一株鸡源唾液乳杆菌的分离鉴定及其对育雏早期蛋鸡肠道健康的影响

doi:10.11843/j.issn.0366-6964.2024.09.040

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4161-4171.doi: 10.11843/j.issn.0366-6964.2024.09.040

一株鸡源唾液乳杆菌的分离鉴定及其对育雏早期蛋鸡肠道健康的影响

于秀菊¹(), 胡燕姣¹(), 刘佳悦¹, 王海东¹, 朱芷葳², 范阔海¹, 王蓉蓉², 段承昊², 石佳炜², 杨丽华¹^,*()

1. 山西农业大学动物医学学院, 太谷 030801
2. 山西农业大学生命科学学院, 太谷 030801

收稿日期:2024-01-29 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 杨丽华 E-mail:yxjfkh@126.com;18328305198@163.com;13834526215@163.com
作者简介:于秀菊(1983-), 女, 山西大同人, 高级实验师, 博士, 主要从事动物益生菌及其细菌素研究, E-mail: yxjfkh@126.com
胡燕姣(1997-), 女, 四川成都人, 硕士生, 主要从事动物益生菌筛选及其功能研究, E-mail: 18328305198@163.com
第一联系人：
于秀菊和胡燕姣为同等贡献作者
基金资助:
山西农业大学科技创新提升工程(CXGC2023020);山西农业大学博士科研启动项目(2021BQ74)

Isolation and Identification of a Chicken Source Lactobacillus salivary Strain and Its Effect on Intestinal Health of Laying Hens in Early Brood Period

Xiuju YU¹(), Yanjiao HU¹(), Jiayue LIU¹, Haidong WANG¹, Zhiwei ZHU², Kuohai FAN¹, Rongrong WANG², Chenghao DUAN², Jiawei SHI², Lihua YANG¹^,*()

1. College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China
2. College of Life Sciences, Shanxi Agricultural University, Taigu 030801, China

Received:2024-01-29 Online:2024-09-23 Published:2024-09-27
Contact: Lihua YANG E-mail:yxjfkh@126.com;18328305198@163.com;13834526215@163.com

摘要/Abstract

摘要：

旨在从健康海兰褐鸡肠道中分离鉴定乳酸菌, 并研究其对育雏早期蛋鸡肠道健康的影响。本研究采用细菌培养技术、16S rRNA测序和牛津杯扩散法, 分离、筛选、鉴定可产细菌素的乳酸菌菌株, 检测其对高温、pH和胆盐的耐受性; 动物试验选取240只1日龄健康、体重相近的海兰褐鸡, 随机分为4组, 每组60只鸡, 包括对照组(C)、低剂量唾液乳杆菌CL09组(L)、中剂量唾液乳杆菌CL09组(M)和高剂量唾液乳杆菌CL09组(H), 研究该乳酸菌菌株对育雏早期蛋鸡生长性能、免疫器官指数及肠道健康相关指标的影响。试验周期为21 d。结果表明: 分离菌为可产细菌素的革兰阳性菌, 结合16S rRNA鉴定结果将其命名为唾液乳杆菌CL09, 该菌的无细胞上清液可抑制大肠杆菌、沙门菌、金黄色葡萄球菌和李斯特菌的生长。该菌的耐受温度为60 ℃, 具有耐酸、耐胆盐的特性。动物试验结果显示: 与对照组相比, 灌服唾液乳杆菌CL09可以增加雏鸡体重和免疫器官重量; H组十二指肠、空肠的隐窝深度变化最为显著, L和M组十二指肠绒毛高度显著增加, 且H组十二指肠绒毛呈“波浪形”; M和H组肠道干细胞标记基因Lgr5和Bmi1表达水平升高极显著(P＜0.001);H组的黏膜屏障基因Zo-1和Ocln的mRNA表达水平升高极显著(P＜0.01);M组潘氏细胞标记性基因Lyz的mRNA表达水平最高。L、M和H组的杯状细胞标记基因Muc的mRNA表达水平均升高, 且差异极显著(P＜0.01)。综上所述, 源自健康海兰褐鸡肠道的唾液乳杆菌CL09, 通过促进雏鸡肠道干细胞分化为潘氏细胞和杯状细胞, 增加绒毛高度, 降低隐窝深度, 从而增大肠道的黏膜面积。该菌具有调节育雏早期鸡肠道健康的良好特性, 可作为益生菌的备选菌株。

关键词: 蛋鸡, 唾液乳杆菌, 肠道健康, 绒毛高度, 隐窝深度

Abstract:

This experiment mainly aimed at isolating and identifying lactic acid bacteria from the gut of healthy Hyline brown laying hens, and detected the effect of the isolated lactic acid bacteria on intestinal health of laying hens in early brood period. Bacterial culture, 16S rRNA and Oxford Cup diffusion methods were used to isolate, screen and identify Lactobacillus strains that could producing bacteriocins. The tolerance to high temperature, pH and bile salt was assessed. Two hundred and forty healthy one-day-old Hyline brown laying hens with similar body weight were randomly divided into four groups: control group (C), low dose Lactobacillus salivarius CL09 group (L), medium dose Lactobacillus salivarius CL09 group (M) and high dose Lactobacillus salivarius CL09 group (H). The effects of this strain on growth performance, immune organ index and intestinal health were analyzed. The experiment lasted for 21 days. The results showed that the isolated strain was a bacteriocin-producing Gram-positive bacteria, identified as Lactobacillus salivarius CL09 based on 16S rRNA identification. The cell-free supernatant of Lactobacillus salivarius CL09 demonstrated inhibitory effects on the growth of Escherichia coli, Salmonella, Staphylococcus aureus and Listeria. The resistant temperature of Lactobacillus salivarius CL09 was 60 ℃, and possessed the characteristics of acid and bile salt tolerance. The results of animal testing showed the Lactobacillus salivary CL09 supplementation could improved body weight and immune organ weight, with noticeable decreases in crypt depth of duodenum and jejunum observed in the H group, and significant increase in villus height of duodenum observed in L and M groups. The intestinal villi in the group H showed a wavy trend. Additionally, the mRNA levels of intestinal stem cell marker genes Lgr5 and Bmi1 were significantly increased in the M and H group(P < 0.001), while the mRNA level of mucosal barrier genes Zo-1 and Ocln was significantly increased in the H group(P < 0.01). Lactobacillus salivarius CL09 supplementation also led to the increasing of mRNA levels of Pan's cell marker gene Lyz and goblet cell marker gene Muc (P < 0.01). In summary, Lactobacillus salivary CL09 isolated from the gut of healthy Hyline brown laying hens, has the potential to enhance villi length and crypt depth by promoting differentiation of intestinal stem cells into Paneth and goblet cells, thereby increasing the mucosal surface area in the intestine. Lactobacillus salivary CL09 exhibits favorable characteristics for regulating the intestinal health of chicks in the early brood period, and could be considered as a candidate probiotic strain.

Key words: laying hens, Lactobacillus salivarius, intestinal health, villus height, crypt depth

中图分类号:

S831

于秀菊, 胡燕姣, 刘佳悦, 王海东, 朱芷葳, 范阔海, 王蓉蓉, 段承昊, 石佳炜, 杨丽华. 一株鸡源唾液乳杆菌的分离鉴定及其对育雏早期蛋鸡肠道健康的影响[J]. 畜牧兽医学报, 2024, 55(9): 4161-4171.

Xiuju YU, Yanjiao HU, Jiayue LIU, Haidong WANG, Zhiwei ZHU, Kuohai FAN, Rongrong WANG, Chenghao DUAN, Jiawei SHI, Lihua YANG. Isolation and Identification of a Chicken Source Lactobacillus salivary Strain and Its Effect on Intestinal Health of Laying Hens in Early Brood Period[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4161-4171.

图/表 7

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基因 Gene	序列(5′→3′) Primer sequence	产物大小/bp Product size	登录号/参考文献 GenBank accession number/References
Lgr5	F: CTTCTCTTCGTTCTCTGGATTTAGC	115	XM_425441.4
Lgr5	R: GAGGACAAAAGGTTGGATGAC	115	XM_425441.4
Bmi1	F: AGTTCCTGCGGAGTAAGATGG	104	NM_001007988.1
Bmi1	R: GTAGGCAATGTCCATCAGCGT	104	NM_001007988.1
Zo-1	F: GCGCCTCCCTATGAGGAGCA	160	[10]
Zo-1	R: CAAATCGGGGTTGTGCCGGA	160	[10]
Ocln	F: CCGTAACCCCGAGTTGGAT	214	[11]
Ocln	R: ATTGAGGCGGTCGTTGATG	214	[11]
Lyz	F: TTTGCTAATCTTGGTGCT	128	FJ542373.1
Lyz	R: AGTTTCCCAGGCTGTATC	128	FJ542373.1
Muc	F: GTGAAGACCCTGATGAAAC	156	JX284122.1
Muc	R: TGAACACTGGCGAGAATA	156	JX284122.1
GAPDH	F: GATGGGTGTCAACCATGAGAAA	116	NM_204305
GAPDH	R: CAATGCCAAAGTTGTCATGGA	116	NM_204305

组别 Group	体重/g Body weight	径长/mm Tibia length	免疫器官重量/g Immune organ weight
组别 Group	体重/g Body weight	径长/mm Tibia length	法氏囊 Bursa of Fabricius	脾 Spleen
对照组 C	193.57±11.41	52.60±0.601 2	1.111±0.092 85	0.418±0.026 60
低剂量唾液乳杆菌CL09组 L	200.94±7.653	53.44±1.222	1.117±0.082 02	0.415 9±0.012 73
中剂量唾液乳杆菌CL09组 M	203.57±8.982	53.78±0.409 5	1.165±0.129 0	0.435 5±0.037 60
高剂量唾液乳杆菌CL09组 H	211.54±11.41	54.09±1.397	1.256±0.091 33	1.941±0.019 22

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一株鸡源唾液乳杆菌的分离鉴定及其对育雏早期蛋鸡肠道健康的影响

Isolation and Identification of a Chicken Source Lactobacillus salivary Strain and Its Effect on Intestinal Health of Laying Hens in Early Brood Period

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