罗非鱼腐败过程菌群结构分析及腐败菌的分离、鉴定与调控
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摘要
罗非鱼是中国最重要的养殖和出口鱼类,但由于罗非鱼及相关产品货架期短,因此影响了罗非鱼养殖业的进一步发展。由于微生物在鱼类腐败过程中起主导作用,因此分析鱼类腐败过程中菌群结构变化及腐败菌群间的群体感应,有助于我们研究延长水产鱼类保藏时间、减少经济损失的方法。
本研究中以新鲜罗非鱼为试验对象,分别设置剔除鱼骨和肠道(JY)和只剔除鱼骨(QY)(每组有3个重复)两组试验样品,并分别在4℃和25℃贮存;以相同的时间(1、3、7和14天)取样并检测样品中的菌群变化。应用变性梯度凝胶电泳技术(DGGE)、平板计数、pH的测定以及腐败菌的分离鉴定等方法对腐败过程中的菌群变化进行分析。
应用DGGE技术分析表明JY样品腐败过程中的细菌属于α-变形杆菌亚门(α-proteobacteria)、β-变形杆菌亚门(β-proteobacteria)、γ-变形杆菌亚门(γ-proteobacteria)、厚壁门(Firmicutes)和未分类菌(Unclassified);其中γ-变形杆菌门细菌是样品腐败过程中的主要优势菌。QY样品腐败过程中的细菌属于变形杆菌门(Proteobacteria)、厚壁门(Firmicutes)和未分类菌(Unclassified);其中变形杆菌门细菌是样品腐败过程中的主要优势菌。
有研究表明DGGE技术只能检测到样品中高于总菌量1%的优势菌群,但是鱼肉中部分低丰度的致病菌仍能引起食品安全风险,因此用传统纯培养方法对样品中的细菌进行分离鉴定是对DGGE的有益补充。本研究采用在25℃储存3天的样品对可培养细菌进行分离鉴定,共获得23株细菌。其分别属于放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁门(Firmicutes)和变形杆菌亚门(Proteobacteria)。
目前已有研究表明群体感应的N-酰基高丝氨酸内酯类信号分子(AHLs)能够调控多种革兰氏阴性致病菌的基因的表达。本研究应用一种来源于芽孢杆菌的高丝氨酸内酯酶(AiiAB546)降解AHLs,进一步分析群体感应信号分子和腐败相关的酶基因表达的关系。由于摩氏摩根菌(M. morganii)、普通变形杆菌(Proteus vulgaris)、黄杆菌(Flavobacteriacea)在样品的腐败过程中起关键作用,并且都能够产生AHLs,因此本研究中以这三株菌为实验菌株,研究高丝氨酸内酯酶对胞外蛋白酶和组氨酸脱羧酶的调控作用。试验结果表明AiiA B546对黄杆菌所产的信号分子有降解作用且能降低其胞外蛋白酶酶活,但是对摩氏摩根菌、普通变形杆菌所产信号分子没有作用。
Tilapia is the most important fish among China aquaculture and exports. Due to the short shelf life of live tilapia and tilapia product, further development of tilapia culture has been limited. Because microorganisms are the major cause of fish spoilage, studies of the bacterial community structure change during spoilage and quorum-sensing system of spoilage organisms will facilitate us to find a way to lengthen the shelf life of fresh fish and reduce economic loss.
In this study, fresh tilapia was selected and treated by two different processing methods (3 replicates): removing bone and intestinal tract (JY) and only removing bone (QY). The samples were stored at 4°C and 25°C, respectively. Each treatment was sampled at 1, 3, 7, 14 days. Using DGGE, plate count, pH test, and isolation and identification of spoilage bacteria, the change of bacterial community structure during spoilage was explored.
The DGGE analysis showed that the bacteria in group JY were confined intoα-proteobacteria,β-proteobacteria,γ-proteobacteria, Firmicutes and unclassified. Among them,γ-proteobacteria was the predominant bacteria. The bacteria in group QY were mainly proteobacteria, Firmicutes and Unclassified.
It has been reported that the DGGE technology can only detect the bacteria of more than 1% of the total bacterial community. However, some pathogenetic bacteria in fish are lower than 1% of the total bacteria, thus isolation and identification of bacteria using the traditional pure culture approach was supplementary to the DGGE analysis. The samples stocked at 25°C for 3 days were subjected to isolation and identification of spoilage organisms. A total of 23 bacterial strains were isolated and classified into Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria.
N-Acyl homoserine lactones (AHLs) have been identified to regulate the expression of pathogenetic genes in Gram-negative bacteria by quorum-sensing system. AiiAB546 from Bacillus sp. B546, an N-acylhomoserine lactone lactonase, was used to study the relationship between AHLs and expression of genes related with spoilage. M. morganii, Proteus vulgaris, and Flavobacteriacea sp. playing key roles in fish spoilage and having ability to produce AHLs were selected as spoilage organisms. Results showed that AiiAB546 could degrade the AHLs produced by Flavobacteriacea sp., resulting in lower extracellular proteinase activity. AiiAB546 had no effect on the AHLs produced by M. morganii and P. vulgaris.
本研究中以新鲜罗非鱼为试验对象,分别设置剔除鱼骨和肠道(JY)和只剔除鱼骨(QY)(每组有3个重复)两组试验样品,并分别在4℃和25℃贮存;以相同的时间(1、3、7和14天)取样并检测样品中的菌群变化。应用变性梯度凝胶电泳技术(DGGE)、平板计数、pH的测定以及腐败菌的分离鉴定等方法对腐败过程中的菌群变化进行分析。
应用DGGE技术分析表明JY样品腐败过程中的细菌属于α-变形杆菌亚门(α-proteobacteria)、β-变形杆菌亚门(β-proteobacteria)、γ-变形杆菌亚门(γ-proteobacteria)、厚壁门(Firmicutes)和未分类菌(Unclassified);其中γ-变形杆菌门细菌是样品腐败过程中的主要优势菌。QY样品腐败过程中的细菌属于变形杆菌门(Proteobacteria)、厚壁门(Firmicutes)和未分类菌(Unclassified);其中变形杆菌门细菌是样品腐败过程中的主要优势菌。
有研究表明DGGE技术只能检测到样品中高于总菌量1%的优势菌群,但是鱼肉中部分低丰度的致病菌仍能引起食品安全风险,因此用传统纯培养方法对样品中的细菌进行分离鉴定是对DGGE的有益补充。本研究采用在25℃储存3天的样品对可培养细菌进行分离鉴定,共获得23株细菌。其分别属于放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁门(Firmicutes)和变形杆菌亚门(Proteobacteria)。
目前已有研究表明群体感应的N-酰基高丝氨酸内酯类信号分子(AHLs)能够调控多种革兰氏阴性致病菌的基因的表达。本研究应用一种来源于芽孢杆菌的高丝氨酸内酯酶(AiiAB546)降解AHLs,进一步分析群体感应信号分子和腐败相关的酶基因表达的关系。由于摩氏摩根菌(M. morganii)、普通变形杆菌(Proteus vulgaris)、黄杆菌(Flavobacteriacea)在样品的腐败过程中起关键作用,并且都能够产生AHLs,因此本研究中以这三株菌为实验菌株,研究高丝氨酸内酯酶对胞外蛋白酶和组氨酸脱羧酶的调控作用。试验结果表明AiiA B546对黄杆菌所产的信号分子有降解作用且能降低其胞外蛋白酶酶活,但是对摩氏摩根菌、普通变形杆菌所产信号分子没有作用。
Tilapia is the most important fish among China aquaculture and exports. Due to the short shelf life of live tilapia and tilapia product, further development of tilapia culture has been limited. Because microorganisms are the major cause of fish spoilage, studies of the bacterial community structure change during spoilage and quorum-sensing system of spoilage organisms will facilitate us to find a way to lengthen the shelf life of fresh fish and reduce economic loss.
In this study, fresh tilapia was selected and treated by two different processing methods (3 replicates): removing bone and intestinal tract (JY) and only removing bone (QY). The samples were stored at 4°C and 25°C, respectively. Each treatment was sampled at 1, 3, 7, 14 days. Using DGGE, plate count, pH test, and isolation and identification of spoilage bacteria, the change of bacterial community structure during spoilage was explored.
The DGGE analysis showed that the bacteria in group JY were confined intoα-proteobacteria,β-proteobacteria,γ-proteobacteria, Firmicutes and unclassified. Among them,γ-proteobacteria was the predominant bacteria. The bacteria in group QY were mainly proteobacteria, Firmicutes and Unclassified.
It has been reported that the DGGE technology can only detect the bacteria of more than 1% of the total bacterial community. However, some pathogenetic bacteria in fish are lower than 1% of the total bacteria, thus isolation and identification of bacteria using the traditional pure culture approach was supplementary to the DGGE analysis. The samples stocked at 25°C for 3 days were subjected to isolation and identification of spoilage organisms. A total of 23 bacterial strains were isolated and classified into Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria.
N-Acyl homoserine lactones (AHLs) have been identified to regulate the expression of pathogenetic genes in Gram-negative bacteria by quorum-sensing system. AiiAB546 from Bacillus sp. B546, an N-acylhomoserine lactone lactonase, was used to study the relationship between AHLs and expression of genes related with spoilage. M. morganii, Proteus vulgaris, and Flavobacteriacea sp. playing key roles in fish spoilage and having ability to produce AHLs were selected as spoilage organisms. Results showed that AiiAB546 could degrade the AHLs produced by Flavobacteriacea sp., resulting in lower extracellular proteinase activity. AiiAB546 had no effect on the AHLs produced by M. morganii and P. vulgaris.
引文
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