CN111685235B - 一种仔猪用植物乳杆菌发酵液体全价饲料的制备方法 - Google Patents
一种仔猪用植物乳杆菌发酵液体全价饲料的制备方法 Download PDFInfo
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- CN111685235B CN111685235B CN202010680995.6A CN202010680995A CN111685235B CN 111685235 B CN111685235 B CN 111685235B CN 202010680995 A CN202010680995 A CN 202010680995A CN 111685235 B CN111685235 B CN 111685235B
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- lactobacillus plantarum
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- A—HUMAN NECESSITIES
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- Fodder In General (AREA)
Abstract
本发明涉及仔猪用植物乳杆菌发酵液体全价饲料的制备方法,可有效解决现有仔猪饲养成本高,断奶仔猪胃肠健康欠佳,腹泻率高,传统的干料适口性不好,营养物质利用率低的问题,其解决的技术方案是,是将植物乳杆菌接种至配制好的液体全价饲料中,接种量4~6%,于26~33℃摇床160~200rpm发酵3~6d,即得植物乳杆菌发酵液体全价饲料,本发明的植物乳杆菌是适宜发酵液体饲料的优良菌种,能够有效抑制饲料原料中杂菌、有害菌的生长繁殖,保证发酵液体饲料的良好发酵,并且制备发酵液体饲料的工艺简单,易于操作,特别是对仔猪的生长性能效果显著,是发酵饲料上的创新。
Description
技术领域
本发明涉及发酵饲料技术领域,特别是一种仔猪用植物乳杆菌发酵液体全价饲料的制备方法。
背景技术
近年来,国内食品安全事件频繁发生,特别是抗生素的滥用造成药物残留及细菌耐药性,已严重威胁到食品安全及人类的健康,饲料的安全也越来越受到国家和社会的关注。微生物发酵饲料作为一种新型绿色环保饲料,为我们人类带来了新的研究思路。因此,在提倡食品安全及绿色养殖的新形势下,我们更需要大力发展发酵液体饲料,从源头上保证肉类食品的安全。
发酵液体饲料(Fermented liquid Feed,FLF)是通过人为控制发酵条件下,有益微生物通过代谢活动将饲料中的抗营养因子分解或转化,产生适口性好,营养水平高的饲料原料。FLF在应用方面与传统的干料和液体饲料相比有巨大的优势:FLF可以大大节省饲料成本,提高饲料的消化利用率,改善动物的生长性能,液体发酵过程中产生大量的酸性物质,使饲料酸度增强,从而抑制或杀灭沙门氏菌和大肠杆菌,最终促进动物健康,保证人类的健康,具有良好的应用前景。
发明内容
针对上述情况,为解决现有技术之缺陷,本发明之目的就是提供一种仔猪用植物乳杆菌发酵液体全价饲料的制备方法,可有效解决现有仔猪饲养成本高,断奶仔猪胃肠健康欠佳,腹泻率高,传统的干料适口性不好,营养物质利用率低等问题。
本发明解决的技术方案是,本发明所述植物乳杆菌UR-3,其分类命名为植物乳杆菌(Lactobacillus plantarum),已于2019年12月16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,保藏编号为:CGMCC No.19131。保藏地址:北京市朝阳区北辰西路1号院3号。
利用植物乳杆菌UR-3发酵液体全价饲料的方法,是将植物乳杆菌接种至配制好的液体全价饲料中,接种量4~6%,于26~33℃摇床160~200rpm发酵3~6d,即得植物乳杆菌发酵液体全价饲料。
所述的全价饲料是由以下质量百分比的原料制成:玉米47~52%、豆粕16~24%、膨化大豆16.5~20%、乳清粉1~4%、进口鱼粉1~5%、豆油0.5~1.8%,赖氨酸0.1~0.14%、蛋氨酸0.1~0.14%、食盐0.1~0.2%、石粉1.0~1.2%、磷酸氢钙0.1~0.9%和预混料2.0%,所述的预混料每千克是由(铜)Cu 200mg、(铁)Fe 2300mg、(锌)Zn2800 mg、(锰)Mn 450mg、(维生素A)VA 95000IU、(维生素D3)VD3 13000IU、(维生素E)VE 3000IU、(维生素K3)VK3 90mg、(维生素B2)VB2 300mg和余量为淀粉混合均匀制成。
本发明的植物乳杆菌是适宜发酵液体饲料的优良菌种,能够有效抑制饲料原料中杂菌、有害菌的生长繁殖,保证发酵液体饲料的良好发酵,并且制备发酵液体饲料的工艺简单,易于操作,能够在粗放的猪场环境中进行发酵,进一步增强仔猪的肠道健康,加快仔猪的生长增重,维持机体正常的生理功能,改善仔猪的免疫功能,效果良好,特别是对仔猪的生长性能效果显著,是发酵饲料上的创新。
具体实施方式
以下结合实施例对本发明的具体实施方式作进一步详细说明。
实施例1
本发明利用植物乳杆菌制备发酵液体全价饲料的具体步骤如下:
1)种子液的制备:取植物乳杆菌UR-3接种到制备好的已灭菌的MRS液体培养基中,26~33℃摇床160~180rpm发酵2~4d,再吸取2~4%的菌种,转接到另一瓶新制备好MRS液体培养基中,26~33℃摇床160~180rpm发酵2~4d,最终制成植物乳杆菌种子液,种子液活菌数≥1×109cfu/mL;
2)原料预处理:将全价饲料原料粉碎后充分混合均匀,将混合后的原料细粉碎至粒度为60目,制成发酵液体饲料干粉物料,将发酵液体饲料干粉物料按水料比1:2~3的比例混合均匀,加入三角瓶中;
3)发酵:将步骤1)中制备好的植物乳杆菌种子液接种到步骤2)中已混匀装好的发酵液体饲料干粉物料的三角瓶中,接种量4~6%,完全密封,26~37℃摇床160~200rpm发酵3~6d,当发酵液pH降到4.0以下时结束发酵,即得植物乳杆菌发酵液体全价饲料。
实施例2
1)种子液的制备:取植物乳杆菌UR-3接种到制备好的已灭菌的MRS液体培养基中,30℃摇床170rpm发酵3d,再吸取3%的菌种,转接到另一瓶新制备好MRS液体培养基中,30℃摇床170rpm发酵3d,最终制成植物乳杆菌种子液,种子液活菌数≥1×109cfu/mL;
2)原料预处理:将玉米50%、豆粕20%、膨化大豆19.01%、乳清粉3%、进口鱼粉3%、豆油1%,赖氨酸0.12%、蛋氨酸0.12%、食盐0.15%、石粉1.1%、磷酸氢钙0.5%和预混料2.0%粉碎后充分混合均匀,将混合后的原料细粉碎至粒度为60目,制成发酵液体饲料干粉物料,将发酵液体饲料干粉物料按水料比1:2~3的比例混合均匀,加入三角瓶中;
3)发酵:将步骤1)中制备好的植物乳杆菌种子液接种到步骤2)中已混匀装好的发酵液体饲料干粉物料的三角瓶中,接种量5%,完全密封,30℃摇床180rpm发酵4d,当发酵液pH降到4.0以下时结束发酵,即得植物乳杆菌发酵液体全价饲料。
所述的MRS液体培养基为:牛肉浸粉5.0g、蛋白胨10.0g、酵母浸出粉4.0g、葡萄糖20.0g、柠檬酸三铵2.0g、吐温-80 1.0ml、硫酸锰0.05g、硫酸镁0.2g、乙酸钠5.0g、磷酸氢二钾2.0g、100ml蒸馏水,加热煮沸溶解分装,调pH值至6.2±0.2,121℃高压灭菌15min备用。
所述的预混料每千克是由(铜)Cu 200mg、(铁)Fe 2300mg、(锌)Zn2800 mg、(锰)Mn450mg、(维生素A)VA 95000IU、(维生素D3)VD3 13000IU、(维生素E)VE 3000IU、(维生素K3)VK3 90mg、(维生素B2)VB2 300mg和余量为淀粉混合均匀制成。
对植物乳杆菌发酵液体全价饲料各项指标进行检测,经指标检测,发酵饲料产品满足产品技术标准:乳酸浓度≥150mmol/L,乳酸菌活菌数≥1.0×109cfu/mL,pH≤4.5,乙酸浓度≤40mmol/L,乙酸浓度≤0.8mmol/L。
一、本发明植物乳杆菌的筛选方法如下:
1:菌株的紫外诱变:取原始菌株(CGMCC编号1.1856,植物乳杆菌Lactobacillusplantarum)制成菌悬液,稀释为不同浓度梯度后,经紫外照射后涂布于MRS固体培养基上,并用黑布裹好,26~33℃避光培养3~6d,取出进行计数,算出致死率,找出最佳照射剂量。
2:筛选诱变菌株:①在含有碳酸钙的分离培养基上通过溶钙圈进行初筛;②通过复筛最终得到一株乳酸高产菌株UR-3,显著提高了产乳酸量。
所述的MRS固体培养基为:牛肉浸粉5.0g、蛋白胨10.0g、酵母浸出粉4.0g、葡萄糖20.0g、柠檬酸三铵2.0g、吐温-80 1.0ml、硫酸锰0.05g、硫酸镁0.2g、乙酸钠5.0g、琼脂粉1.8g、磷酸氢二钾2.0g、碳酸钙2.0g、100ml蒸馏水,加热煮沸溶解分装,调pH值至6.2±0.2,121℃高压灭菌15min备用。
本发明提供的诱变菌种UR-3与其它发酵液体饲料的菌种相比,在生长、产乳酸等方面表现出很好的性能,在发酵液体饲料中,UR-3菌株产乳酸能力较原始菌株提高了44.53%,乳酸浓度≥200mmol/L,生物量明显提高,粗蛋白量也相对提高。
3:优良菌种的鉴定:
挑取诱变的优良菌株UR-3的菌落划线接种到MRS培养基平板上作纯培养,26-33℃培养24h-72h后,进行镜检,观察分离细菌的颜色及形态特征:形成直径1.0mm-3.0mm白色圆形凸起的菌落,表面光滑湿润,边缘整齐,革兰氏染色阳性,无运动性。结合参照《伯杰细菌鉴定手册》,其中菌株生理生化实验结果如表1所示,碳源发酵实验结果如表2所示,根据生理生化及碳源发酵实验结果并对菌株UR-3进行16SrRNA测序数据比对,得出菌株UR-3为植物乳杆菌(Lactobacillus plantarum),已于2019年12月16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,保藏编号为:CGMCC No.19131。保藏地址:北京市朝阳区北辰西路1号院3号。
表1菌株UR-3生理生化实验结果
注:“-”代表阴性,“+”代表阳性。
表2菌株UR-3碳源发酵实验结果
注:“+”代表强发酵,“-”代表不发酵,“d”表示略发酵。
二、饲喂仔猪实验:
本发明提供的植物乳杆菌发酵液体猪全价饲料应用于饲喂仔猪,能够显著提高仔猪的平均日增重和平均日采食量,降低仔猪的腹泻率和料肉比,进而改善仔猪的血液生化指标,并经试验资料得到充分证明,相关试验资料如下:
本发明提供的植物乳杆菌发酵液体猪全价饲料应用于仔猪的饲喂:
选用35日龄杜长大三元杂交仔猪90头,随机进行分成3组,试验周期为21天。
试验方案
Ⅰ组:对照组,饲喂基础饲粮,基础饲粮为:玉米50%、豆粕20%、膨化大豆19.01%、乳清粉3%、进口鱼粉3%、豆油1%,赖氨酸0.12%、蛋氨酸0.12%、食盐0.15%、石粉1.1%、磷酸氢钙0.5%和预混料2.0%。
Ⅱ组:饲喂含抗生物的基础饲粮;
Ⅲ组:饲喂本发明实施例2得到的植物乳杆菌发酵液体全价饲料。
试验组和对照组均自由采食和饮水,试验开始和结束时空腹称重,统计耗料量,注意观察腹泻情况,计算平均日增重、采食量、腹泻发生率。
试验结果如表1所示。
表1乳酸菌发酵液体饲料对仔猪生产性能的影响
从表1中可以看出:与对照组相比,试验Ⅱ组和试验Ⅲ组的平均日增重分别提高了17.68%(P<0.05)和26.88%(P<0.05),试验Ⅱ组和试验Ⅲ组间差异不显著(P>0.05),试验Ⅱ组和试验Ⅲ组的平均日采食量分别提高了5.34%(P<0.05)和10.68%(P<0.05),试验Ⅱ组和试验Ⅲ组的料肉比分别降低了12.75%(P<0.05)和11.74%(P<0.05)。可见该植物乳杆菌发酵液体饲料可增加仔猪日采食量,减少腹泻率。
表2植物乳杆菌发酵液体猪全价饲料对仔猪血液生化指标的影响
由表2得知,与对照组相比,试验Ⅱ组和试验Ⅲ组血样中总蛋白、免疫球蛋白A、免疫球蛋白G指标明显提高,差异显著(P<0.05),且试验Ⅱ组和试验Ⅲ组间差异显著(P<0.05),试验Ⅱ组和试验Ⅲ组血样中尿素氮含量明显降低,差异显著(P<0.05),且试验Ⅱ组与试验Ⅲ组间差异显著(P<0.05)。
经多批次重复实验,由本发明其他实施例制得的产品均能取得和以上实验结果相同或相近似的有益效果。
本发明提供的植物乳杆菌发酵液体全价饲料主要应用于饲喂仔猪,在整个养猪过程中难度最大且复杂的环节是仔猪生长阶段,对于仔猪来说,植物乳杆菌发酵液体猪全价饲料在形态方面比较接近母乳,可以提供充足的营养和水,从饲喂试验我们可以看出本发明提供的植物乳杆菌发酵液体猪全价饲料具有良好的饲喂效果,能提高仔猪的采食量,减少腹泻率;在发酵过程中产生的乳酸可抑制肠道病原菌生长,能够显著提升仔猪的免疫力,有效促进它的健康生长,是仔猪阶段的理想饲粮;另外,植物乳杆菌发酵液体猪全价饲料对仔猪血液生化指标影响的相关研究较少,因此,研发植物乳杆菌发酵液体猪全价饲料对我国畜牧业的发展意义重大,植物乳杆菌发酵液体猪全价饲料的工艺及其在仔猪上的应用的研究是发酵饲料上的一大创新,为植物乳杆菌发酵液体猪全价饲料在生猪无抗养殖应用生产中替代抗生素提供了试验理论依据,为进一步研发安全、高效、环保的发酵饲料奠定了理论基础,作为一种高效、绿色、生态和安全的饲料具有在各类养猪场推广应用的潜力。
本发明提供的植物乳杆菌发酵液体猪全价饲料作为一种新型绿色环保饲料优点是:易生产制备、成本低、安全、无污染、无药物残留、易消化、对饲料原料营养成分破坏小,应用效果好,完全满足发酵液体饲料产品标准,经乳酸菌发酵的饲料,含有丰富的乳酸菌及其代谢产物、乳酸菌素、小肽、维生素等,并具有酸香气味,适口性好,对动物有诱食效果,从而增加仔猪的采食量,饲喂畜禽后可抑制肠道有害菌的生长,可维持动物消化道微生态平衡,增强动物机体的免疫力,有效防止畜禽多种疾病的发生,是发酵饲料上的创新,具有良好的推广和应用价值。
Claims (3)
1.一种植物乳杆菌UR-3,其特征在于,其分类命名为植物乳杆菌(Lactobacillusplantarum),已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为:CGMCC No.19131。
2.权利要求1所述的植物乳杆菌UR-3在制备液体全价饲料中的应用,其特征在于,是将植物乳杆菌接种至配制好的液体全价饲料中,接种量4~6%,于26~33℃摇床160~200rpm发酵3~6d,即得植物乳杆菌发酵液体全价饲料;所述的全价饲料是由以下质量百分比的原料制成: 玉米47~52%、豆粕16~24%、膨化大豆16.5~20%、乳清粉1~4%、进口鱼粉1~5%、豆油0.5~1.8%,赖氨酸0.1~0.14%、蛋氨酸0.1~0.14%、食盐0.1~0.2%、石粉1.0~1.2%、磷酸氢钙0.1~0.9%和预混料2.0%,所述的预混料每千克是由Cu 200 mg、Fe 2300mg、Zn 2800 mg、Mn 450 mg、VA 95000 IU、VD3 13000 IU、VE 3000 IU、VK3 90 mg、VB2 300 mg和余量为淀粉混合均匀制成;
包括以下步骤:
1)种子液的制备:取植物乳杆菌UR-3接种到制备好的已灭菌的MRS液体培养基中,26~33℃摇床160~180rpm发酵2~4d,再吸取2~4%的菌种,转接到另一瓶新制备好MRS 液体培养基中,26~33℃摇床160~180rpm发酵2~4d,最终制成植物乳杆菌种子液,种子液活菌数≥1×109cfu/mL;所述的MRS液体培养基为:牛肉浸粉5.0g、蛋白胨10.0g、酵母浸出粉4.0g、葡萄糖20.0g、柠檬酸三铵2.0g、吐温-80 1.0ml、硫酸锰0.05g、硫酸镁0.2g、乙酸钠5.0g、磷酸氢二钾2.0g、100ml蒸馏水,加热煮沸溶解分装,调pH值至6.2±0.2,121℃高压灭菌15min备用;
2)原料预处理:将全价饲料原料粉碎后充分混合均匀,将混合后的原料细粉碎至粒度为60目,制成发酵液体饲料干粉物料,将发酵液体饲料干粉物料按水料比1:2~3的比例混合均匀,加入三角瓶中;
3)发酵:将步骤1)中制备好的植物乳杆菌种子液接种到步骤2)中已混匀装好的发酵液体饲料干粉物料的三角瓶中,接种量4~6%,完全密封,26~37℃摇床160~200rpm发酵3~6d,当发酵液pH降到4.0以下时结束发酵,即得植物乳杆菌发酵液体全价饲料。
3.根据权利要求2所述的植物乳杆菌UR-3在制备液体全价饲料中的应用,其特征在于,包括以下步骤:
1)种子液的制备:取植物乳杆菌UR-3接种到制备好的已灭菌的MRS液体培养基中,30℃摇床170rpm发酵3d,再吸取3%的菌种,转接到另一瓶新制备好MRS 液体培养基中,30℃摇床170rpm发酵3d,最终制成植物乳杆菌种子液,种子液活菌数≥1×109cfu/mL;所述的MRS液体培养基为:牛肉浸粉5.0g、蛋白胨10.0g、酵母浸出粉4.0g、葡萄糖20.0g、柠檬酸三铵2.0g、吐温-80 1.0ml、硫酸锰0.05g、硫酸镁0.2g、乙酸钠5.0g、磷酸氢二钾2.0g、100ml蒸馏水,加热煮沸溶解分装,调pH值至6.2±0.2,121℃高压灭菌15min备用;
2)原料预处理:将玉米50%、豆粕20%、膨化大豆19.01%、乳清粉3%、进口鱼粉3%、豆油1%,赖氨酸0.12%、蛋氨酸0.12%、食盐0.15%、石粉1.1%、磷酸氢钙0.5%和预混料2.0%粉碎后充分混合均匀,将混合后的原料细粉碎至粒度为60目,制成发酵液体饲料干粉物料,将发酵液体饲料干粉物料按水料比1:2~3的比例混合均匀,加入三角瓶中;
3)发酵:将步骤1)中制备好的植物乳杆菌种子液接种到步骤2)中已混匀装好的发酵液体饲料干粉物料的三角瓶中,接种量5%,完全密封,30℃摇床180rpm发酵4d,当发酵液pH降到4.0以下时结束发酵,即得植物乳杆菌发酵液体全价饲料。
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