青贮发酵体系中乳酸菌多样性的研究
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摘要
本文以实验室自制袋装青贮和不同地区青贮为样品,采用PCR-DGGE技术与传统的纯培养技术相结合,比较了玉米青贮和苜蓿青贮动态发酵过程中菌群多样性的变化;研究了不同地区青贮原料表面附着菌和半年以上青贮中菌群的多样性。得出以下结论:
1.玉米青贮和苜蓿青贮中乳酸菌多样性研究
玉米青贮pH值迅速降低,至试验结束,青贮各项营养指标均达到优质青贮指标;而苜蓿青贮pH值一直维持在5.5以上,其余各项营养指标均达到优质指标。利用细菌V3区通用引物扩增玉米青贮和苜蓿青贮中微生物16S rDNA,产物经DGGE分析发现,玉米青贮和苜蓿青贮发酵过程中优势条带不同;其中?-Acinetobacter、?-Pantoea、?-Escherichia、?-Lactococcus、?-Weissella koreensis、?-Enterobacter等类细菌一直活跃在青贮中;?-Streptococcus、?-Endophytic、?-Enterobacter等细菌7d后从玉米青贮体系中消失,而有些条带(克隆未成功)是在苜蓿青贮后期逐渐变亮,逐渐占据优势。
利用细菌LAB类通用引物扩增玉米青贮和苜蓿青贮中微生物总16S rDNA,产物经DGGE分析发现,在青贮发酵过程中以?-Lactobacillus类居多,其次为?- Lactococcus和?- Weissella类;Lactobacillus brevis、?-Acetobacter、?-Leuconostoc在玉米青贮7d后有逐渐消失的趋势,而这几株菌在苜蓿青贮中一直存在,且没有显著变化。杆菌作为启动菌株一直存在于玉米及苜蓿青贮发酵体系中,且在青贮前(0d)及青贮后各时间点,条带一直较亮。
利用传统纯培养技术对玉米青贮和苜蓿青贮各时间点乳酸菌进行分离、鉴定,共发现118株,其中乳酸杆菌57株、球菌61株。这些菌分别出现在两种原料中,并在各个时间分离出的LAB不同,前期以球菌居多,但随着青贮发酵时间的延长,青贮中的LAB种类由开始的球菌逐渐演替成杆菌。
2.不同地区青贮原料表面附着菌及青贮中乳酸菌多样性研究
不同地区青贮原料及青贮的营养成分差异显著,青贮中发酵产物差异性显著,原料发酵后符合优质青贮要求。
利用细菌V3区通用引物扩增不同地区青贮原料表面附着菌及青贮中微生物16S rDNA,产物经DGGE方法分析发现:同一地区青贮原料表面附着菌的种类相似性比较高,最高达到93%最低也在80%以上;植株表面附着菌多为与青贮发酵无关的“杂菌”,LAB类较少,仅发现Lactococcus lactis subsp. Cremoris、Weissella koreensis、Lactobacillus buchneri等三种乳酸菌。不同地区青贮中细菌的种类相似性不高,仅在60%左右。检测出的菌群含有?-Acinetobacter、?-Pantoea、?-Escherichia、?-Pseudomonas等类“杂菌”,同时还检测出?-Lactobacillus、?-Lactococcus、?-Weissella等乳酸菌,这些菌群分布在各地区牧场青贮中。
利用LAB类通用引物扩增不同地区青贮原料表面附着菌及青贮中微生物16S rDNA,产物经DGGE方法分析发现:不同地区青贮原料表面附着菌的种类相似性比较低,最高也只在75%左右,同一地区的相似性比较高,均在63%至94%之间;使用该LAB类通用引物扩增,发现的绝大部分是LAB,且以杆菌为主,球菌较少;其中Weissella confusa、Lactobacillus brevis、Acetobacter ghanensis、Lactococcus lactis、Weissella hellenica、Lactobacillus plantarum等菌种在九个牧场中均出现。不同地区青贮中乳酸菌经聚类分析发现九个牧场相似性不高,在67%至90%之间;且半年以上的青贮窖中以乳杆菌为主要类群(?- Lactobacillus),同时仍有球菌(Lactococcus、Weissella、Leuconostoc)出现。
利用纯培养技术对不同地区青贮原料及半年以上青贮中LAB进行分离鉴定,不同地区青贮原料表面共分离出69株为乳酸菌,其中乳杆菌40株,乳球菌29株,地区不同乳酸菌种类有差异,而同一个地区植株表面的乳酸菌具有较高的相似性;于发酵半年以上的青贮中分离出46株为乳酸菌,均为乳杆菌,地区不同种类差异性较大,且同一个地区的三个牧场之间相似性也很低。
传统纯培养技术与PCR-DGGE方法得出的结论并不完全一致,传统培养技术对LAB具有选择性培养,而DGGE更能真实的反映发酵体系中微生物结构。
In this paper, we use the laboratory-made bag silage and silage samples in different regions, employing PCR-DGGE technology and traditional pure culture technology, comparing corn silage and alfalfa silage fermentation process of dynamic changes in flora diversity, and study the diversity of different surface areas of raw materials attached bacteria silage and silage more than half a year. And we come to the following conclusions.
1. Research of corn silage and alfalfa silage in the diversity of lactic acid bacteria.
pH value of corn silage reduced rapidly to the end of the experiment, and silage of the nutrition indicators all got to quality indicators. pH value of alfalfa silage and was maintained at above 5.5, and the rest of the nutrition indicators all got to quality indicators.
We use bacterial universal primers V3 area expansion of corn silage and alfalfa silage microbial 16S rDNA and find by DGGE analysis of the product, corn silage and alfalfa silage fermentation with different advantages strips; Among them, ?-Acinetobacter、?-Pantoea、?-Escherichia、?-Lactococcus、?-Weissella koreensis、?-Enterobacter and other types of bacteria have been active in the silage; ? -Streptococcus、?-Endophytic、?-Enterobacter and other types of bacteria disappeare from the corn silage system 7days later. And some bands (cloning was not successful) of alfalfa silage in the late light became gradually dominated.
LAB using bacterial universal primer amplification type of corn silage and alfalfa silage in the total microbial 16S rDNA, product analysis by DGGE, fermentation process in silage-Lactobacillus category was the most, followed by?-Lactococcus, and? Class-Weissella。There was trend that Lactobacillus brevis、?–Acetobacter、?- Leuconostoc corn silage in the gradual disappeared after 7days. Several Strains of bacteria were in the existence of alfalfa silage, and there are no significant change. Bacillus strains as a start was found in corn and alfalfa silage fermentation system, and in silage before (0 days) and silage at different time points, and it had a bright strip.
After the use of traditional techniques of pure culture of corn silage and alfalfa silage lactic acid bacteria at different time points were isolated and identified, 118 were found, of which 57 bacilli, cocci 61. These bacteria appeared in the two materials, respectively, and in various time of the LAB isolated from different bacteria in the early majority, but as time silage fermentation, silage types of LAB cocci by the beginning of a gradual succession as Bacillus.
2. Research of the diversity of materials attached to different parts of silage and silage strains of lactic acid bacteria
Silage in different regions of the raw materials and silage significant differences in nutrient composition, the product of silage fermentation was significantly higher, in line with the quality of raw materials after the silage fermentation requirements.
The use of common areas of bacterial V3 amplified different regions of the surface material attached bacteria silage and silage microbial 16S rDNA, the product analysis by DGGE is as followed, the same surface area of raw materials silage bacteria attached to the type of similarity is relatively high, up to 93%, at least more than 80%; Plant surface attachment for silage fermentation bacteria unrelated to "bacteria", less LAB type, only found Lactococcus lactis subsp. Cremoris, Weissella koreennsis, Lactobacillus buchneri three lactic acid bacteria. Silage in different parts of similar types of bacteria is not high, only around 60%. Containing the bacteria detected ?-Acinetobacter,?–Pantoea,? -Escherchia,?-Pseudomonas and other types of bacteria. We also detected?-Lactobacillus,?-Lactococcus, ?-Weissella and other types of bacteria. These bacteria are located in various regions of farm silage.
The use of common areas of bacterial LAB amplified different regions of the surface material attached bacteria silage and silage microbial 16S rDNA, the product analysis by DGGE is as followed, the same surface area of raw materials silage bacteria attached to the type of similarity was relatively low, up to only 75%, similarity of the same area was relatively high, both in the 63-94% between. LAB using the universal primer amplification type found most of the LAB, as well as bacteria-based, less cocci; among them Weissella confuse, Lactbacillus brevis, Acetobacter ghanensis, Lactococcus lactis, Weissella hellenica, Lactobacillus plantarum were found in 9 farms. Silage in different parts of lactic acid bacteria found by the cluster analysis of nine similar farm was not high, between 67% and 90%. More than half a year to the silo in the main group were ?-Lactobacillus ,and there are Lactococcus、Weissella、Leuconostoc in silage .
The use of pure culture techniques in different regions of more than half a year of raw materials and silage analysis in identification of LAB, silage materials in different regions of the surface of a total of 69 lactic acid bacteria isolated, of which 40 were Lactobacillus, 29 were cocci. Lactic acid bacteria were different in difference regions, and plants in the same area with the surface of the lactic acid bacteria with high similarity. in the fermentation of silage 46 of more than half a year lactic acid bacteria were isolated. Lactobacillus were regional different between different types of large, and a regional three of the similarity between the farm was also low.
Traditional pure culture method and PCR-DGGE conclusions do not correspond exactly. The traditional culture trains LAB selectively, and DGGE can better reflect the real structure of microbial fermentation system.
1.玉米青贮和苜蓿青贮中乳酸菌多样性研究
玉米青贮pH值迅速降低,至试验结束,青贮各项营养指标均达到优质青贮指标;而苜蓿青贮pH值一直维持在5.5以上,其余各项营养指标均达到优质指标。利用细菌V3区通用引物扩增玉米青贮和苜蓿青贮中微生物16S rDNA,产物经DGGE分析发现,玉米青贮和苜蓿青贮发酵过程中优势条带不同;其中?-Acinetobacter、?-Pantoea、?-Escherichia、?-Lactococcus、?-Weissella koreensis、?-Enterobacter等类细菌一直活跃在青贮中;?-Streptococcus、?-Endophytic、?-Enterobacter等细菌7d后从玉米青贮体系中消失,而有些条带(克隆未成功)是在苜蓿青贮后期逐渐变亮,逐渐占据优势。
利用细菌LAB类通用引物扩增玉米青贮和苜蓿青贮中微生物总16S rDNA,产物经DGGE分析发现,在青贮发酵过程中以?-Lactobacillus类居多,其次为?- Lactococcus和?- Weissella类;Lactobacillus brevis、?-Acetobacter、?-Leuconostoc在玉米青贮7d后有逐渐消失的趋势,而这几株菌在苜蓿青贮中一直存在,且没有显著变化。杆菌作为启动菌株一直存在于玉米及苜蓿青贮发酵体系中,且在青贮前(0d)及青贮后各时间点,条带一直较亮。
利用传统纯培养技术对玉米青贮和苜蓿青贮各时间点乳酸菌进行分离、鉴定,共发现118株,其中乳酸杆菌57株、球菌61株。这些菌分别出现在两种原料中,并在各个时间分离出的LAB不同,前期以球菌居多,但随着青贮发酵时间的延长,青贮中的LAB种类由开始的球菌逐渐演替成杆菌。
2.不同地区青贮原料表面附着菌及青贮中乳酸菌多样性研究
不同地区青贮原料及青贮的营养成分差异显著,青贮中发酵产物差异性显著,原料发酵后符合优质青贮要求。
利用细菌V3区通用引物扩增不同地区青贮原料表面附着菌及青贮中微生物16S rDNA,产物经DGGE方法分析发现:同一地区青贮原料表面附着菌的种类相似性比较高,最高达到93%最低也在80%以上;植株表面附着菌多为与青贮发酵无关的“杂菌”,LAB类较少,仅发现Lactococcus lactis subsp. Cremoris、Weissella koreensis、Lactobacillus buchneri等三种乳酸菌。不同地区青贮中细菌的种类相似性不高,仅在60%左右。检测出的菌群含有?-Acinetobacter、?-Pantoea、?-Escherichia、?-Pseudomonas等类“杂菌”,同时还检测出?-Lactobacillus、?-Lactococcus、?-Weissella等乳酸菌,这些菌群分布在各地区牧场青贮中。
利用LAB类通用引物扩增不同地区青贮原料表面附着菌及青贮中微生物16S rDNA,产物经DGGE方法分析发现:不同地区青贮原料表面附着菌的种类相似性比较低,最高也只在75%左右,同一地区的相似性比较高,均在63%至94%之间;使用该LAB类通用引物扩增,发现的绝大部分是LAB,且以杆菌为主,球菌较少;其中Weissella confusa、Lactobacillus brevis、Acetobacter ghanensis、Lactococcus lactis、Weissella hellenica、Lactobacillus plantarum等菌种在九个牧场中均出现。不同地区青贮中乳酸菌经聚类分析发现九个牧场相似性不高,在67%至90%之间;且半年以上的青贮窖中以乳杆菌为主要类群(?- Lactobacillus),同时仍有球菌(Lactococcus、Weissella、Leuconostoc)出现。
利用纯培养技术对不同地区青贮原料及半年以上青贮中LAB进行分离鉴定,不同地区青贮原料表面共分离出69株为乳酸菌,其中乳杆菌40株,乳球菌29株,地区不同乳酸菌种类有差异,而同一个地区植株表面的乳酸菌具有较高的相似性;于发酵半年以上的青贮中分离出46株为乳酸菌,均为乳杆菌,地区不同种类差异性较大,且同一个地区的三个牧场之间相似性也很低。
传统纯培养技术与PCR-DGGE方法得出的结论并不完全一致,传统培养技术对LAB具有选择性培养,而DGGE更能真实的反映发酵体系中微生物结构。
In this paper, we use the laboratory-made bag silage and silage samples in different regions, employing PCR-DGGE technology and traditional pure culture technology, comparing corn silage and alfalfa silage fermentation process of dynamic changes in flora diversity, and study the diversity of different surface areas of raw materials attached bacteria silage and silage more than half a year. And we come to the following conclusions.
1. Research of corn silage and alfalfa silage in the diversity of lactic acid bacteria.
pH value of corn silage reduced rapidly to the end of the experiment, and silage of the nutrition indicators all got to quality indicators. pH value of alfalfa silage and was maintained at above 5.5, and the rest of the nutrition indicators all got to quality indicators.
We use bacterial universal primers V3 area expansion of corn silage and alfalfa silage microbial 16S rDNA and find by DGGE analysis of the product, corn silage and alfalfa silage fermentation with different advantages strips; Among them, ?-Acinetobacter、?-Pantoea、?-Escherichia、?-Lactococcus、?-Weissella koreensis、?-Enterobacter and other types of bacteria have been active in the silage; ? -Streptococcus、?-Endophytic、?-Enterobacter and other types of bacteria disappeare from the corn silage system 7days later. And some bands (cloning was not successful) of alfalfa silage in the late light became gradually dominated.
LAB using bacterial universal primer amplification type of corn silage and alfalfa silage in the total microbial 16S rDNA, product analysis by DGGE, fermentation process in silage-Lactobacillus category was the most, followed by?-Lactococcus, and? Class-Weissella。There was trend that Lactobacillus brevis、?–Acetobacter、?- Leuconostoc corn silage in the gradual disappeared after 7days. Several Strains of bacteria were in the existence of alfalfa silage, and there are no significant change. Bacillus strains as a start was found in corn and alfalfa silage fermentation system, and in silage before (0 days) and silage at different time points, and it had a bright strip.
After the use of traditional techniques of pure culture of corn silage and alfalfa silage lactic acid bacteria at different time points were isolated and identified, 118 were found, of which 57 bacilli, cocci 61. These bacteria appeared in the two materials, respectively, and in various time of the LAB isolated from different bacteria in the early majority, but as time silage fermentation, silage types of LAB cocci by the beginning of a gradual succession as Bacillus.
2. Research of the diversity of materials attached to different parts of silage and silage strains of lactic acid bacteria
Silage in different regions of the raw materials and silage significant differences in nutrient composition, the product of silage fermentation was significantly higher, in line with the quality of raw materials after the silage fermentation requirements.
The use of common areas of bacterial V3 amplified different regions of the surface material attached bacteria silage and silage microbial 16S rDNA, the product analysis by DGGE is as followed, the same surface area of raw materials silage bacteria attached to the type of similarity is relatively high, up to 93%, at least more than 80%; Plant surface attachment for silage fermentation bacteria unrelated to "bacteria", less LAB type, only found Lactococcus lactis subsp. Cremoris, Weissella koreennsis, Lactobacillus buchneri three lactic acid bacteria. Silage in different parts of similar types of bacteria is not high, only around 60%. Containing the bacteria detected ?-Acinetobacter,?–Pantoea,? -Escherchia,?-Pseudomonas and other types of bacteria. We also detected?-Lactobacillus,?-Lactococcus, ?-Weissella and other types of bacteria. These bacteria are located in various regions of farm silage.
The use of common areas of bacterial LAB amplified different regions of the surface material attached bacteria silage and silage microbial 16S rDNA, the product analysis by DGGE is as followed, the same surface area of raw materials silage bacteria attached to the type of similarity was relatively low, up to only 75%, similarity of the same area was relatively high, both in the 63-94% between. LAB using the universal primer amplification type found most of the LAB, as well as bacteria-based, less cocci; among them Weissella confuse, Lactbacillus brevis, Acetobacter ghanensis, Lactococcus lactis, Weissella hellenica, Lactobacillus plantarum were found in 9 farms. Silage in different parts of lactic acid bacteria found by the cluster analysis of nine similar farm was not high, between 67% and 90%. More than half a year to the silo in the main group were ?-Lactobacillus ,and there are Lactococcus、Weissella、Leuconostoc in silage .
The use of pure culture techniques in different regions of more than half a year of raw materials and silage analysis in identification of LAB, silage materials in different regions of the surface of a total of 69 lactic acid bacteria isolated, of which 40 were Lactobacillus, 29 were cocci. Lactic acid bacteria were different in difference regions, and plants in the same area with the surface of the lactic acid bacteria with high similarity. in the fermentation of silage 46 of more than half a year lactic acid bacteria were isolated. Lactobacillus were regional different between different types of large, and a regional three of the similarity between the farm was also low.
Traditional pure culture method and PCR-DGGE conclusions do not correspond exactly. The traditional culture trains LAB selectively, and DGGE can better reflect the real structure of microbial fermentation system.
引文
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