兰州大学机构库 >生命科学学院
产细菌素乳酸菌影响饲草青贮及其瘤胃发酵的微生物机制
Alternative TitleMicrobial mechanism of bacteriocin-producing lactic acid bacteria affecting forage silage and rumen fermentation
李子谦
Subtype博士
Thesis Advisor郭旭生
2023-09-03
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学博士
Degree Discipline微生物学
Keyword植物乳杆菌 Lactiplanatibaillus Plantarum, bacteriocin, silage, rumen, microbiome 细菌素 青贮饲料 瘤胃 微生物组
Abstract

青贮饲料是反刍动物日粮的主要成分之一,对促进草食家畜健康、高效养殖具有重要作用。青贮饲料发酵品质通常由青贮原料、附着菌群、厌氧条件及添加剂种类所决定。大量研究表明,优良乳酸菌青贮添加剂不仅能改善饲草适口性、提高营养价值,还能提高草食家畜生产性能和畜产品品质,同时有利于缓解瘤胃甲烷(CH4)排放。另外,开发代替抗生素的新型天然饲料添加剂也已成为近十年的研究热点。而产细菌素乳酸菌因其所产细菌素具有高效、耐高温、无毒、无残留和无耐药性等特点,已成为极具发展潜力的青贮饲料添加剂之一。因此,探讨产细菌素乳酸菌对饲草青贮与瘤胃发酵特性的影响及其微生物机制对于新型乳酸菌饲料添加剂的研发及应用具有重要意义。本研究以三株产细菌素植物乳杆菌(Lactiplantibacillus plantarum)为试验对象,以青贮苜蓿和全株玉米青贮饲料为试验材料,根据三株产细菌素L. plantarum的抑菌特性,探讨产细菌素L. plantarum对青贮饲料发酵品质,及其瘤胃体外发酵特性、CH4产量与微生物区系的影响。

本研究的主要结果如下:

1. 三株产细菌素L. plantarum抑菌特性比较

三株产细菌素L. plantarum ATCC14917、CICC24194和LP1-4的生长曲线、产酸曲线、抑菌效果、抑菌谱、抑菌曲线及其所产细菌素抑菌活性pH稳定性的研究结果表明,产Ⅱa类细菌素L. plantarum ATCC14917的长势最好,产酸最高。产Ⅱa类细菌素L. plantarum CICC24194抑制革兰氏阳性细菌的效果最好,产类细菌素L. plantarum LP1-4抑制革兰氏阳性细菌和革兰氏阴性细菌的效果相似。蜡样芽胞杆菌(Bacillus cereus)和大肠杆菌(Escherichia coli)在CICC24194处理组中生长最为缓慢,且到达稳定期后数量保持最低。三株L. plantarum所产细菌素的活性均在pH为2.5时最好,并随着pH升高而逐渐降低;在pH到达8时逐渐失活,在pH为10时彻底失活。

2. 产细菌素L. plantarum对不同干物质青贮苜蓿发酵品质、化学组成及细菌群落演替的影响

产Ⅱa类细菌素L. plantarum ATCC14917和产类细菌素L. plantarum LP1-4在青贮苜蓿中应用结果表明,与中等干物质(DM,35.5%)含量相比,高DM(42.8%)处理组中pH显著增加,且乳酸浓度显著降低(P < 0.05)。在中等DM含量下,ATCC14917显著提高青贮发酵初期(3 d)的乳酸浓度(P < 0.05)。与对照组和商品乳酸菌青贮剂L. plantarum MTD/1(不产细菌素)处理组相比,ATCC14917显著降低两种DM含量下青贮苜蓿DM损失和非蛋白氮浓度(P < 0.05)。在中等DM含量下,ATCC14917和MTD/1在3 ~ 60 d时均提高了L. plantarum的相对丰度,而在90 d时提高了布氏乳杆菌(Lactobacillus buchneri)的相对丰度。在发酵60 d前,ATCC14917降低了苜蓿青贮中细菌群落碳水化合物代谢功能和氨基酸代谢功能。同时,ATCC14917抑制了两种DM含量下青贮过程中Aerococcus(气球菌属)和Enterobacter(肠杆菌属)的生长。因此,产Ⅱa类细菌素L. plantarum ATCC14917抑制了青贮过程中不良细菌的生长,并通过调控青贮过程中细菌群落组成改善青贮苜蓿发酵品质。

3. 产细菌素L. plantarum对不同干物质青贮苜蓿瘤胃体外发酵特性、甲烷排放及微生物区系的影响

产Ⅱa类细菌素L. plantarum ATCC14917和产类细菌素plantarum LP1-4在青贮苜蓿瘤胃体外发酵中应用的结果表明,与商品青贮饲料接种剂MTD/1相比,ATCC14917和LP1-4显著提高体外干物质消化率(P < 0.05),并显著降低CH4产量(P < 0.05),以ATCC14917的效果最佳。青贮苜蓿的DM含量影响了瘤胃发酵模式,中等DM含量以乙酸发酵为主,高DM含量以丙酸发酵为主。两株产细菌素L. plantarum通过提高与丙酸代谢途径相关纤维降解菌相对丰度,并通过降低瘤胃总细菌数量,间接调控原生动物与产甲烷菌数量,改善瘤胃体外发酵特性并降低CH4产量,以ATCC14917的效果最佳。

4. 产Ⅱa类细菌素L. plantarum对全株玉米青贮饲料发酵品质、化学组成及细菌群落演替的影响

产Ⅱa类细菌素L. plantarum ATCC14917和CICC24194在全株玉米青贮饲料中应用的结果表明,除发酵3 d外,CICC24194处理组中乳酸浓度在青贮发酵期间均最高(P < 0.05)。DM含量在CICC24194处理组中最高(P < 0.05),两株产细菌素菌株处理组中DM损失均显著降低(P < 0.05)。与此同时,两株产细菌素菌株均促进了Lactobacillus brevis(短乳杆菌)的发酵。由细菌群落随机森林和物种丰度聚类分析的结果可知,MTD/1和ATCC14917降低了关键变量uncultured_Bacillus_sp的相对丰度,而CICC24194降低了关键变量uncultured_bacterium的相对丰度。因此,两株产Ⅱa类细菌素L. plantarum均能通过调控青贮过程中细菌群落组成改善全株玉米青贮饲料发酵品质,以CICC24194的效果最佳。

5. 产Ⅱa类细菌素L. plantarum对全株玉米青贮饲料瘤胃体外发酵特性、甲烷排放及微生物区系的影响

产Ⅱa类细菌素L. plantarum ATCC14917和CICC24194在全株玉米青贮饲料瘤胃体外发酵中应用的结果表明,两株产细菌素菌株均显著提高体外干物质消化率(P < 0.05)。与商品青贮饲料接种剂MTD/1相比,两株产细菌素菌株显著降低CH4产量(P < 0.05)。解木聚糖温暖微菌属(Tepidimicrobium)的相对丰度在ATCC14917处理中最高。CICC24194增加了密螺旋体菌属(Treponema_2)的相对丰度。结合细菌群落随机森林和物种丰度聚类分析的结果可知,与对照组相比,ATCC14917增加了关键变量芽孢杆菌属(Bacillus)的相对丰度。CICC24194增加了关键变量胃球菌科(Ruminococcaceae_UCG.010)的相对丰度。总细菌、真菌和原虫数量在CICC24194处理组中最低(P < 0.05)。仅CICC24194降低了产甲烷菌数量(P < 0.05)。两株产Ⅱa类细菌素L. plantarum通过调控瘤胃细菌、原生动物与产甲烷菌的相互作用改善瘤胃体外发酵特性并降低CH4产量。

综合以上研究结果,产Ⅱa类细菌素L. plantarum ATCC14917、CICC24194和产类细菌素L. plantarum LP1-4均通过调控青贮过程中细菌群落组成改善青贮饲料发酵品质。同时,三株产细菌素菌株均通过调控瘤胃微生物之间的相互作用及瘤胃纤维降解菌的组成提高青贮饲料瘤胃体外干物质消化率,并降低CH4排放。 ATCC14917在青贮苜蓿及其瘤胃体外发酵中应用的效果优于LP1-4。CICC24194在全株玉米青贮饲料中应用的效果优于ATCC14917。本论文揭示了产细菌素L. plantarum调控青贮过程中细菌群落组成及其演替方式,以及青贮饲料瘤胃发酵微生物组成及其相互作用,从而改善了瘤胃体外干物质消化率与CH4排放。研究结果为产细菌素乳酸菌在青贮饲料中的应用,及其调控饲草青贮与瘤胃发酵的机制提供了重要科学依据。

Other Abstract

Silage is one of the main ingredients of ruminant diets and plays a critical role in promoting the health and efficient breeding of grass-feeding livestock. The fermentation quality of silage is usually determined by the raw materials, attached microorganism, anaerobic conditions and additives. A large number of studies have shown that the excellent lactic acid bacteria silage additive can not only improve the palatability and nutritional value of forage, but also improve the production performance of grass-feeding livestock and the quality of livestock products, and is conducive to alleviating rumen methane (CH4) emissions. In addition, the development of new natural feed additives that substitute for antibiotics has also become a research hotspot in the past decade. Bacteriocin has become one of the silage additives with great potential for development due to its high efficiency, high temperature resistance, non-toxic, non-residue and non-drug resistance. Therefore, exploring the effects of bacteriocin-producing lactic acid bacteria on forage silage and rumen fermentation characteristics, and their microbial mechanisms is of great significance for the development and application of new lactic acid bacteria feed additives. In the present study, the three bacteriocin-producing Lactiplanatibaillus plantarum strains were used as trial subject, and the alfalfa and whole crop-corn silage were used as trial materials to explore the antibacterial properties of the three bacteriocin-producing L. plantarum strains, and its effects on the silage fermentation quality, in vitro ruminal fermentation properties, microbiota, and CH4 productions.

The main results are as follows:

1. Comparison of antibacterial properties of bacteriocin-producing L. plantarum

The results of growth curve, acid production curve, antibacterial effect, inhibition spectrum, antibacterial curve and pH stability of antibacterial activity of bacteriocin of the three bacteriocin-producing L. plantarum ATCC14917, CICC24194 and LP1-4 showed that Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 had the best activity and highest acid production. Class Ⅱa bacteriocin-producing L. plantarum CICC24194 showed the best antibacterial effect on gram-positive bacteria, while L. plantarum LP1-4 that produced bacteriocin-like showed similar antibacterial effects on both Gram-positive and Gram-negative bacteria. Bacillus cereus and Escherichia coli grew the slowest in the CICC24194 treatment, and their numbers remained the lowest after reaching the stable period. The bacteriocin produced by the three L. plantarum strains showed the best antibacterial effect at pH 2.5, and gradually decreased with the increase of pH. It was gradually inactivated when pH reached 8 and completely inactivated when pH reached 10.

2. Effects of bacteriocin-producing L. plantarum on fermentation quality, chemical composition and bacterial community successions of alfalfa silage with different dry matter contents

The results of application of Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 and L. plantarum LP1-4 that produced bacteriocin-like in alfalfa silage showed that compared with the moderate dry matter (DM, 35.5%) content, high DM content (42.8%) had higher pH and lower lactic acid concentration (P < 0.05). At moderate DM content, ATCC14917 had the highest lactic acid concentration in the early stage of fermentation (3 d). Compared with the control group and MTD/1 treatment (without producing bacteriocin), ATCC14917 decreased the DM loss and non-protein nitrogen concentrations of alfalfa silage at both two DM contents (P < 0.05). At moderate DM content, ATCC14917 and MTD/1 increased the relative abundance of L. plantarum from 3 to 60 d, while increased the relative abundance of Lactobacillus buchneri on 90 d. Before 60 d of fermentation, ATCC14917 decreased the functions of carbohydrate and amino acid metabolism of bacterial community at both DM contents. ATCC14917 inhibited the growth of Aerococcus and Enterobacter at both DM contents during ensiling. Therefore, Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 could inhibit the growth of undesirable bacteria and improve the fermentation quality of alfalfa silage by regulating bacterial community composition during ensiling.

3. Effects of bacteriocin-producing L. plantarum on in vitro ruminal fermentation characteristics, methane emissions and microbiota of alfalfa silage with different dry matter contents

The results of application of Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 and L. plantarum LP1-4 that produced bacteriocin-like in in vitro rumen fermentation of alfalfa silage showed that compared with MTD/1, a commercial silage inoculant, ATCC14917 and LP1-4 significantly increased the in vitro ruminal dry matter digestibility (P < 0.05), and significantly decreased the CH4 productions (P < 0.05), and ATCC14917 had the most efficient effect. The DM content of alfalfa silage influenced the ruminal fermentation pattern, with acetic acid fermentation being the dominant fermentation for moderate DM content and propionate fermentation for high DM content. Both the two bacteriocin-producing strains could increase the relative abundance of ruminal fibrolytic bacteria related to the propionic acid metabolism pathway, indirectly regulate the populations of protozoa and methanogens by reducing the populations of total bacteria, improve the in vitro rumen fermentation characteristics and reduce the CH4 production, with ATCC14917 the most efficient.

4. Effects of Class Ⅱa bacteriocin-producing L. plantarum on fermentation quality, chemical composition and bacterial community successions of whole crop-corn silage

The results of application of Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 and CICC24194 in whole crop-corn silage showed that the lactic acid concentration in the CICC24194 treatment was the highest during fermentation except for 3 d (P < 0.05). DM content in the CICC24194 treatment was the highest (P < 0.05), while DM loss was significantly decreased in the two bacteriocin-producing strains treatments (P < 0.05). Moreover, both the two bacteriocin-producing strains promoted the fermentation of Lactobacillus brevis. Based on the results of Random Forest and clustering analysis of species abundance, MTD/1 and ATCC14917 reduced the relative abundance of the influential variable uncultured_Bacillus_sp., while CICC24194 reduced the relative abundance of the influential variable uncultured_bacterium. Therefore, both the two Class Ⅱa bacteriocin-producing L. plantarum strains could improve the fermentation quality of whole crop-corn silage by regulating the bacterial community composition during ensiling, with CICC24194 being the most effective.

5. Effects of Class Ⅱa bacteriocin-producing L. plantarum on in vitro ruminal fermentation characteristics, methane emissions and microbiota of whole crop-corn silage.

The results of application of Class Ⅱa bacteriocin-producing L. plantarum ATCC14917 and CICC24194 in in vitro rumen fermentation of whole crop-corn silage showed that both the two bacteriocin-producing strains significantly increased the in vitro rumen dry matter digestibly (P < 0.05). Compared with the commercial silage inoculant MTD/1, CH4 productions were significantly decreased by the two bacteriocin-producing strains (P < 0.05). The relative abundance of Tepidimicrobium was the highest in the ATCC14917 treatment. CICC24194 increased the relative abundance of Treponema_2. Combining the results of Random Forest and clustering analysis of genus levels showed that ATCC14917 increased the relative abundance of the influential variable Bacillus compared with the control group. CICC24194 increased the relative abundance of the influential variable Ruminococcaceae_UCG.010. The populations of total bacteria, fungi and protozoa was the lowest in the CICC24194 treatment (P < 0.05). Only CICC24194 reduced the methanogen population (P < 0.05). Therefore, both the two Class Ⅱa bacteriocin-producing L. plantarum strains could improve the in vitro ruminal fermentation characteristics and reduce CH4 productions by regulating the interactions among the rumen bacteria, protozoa and methanogens.

In conclusion, Class Ⅱa bacteriocin-producing L. plantarum ATCC14917, CICC24194 and bacteriocin-producing L. plantarum LP1-4 improved the silage fermentation quality by regulating bacterial community composition during ensiling. Moreover, all the three bacteriocin-producing strains could improve the in vitro rumen dry matter digestibility and reduce CH4 emissions of forage silage by regulating the interactions among the rumen microorganisms and the composition of ruminal fibrolytic bacteria. ATCC14917 was more effective than LP1-4 in alfalfa silage and its in vitro rumen fermentation. CICC24194 was more effective than ATCC14917 in whole crop-cron silage. This study revealed that bacteriocin-producing L. plantarum regulated bacterial community compositions and their succession mode during ensiling, as well as microbial compositions and their interactions in rumen fermentation of silage, thereby improving in vitro rumen dry matter digestibility and CH4 emission. The results provided an important scientific basis for the application of bacteriocin-producing lactic acid bacteria in silage and the mechanism of regulating forage silage and rumen fermentation.

Subject Area应用微生物
MOST Discipline Catalogue理学 - 生物学 - 微生物学
URL查看原文
Language中文
Other Code262010_120190905430
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/539771
Collection生命科学学院
Affiliation
兰州大学生命科学学院
Recommended Citation
GB/T 7714
李子谦. 产细菌素乳酸菌影响饲草青贮及其瘤胃发酵的微生物机制[D]. 兰州. 兰州大学,2023.
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