(1)从平板上挑取适量菌株的纯菌落,接种到PDB液体培养基中扩大培养,28℃培养3d后,使用高速冷冻离心机离心收集菌体,进行全基因测序;(2)菌株B1的基因组序列使用IlluminaNovaseq进行测定分析;(3)使用SOAPdenovo对指控处理后的数据进行组装,采用krskgf、gapclose等软件对组装结果进行优化和校对,从而得到最终的组装结果。使用从头Augustus方法预测编码基因,通过队中方法对非编码RNA进行预测。使用Circos软件结合基因的预测结果,构建全基因组图谱,如图3。
4.红酵母菌株B1的细胞组成分析:
对制备得到的色素溶液进行薄层层析,选用石油醚:丙酮:氯仿=50:1:1(v:v:v)为展层剂对色素溶液样品和β-胡萝卜素标准品进行展层,薄层层析色谱分离后的结果如图4所示,色素溶液样品分离出两种色素成分,色素a和色素b,色素a和β-胡萝卜素标准品c展层后的位置抑制。将斑点a和斑点c回收过滤,利用高效液相色谱检测,结果如图5所示,β-胡萝卜素与色素溶液样品在24.75分钟450nm处有相同的吸收峰,因此确定色素a为β-胡萝卜素。根据文献报道,推测色素b为圆酵母素。
5.(1)采用食品安全国脚标准中氨基酸的测定方法(GB 5009.124-2016),对酵母菌进行测定,结果如图6所示,16中氨基酸总量为26.70%,其中谷氨酸含量最大,占总氨基酸的17.49%。动物所必需的九种氨基酸占总氨基酸的38.1%;(2)通过皂化、甲酯化等处理提取脂肪酸,利用气相色谱-质谱联用仪对脂肪酸成分进行测定,最后利用面积归一化法进行定量,结果如图7所示,其中油酸含量最高,占52%,油酸、亚麻酸、亚油酸等不饱和脂肪酸占比大,占总量的75.64%。
实施例二,红酵母菌剂的制备:
一种动物生长促进剂,是用红酵母菌剂制备,其制备方法如下:
(1)胶红酵母B1,活化后经30℃、200r/min发酵培养72h,即得红酵母菌剂;
(2)其中,活化培养得培养基组分是:PDA液体培养基:马铃薯200g,葡萄糖20g,蒸馏水1000mL;
(3)经测定,红酵母菌剂中胶红酵母得活菌数为3.33X1010CFU/mL。
实施例三,动物饲料添加剂的制备:
一种用于罗非鱼的饲料添加剂,是用红酵母菌剂制备,其制备方法如下:
(1)按实施例2制备红酵母菌剂;
(2)将上述红酵母菌剂与普通饲料混合,二者重量比为1:50,饲料压缩器压缩制成颗粒状,孔径1.5mm,自然风干,4℃保存,即得红酵母饲料,其中的胶红酵母活菌数为16.77X108CFU/g。
实施例四,红酵母饲料对罗非鱼生长、生产性能的影响:
1、购买一批幼鱼,在一个大缸中驯化一周后,选取200尾健康、活泼、摄食良好、体色正常、大小一致的幼鱼,随机选取20尾幼鱼用0周样品,剩余180尾幼鱼随机分组,随机分为4组,每组3个平行,共12个养殖槽,每个养殖槽饲喂15幼鱼:
(1)对照组罗非鱼饲喂基础日粮,正常饮水;
(2)试验1组罗非鱼饲喂基础饲料中添加0.5%红酵母冻干粉;
(3)试验2组罗非鱼饲喂基础饲料中添加1%红酵母冻干粉;
(4)试验3组罗非鱼饲喂基础饲料中添加2%红酵母冻干粉。
2、试验结果如图8、图9
(1)如图8所示,试验1组、试验2组和试验3组的罗非鱼的增重率为24.22%、71.7%、46.56%,试验2组和试验3组和对照组相比较,增重率提高了30.42%、5.28%,试验1没有添加红酵母和对照组相比不显著。
(2)如图9所示, 相对于对照组,1%和2%组SOD含量分别增加14.69%和10.38%,GSH-PX相对于对照组,有明显的差异。
尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种菌株,其特征在于:所述菌株系胶红酵母B1,其菌落呈圆形,凸起,边缘整齐,不透明,表面光滑或较光滑,颜色呈粉红色,直径为2-4mm,扫描电镜下,细胞长度为2-5um,球形,出牙生殖,最适生长温度为28-30℃。
2.根据权利要求1所述的一种菌株,其特征在于:所述胶红酵母菌株B1在制备用于罗非鱼幼体存活生长影响的应用。
3.根据权利要求2所述的胶红酵母菌株B1的应用,其特征在于:所述的罗非鱼幼体红酵母饲料的制备方法步骤如下:
(1)将所述的胶红酵母B1接种于种子培养基中,摇瓶培养得到红酵母B1种子液;
(2)在发酵罐中加入所述的红酵母B1的最佳发酵培养基并灭菌,接种上红酵母B1种子液并进行发酵,得到发酵菌液;
(3)将所述发酵菌液4℃冷冻离心后,去除上清液,收集沉淀,重复上述操作三次,最后一次离心后收集沉淀,所得沉淀即为红酵母菌剂;
(4)将所述红酵母菌剂冷冻干燥,所得粉末即为红酵母冻干粉;
(5)将所述红酵母冻干粉添加于基础饲料中,即得红酵母饲料。
4.根据权利要求3所述的胶红酵母菌株B1的应用,其特征在于:所述步骤(2)胶红酵母B1种子液最适接种量为1%。
5.根据权利要求3所述的胶红酵母菌株B1的应用,其特征在于:所述步骤(2)基础饲料中添加的红酵母冻干粉设实验组,对照组不添加红酵母,实验组1添加量为0.5%,实验组2添加量为1%,试验组3添加量为2%。
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