CN112574919B - 一种菌丝霉素分泌型益生菌剂及其制备方法与应用 - Google Patents
一种菌丝霉素分泌型益生菌剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明属于饲料添加剂技术领域,涉及一种菌丝霉素分泌型益生菌剂及其制备方法与应用。本发明将菌丝霉素分泌型酪丁酸梭菌、植物乳杆菌、罗伊氏乳杆菌、低聚果糖和乳糖进行科学配伍,得到菌丝霉素分泌型益生菌剂。菌丝霉素分泌型酪丁酸梭菌和植物乳杆菌、罗伊氏乳杆菌配伍使用,既能减少抗生素的使用所引起的抗药性、二重感染等,又能提高饲料利用率以及动物的自然防御机制和动物产品质量。在广东罗非鱼良种场试用该益生菌剂,结果表明其具有明显的抗链球菌感染功效,成活率提高,发病率显著低于抗生素组。该菌丝霉素分泌型益生菌剂在作为或制备水产动物饲料添加剂中具有广阔的应用前景。
Description
技术领域
本发明涉及饲料添加剂技术领域,更具体地,涉及一种菌丝霉素分泌型益生菌剂及其制备方法与应用。
背景技术
随着罗非鱼高密度集约化养殖的发展,罗非鱼发病率越来越高,其中链球菌病防治难度较大,对罗非鱼养殖造成了重大威胁,己成为罗非鱼养殖可持续发展的最主要制约因素。该病传播快,致死率高,链球菌病爆发往往与水质恶化、养殖密度过高等有关。目前仍然缺乏控制链球菌引起的罗非鱼链球菌病的安全高效的药物。罗非鱼是主要的水产品出口品种,对我国的农业国际贸易极其重要,罗非鱼链球菌病的爆发,渔农频繁使用一些违禁药物,对我国水产品的质量安全造成极大威胁。
菌丝霉素(Plectasin)是一种新型真菌源抗菌肽,可克服抗生素类药物在罗非鱼链球菌病防治中存在的缺陷,具有良好的研究前景。体外抗菌活性实验显示对革兰氏阳性菌肺炎链球菌有显著的抑菌活性,而其同时能促进细胞的增殖,增强巨噬细胞的吞噬活性,刺激免疫应答,提高抗体水平,增强抗病能力。酪丁酸梭菌具有抑制肠道有害细菌和促进肠道有益菌群增殖的双重作用,从而维持肠道的正常微生态平衡。临床研究表明酪丁酸梭菌能够增强动物的免疫力,其发酵动物饲料能降低饲料中的抗营养因子,从而改善动物的生产性能和产品质量,减少抗生素用量,提高经济效益。贾军皓等将启动子thl克隆到载体pMTL82151上,得到表达型质粒pMTL82151-thl,然后根据酪丁酸梭菌表达***对密码子的偏爱性,人工合成优化后的Pt基因,再将产菌丝霉素基因Pt克隆到启动子thl的下游,构建表达质粒pMTL82151-Pt,并接合转化入酪丁酸梭菌,构建并筛选一株能高水平表达出菌丝霉素的丁酸梭菌工程菌株,即菌丝霉素分泌型酪丁酸梭菌株(贾军皓,曹丁,陈勉华等.菌丝霉素在酪丁酸梭菌中的表达研究[J].食品工业科技,2020(18))。该菌株兼具有菌丝霉素抗菌肽和酪丁酸梭菌的双重功能,虽然其对细菌具有一定的抑制作用,但其抗菌活性和提高免疫力的效果仍然有待进一步提高。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供一种菌丝霉素分泌型益生菌剂。
本发明的第二个目的在于提供所述菌丝霉素分泌型益生菌剂的制备方法。
本发明的第三个目的在于提供所述菌丝霉素分泌型益生菌剂的应用。
本发明的上述目的是通过以下技术方案给予实现的:
一种菌丝霉素分泌型益生菌剂,包括菌丝霉素分泌型酪丁酸梭菌粉、植物乳杆菌粉、罗伊氏乳杆菌粉、低聚果糖、乳糖。
本发明创造性地将菌丝霉素分泌型酪丁酸梭菌、植物乳杆菌、罗伊氏乳杆菌、益生元低聚果糖和乳糖进行科学配伍,得到菌丝霉素分泌型益生菌剂。其中,酪丁酸梭菌特异性表达的菌丝霉素作为一种真菌源抗菌肽,其对罗非鱼链球菌、肠毒性大肠杆196、金黄色葡萄球菌、蜡样芽孢杆菌、伤寒沙门氏菌等均具有明显的抑制。菌丝霉素分泌型酪丁酸梭菌和植物乳杆菌、罗伊氏乳杆菌配伍使用,可协同发挥益生菌的调理肠道微生态平衡,提高动物生产性能和饲料利用率。该菌丝霉素分泌型益生菌剂既能减少抗生素的使用所引起的抗药性、二重感染等,又能提高动物的自然防御机制,还能改善动物产品质量。
优选地,所述菌丝霉素分泌型益生菌剂,包括以下重量百分比的组分:菌丝霉素分泌型酪丁酸梭菌粉35~45%、植物乳杆菌粉20~30%、罗伊氏乳杆菌粉10~20%,低聚果糖1~10%、乳糖10~20%。
更优选地,所述菌丝霉素分泌型益生菌剂,包括以下重量百分比的组分:菌丝霉素分泌型酪丁酸梭菌40%、植物乳杆菌粉25%、罗伊氏乳杆菌粉15%,低聚果糖5%、乳糖15%。
更优选地,所述植物乳杆菌粉包括植物乳杆菌和保护剂;所述植物乳杆菌的活菌数为1×1011~5×1011cfu/g;所述保护剂的组分包括脱脂奶粉、海藻糖、谷氨酸钠组成,脱脂奶粉、海藻糖、谷氨酸的质量比为(20~30):(5~10):(0.5~5)。
更优选地,所述罗伊氏乳杆菌粉包括罗伊氏乳杆菌和保护剂;所述罗伊氏乳杆菌的活菌数为5×1010~2×1011cfu/g,保护剂的组分包括脱脂奶粉、乳糖、海藻糖、低聚果糖,脱脂奶粉、乳糖、海藻糖、低聚果糖的质量比为(20~30):(5~10):(1~10):(1~5)。
参考公开号为CN107164274A的中国专利“一种乳酸菌复合菌剂及其制备方法和应用”制备上述植物乳杆菌粉和罗伊氏乳杆菌杆菌粉。
更优选地,所述菌丝霉素分泌型酪丁酸梭菌粉包括菌丝霉素分泌型酪丁酸梭菌和保护剂;所述菌丝霉素分泌型酪丁酸梭菌的活菌数为3×108~3×109cfu/g;所述保护剂由辅美粉、米糠、玉米淀粉组成,辅美粉、米糠、玉米淀粉的质量比(70~90):(5~15):(5~15)。
优选地,所述菌丝霉素分泌型酪丁酸梭菌粉的制备方法包括以下步骤:
S1.菌株发酵培养:将表达菌丝霉素的重组酪丁酸梭菌工程菌株发酵,得到发酵液;
S2.在发酵液体中加入质量体积比5%~15%的辅美粉溶解,在进风口120~170℃、出风口60~80℃条件下进行喷雾干燥,然后按照辅美粉、米糠、玉米淀粉质量比为(70~90):(5~15):(5~15)的比例进行复配,制得所述菌丝霉素分泌型酪丁酸梭菌粉。
更优选地,所述步骤S1菌株发酵培养的步骤如下:将酪丁酸梭菌工程菌株接种在种子培养基中,于36~38℃的厌氧箱静止培养16~18小时;再转接到新的种子培养基中,于36~38℃的厌氧箱静止培养12~16小时,得到种子液;接种到发酵培养基中,在37℃下严格厌氧发酵,中后期间歇补料,即在12小时、16小时、20小时、24小时和28小时各补料一次,发酵32h结束培养。
更优选地,所述种子培养基组分为:葡萄糖25g/L,酵母浸膏20g/L,胰蛋白胨10g/L,硫酸铵1.5g/L,碳酸氢钠1.5g/L,一水合硫酸锰0.23g/L,七水合硫酸镁0.24g/L,氯化钙0.021g/L。
更优选地,所述发酵培养基组分为:葡萄糖20g/L,豆饼粉提取液40g/L,硫酸铵1g/L,碳酸氢钠1g/L,玉米浆3g/L,一水合硫酸锰0.2g/L,七水合硫酸镁0.2g/L,氯化钙0.02g/L。
更优选地,所述间歇补料为间歇流加35ml/L质量分数为15%的葡萄糖。
更优选地,所述步骤S2加入质量体积比10%的辅美粉。
更优选地,步骤S2中按照进风口130℃、出风口70℃进行喷雾干燥。
本发明还提供了一种菌丝霉素分泌型益生菌剂的制备方法,其特征在于,将菌丝霉素分泌型酪丁酸梭菌粉、植物乳杆菌粉、罗伊氏乳杆菌粉、低聚果糖和乳糖搅拌混匀,粉碎过100目筛即得。
上述任一所述表达菌丝霉素的重组酪丁酸梭菌工程菌根据已公开文献“贾军皓,曹丁,陈勉华,等.菌丝霉素在酪丁酸梭菌中的表达研究[J].食品工业科技,2020(18).http://kns.cnki.net/kcms/detail/11.1759.TS.20200408.1414.017.html”中的制备方法得到。
本发明还提供了菌丝霉素分泌型益生菌剂在制备防治罗非鱼链球菌病药剂中的应用。
上述菌丝霉素分泌型益生菌剂能够作为水产养殖饲料添加剂,与饲料混合拌料,以投喂的形式给鱼用药,降低罗非鱼链球菌病的发病率,减少或不用抗生素等药物,提高罗非鱼产品的品质,保障食品安全,推动水产养殖业的健康持续发展。因此,菌丝霉素分泌型益生菌剂在作为或制备水产动物饲料添加剂方面的应用,也应在本发明的保护范围之内。
优选地,所述水产动物饲料添加剂是指具备调节水生动物胃肠道微生态平衡,抑制致病菌生长,提高动物生长水平和饲料利用率,并能发挥抗菌抗病毒和免疫调节作用的水产动物饲料添加剂。
优选地,所述菌丝霉素分泌型益生菌剂在水产养殖动物饲料添加剂中的添加量为每吨饲料中添加200~1000g。
与现有技术相比,本发明具有以下有益效果:
本发明提供的菌丝霉素分泌型益生菌剂,菌丝霉素分泌型酪丁酸梭菌具有分泌抗菌肽菌丝霉素的功能,通过复配植物乳杆菌粉和罗伊氏乳杆菌,能够在水生动物体内发挥协同效果,既能高效杀灭链球菌等致病菌,还能调节动物胃肠道微生态平衡,显著提高罗非鱼生长水平和饲料利用率,提高罗非鱼的免疫力。该益生菌剂的稳定性高,肠道定殖能力强,成本低廉,无环境污染,无毒副作用,无耐药性,无残留,能够减少或替代抗生素等药物,减少养殖水体有害物质的积累,提高罗非鱼水产品的品质,保证水产品的质量安全;在广东罗非鱼良种场试用该菌丝霉素分泌型益生菌剂,结果表明其具有明显的抗链球菌感染,提高饲料利用率和存活率的功效,与抗生素组和单独使用菌丝霉素分泌型酪丁酸梭菌相比,罗非鱼的免疫力得到提高,链球菌感染的发病率显著下降。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1菌丝霉素分泌型益生菌剂的制备
1.菌丝霉素分泌型酪丁酸梭菌粉的制备
(1)根据现有技术“贾军皓,曹丁,陈勉华,等.菌丝霉素在酪丁酸梭菌中的表达研究[J].食品工业科技,2020(18).http://kns.cnki.net/kcms/detail/11.1759.TS.20200408.1414.017.html”制备菌丝霉素分泌型酪丁酸梭菌株。
(2)扩大培养后,在50L的发酵罐中配制30L发酵培养基,以接种量8%接种菌丝霉素分泌型酪丁酸梭菌株,发酵过程控制pH值为6.5,生长温度为37℃,全程严格厌氧,装液量60%,发酵中后期用15%的葡萄糖采用间歇补料方式,从发酵最旺盛的12h开始流加15%质量浓度的葡萄糖,每4小时间歇流加35ml/L,即在12小时、16小时、20小时、24小时和28小时各补料一次,发酵32h结束培养。发酵培养基的成分为:葡萄糖20g/L,豆饼粉提取液40g/L,硫酸铵1g/L,碳酸氢钠1g/L,玉米浆3g/L,一水合硫酸锰0.2g/L,七水合硫酸镁0.2g/L,氯化钙0.02g/L。
(3)在发酵液体中加入质量体积比10%的辅美粉溶解,按照进风口130℃、出风口70℃进行喷雾干燥,按照辅美粉与米糠、玉米淀粉的质量比为70:15:15的比例添加米糠、玉米淀粉,即为所述菌丝霉素分泌型酪丁酸梭菌粉,封好口放于阴凉干燥处保存。
参考公开号为CN107164274A的中国专利“一种乳酸菌复合菌剂及其制备方法和应用”制备植物乳杆菌粉和罗伊氏乳杆菌杆菌粉。
2.菌丝霉素分泌型益生菌剂的制备
称量以下质量百分比的各个组分:菌丝霉素分泌型酪丁酸梭菌粉40%、植物乳杆菌粉25%、罗伊氏乳杆菌杆菌粉15%,低聚果糖5%、乳糖15%。其中,菌丝霉素分泌型酪丁酸梭菌粉的活菌数为3×109cfu/g,保护剂配方按质量比为辅美粉:米糠:玉米淀粉=70:15:15;植物乳杆菌粉的活菌数为2×1011cfu/g,保护剂配方按质量比为脱脂奶粉:海藻糖:谷氨酸钠=25:10:3;罗伊氏乳杆菌粉的活菌数为1×1011cfu/g,保护剂配方按质量比为脱脂奶粉:乳糖:海藻糖:低聚果糖=25:10:10:5;将以上组分用混料机搅拌混合后,粉碎后过100目筛,即得菌丝霉素分泌型复合益生菌剂。
实施例2菌丝霉素分泌型益生菌剂的制备
称量以下质量百分比的各个组分:菌丝霉素分泌型酪丁酸梭菌粉35%、植物乳杆菌粉30%、罗伊氏乳杆菌粉20%,低聚果糖5%、乳糖10%。其中,菌丝霉素分泌型酪丁酸梭菌粉的活菌数为3×108cfu/g,保护剂配方按质量比为辅美粉:米糠:玉米淀粉=75:10:15;植物乳杆菌粉的活菌数为5×1011cfu/g,保护剂配方按质量比为脱脂奶粉:海藻糖:谷氨酸钠=30:5:5;所述罗伊氏乳杆菌粉的活菌数为2×1011cfu/g,保护剂配方按质量比为脱脂奶粉:乳糖:海藻糖:低聚果糖=30:5:1:1。将以上组分用混料机搅拌混合后,粉碎后过100目筛,即得菌丝霉素分泌型复合益生菌剂。
实施例3菌丝霉素分泌型益生菌剂的制备
称量以下质量百分比的各个组分:菌丝霉素分泌型酪丁酸梭菌粉45%、植物乳杆菌粉20%、罗伊氏乳杆菌粉10%,低聚果糖10%、乳糖15%。其中,菌丝霉素分泌型酪丁酸梭菌粉的活菌数为3×109cfu/g,保护剂配方按质量比为辅美粉:米糠:玉米淀粉=80:10:10;植物乳杆菌粉的活菌数为1×1011cfu/g,保护剂配方按质量比为脱脂奶粉:海藻糖:谷氨酸钠=20:10:5;所述罗伊氏乳杆菌粉的活菌数为5×1010cfu/g,保护剂配方按质量比为脱脂奶粉:乳糖:海藻糖:低聚果糖20:10:10:5。将以上组分用混料机搅拌混合后,粉碎后过100目筛,即得菌丝霉素分泌型复合益生菌剂。
对比例1菌丝霉素分泌型酪丁酸梭菌粉的制备
仅含实施例1制备的菌丝霉素分泌型酪丁酸梭菌粉。
对比例2对比菌丝霉素分泌型益生菌剂的制备
称量以下质量百分比的各个组分:菌丝霉素分泌型酪丁酸梭菌粉10%、植物乳杆菌粉10%、罗伊氏乳杆菌粉40%,低聚果糖15%、乳糖25%。其中,菌丝霉素分泌型酪丁酸梭菌粉的活菌数为3×109cfu/g,保护剂配方按质量比为辅美粉:米糠:玉米淀粉=70:15:15。植物乳杆菌粉的活菌数为2×1011cfu/g,保护剂配方按质量比为脱脂奶粉:海藻糖:谷氨酸钠=25:10:3。所述罗伊氏乳杆菌粉的活菌数为1×1011cfu/g,保护剂配方按质量比为脱脂奶粉:乳糖:海藻糖:低聚果糖=25:10:10:5;将以上组分用混料机搅拌混合后,粉碎后过100目筛,即得菌丝霉素分泌型复合益生菌剂。
对比例3不含菌丝霉素分泌型酪丁酸梭菌的益生菌剂的制备
将实施例1的菌丝霉素分泌型酪丁酸梭菌粉替换为等量的米糠,其余组分及含量与实施例1相同。
对比例4不含植物乳杆菌的益生菌剂的制备
将实施例1的植物乳杆菌粉替换为等量的米糠,其余组分及含量与实施例1相同。
对比例5不含罗伊氏乳杆菌的益生菌剂的制备
将实施例1的罗伊氏乳杆菌粉替换为等量的米糠,其余组分及含量与实施例1相同。
测试例菌丝霉素分泌型益生菌剂对罗非鱼链球菌病发病率及生长性能的影响
1、实验方法:
(1)实验分组及处理
选择罗非鱼链球菌病发病严重的水域,实验鱼为阳江罗非鱼良种基地自行繁育的吉富罗非鱼幼鱼,体重在3克左右,选择无病无伤、活力强的个体作为实验用鱼。实验前在网箱中暂养10天,池塘水温为(35±2)℃,自然光周期。暂养期间用增氧机不间断增氧,每天8:00和16:00各投喂浮性饲料1次(粗蛋白质为27~30.0%、脂肪4~5.5%),投喂量为体重的8~10%。
实验共分8组,在2米×2米×1米的网箱中进行。在同一水塘中搭建4个同等规格的网箱,水深为0.8米,每组饲养吉富罗非鱼苗200尾,实验共计60天。实验间的平均水温为35℃,养殖期间若发现死鱼及时捞出并解剖记录,分析死亡原因,并记录观察罗非鱼在实验期的健康状况。
对照组:饲喂基础日粮,各组随机分配,每组50尾,试验期60天,每周饲喂2次罗非鱼,每组50尾,试验期60天。
抗生素组:按照罗非鱼常规养殖抗生素添加量在基础日粮中添加磺胺嘧啶钠。
试验组:分为实施例1、对比例1、对比例2、对比例3、对比例4以及对比例5共6组。在对照组的基础上,将上述6组的益生菌剂作为饲料添加剂,按照每吨饲料里面200g的添加量拌料,每周饲喂2次罗非鱼,每组50尾,试验期60天。
(2)结果统计
试验期结束后,统计所有罗非鱼的周增重,料重比和链球菌病的发病率和成活率。
2、测试结果
结果如表1:
表1不同益生菌剂对罗非鱼生长性能和免疫性能的影响
名称 | 周增重(g) | 料重比 | 发病率(%) | 成活率(%) |
对照组 | 2.23 | 1.15 | 26.0 | 75.5 |
抗生素组 | 3.35 | 0.79 | 12.5 | 90.0 |
实施例1 | 3.36 | 0.81 | 1.5 | 99.5 |
对比例1 | 2.93 | 0.87 | 8.0 | 93.5 |
对比例2 | 3.12 | 0.92 | 12.5 | 90.0 |
对比例3 | 2.85 | 0.89 | 16.5 | 85.5 |
对比例4 | 3.09 | 0.90 | 6.5 | 95.0 |
对比例5 | 3.23 | 0.92 | 6.0 | 94.5 |
注:表1中数据均为各组相应指标的平均值。
由表1可知,抗生素组与对照组相比,平均周增重提高,料重比降低,降低罗非鱼链球菌病发病率,说明抗生素作为罗非鱼饲料添加剂具有一定的促生长和免疫力提高作用;在饲料中不添加抗生素的前提下,仅用菌丝霉素分泌型酪丁酸梭菌(对比例1)饲喂罗非鱼,与对照组相比,提高了罗非鱼的生长水平和饲料的利用率,增加了周增重,降低料肉比和发病率,且由于菌丝霉素分泌型酪丁酸梭菌分泌抗菌肽,其对罗非鱼链球菌病的控制效果优于抗生素组,但促生长效果不如抗生素;实施例1的罗非鱼发病率最低,成活率最高,即疾病预防效果显著优于抗生素组和菌丝霉素分泌型酪丁酸梭菌组(对比例1),且促生长效果与抗生素组相当,说明在益生菌剂中菌丝霉素分泌型酪丁酸梭菌与植物乳杆菌、罗伊氏乳杆菌经过一定的比例配伍使用具有协同增效的作用,能够显著降低罗非鱼链球菌病的发病率,提高其生长水平和存活率。与对照组比较,对比例2、对比例3、对比例4和对比例5虽然有一定的促生长、降低发病率和提高成活率的作用,但促生长、降低发病率以及提高成活率均没有实施例1好,进一步说明实施例1的益生菌剂中的各组分经过科学配伍优化后起到了协同增效的作用。
Claims (4)
1.一种菌丝霉素分泌型益生菌剂,其特征在于,包括以下重量百分比的组分:菌丝霉素分泌型酪丁酸梭菌粉40%、植物乳杆菌粉25%、罗伊氏乳杆菌粉15%、低聚果糖5%、乳糖15%;其中,菌丝霉素分泌型酪丁酸梭菌粉的活菌数为3×109cfu/g,菌丝霉素分泌型酪丁酸梭菌粉的保护剂由质量比为辅美粉:米糠:玉米淀粉=70:15:15的辅美粉、米糠、玉米淀粉组成;
植物乳杆菌粉的活菌数为2×1011cfu/g,植物乳杆菌粉的保护剂由质量比为脱脂奶粉:海藻糖:谷氨酸钠=25:10:3的脱脂奶粉、海藻糖、谷氨酸钠组成;
罗伊氏乳杆菌粉的活菌数为1×1011cfu/g,罗伊氏乳杆菌粉的保护剂由质量比为脱脂奶粉:乳糖:海藻糖:低聚果糖=25:10:10:5的脱脂奶粉、乳糖、海藻糖、低聚果糖组成。
2.权利要求1所述菌丝霉素分泌型益生菌剂的制备方法,其特征在于,按重量百分比,将菌丝霉素分泌型酪丁酸梭菌粉、植物乳杆菌粉、罗伊氏乳杆菌粉、低聚果糖和乳糖搅拌混匀,粉碎过100目筛即得。
3.根据权利要求1所述菌丝霉素分泌型益生菌剂在制备防治罗非鱼链球菌病药剂中的应用。
4.根据权利要求2所述制备方法制得的菌丝霉素分泌型益生菌剂在制备水产动物饲料添加剂方面的应用。
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