CN116083273B - 一株植物乳杆菌NHE-LpE15及应用 - Google Patents
一株植物乳杆菌NHE-LpE15及应用 Download PDFInfo
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- CN116083273B CN116083273B CN202211079595.5A CN202211079595A CN116083273B CN 116083273 B CN116083273 B CN 116083273B CN 202211079595 A CN202211079595 A CN 202211079595A CN 116083273 B CN116083273 B CN 116083273B
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Abstract
本发明提供了一株植物乳杆菌(Lactobacillus plantarum)NHE‑LpE15,该菌分离自仔猪肠道,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.24434,保藏日期为2022年2月28日。本发明的植物乳杆菌NHE‑LpE15具有显著的产植酸酶性能,发酵性能强,制备液体发酵饲料不损失饲料游离氨基酸及营养物质,改善饲料利用率吗,改善动物健康,可用于猪用液体发酵饲料的制备,拓宽猪用液体发酵饲料的微生物菌剂。
Description
技术领域
本发明属于畜牧饲用益生菌技术领域,尤其涉及一株植物乳杆菌NHE-LpE15及应用。
背景技术
目前,在畜牧业生产中,通常是采用抗生素来治疗和预防疾病,从而实现减缓应激和促进生长的目的。
但是,抗生素在规模化养殖中的长期使用会导致病原体的耐药性,并且会导致肉食品中的兽药残留和环境污染。因此需要新的抗生素替代品来替代抗生素。
益生菌是常见的抗生素的替代品之一,其对于猪的生长性能和疾病的防治具有积极的作用。现有的研究表明,益生菌可以有效的补充肠道内微生物的不足,提高仔猪的抗病能力。但是现有的菌株功能多较为单一,依然存在着较多的不足,因此,开发更多性能优良的菌株依然是目前急需解决的问题。
发明内容
本发明的目的在于提供一株植物乳杆菌NHE-LpE15及应用。
为实现上述目的,本发明提供了如下技术方案:
一株植物乳杆菌NHE-LpE15,分类命名为植物乳杆菌(Lactobacillus plantarum)NHE-LpE15,于2022年2月28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,其保藏编号为CGMCC NO.24434,保藏地址为中国北京。
优选地,所述植物乳杆菌NHE-LpE15是从新鲜仔猪肠道内容物中分离的植物乳杆菌,通过菌落形态观察、分子生物学鉴定等确定其为植物乳杆菌,其16S rDNA序列如SEQ IDNO.1所示。
优选地,所述植物乳杆菌NHE-LpE15具有以下微生物学特征:植物乳杆菌NHE-LpE15是革兰氏阳性杆菌,在MRS琼脂培养基上生长良好,培养48h,形成圆形菌落,菌落直径2-3mm,呈乳白色,表明不光滑,不透明,中间凸起,边缘不整齐,菌落形态如图1,显微形态如图2,革兰氏阳性,呈杆状,无芽孢,兼性厌氧,生长适宜温度范围为20℃-40℃,最佳生长温度为25℃-37℃,生长pH为3.0-7.0,最适pH为4.0-7.0。
优选地,所述植物乳杆菌NHE-LpE15的培养方法如下:
取植物乳杆菌NHE-LpE15(保藏编号为CGMCC No.24434)种子液(活菌浓度为109CFU/mL)3mL,接种于300mL摇瓶发酵培养基中进行摇瓶发酵培养;摇瓶发酵结束后进行发酵罐发酵培养,取300mL摇瓶发酵种子液接种到50L发酵罐中的发酵培养基中进行发酵培养,50L发酵罐装液量为35L发酵培养培养基。
所述摇瓶发酵培养基由下述组分组成:蔗糖0.5-4%,葡萄糖0.5-2.5%,酵母浸粉0.5-3.0%,牛肉浸粉0.5-2.5%,氯化镁0.01-0.5%,碳酸钙0.01-1.0%,硫酸锰0.01-0.5%,余量为水。
优选为:蔗糖1%,葡萄糖1%,酵母浸粉0.5%,牛肉浸粉1%,氯化镁0.1%,碳酸钙0.1%,硫酸锰0.02%,余量为水。
所述摇瓶发酵条件为:接种量1%(体积比),发酵温度为35℃、pH6.8、200r/min,发酵时间10h。
所述50L发酵罐的培养基成分及发酵条件为:绵白糖1.0-4.0%,葡萄糖0.2-2.0%,酵母膏0.5-3.5%,牛肉浸粉0.5-2.5%,玉米将干粉0.5-2.5%,氯化镁0.01-0.5%,碳酸钙0.01-1.0%,硫酸锰0.01-0.5%,吐温-80 0.05-0.2%,余量为水。
优选为:绵白糖1.5%,葡萄糖0.5%,酵母膏0.5%,牛肉浸粉0.5%,玉米将干粉0.5%,氯化镁0.1%,碳酸钙0.1%,硫酸锰0.02%,吐温-80 0.1%,余量为水。
所述摇瓶发酵条件为:50L发酵罐装液量为35L培养基,控制罐压0.05-0.06MPa,接种量为300mL,发酵温度为35℃,发酵时间8h,pH值6.0,搅拌转速200r/min。
植物乳杆菌NHE-LpE15在制备为生态制剂中的应用。
优选地,所述微生态制剂中植物乳杆菌NHE-LpE15的活菌数为3.6×1010CFU/mL。
植物乳杆菌NHE-LpE15制备的微生态制剂在制备沙门氏菌感染免疫预防药物中的应用。
优选地,所述微生态制剂中植物乳杆菌NHE-LpE15的活菌数为3.6×1010CFU/mL。
植物乳杆菌NHE-LpE15在制备广谱杀菌剂中的应用。
优选地,所述广谱杀菌剂的杀菌剂如下:猪丹毒丝菌、绿脓假单胞菌、肺炎克雷伯氏菌、伊氏李斯特菌、肺炎链球菌、肠道致病大肠杆菌、金黄色葡萄球菌、伤寒沙门氏菌、沙门氏菌、产气荚膜杆菌、彭氏变形杆菌、嗜水气单胞菌、副溶血弧菌。
植物乳杆菌NHE-LpE15在制备加州鲈鱼饲料中的应用。
植物乳杆菌NHE-LpE15在制备液体发酵饲料中的应用。
本发明的有益效果是:
(1)本发明的植物乳杆菌NHE-LpE15产酸能力强、发酵不利用游离氨基酸、产植酸酶能力强。经发酵培养基和工艺优化,35℃发酵8h,发酵液活菌数可达到3.6×1010CFU/mL,可用于液体发酵饲料的生产,能改善液体发酵饲料特性,增加液体发酵饲料中植酸磷的释放。
(2)本发明中的植物乳杆菌NHE-LpE15可用于制备液体发酵饲料,制备的液体发酵饲料富含乳酸菌,pH值明显降低,发酵前后及液体发酵饲料贮藏过程中不损失游离氨基酸,不产生有害成分,制备的液体发酵饲料性质稳定,富含有益微生物。
(3)本发明中的植物乳杆菌NHE-LpE15具有广谱的杀菌活性可以抑制多种有害菌株。
附图说明
图1是植物乳杆菌NHE-LpE15在MRS培养基上的菌落形态图;
图2是植物乳杆菌NHE-LpE15菌株的革兰氏染色图。
具体实施方式
下面通过具体的实施方案叙述本发明。实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
若未特别指明,实施例中所用的化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
本发明中涉及到的百分号“%”,若未特别说明,是指质量百分比;但溶液的百分比,除另有规定外,是指100mL溶液中含有溶质的克数。
实施例1植物乳杆菌NHE-LpE15的分离、筛选及鉴定
1、乳酸菌的分离纯化
将采集的新鲜仔猪肠道内容物带回实验室,在无菌条件下称取样品10克,置于盛有90mL灭菌生理盐水的三角瓶中,于37℃下恒温震荡1h后,采用10倍倍比稀释的方法依次稀释至100万倍,选择1万倍、10万倍和100万倍三个稀释度,吸取0.1mL涂布于改良的MRS琼脂平板上;
(2)平板涂布后37℃倒置培养48h,用接种环挑取溶钙圈明显且大于5mm的疑似乳酸菌的菌落在MRS琼脂平板上进行划线分离培养;
(3)经48h培养后,挑取分离效果较好的菌落,用接种环转接于MRS琼脂斜面上作纯培养,重复传代纯培养3次,而后将菌种细胞悬浮于20%甘油溶液中,于-80℃冰箱保存备用。
2、菌落形态的观察
根据步骤1中溶钙圈大小,初步筛选出产酸能力强的菌株可初步确定为乳酸菌,共182株。将步骤1保存的甘油管菌种,经MRS琼脂平板2-3次活化,然后接种于MRS肉汤培养基中,37℃恒温180转/分钟震荡培养1-20h,取干净的载玻片进行革兰氏染色,镜检,观察菌株显微形态,选取革兰氏染色为阳性的不产芽胞细菌作为备用。
3、乳酸菌悬液和发酵液制备
将步骤1获得的乳酸菌在MRS琼脂平板上划线35℃培养48h,从平板上挑取单菌落于100mL MRS液体培养基中,35℃,180r/min震荡培养24h,获得乳酸菌悬液备用;继续35℃,180r/min震荡培养96h,获得乳酸菌发酵液备用。
4、乳酸菌产酸能力的筛选
将步骤3种获得的乳酸菌悬液按1%(体积比)的接种量接种于MRS液体培养基中,35℃培养12h,筛选出12h将发酵液pH降至4.0以下的乳酸菌,共获得39株乳酸菌。
5、乳酸菌降解赖氨酸的筛选
将步骤3中获得的乳酸菌悬液按1%(体积比)的接种量接种于添加1%赖氨酸的MRS液体培养基中,35℃培养24h,每4小时测一次游离赖氨酸含量,选择发酵过程中不降解赖氨酸的菌株26株。
6、乳酸菌产植酸酶能力的筛选
将步骤1获得的乳酸菌在MRS琼脂平板上划线35℃培养48h,从平板上挑取单菌落接种于植酸酶筛选培养基平板上,35℃恒温培养48h,然后用植酸酶筛选染色液染色,选择透明圈大的菌株E15、E28、E76。然后将以上3株菌进行液体发酵培养,培养完成后进行植酸酶的酶活测定,菌株E15植酸酶活最高,为4.2U/mL,作为下一步研究菌株。
7、菌株种属的鉴定
对菌株E15进行了形态和生理生化鉴定,菌株E15在MRS平板上能良好生长,培养48h,形成圆形菌落,菌落直径2-3mm,呈乳白色,表明不光滑,不透明,中间凸起,边缘不整齐,菌落形态如图1,显微形态如图2,革兰氏阳性,呈杆状,无芽孢,兼性厌氧,生长适宜温度范围为20℃-40℃,最佳生长温度为25℃-37℃,生长pH为3.0-7.0,最适pH为4.0-7.0。采用细菌DNA提取的试剂盒提取E15菌株基因组DNA。通过引物F和R对E15菌株的16S rDNA基因片段进行测序,所得序列如SEQ ID NO.1所示,将所测得的序列与GenBank中的16S rDNA序列进行BLAST分析比对,可以看出菌株E15与植物乳杆菌的同源性达到100%。通过菌株E15的形态特征、16S rDNA特征,确定菌株E15为植物乳杆菌(Lactobacillus plantarum),并正式标号为NHE-LpE15。
8、菌株保藏
上述经分离、纯化、筛选得到的植物乳杆菌(Lactobacillus plantarum)NHE-LpE15已于2022年2月28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,地址为: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,邮政编码:100101),保藏编号为CGMCC NO.24434,分类命名为植物乳杆菌(Lactobacillusplantarum)。
改良MRS琼脂培养基组成为:蛋白胨1.0%,乙酸钠0.5%,牛肉膏1.0%,葡萄糖2%,酵母膏0.5%,吐温80 0.1%,K2HPO4 0.2%,MgSO4 0.058%,柠檬酸二铵0.2%,MnSO40.025%,琼脂1.8%,碳酸钙1%,余量为水,pH 7.0±0.2。
MRS琼脂培养基组成为:蛋白胨1.0%,乙酸钠0.5%,牛肉膏1.0%,葡萄糖2%,酵母膏0.5%,吐温80 0.1%,K2HPO4 0.2%,MgSO4 0.058%,柠檬酸二铵0.2%,MnSO40.025%,琼脂1.8%,余量为水,pH 7.0±0.2。
MRS肉汤培养基组成为:蛋白胨1.0%,乙酸钠0.5%,牛肉膏1.0%,葡萄糖2%,酵母膏0.5%,吐温80 0.1%,K2HPO4 0.2%,MgSO4 0.058%,柠檬酸二铵0.2%,MnSO40.025%,余量为水,pH 7.0±0.2。
植酸酶筛选培养基组成为:胰蛋白胨1%,酵母粉0.5%,氯化钠1%,植酸钙0.2%,琼脂1.8%,余量为水,pH 7.0±0.2。
植酸酶筛选染色液组成为:硝酸溶液2份,钼酸铵溶液1份,偏钒酸铵溶液1份,混合后使用,现用现配。
钼酸铵溶液组成为:钼酸铵10%,25%氨水0.1%,余量为水。
偏钒酸铵溶液组成为:偏钒酸铵0.235%,硝酸铵溶液2%,余量为水。
硝酸溶液组成为:硝酸33%,余量为水。
植酸酶活测定方法参照GB/T 18634-2009饲用植酸酶活性的测定-分光光度法。
实施例2植物乳杆菌NHE-LpE15发酵液的制备
取植物乳杆菌NHE-LpE15(保藏编号为CGMCC No.24434)种子液(活菌浓度为109CFU/mL)3mL,接种于300mL摇瓶发酵培养基中进行摇瓶发酵培养;摇瓶发酵结束后进行发酵罐发酵培养,取300mL摇瓶发酵种子液接种到50L发酵罐中的发酵培养基中进行发酵培养,50L发酵罐装液量为35L发酵培养培养基。发酵结束后,检测发酵液活菌数为3.6×1010CFU/mL。
摇瓶发酵培养基由下述组分组成:蔗糖1%,葡萄糖1%,酵母浸粉0.5%,牛肉浸粉1%,氯化镁0.1%,碳酸钙0.1%,硫酸锰0.02%,余量为水。
摇瓶发酵条件为:接种量1%(体积比),发酵温度为35℃、pH6.8、200r/min,发酵时间10h。
50L发酵罐的培养基成分及发酵条件为:绵白糖1.5%,葡萄糖0.5%,酵母膏0.5%,牛肉浸粉0.5%,玉米将干粉0.5%,氯化镁0.1%,碳酸钙0.1%,硫酸锰0.02%,吐温-80 0.1%,余量为水。
摇瓶发酵条件为:50L发酵罐装液量为35L培养基,控制罐压0.05-0.06MPa,接种量为300mL,发酵温度为35℃,发酵时间8h,pH值6.0,搅拌转速200r/min。
实施例3植物乳杆菌NHE-LpE15益生性验证
(1)取致病菌(猪丹毒丝菌、绿脓假单胞菌、肺炎克雷伯氏菌、伊氏李斯特菌、肺炎链球菌、肠道致病大肠杆菌、金黄色葡萄球菌、伤寒沙门氏菌、沙门氏菌、产气荚膜杆菌、彭氏变形杆菌、嗜水气单胞菌、副溶血弧菌)浓度为109CFU/mL的菌悬液2mL,加入装有200mL灭菌后冷至45℃左右的致病菌培养基中,然后吸取10mL未凝固的带菌培养基,转入倒有10mL底层平板的营养琼脂平板上,制备成致病菌平板若干个。
(2)在超净工作台上每个病原菌营养琼脂平板用无菌镊子夹取1个灭菌牛津杯(内径6mm、外径8mm、高10mm的圆形小管,管中可加入200μL液体,管的两端要光滑)放在平皿上,使其与培养基接触无空隙,待10分钟后,分别向各小管中滴加200μL保存好的实施例2制备的发酵液,勿使其外溢,37℃培养36-96h,然后测量抑菌圈直径。每个实验三个重复,取平均值,结果如表1。
致病菌培养基分别为:猪丹毒丝菌、伊氏李斯特菌和肺炎链球菌为TSA+5%脱纤维羊血琼脂,绿脓假单胞菌、肺炎克雷伯氏菌、肠道致病大肠杆菌、金黄色葡萄球菌、伤寒沙门氏菌、沙门氏菌、彭氏变形杆菌和嗜水气单胞菌为营养琼脂培养基,副溶血弧菌为TCBS培养基,产气荚膜杆菌为胰胨-亚硫酸盐-环丝氨酸琼脂培养基。
TSA+5%脱纤维羊血琼脂组成为:胰蛋白胨1.5%,大豆胨0.5%,氯化钠0.5%,琼脂1.5%,余量为水,pH 7.2±0.2,使用时冷却至50℃时加入5%脱纤维羊血。
营养琼脂培养基组成为:蛋白胨1%,牛肉膏0.3%,琼脂2%,NaCl 0.5%,余量为水,pH 7.2±0.2。
TCBS琼脂培养基组成为:酵母粉0.5%,蛋白胨1%,硫代硫酸钠1%,枸橼酸钠1%,牛胆粉0.5%,牛胆酸钠0.3%,蔗糖2%,氯化钠1%,柠檬酸铁0.1%,麝香草酚兰0.0004%,琼脂1.5%,pH 8.6±0.1。
胰胨-亚硫酸盐-环丝氨酸琼脂培养基组成为:胰胨1.5%,大豆胨0.5%,酵母粉0.5%,焦亚硫酸钠0.1%,柠檬酸铁铵0.1%,琼脂2%,余量为水,pH 7.6±0.2,使用时冷却至50℃时加入过滤除菌的0.5%D-环丝氨酸溶液20ml/250mL。
表1植物乳杆菌NHE-LpE15对病原菌的抑菌效果
实施例4植物乳杆菌NHE-LpE15抗逆性验证
1.耐人工胃液性能测定:
取10mL植物乳杆菌NHE-LpE15菌悬液(按照实施例1第3步骤中描述的方法制备)置于90mL(250mL三角瓶)配置的人工胃液中,37℃,200r/min恒温震荡180min;震荡完成后取10mL样液调节pH值至7.0,加入90mL生理盐水,37℃,200r/min恒温震荡30min,然后进行稀释平板菌落培养计数,结果如表2所示。
从表2中可以看出,植物乳杆菌NHE-LpE15在pH1.5、pH2.0、pH2.5的人工胃液(含酶)中分别处理3h存活率均在95%以上,表明该菌株具有较高的耐酸性,可以耐受胃酸,能顺利到达肠道发挥作用。
人工胃液制备方法:人工胃液的制备参照《中华人民共和国药典》2010版中的制备方法,取稀盐酸16.4mL,加水约800mL与胃蛋白酶10g,摇匀后,加水称释成1000mL,调节pH值分别为1.5、2.0和2.5,微孔滤膜除菌(0.22μm)备用。
表2植物乳杆菌NHE-LpE15在人工胃液中处理后3h的存活情况
处理 | pH1.5 | pH2.0 | pH2.5 |
初始活性CFU/mL | 3.6×1010 | 3.6×1010 | 3.6×1010 |
处理后活性CFU/mL | 3.5×1010 | 3.6×1010 | 3.6×1010 |
处理后存活率% | 97.22 | 100 | 100 |
2.耐人工肠液性能测定
取1mL植物乳杆菌NHE-LpE15菌悬液(按照实施例1第3步骤中描述的方法制备)置于99mL(250mL三角瓶)人工肠液中,37℃,200r/min恒温震荡5h,震荡完成后取1mL样液加入99mL生理盐水,37℃,200r/min恒温震荡30min,然后进行稀释平板菌落培养计数。结果显示,植物乳杆菌NHE-LpE15在人工肠液中活性不减少,存活率100%,表明该菌株能很好在肠液中生存并保存活力,从而发挥其益生作用。
人工肠液制备:人工肠液的制备参照《中华人民共和国药典》2010版中的制备方法,磷酸盐缓冲液(含胰酶)(pH6.8),取磷酸二氢钾6.8g,加水500mL使溶解,用0.1mol/L氢氧化钠溶液调节pH值至6.8;另取胰酶10g,加水适量溶解,将两液混合后,加水稀释至1000mL,然后用0.22μm微滤膜过滤除菌,即得。
3.耐胆盐性能测定
取1mL植物乳杆菌NHE-LpE15菌悬液(按照实施例1第3步骤中描述的方法制备)置于99mL(250mL三角瓶)不同胆盐浓度的溶液中,胆盐浓度分别为0.03%、0.15%、0.3%,然后37℃,200r/min恒温震荡120min,震荡完成后取1mL样液加入99mL生理盐水,37℃,200r/min恒温震荡30min,然后进行稀释平板菌落培养计数。结果如表3所示。植物乳杆菌NHE-LpE15在0.3%胆盐溶度的溶液中处理2小时,存活率为86.11%,表明该菌株具有较高的耐胆盐性,可以耐受十二指肠液中的胆盐,使菌株能到达肠道去发挥作用。
所述不同浓度胆盐溶液制备方法:分别取1.8mL、9mL、18mL的5%胆盐溶液加入pH7.4的PBS溶液中,定容至300mL,混合均匀,此为含胆盐0.03%、0.15%、0.30%的PBS溶液。
所述5%胆盐溶液制备方法:准确称取5.0g胆盐,用100mLPBS溶液溶解定容,121℃灭菌20min。
所述PBS溶液制备方法:氯化钠0.8%,氯化钾0.02%,磷酸氢二钠0.363%,磷酸二氢钾0.024%,余量为水。用6mol/L的HCl调pH至7.4,121℃灭菌20min,备用。
表3植物乳杆菌NHE-LpE15在不同浓度胆盐溶液中处理2h后的存活情况
实施例5植物乳杆菌NHE-LpE15急性毒性试验
植物乳杆菌的安全性评价采用急性毒性试验,参照国标GB15193.3-2003最大耐受剂量法进行。取普通昆明小白鼠60只,雌雄各半,18-20g,常规饲养1周后,一日分三次对小白鼠进行灌胃,0.25g/mL植物乳杆菌NHE-LpE15菌液(相当于15000mg/kg体重),连续灌服2周,观察小鼠是否有中毒或死亡的现象。
试验期间,小鼠精神状态良好,无中毒和死亡现象,因此本发明菌株的急性毒性试验最大耐受剂量MTD>15000mg/kg,根据分级标准,可确定该菌种为无毒级,具有较高的安全性。
实施例6小鼠攻毒试验
选取C57小鼠60只,雌性,11-13g,常规饲养。随机分为三组,每个处理饲喂基础日粮,饲喂5天,使小鼠尽快适应。开始正式试验,正式试验分两个阶段。第一阶段生长性能观察试验,实验组在饮水中添加植物乳杆菌发酵液,添加浓度为1×107CFU/mL。对照组和负对照组饮用未加植物乳杆菌的纯净水,连续饮水两周。试验前后分别称重1次,试验期间观察小鼠生长情况,计算各组体重平均增长率,结果如表4。第二阶段沙门氏菌攻毒试验,对负对照组和试验组进行沙门氏菌攻毒,攻毒方式采用灌胃的方式,具体为灌胃浓度为108数量级的沙门氏菌0.5mL/只,对照组则灌胃0.5mL/只生理盐水。攻毒后观察小鼠精神状态及死亡情况,攻毒后试验组继续饮用1×107CFU/mL浓度的植物乳杆菌NHE-LpE15发酵液,对照组和负对照组正常饮用自来水。试验期间小鼠同室分笼饲养,自然光照,自由采食。环境温度控制在25±2℃,湿度60%,植物乳杆菌发酵液为实施例2制备的发酵液。
沙门氏菌攻毒后,通过每日观察,小鼠的发病症状有:基本不进食,不活泼,精神萎靡不振,蜷缩在一起,毛色不光泽,且背毛凌乱,眼睛充血。攻毒后,第1天,负对照组开始有发病症状,且死亡2只,其它存活的也不同程度的出现了发病症状。试验组在第3天死亡1只,第5天死亡1只,其它小鼠5天内,精神不佳,不进食,之后逐渐恢复正常。
攻毒后各组死亡时间、死亡数及存活率如表5所示,试验组小鼠攻毒后中毒现象不严重,表明植物乳杆菌NHE-LpE15对沙门氏菌感染有一定的免疫预防效果,有效地降低了小鼠受沙门氏菌感染时的发病率,延缓攻毒后的发病时间,从而有效提高了存活率。
表4小鼠增重情况
分组 | 平均始重(g/只) | 平均末重(g/只) | 平均增长率(%) |
对照组 | 11.25±0.16 | 22.14±0.78 | 96.80b |
负对照组 | 11.43±0.24 | 22.42±0.49 | 96.15b |
试验组 | 11.55±0.23 | 26.56±0.87 | 129.96a |
表5攻毒前、后的小鼠死亡情况
实施例7植物乳杆菌NHE-LpE15液体发酵饲料制备
将保藏的4株乳酸菌菌种,经MRS琼脂平板2-3次活化,然后接种于MRS肉汤培养基中,37℃恒温180转/分钟震荡培养24h,获得4株乳酸菌种子液备用。将5株乳酸菌种子液混合均匀,获得菌种混合液。然后按照5%接种量加入液体饲料中,混合转速200r/min,混合30min,然后装入发酵桶中,密闭发酵14h,获得植物乳杆菌NHE-LpE15液体发酵饲料。液体发酵饲料经过检测,发酵前后相关指标检测结果如表1所示,发酵前后明显降低液体饲料pH值,降低氧化还原电位,明显提升微生物含量,营养物质不损失。
表1液体发酵饲料发酵前后相关指标检测情况
项目 | 发酵前 | 发酵后 |
pH | 6.82±0.12a | 3.95±0.23b |
氧化还原电位(mV) | 179±10.21a | -181±8.34b |
乳酸菌有效活菌数(CFU/mL) | 2.1a×106 | 4.9b×109 |
游离赖氨酸含量(mg/mL) | 1.29±0.03 | 1.29±0.09 |
总游离氨基酸含量(%) | 1.34±0.08 | 1.62±0.13 |
氨基酸总和(%) | 18.53±0.34 | 18.57±0.21 |
尸胺含量(μg/mL) | 4.12±0.23 | 4.21±0.12 |
所述菌种混合液由下述组分组成:
乳酸片球菌NHB-PaA4种子液30%,副干酪乳杆菌NHB-LpD2种子液20%,植物乳杆菌NHE-LpE5种子液20%,植物乳杆菌NHE-LpE9种子液20%,植物乳杆菌NHE-LpE15种子液10%(实施例2制备的发酵液)。
所述液体饲料由下述组分组成:固体饲料50%,余量为水。
优选为:固体饲料30%,余量为水
所述固体饲料由下述组分组成:玉米(中大猪用)51.43份,小麦15份,小麦胚(二级)5份,豆粕(蛋白含量大于等于46%)11,4份,麸皮(粗灰分小于等于5.5)8.1份,小麦次粉(二级)5份,石粉1.03份,L-赖氨酸硫酸盐及其发酵副产物(70%)0.89份,磷酸氢钙(Ⅲ)0.64份,氯化钠(98.5%)0.41份,蒙脱石(95)0.4份,L-苏氨酸(98.5%)0.23份,佳尔富(M-S220D)0.2份,DL-蛋氨酸(98.5%进口)0.18份,L-色氨酸(98%)0.04份,佳尔富(V-S203)0.03份,植酸酶(耐高温20000)0.02份。
副干酪乳杆菌NHB-LpD2的保藏编号为CGMCC NO.24436,该菌已于2022年2月28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。
植物乳杆菌NHE-LpE5的保藏编号为CGMCC NO.24432,该菌已于2022年2月28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。
植物乳杆菌NHE-LpE9的保藏编号为CGMCC NO.24433,该菌已于2022年2月28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。
植物乳杆菌NHE-PaA4的保藏编号为CGMCC NO.24435,该菌已于2022年2月28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。
实施例8液体发酵饲料对断奶仔猪生长性能影响
(1)试验选用同胎次、体重相近(杜洛克×长白×大白)三元杂交断奶仔猪(公母各半),28天断奶仔猪200头,随机分成两组(对照组和试验组),每处理10栏,每栏10头,试验43天,在相同环境下进行饲养试验。对照组饲粮为基础饲粮(参照中国猪饲养标准),试验组饲粮为实施例6制备的液体发酵饲料。试验开始前,对猪舍进行全面的消毒。
(2)对照组猪群采用干料桶饲喂模式,试验猪群采用液态粥料机,均自由采食和饮水。每天记录投料量及健康状况,定期观察猪只皮毛亮度及粪便状况,其他饲养管理均相同。在试验过程中,试验组仔猪形体健美,皮毛光亮,静态时间相对较长,粪便臭味小。与对照组相比,液体发酵饲料组可显著提高试验组仔猪的日增重,显著降低料肉比0.26,显著降低腹泻率。(表5)
表5液体发酵饲料对断奶仔猪生长性能的影响
项目 | 对照组 | 试验组 |
初重(kg) | 6.41±0.17 | 6.41±0.10 |
末重(kg) | 20.20±1.02b | 23.85±1.03a |
平均日增重(g/d) | 306±23.56b | 405±42.18a |
平均日采食量(kg/d) | 563±56.43b | 638±43.21a |
料肉比 | 1.83±0.14a | 1.57±0.21b |
腹泻率 | 7.14±0.14A | 0.95±0.01B |
注:同行数据肩标不同小写字母表示差异显著(p<0.05),肩标相同小写字母或者无字母表示差异不显著(p>0.05)。
实施例9液体发酵饲料对生长育肥猪的影响
选取健康、体重20kg左右、公母比例一致、日龄相近的(杜洛克×长白×大白)三元商品育肥猪80头,完全随机分成2组(对照组和试验组),每组4个栏,每栏10头猪。对照组:试验饲粮为基础日粮(参照中国猪饲养标准);试验组饲粮为实施例6制备的液体发酵饲料。试验猪采用封闭式圈舍,每日喂3次,自由采食和饮水,每日清扫圈舍,饲喂80天。饲养管理及其免疫程序与猪场日常管理相同,定期消毒,发现猪生病及时治疗。试验结果显示,试验组料肉比显著低于对照组,降低0.21,提高了生长性能(表6)。猪肉风味结果显示(表7),液体饲料组猪肉风味显著优于对照组。
表6液体发酵饲料对育肥猪生长性能的影响
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表7液体发酵饲料对育肥猪肉风味的影响
指标 | 对照组 | 试验组 |
醛类 | 226.88±15.54b | 308.56±14.91a |
醇类 | 28.71±2.89b | 47.84±4.97a |
酯类 | 1.31±0.98b | 2.42±1.42a |
酸类 | 61.48±5.70b | 106.86±10.03a |
酮类 | 50.55±3.21b | 92.60±3.49a |
其它 | 9.53±1.99 | 9.39±4.14 |
总计 | 378.47±30.31b | 567.66±38.97a |
实施例10植物乳杆菌NHE-LpE15发酵软颗粒料饲料制备
将植物乳杆菌发酵液按照20%接种量接入加州鲈鱼膨化饲料中,采用喷涂方式接种,然后30℃密闭发酵5天,获得植物乳杆菌NHE-LpE15发酵软颗粒料饲料。发酵结束后检测饲料中植物乳杆菌有效活菌数为7.8×108CFU/g,发酵前后营养成分检测显示营养不损失。
植物乳杆菌NHE-LpE15种子液由实施例2制备的发酵液。
发酵含水量为32%。
所述加州鲈鱼膨化饲料由下述组分组成:进口鱼粉350份,鱼排粉(巴沙或罗非)154份,鸡肉粉(猪肉粉)100份,发酵豆粕50份,豆粕54份,棉粕77份,高筋面粉100份,大豆油40份,谷朊粉30份,酵母提取物20份,磷酸二氢钙15份,预混料及添加剂10份。
所述预混料及添加剂由下述组分组成:FM01 0.25份,氯化胆碱(60)
0.30份,FV01 0.15份,赖氨酸(98)0.3份,蛋氨酸(98)0.15份,防霉剂0.10份,VC酯0.05份,抗氧化剂0.03份,L-肉碱0.01份,爱克多0.02份VE 0.02份。
实施例11发酵软颗粒料对加州鲈鱼生长的影响
(1)选取健康、体重172.5g左右当年加州鲈鱼鱼种,按70尾每箱分成10箱(2m×2m×1.5m),每箱初重基本一致,将10口网箱鱼随机分为2个处理,每个处理5个重复。
(2)正式试验每天投喂3次,投喂时间分别为7:30、14:00、19:00,投喂量为2-4%,根据天气适当调整,每隔一周投喂量增加10%。定时开启充氧系统,保证溶氧大于4mg/L;每天定时测定水温;每周定时测定溶氧、氨氮及亚硝酸盐,根据测定情况进行预防用药及调节水质。养殖周期56天。
试验结果显示,试验组增重率高于对照组,提高了20.53%,试验组加州鲈鱼健康状况明显高于对照组,死亡率明显降低。
表6液体发酵饲料对加州鲈鱼生长性能的影响
指标 | 对照组 | 试验组 |
初重(g) | 172.45±3.41 | 172.45±2.32 |
末重(g) | 252.31±6.79b | 287.72±9.45a |
增重率(%) | 46.31 | 66.84% |
死亡率% | 35.24 | 23.33 |
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 (8)
1.一株植物乳杆菌NHE-LpE15,分类命名为植物乳杆菌(Lactobacillus plantarum)NHE-LpE15,于2022年2月28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,其保藏编号为CGMCC NO.24434,保藏地址为中国北京。
2.如权利要求1所述的植物乳杆菌NHE-LpE15在制备为微生态制剂中的应用。
3.根据权利要求2所述的应用,其特征在于,所述微生态制剂中植物乳杆菌NHE-LpE15的活菌数为3.6×1010CFU/mL。
4.如权利要求1所述的植物乳杆菌NHE-LpE15制备的微生态制剂在制备沙门氏菌感染免疫预防药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述微生态制剂中植物乳杆菌NHE-LpE15的活菌数为3.6×1010CFU/mL。
6.如权利要求1所述的植物乳杆菌NHE-LpE15在制备广谱杀菌剂中的应用,其特征在于,所述广谱杀菌剂的杀菌谱如下:猪丹毒丝菌、绿脓假单胞菌、肺炎克雷伯氏菌、伊氏李斯特菌、肺炎链球菌、肠道致病大肠杆菌、金黄色葡萄球菌、伤寒沙门氏菌、产气荚膜杆菌、彭氏变形杆菌、嗜水气单胞菌和副溶血弧菌。
7.如权利要求1所述的植物乳杆菌NHE-LpE15在制备加州鲈鱼饲料中的应用。
8.如权利要求1所述的植物乳杆菌NHE-LpE15在制备液体发酵饲料中的应用。
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