CN113717887A - 一种鹅源植物乳杆菌及其应用 - Google Patents
一种鹅源植物乳杆菌及其应用 Download PDFInfo
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- CN113717887A CN113717887A CN202111001533.8A CN202111001533A CN113717887A CN 113717887 A CN113717887 A CN 113717887A CN 202111001533 A CN202111001533 A CN 202111001533A CN 113717887 A CN113717887 A CN 113717887A
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- intestinal
- lactobacillus plantarum
- goose
- improve
- lactobacillus
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- A—HUMAN NECESSITIES
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Abstract
本发明提供了一种鹅源植物乳杆菌ER41(Lactobacillus plantarum ER41),其保藏编号为CCTCC No:M 2021932。本发明的植物乳杆菌ER41同时具有如下技术效果:1)能够快速产酸,8h的pH值降到4.0以下,24h产乳酸量为165.87mmol/L,产酸快且产乳酸能力强。2)对大肠杆菌、金黄色葡萄球菌、沙门氏菌和李斯特菌均具有抑菌效果。3)能够更有效地黏附鹅肠道上皮细胞,在鹅肠道内定植。4)能够提高空肠、回肠和盲肠的绒毛高度,显著降低回肠和盲肠的隐窝深度,显著提高盲肠的绒隐比,促进肠道黏膜的健康发育。5)能够提高肠道乙酸、丙酸等短链脂肪酸的含量,促进肠道健康。6)能够显著降低大肠杆菌志贺菌属、肠球菌属等致病菌丰度,提高乳杆菌丰度,调节肠道菌群。
Description
技术领域
本发明属于家禽饲料中的益生菌技术领域,具体公开了一种鹅源植物乳杆菌及其应用。
背景技术
近年来,鹅肉食品持续走俏,肉鹅规模化养殖高速增长,鹅产业成为推动畜牧业发展的重要途径之一。但在“禁抗、禁水养”等政策实施后,肉鹅肠道疾病增加,免疫力下降。运用益生菌调控肠道稳态平衡,提升肠黏膜微生物的屏障保护功能,增强宿主肠道健康、抵御应激能力,是国内外肠道健康研究的热点之一。
肠道微生物会随着食物种类的差异及宿主年龄的增长等生理因素而改变,但总体上处于相对稳态的状态。各种细菌依据其特性及其与宿主形成的适应性,定植在不同的生境中,形成各自的群落。根据细菌与宿主关系的不同,肠道菌群又分为原籍菌群和外籍菌群。原籍菌群是在长期进化过程中形成的,与宿主的关系极为密切和稳定,数量多且有利于维持宿主的生长和健康,又称之为固有菌群或常驻菌。外籍菌群是指与宿主的关系不密切,数量少,不定植或仅短时间定植于动物消化道的细菌,所以也被称为过路菌。
倪世勤在“原籍菌在肉鸡饲料中的研究与应用进展”(《饲料工业》,2021,42(5))指出原籍菌一方面可发挥空间占位效应,占据致病菌的黏附位点,阻断其与肠黏膜的接触,抑制定植和转位,维护肠黏膜的完整性;另一方面,通过争夺营养或产生拮抗作用,阻止病原菌的侵入。因此,将原籍菌分离筛选并制成益生菌后,更易于在对应动物体内定植、繁殖及发挥特定的益生功效。
在菌株筛选方面,中国专利申请CN112877262A提供了一种植物乳杆菌及其应用,其中的菌株NKK20是分离自人体肠道的原籍菌,应用于人类食品中,未研究其对动物肠道健康的作用。中国专利申请CN106399196A提供了一株植物乳杆菌HEW-A490及其应用,其中的菌株HEW-A490分离自鸡肠道,显著提高了鸡的生长性能,但并未研究对肠道结构、肠道菌群等肠道健康的作用,也未提及对动物肠道细胞的黏附作用。
在菌株黏附研究方面,中国专利申请CN109504619A提供了一种植物乳杆菌及其应用,中国专利申请CN109706103A提供了具有高黏附性植物乳杆菌RS-09及其应用,这两个专利中的菌株PA01和RS-09的菌株分离自泡菜和苹果汁等植物,分别对人结肠细胞CAO-2和HT-29有较强的黏附作用,未提及对禽类肠道细胞的黏附作用,也未研究对肠道结构、肠道菌群、肠道代谢物的作用。
因此,基于根据微生物对生境的适应性和特异性原理,运用鹅源原籍菌调控肠道稳态平衡,从肠道结构、肠道菌群、肠道代谢物等方面提升肉鹅肠黏膜微生物的屏障保护功能,可有效缓解当前肉鹅产业中的肠道菌群失衡、肠道亚健康等问题,为肉鹅产业的健康发展,实现无抗养殖提供有力支撑。
发明内容
本发明针对现有技术的不足,其目的在于提供一种鹅源植物乳杆菌及其应用。
本发明的第一个目的是提供一种鹅源植物乳杆菌ER41(Lactobacillusplantarum ER41),所述的植物乳杆菌是从种鹅肠道筛选出来的,具有鹅肠道黏附力强、产酸速度快、抑菌能力强的乳杆菌;该菌株已于2021年7月26日保藏于中国典型培养物保藏中心,其保藏编号为CCTCC NO:M 2021932。
菌株特征:植物乳杆菌(Lactobacillu splantarum)ER41的菌落呈乳白色,边缘整齐;细胞显微形态为短杆状,无芽孢,革兰氏染色阳性;接触酶阴性,氧化酶阴性;具有较强的生长及产酸性能,在起始pH值为3.0-8.0的培养基上能生长,在10℃条件下呈现微弱生长,在15℃—40℃条件下生长状况良好,在不大于6.5%NaCl的条件下生长状况良好。
本发明的第二个目的是提供含有所述植物乳杆菌ER41的菌剂。
本发明的第三个目的是提供所述植物乳杆菌ER41或其发酵产物或所述菌剂在肉鹅养殖生产中的应用。
根据微生物对生境的适应性和特异性原理,本发明从种鹅肠道筛选具有鹅肠道黏附力强、产酸速度快、抑菌能力强的乳杆菌ER41,相对于现有技术,本发明的植物乳杆菌同时具有如下预料不到的技术效果:
1)从实施例2的产酸能力的检测结果表1可以看出,本发明的植物乳杆菌ER41能够快速产酸,8h的pH值降到4.0以下,24h产乳酸量为165.87mmol/L,产酸快且产乳酸能力强。
2)从实施例3的抑菌活性的检测结果表2可以看出,本发明的植物乳杆菌ER41对大肠杆菌、金黄色葡萄球菌、沙门氏菌和李斯特菌均具有抑菌效果。
3)从实施例4的肠道黏附力的测定结果表3可以看出,本发明的植物乳杆菌ER41能够更有效地黏附鹅肠道上皮细胞,在鹅肠道内定植。
4)从实施例5的肉鹅肠道黏膜结构结果表4可以看出,本发明的植物乳杆菌ER41能够提高空肠、回肠和盲肠的绒毛高度,显著降低回肠和盲肠的隐窝深度,显著提高盲肠的绒隐比,促进肠道黏膜的健康发育。
5)从实施例5的肉鹅肠道短链脂肪酸结果表5可以看出,本发明的植物乳杆菌ER41能够提高肠道乙酸、丙酸等短链脂肪酸的含量,促进肠道健康。
6)从实施例5的肉鹅肠道优势菌群相对丰度结果表6可以看出,本发明的植物乳杆菌ER41能够显著降低大肠杆菌志贺菌属、肠球菌属等致病菌丰度,提高乳杆菌丰度,调节肠道菌群。
综上,本发明的植物乳杆菌能有效黏附鹅肠道上皮细胞,在鹅肠道内定植,显著抑制肠道病原菌,从肠道黏膜结构、肠道菌群、短链脂肪酸等方面促进了鹅肠道健康,可广泛应用于肉鹅养殖,提高养殖效益。
本发明涉及到的微生物的保藏信息如下:
保藏单位:中国典型培养物保藏中心(CCTCC);
保藏单位地址:中国武汉;
分类命名:植物乳杆菌ER41(Lactobacillus plantarum ER41);
保藏编号为CCTCC NO:M 2021932;
保藏日期为:2021年7月26日。
具体实施方式
下面结合实施例对本发明做进一步说明,以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。所用的培养基购自北京奥博星生物技术有限公司。其他试剂等,如无特殊说明,均可从商业途径得到。
实施例1植物乳杆菌(Lactobacillus plantarum)ER41的分离与鉴定
1、分离纯化
从鹅肠道中分离纯化到植物乳杆菌ER41,具体如下:称取1g肠道食糜样品加入灭菌的99ml蒸馏水中,利用恒温摇床震荡1h,然后10倍梯度稀释,分别取10-3、10-4的样品稀释液涂布在MRS固体培养基上,培养48h,然后将其取出,依据菌落的大小、形态和颜色,挑取MRS上生长的单菌落,进行过氧化氢酶实验及革兰氏染色。将过氧化氢酶阴性及革兰氏染色阳性者暂定为乳杆菌,于MRS固体培养基上继续划线纯化2次。-80℃保存于25%甘油的MRS培养基中,菌株命名为ER41。
2、生理生化检测
乳杆菌ER41在起始pH值为3.0-8.0的培养基上能生长,在25℃-37℃条件下生长状况良好,在不大于6.5%NaCl的条件下生长状况良好。菌株ER41兼性异型发酵乳糖,能较好地利用葡萄糖、乳糖、果糖、半乳糖、纤维二糖、麦芽糖、甘露糖、松三糖、核糖、蔗糖、海藻糖、山梨醇、甘露醇,可缓慢利用淀粉,不能利用蜜二糖、棉籽糖、鼠李糖、木糖、阿拉伯糖,H2S试验、吲哚试验、明胶液化试验、硝酸盐还原试验均为阴性。
3、分子鉴定
将固体平板上培养48h的乳杆菌菌株ER41,采用菌落PCR扩增,进行16rRNA基因序列鉴定。引物选用细菌的16S rRNA基因扩增通用引物:27F和1492R。PCR反应体系为50μl:Premix Taq25μl,27F和1492R(均为20μM)1μl,补充灭菌蒸馏水至50μl,直接将菌落加入反应体系,进行PCR扩增,得到PCR产物。将PCR产物送去生工生物工程有限公司测序,结果为序列表中SEQ No.1所示。
经过上述鉴定,植物乳杆菌ER41于2021年7月26日,已保藏于中国典型培养物保藏中心(CCTCC),保藏地址:湖北省武汉市武昌区八一路299号武汉大学校内,植物乳杆菌ER41(Lactobacillus plantarum ER41),保藏号:CCTCC NO:M 2021932,保藏日期为:2021年7月26日。
实施例2植物乳杆菌ER41(Lactobacillus plantarum ER41)产酸能力的检测
菌株活化:将冷冻保存的Lactobacillus plantarum ER41接种于MRS液体培养基中,在温度37℃下培养24h,如此传代培养2次得到所述的活化Lactobacillus plantarumER41;活化后的菌株按3%体积比的接种量重新转接MRS液体培养基中,培养24小时,分别在4h、8h、12h、24h取样,测定上清液的pH值和产乳酸量。结果如表1所示。由表1可知,ER41能快速产酸,8h的pH值降到4.0以下,24h产乳酸量为165.87mmol/L,产酸快且产乳酸能力强。
表1:植物乳杆菌ER41的产酸能力
时间 | pH | 产乳酸量mmol/L |
4h | 4.55 | 63.49 |
8h | 3.96 | 97.69 |
12h | 3.85 | 134.55 |
24h | 3.71 | 165.87 |
实施例3:植物乳杆菌ER41(Lactobacillus plantarum ER41)抑菌活性的检测
菌株活化:将冷冻保存的Lactobacillus plantarum ER41接种于MRS液体培养基中,在温度37℃下培养24h,如此传代培养2次得到所述的活化Lactobacillus plantarumER41;活化后的菌株按3%体积比的接种量重新转接MRS液体培养基中,培养24小时,菌液离心得上清液。
细菌(大肠杆菌、金黄色葡萄球菌、沙门氏菌、李斯特菌)为指示菌,以LB培养基作为指示细菌的培养基。将甘油保存的指示菌株活化。活化后的菌株按3%的接种量重新转接LA肉汤中,指示细菌37℃条件下培养24h,用生理盐水分别将指示菌液稀释至OD600吸光值为0.1。向培养皿中倾注10mL的高压灭菌过的水琼脂(1.5%琼脂)进行铺底,每个培养皿厚度均一,待素琼脂冷却凝固后,将灭菌后的牛津杯置于琼脂之上。吸取1mL指示菌菌液加入到100mL恒温至50±5℃的LB培养基中,轻轻摇匀。将上述加过菌液的琼脂培养基倒入放有牛津杯的凝固的琼脂培养皿中,每个平皿倒20mL,待其冷却凝固。用镊子取出牛津杯,吸取200μL的ER41菌液上清液注入到相应的孔内,设立MRS培养液为空白对照,同时在培养皿上做好标记,将培养皿放入4℃冰箱内放置2h进行样品的扩散。将培养皿分别移入37℃培养箱培养。在接下来的18h内观察植物乳杆菌ER41对指示病原菌的抑菌效果,结果见表2。从表2可以看出,对不同病原菌的抑菌圈都大于18mm,表明植物乳杆菌对病原菌有很强的抑菌能力。
表2:植物乳杆菌ER41的抑菌能力
病原菌 | 大肠杆菌 | 沙门氏菌 | 金黄色葡萄球菌 | 李斯特菌 |
抑菌圈(mm) | 20.8±0.6 | 22.1±0.5 | 18.3±0.3 | 19.1±0.4 |
实施例4:植物乳杆菌ER41(Lactobacillus plantarum ER41)肠道黏附力的测定
鹅肠上皮细胞的制备:取25~26日胚龄发育良好鹅胚,75%酒精棉擦拭外壳,用灭菌解剖器械在超净台内取出小肠组织,暂置于PBS,剥离肠系膜,使用PBS反复冲洗肠腔内容物,直至上清澄清。将小肠组织剪成<1mm碎块,使用比例为9:1胶原酶Ⅺ和中性蛋白酶混合酶,37℃消化30min后,4℃离心10min弃上清,即获得鹅小肠上皮细胞沉淀。分离得到的细胞沉淀用含双抗和10%血清的DMEM/F12培养基吹匀,随后将悬液接种至第1个培养瓶中,置于37℃、5%CO2培养箱培养90min;然后轻轻吸取全部培养液(含未贴壁上皮细胞),接种到第2个培养瓶中,置于37℃、5%CO2培养箱继续培养24h后,更换新鲜细胞培养液,即获得纯化的鹅小肠上皮细胞。
将生长至对数期的细胞接种至24孔细胞培养板,待细胞生长密度至80%,再分别加入不同的乳杆菌菌液,每个处理组做3个重复孔。待乳杆菌处理细胞3h后,吸取培养液,用PBS清洗3次细胞以去除未黏附的细菌,将培养液和洗涤液一并收集,经系列稀释后选择合适稀释度涂布于MRS培养平板上,37℃生化培养箱中培养48h后计数,初始菌悬液样品计数结果为初始菌数,细胞细菌悬液样品计数结果为未黏附菌数。按照下式计算乳杆菌在鹅肠上皮细胞的黏附率:黏附率=(初始菌数-未黏附菌数)/初始菌数×100%。分别选择不同来源的乳杆菌:植物乳杆菌ZRR、植物乳杆菌ER41分别来源于牧草和鹅肠道。植物乳杆菌ZRR,保藏编号CCTCC No:M2016281(其已经在中国专利CN106148249A中公开)。结果如表3所示。
表3:不同乳杆菌在鹅肠上皮细胞的黏附能力
菌株 | ZRR | ER41 |
黏附率(%) | 50.8±4.6<sup>a</sup> | 71.3±2.5<sup>b</sup> |
由表3可知,来源于鹅肠道的植物乳杆菌ER41在鹅肠道上皮细胞的黏附率最高,达71.3%,远高于牧草附属菌ZRR的50.8%。
实施例5:植物乳杆菌ER41(Lactobacillus plantarum ER41)在肉鹅生产中的应用
菌粉的制备:将冷冻保存的ER41接种于MRS液体培养基中,在温度37℃下培养24h,如此传代培2次得到所述的活化ER41菌种。将活化的菌种接种在MRS液体培养基中37℃培养,菌液经离心,冷冻干燥、粉碎、辅料混合制成ER41的冻干粉,使乳杆菌冻干粉的活菌数达到1×1010cfu/g。
选取健康1日龄的三花鹅660只,随机分为2组,每组6个重复,每重复55只,至42日出栏。以正常饮水为对照,试验组在饮水中添加植物乳杆菌ER41,即依次设为对照和试验组,每毫升水中添加3*105CFU,预试期7天,试验期为14天。样品采集:于试验结束的最后一天对试验鹅进行屠宰取其各个肠段,开展肠道健康研究。
取其空肠、回肠和盲肠,并放入4%的多聚甲醛固定液中进行固定和组织切片的HE染色,显微镜观察肠道黏膜发育,结果如表4所示。黏膜结构是肠道健康的一个重要指标。绒毛高度的增加会增强肠道对于营养物质的吸收利用,而隐窝深度的降低会增强肠道对于营养物质的消化吸收能力,绒隐比可综合反映动物肠道的消化吸收能力,其比值越大表明肠道内膜面积相对越大。
采取肠道食糜,通过气相色谱仪测定肠道食糜的短链脂肪酸。色谱毛细柱参数:色谱柱毛细吸管柱(Agilent,US),柱温为130℃,汽化温度为180℃,检测温度为180℃,应用氢离子火焰检测器,载气为氮气压力为60kpa,氢气压力为50kPa,氧气压力50kPa。结果如表5所示。短链脂肪酸主要包括乙酸、丙酸、丁酸等。肠道短链脂肪酸参与肠上皮细胞的能量供应,可影响肠腔pH和电解质平衡,肠黏膜屏障的通透性,肠道高敏感和肠道动力的调节,具有抗炎等作用,是肠道健康另一重要指标。
采用分子微生物学方法系统研究肠道的微生物组成,提取盲肠的微生物DNA,进行了细菌16SrDNAV3的扩增,采用Illumina Nova Seq平台测序,开展Metastats分析,研究组间差异显著的菌属,结果如表6所示。
表4植物乳杆菌ER41对肉鹅肠道黏膜结构的影响
表5植物乳杆菌ER41对肉鹅肠道短链脂肪酸的影响(mmol/L)
组别 | 乙酸 | 丙酸 | 丁酸 |
对照组 | 4.27<sup>a</sup> | 1.32<sup>a</sup> | 0.62 |
试验组 | 6.68<sup>b</sup> | 2.69<sup>b</sup> | 0.74 |
表6植物乳杆菌ER41对肉鹅肠道优势菌群相对丰度的影响(%)
组别 | Escherichia-Shigella | Enterococcus | Klebsiella | Lactobacillus |
对照组 | 1.25<sup>b</sup> | 0.018<sup>b</sup> | 0.28 | 0.0067 |
试验组 | 0.38<sup>a</sup> | 0.0012<sup>a</sup> | 0.10 | 0.030 |
结论:
1、由表4可知,本发明植物乳杆菌ER41提高空肠、回肠和盲肠的绒毛高度,显著降低回肠和盲肠的隐窝深度,显著提高了盲肠的绒隐比,促进了肠道黏膜的健康发育。
2、由表5可知,植物乳杆菌ER41可以提高肠道乙酸、丙酸等短链脂肪酸的含量,促进肠道健康。
3、由表6可知,植物乳杆菌ER41显著降低大肠杆菌志贺菌属(Escherichia-Shigella)、肠球菌属(Enterococcus)等致病菌丰度,提高乳杆菌属(Lactobacillus)丰度,调节肠道菌群。
以上仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效变换,或直接或间接运用在其它相关的技术领域,均同理包括在本发明的专利保护范围内。
序列表
<110> 江苏省农业科学院
<120> 一种鹅源植物乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1467
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
gaccggcgcg tgctatactg cagtcgaacg aactctggta ttgattggtg cttgcatcat 60
gatttacatt tgagtgagtg gcgaactggt gagtaacacg tgggaaacct gcccagaagc 120
gggggataac acctggaaac agatgctaat accgcataac aacttggacc gcatggtccg 180
agcttgaaag atggcttcgg ctatcacttt tggatggtcc cgcggcgtat tagctagatg 240
gtggggtaac ggctcaccat ggcaatgata cgtagccgac ctgagagggt aatcggccac 300
attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360
tggacgaaag tctgatggag caacgccgcg tgagtgaaga agggtttcgg ctcgtaaaac 420
tctgttgtta aagaagaaca tatctgagag taactgttca ggtattgacg gtatttaacc 480
agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540
ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt aagtctgatg tgaaagcctt 600
cggctcaacc gaagaagtgc atcggaaact gggaaacttg agtgcagaag aggacagtgg 660
aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720
ctgtctggtc tgtaactgac gctgaggctc gaaagtatgg gtagcaaaca ggattagata 780
ccctggtagt ccataccgta aacgatgaat gctaagtgtt ggagggtttc cgcccttcag 840
tgctgcagct aacgcattaa gcattccgcc tggggagtac ggccgcaagg ctgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagctacgcg 960
aagaacctta ccaggtcttg acatactatg caaatctaag agattagacg ttcccttcgg 1020
ggacatggat acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttattatca gttgccagca ttaagttggg cactctggtg 1140
agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggat ggtacaacga gttgcgaact cgcgagagta 1260
agctaatctc ttaaagccat tctcagttcg gattgtaggc tgcaactcgc ctacatgaag 1320
tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccat gagagtttgt aacacccaaa gtcggtgggg taacctttta 1440
ggaaccagcc gcctaaggtg acagatt 1467
Claims (3)
1.一种鹅源植物乳杆菌ER41(Lactobacillus plantarum ER41),其保藏编号为CCTCCNO:M 2021932。
2.含有如权利要求1所述植物乳杆菌ER41的菌剂。
3.如权利要求1所述植物乳杆菌ER41或其发酵产物或如权利要求2所述菌剂在肉鹅养殖生产中的应用。
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