CN116064254A - 一种胶红酵母mjh-21091及其发酵产物、菌剂与应用 - Google Patents
一种胶红酵母mjh-21091及其发酵产物、菌剂与应用 Download PDFInfo
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- CN116064254A CN116064254A CN202211085941.0A CN202211085941A CN116064254A CN 116064254 A CN116064254 A CN 116064254A CN 202211085941 A CN202211085941 A CN 202211085941A CN 116064254 A CN116064254 A CN 116064254A
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- rhodotorula mucilaginosa
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- rhodotorula
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Abstract
本发明涉及微生物技术领域,具体涉及一种胶红酵母MJH‑21091及其发酵产物、菌剂与应用。本发明提供的胶红酵母(Rhodotorula mucilaginosa)MJH‑21091,其保藏编号为CGMCC No.23535。本发明的胶红酵母MJH‑21091能够耐100℃的高温、可在pH值2.0以上的酸性环境生长,耐胆盐能力强,具有降解霉菌毒素的作用,同时具有产β‑葡聚糖酶的能力,将其制成饲料添加剂后用于饲喂动物效果安全可靠,在促进营养物质消化吸收,降低腹泻率,提高饲料转化效率,促进生长方面有积极作用。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种胶红酵母MJH-21091及其发酵产物、菌剂与应用。
背景技术
红酵母在动植物体内、江河、湖泊、海洋中都有发现。在海水和淡水环境中,红酵母数量约占到酵母总数的50%左右。红酵母是一类真核细胞型微生物,红酵母属包含粘红酵母、胶红酵母、小红酵母、瘦果红酵母、浅红酵母、乳糖红酵母、橙黄红酵母等。
红酵母是优良的益生菌种,红酵母及其代谢产生的糖、维生素、消化酶、类胡萝卜素等可有效的促进动物的消化吸收、提高免疫力,改善动物性产品的品质。酵母菌在畜禽肠道一般不定值,属于过路菌,它通过呼吸作用,消耗肠道中的氧气。肠道中的大部分有益菌是厌氧菌,致病菌则多数为好氧菌,因此红酵母可以在一定程度上抑制致病菌的生长。但其对动物肠道菌群的具体影响机制报道不多,还需要进一步研究确定。
目前,红酵母作为微生态制剂方面的研究和应用主要集中在水产养殖领域,在畜禽方面的相关报导较少。以往的研究往往集中在红酵母及其代谢产物对水产动物机体抗氧化及免疫力的影响,对红酵母降解霉菌毒素的作用研究较少。
发明内容
本发明的目的是提供一种耐高温、耐酸、耐胆盐且具有降解霉菌毒素能力的胶红酵母及其应用。
为了实现本发明的目的,本发明的技术方案如下:
本发明提供胶红酵母(Rhodotorula mucilaginosa)MJH-21091,其保藏编号为CGMCC No.23535。
本发明的胶红酵母(Rhodotorula mucilaginosa)是从北京市某养猪实验基地附近土壤中筛选得到,命名为MJH-21091。经18S RNA基因序列分析,该菌株MJH-21091为胶红酵母(Rhodotorula mucilaginosa)。该菌株已于2021年10月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址: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,邮编100101),分类命名为胶红酵母Rhodotorula mucilaginosa,保藏编号为CGMCC NO.23535。
胶红酵母(Rhodotorula mucilaginosa)MJH-21091的微生物学特性为:细胞形态为椭圆形,长度约2-4μm,宽度约1-2μm;单菌落大小为3-4mm,颜色呈橙红色,不透明,菌落表面平滑,边缘规则。菌体在80℃处理20min后存活率可达85%以上,100℃处理20min后存活率仍达50%以上,可在pH值2.0以上的酸性环境生长,耐胆盐能力强。且具有较强的降解霉菌毒素的能力,其对呕吐毒素的降解率达到88.62%。
本发明还通过体外法鉴定了胶红酵母MJH-21091的益生效果,结果表明,胶红酵母MJH-21091能够耐酸、酸胆盐,能抵抗胃肠道的内环境,具备益生菌的潜力。
进一步地,本发明还提供胶红酵母(Rhodotorula mucilaginosa)MJH-21091的发酵产物。含有胶红酵母(Rhodotorula mucilaginosa)MJH-21091和/或其发酵产物的菌剂。
本发明所述的菌剂可以为液体菌剂或固体菌剂,可采用常规技术手段、加入微生物制剂领域允许的辅料制备得到。
本发明的菌剂可通过发酵培养胶红酵母MJH-21091获得。
本发明还提供一种产品,其含有胶红酵母(Rhodotorula mucilaginosa)MJH-21091和/或其发酵产物;所述产品为饲料添加剂、动物饲料或食品。
优选,本发明饲料添加剂含有的胶红酵母MJH-21091活菌数为3.6×108CFU/g~3.6×1012CFU/g;更优选地,含有的胶红酵母MJH-21091的活菌数为3.6×109CFU/g~3.6×1011CFU/g。
本发明动物饲料中胶红酵母MJH-21091的活菌数为3.6×107CFU/kg~3.6×1011CFU/kg,优选3.6×108CFU/kg~3.6×1010CFU/kg。
本发明还发现胶红酵母MJH-21091具有降解霉菌毒素的能力,因此本发明还提供一种药品,其含有胶红酵母(Rhodotorula mucilaginosa)MJH-21091和/或其发酵产物。
优选,所述药品为可抑制(降解)霉菌毒素的药品。
更优选,所述霉菌毒素为呕吐毒素。
本发明另提供一种胶红酵母(Rhodotorula mucilaginosa)MJH-21091,或其发酵产物,或上述菌剂在降低食品或饲料中霉菌毒素污染中的应用;在增加动物采食量、提高饲料转化率、促进动物生长、提高动物体重和/或减少动物腹泻率方面的应用,以及在制备饲料、饲料添加剂、食品添加剂或抑制霉菌毒素药品中的应用。
本发明的有益效果在于:
本发明涉及的胶红酵母,其由于出芽生殖的方式对干燥、高温、高压、氧化等不良环境抵抗力很强,耐高温、耐酸、耐胆盐,这种稳定性增加了它作为益生菌的潜力。且本发明的胶红酵母对霉菌毒素有理想的降解效果,可用于制备降解霉菌毒素的多种产品。本发明胶红酵母还可提高饲料利用率,促进饲料中营养物质的消化吸收;增强动物的免疫功能,增加采食量,提高日增重,降低料肉比;具有无污染,无残留,生物环保等特点,在促进动物生长和提高动物体重方面具有显著的效果。
本发明进一步验证了胶红酵母MJH-21091在断奶仔猪饲料添加中的应用效果,发现该菌株具有降低断奶仔猪腹泻率提高饲料转化率的作用。表明胶红酵母MJH-21091可作为一种新型的益生菌添加剂,广泛应用于饲料和食品中。
附图说明
图1为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535的菌落形态图。
图2为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535在光学显微镜下的革兰氏染色形态图。
图3为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535产β葡聚糖酶的示意图。
图4为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535的耐酸性检测结果。
图5为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535的耐胆盐检测结果。
图6为胶红酵母(Rhodotorula mucilaginosa)MJH-21091 CGMCC No.23535的生长曲线。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中所使用的培养基如无特殊说明配置方法如下:YPD培养基:蛋白胨20g,酵母提取物10g,溶解于900ml蒸馏水,葡萄糖20g,溶解于100ml蒸馏水,高压灭菌后混合。
实施例1 胶红酵母(Rhodotorula mucilaginosa)MJH-21091的分离与鉴定
一、菌株MJH-21091的鉴定
1.菌株的分离培养
取1g北京市海淀区中国农业大学西校区养猪基地附近的土壤样品装入有9ml生理盐水的试管中,漩涡器震荡混匀,即为1:10稀释液,再取稀释液进行10倍递增稀释,然后选择3个适宜梯度的稀释液各1ml涂布于YPD培养基上。30℃培养48-72小时,观察并记录菌落形态,挑取长势良好的单菌落,进行划线分离纯化。
2.菌株的革兰氏染色
在载玻片上滴一滴经灭菌的蒸馏水,挑取一个单菌落在水中溶解,经刮片后,在酒精灯上烘干固定。滴加结晶紫染色液,染2min,水洗,自然晾干;滴加碘液媒染2min,水洗,自然晾干;滴加碱性品红乙醇溶液50S,水洗,自然晾干;在普通光学显微镜上观察,菌体呈紫色便于观察其出芽情况。
通过步骤1和2的分离筛选,得到一株通过出芽生殖的方式进行繁殖的菌株,将该菌株编号为MJH-21091。其菌落形态图见图1,在光学显微镜下的革兰氏染色形态见图2。
二、菌株MJH-21091的鉴定
1.形态学鉴定
对处于对数生长期且菌落大小稳定的菌株MJH-21091进行单菌落状态描述,主要包括菌落的大小、颜色、透明度、菌落表面状态及菌落边缘状态。所得单菌落大小为3-4mm,颜色呈橙红色,不透明,菌落表面平滑,边缘规则。
其次,对处于对数生长期的菌株MJH-21091进行染色,采用光学显微镜观察菌体形态。分离并筛选到的菌株MJH-21091,革兰氏染色呈紫色,细胞形态为椭圆形,长度约2-4μm,宽度约1-2μm。
2.18S RNA序列同源性分析
菌株总DNA的提取采用天根生化科技有限公司的基因组DNA提取试剂盒提取。提取后的样品送到上海美吉生物医药科技有限公司进行测序。将测定结果在GenBank数据库中进行BLAST同源性比对,确定菌株类别为胶红酵母(Rhodotorula mucilaginosa)。菌株序列如SEQ ID No.1所示。
上述实验结果表明,该菌株为胶红酵母。该菌株已于2021年10月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC,地址: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,邮编100101),分类命名为胶红酵母(Rhodotorulamucilaginosa),保藏号为CGMCC No.23535。
实施例2 胶红酵母MJH-21091的抗逆性检测
1.耐热性检测:将胶红酵母MJH-21091(CGMCC No.23535)菌液置于水浴锅内20分钟,分别用60℃、80℃、100℃处理,每个处理3个重复,处理结束后采用倾注法测定其活菌数。
胶红酵母MJH-21091在60℃处理20分钟后,存活率可达99%,80℃处理20分钟后可达85%以上,100℃处理20分钟后仍可达到50%以上。
2.耐酸性检测:将胶红酵母MJH-21091分别接种到pH值为2.0、3.0、4.0、5.0的YPD培养基中,分别在0h、1h、2h、3h、4h采用平板倾注法测定其活菌数。
胶红酵母MJH-21091能够在pH为3、4、5的环境中正常生长,结果见图4。当pH为2时,在接种活菌4小时后,活菌数量与接种时相比几乎没有变化,说明胶红酵母MJH-21091具有在pH最低为2的环境下存活的能力,但相对pH为5的环境来说几乎不再繁殖,该菌种对酸性环境有一定的耐受能力。结果提示胶红酵母MJH-21091可以耐受胃酸的影响。
3.耐胆盐检测:将活化好的胶红酵母MJH-21091用无菌生理盐水做倍比稀释,选取合适的稀释梯度并吸取1ml稀释液放于无菌培养皿里,做6个重复,然后用含不同浓度猪胆酸钠(0.1%、0.2%、0.3%、0.4%)的YPD培养基倾注平板,30℃培养4小时,每隔1小时菌落计数,见图5,结果显示不同胆盐浓度下的活菌数随着时间的延长普遍呈上升趋势。0.4%的胆盐作用对胶红酵母MJH-21091的影响比较微弱,几乎不影响其正常生长。说明胶红酵母MJH-21091的耐胆盐能力较强。
4.抗生素敏感性检测:将适宜浓度的胶红酵母MJH-21091涂布于YPD培养基上,每个培养皿中均匀的贴附4个药敏纸片,培养36小时,观察抑菌圈大小。结果见表1。
表1 胶红酵母MJH-21091对不同抗生素敏感性结果
名称 | 药物用量(ug) | 抑菌圈直径(mm) | 敏感度 |
四环素 | 30 | 0 | 不敏 |
链霉素 | 30 | 0 | 不敏 |
青霉素 | 10 | 0 | 不敏 |
庆大霉素 | 10 | 0 | 不敏 |
红霉素 | 15 | 0 | 不敏 |
头孢拉定 | 30 | 0 | 不敏 |
阿莫西林 | 30 | >20 | 极敏 |
氯霉素 | 30 | >20 | 极敏 |
氟苯尼考 | 30 | >20 | 极敏 |
这一结果表明,胶红酵母MJH-21091 CGMCC No.23535并不具有良好的耐药性,因此作为饲用益生菌使用是安全可靠的。
实施例3 胶红酵母MJH-21091霉菌毒素的降解效果实验
将胶红酵母接种在2ml含有60μg/ml呕吐毒素的液体YPD培养基中(胶红酵母浓度为1.0×108cfu/mL),30℃下培养3天(进行3次重复试验),3天后采用高效液相色谱方法检测培养基中呕吐毒素的浓度,发现呕吐毒素浓度为6.81μg/ml(3次实验取平均值),表明胶红酵母MJH-21091具有降解霉菌毒素的能力。其对呕吐毒素的降解率达88.65%。降解率的计算方式为(霉菌毒素添加量-霉菌毒素残留量)/霉菌毒素添加量×100%。
实施例4 胶红酵母MJH-21091的产酶能力测定
利用β-葡聚糖酶选择性培养基鉴定胶红酵母MJH-21091的产酶特性。选择性培养基配方为:β-葡聚糖1.0g/L,刚果红0.4g/L,蛋白胨10.0g/L,磷酸二氢钾1.0g/L,七水合硫酸镁1.0g/L,氯化钙0.4g/L,琼脂20.0g/L。pH 7.0,121℃,灭菌20min,倒入无菌平板备用。挑取单菌落用三区划线法将胶红酵母MJH-21091接种于上述培养皿中。30℃,培养48小时,观察菌落周围是否有透明带,结果如图3所示,从中可知胶红酵母MJH-21091具有产生β-葡聚糖酶的能力。
实施例5 胶红酵母MJH-21091的生长曲线测定
生长曲线代表了菌株在新的适宜的环境中生长繁殖直至衰老死亡全过程的动态变化。将胶红酵母MJH-21091按1%(v/v)的接种量,接种到YPD培养基中,30℃培养36小时,以不加菌液的YPD培养基作为空白对照,每隔2小时测定OD600值,从而计算活菌数。实验设三次重复,结果取其平均值,记录数据并绘制生长曲线。结果如图6所示,在2-18小时,胶红酵母MJH-21091处于对数生长阶段,繁殖速率较高。在18-36小时胶红酵母MJH-21091的生长进入平台期,数目趋于稳定。
实施例6 胶红酵母MJH-21091制剂的制备
1.发酵培养基配方:甘蔗糖蜜15%、硫酸铵1%、无水氯化钙0.008%、磷酸二氢钾0.15%、酵母膏0.16%、硫酸镁0.15%、消泡剂0.05%,加水充分溶解,控制pH在6.5制成发酵培养基。
2.121℃高温蒸汽消毒25min。
3.待发酵培养基降温至30℃时,接种菌龄16小时的菌液1%(v/v)。
4.在30℃条件下,保持溶氧10-20%,发酵培养24小时,放罐,得到胶红酵母的活菌数大于3.0×1010cfu/ml,出芽率达95%以上。
5.将菌泥放入高压均质机进行细胞壁破碎,然后放入低温真空干燥箱内干燥,过筛,收集产品,得到胶红酵母制剂。
实施例7 胶红酵母MJH-21091制剂的安全性评价
本实施例以小鼠作为实验动物,采用灌胃试验的方法,评价胶红酵母的安全性,具体方法如下:
1.实施例6方法制得的胶红酵母MJH-21091菌剂的冻干粉,经平板计数测定,其胶红酵母MJH-21091菌数为3.0×1010cfu/g。
2.选取6周龄左右的小鼠72只,随机分为4组(A组为对照组灌服无菌生理盐水,B组为高剂量组按照3.0×1010cfu/只的量灌服菌液,C组为中剂量组按照3.0×109cfu/只的量灌服菌液,D组为低剂量组按照3.0×108cfu/只的量灌服菌液),每组3个重复,每个重复6只鼠。
3.每天上午九点灌胃一次,连续灌服21天。实验鼠房控制温度湿度恒定,自然光照,小鼠自由采食、饮水,每7天清扫鼠笼一次。实验过程中,每天观察并记录小鼠的状态,存活情况(结果见表2),有无临床异常症状等。检测指标如下:
取完整的心、肝、脾、肾(双侧)称湿重,分别计算心脏指数=(心脏湿重/体重)×100%、肝脏指数=(肝脏湿重/体重)×100%、脾脏指数=(脾脏湿重/体重)×100%、肾脏指数=(肾脏湿重/体重)×100%。结果见表3。
表2 不同处理组小鼠存活情况
A组 | B组 | C组 | D组 | |
7天 | 全部存活 | 全部存活 | 全部存活 | 全部存活 |
14天 | 全部存活 | 全部存活 | 全部存活 | 全部存活 |
21天 | 全部存活 | 全部存活 | 全部存活 | 全部存活 |
从表2可以看出,使用胶红酵母MJH-21091给小鼠灌胃21天后,实验各处理组小鼠均存活,说明上述胶红酵母对动物安全。
表3 不同处理组小鼠的脏器系数
A组,% | B组,% | C组,% | D组,% | |
心脏 | 0.82 | 0.72 | 0.76 | 0.78 |
肝脏 | 5.31 | 5.85 | 5.76 | 5.42 |
脾脏 | 0.41 | 0.42 | 0.44 | 0.42 |
肾脏 | 1.31 | 1.19 | 1.32 | 1.22 |
从表3可以看出,处理组小鼠的脏器指数与对照组相比没有明显变化,说明上述胶红酵母没有造成小鼠各脏器异常。
实施例8 胶红酵母MJH-21091发酵饲料的制备
1.发酵底物配方:35%的小麦麸,35%的玉米粉,30%的豆粕,向其中加水,搅拌混匀,水的加入量为小麦麸、玉米粉和豆粕总质量的一半。
2.121℃高温蒸汽消毒30min。
3.待发酵底物降温至30℃时,接种菌龄18小时的菌液50%(v/v),搅拌混匀,用纱布封口。
4.在30℃条件下,有氧发酵24小时,无氧发酵30小时,得到胶红酵母发酵饲料。
实施例9 胶红酵母MJH-21091制剂的应用
本实验选取28日龄杜长大三元杂交断奶仔猪72头,实验期28天,按照随机区组设计分为2个组,每组6个重复,每个重复6头猪。A组为对照组(基础日粮组),B组为处理组(基础日粮中添加250g/t的实施例6制得的胶红酵母制剂,有效活菌数为3.0×1010cfu/g)。
基础日粮的原料组成和营养水平见表4。
表4
表4中预混料为购自北京同力兴科农业科技有限公司的乳仔猪预混料。
试验期间仔猪饲养在全封闭式保育猪舍内,温度控制在25-27℃,自由采食、饮水。基础日粮中不含任何抗生素,仔猪免疫按照常规免疫程序进行。
测定指标:各处理组断奶仔猪的生产性能,具体包括以下指标:
1.每天记录仔猪的采食量,试验结束后计算平均日采食量;
2.在试验开始和结束的当天记录仔猪体重,计算平均日增重;
3.通过平均日采食量和平均日增重的试验结果计算料肉比,其计算方式为平均日采食量/平均日增重。
4.试验期间每天早上10:00观察并记录仔猪的粪便状况,计算断奶仔猪腹泻率,腹泻率(%)=(腹泻头数×腹泻天数)/(猪只数×试验天数)×100%。
统计结果见表5。
表5 基础日粮中添加胶红酵母制剂对断奶仔猪生产性能及腹泻率的影响
平均日采食量(kg) | 平均日增重(kg) | 料肉比 | 腹泻率(%) | |
A组 | 0.523 | 0.299 | 1.76 | 11.64 |
B组 | 0.572 | 0.364 | 1.56 | 7.35 |
从表5可以看出,处理组仔猪平均日采食量和平均日增重都显著高于对照组(P<0.05),料肉比显著低于对照组(P<0.05),说明添加胶红酵母制剂的饲料效益更好。饲料中添加胶红酵母制剂的仔猪腹泻率显著降低(P<0.05),说明本发明的菌剂具有降低断奶仔猪腹泻率的作用。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.胶红酵母(Rhodotorula mucilaginosa)MJH-21091,其特征在于,保藏编号为CGMCCNo.23535。
2.权利要求1所述的胶红酵母(Rhodotorula mucilaginosa)MJH-21091的发酵产物。
3.一种菌剂,其特征在于,含有权利要求1所述的胶红酵母(Rhodotorulamucilaginosa)MJH-21091和/或权利要求2所述的发酵产物。
4.一种产品,其特征在于,含有权利要求1所述的胶红酵母(Rhodotorulamucilaginosa)MJH-21091和/或权利要求2所述的发酵产物;所述产品为饲料添加剂、动物饲料或食品。
5.一种药品,其特征在于,含有权利要求1所述的胶红酵母(Rhodotorulamucilaginosa)MJH-21091和/或权利要求2所述的发酵产物。
6.根据权利要求5所述的药品,其特征在于,所述药品为可抑制霉菌毒素的药品。
7.根据权利要求6所述的药品,其特征在于,所述霉菌毒素为呕吐毒素。
8.权利要求1所述的胶红酵母(Rhodotorula mucilaginosa)MJH-21091,或权利要求2所述的发酵产物,或权利要求3所述的菌剂在降低食品或饲料中霉菌毒素污染中的应用。
9.权利要求1所述的胶红酵母(Rhodotorula mucilaginosa)MJH-21091,或权利要求2所述的发酵产物,或权利要求3所述的菌剂在增加动物采食量、提高饲料转化率、促进动物生长、提高动物体重和/或减少动物腹泻率方面的应用。
10.权利要求1所述的胶红酵母(Rhodotorula mucilaginosa)MJH-21091,或权利要求2所述的发酵产物,或权利要求3所述的菌剂在制备饲料、饲料添加剂、食品添加剂或抑制霉菌毒素药品中的应用。
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