苹果汁中耐热菌的分离鉴定与PCR快速检测的研究
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摘要
酸土脂环酸芽抱杆菌(A.acidoterrestris),简称耐热菌,是重要的食品工业腐败菌。其显著特点是可以在温度20～70℃和pH2.0～6.0的高温低酸环境下生长。该菌芽孢的耐热能力分别为85℃时56min、90℃时15min、95℃时24min,因此巴氏灭菌不能除去此菌。在浓缩果汁中嗜酸耐热菌不繁殖代谢,但一旦稀释成低浓度的饮用果汁时,在常温下即代谢产生愈创木酚、卤酚,使果汁口感风味变差、浊度升高、形成沉淀导致产品品质下降。欧美各国要求每10克苹果浓缩汁中耐热菌的含量小于1个,耐热菌逐渐成为我国苹果浓缩汁行业产品出口所遭遇的主要的技术壁垒。我国对耐热菌研究属于起步阶段,主要集中在分离方法和控制上。果汁中芽孢杆菌菌种分布的研究和快速检测方面的研究也比较缺乏。
PCR技术是聚合酶链反应(Polymerase Chain Reaction , PCR),80年代逐渐发展起来的体外核酸扩增技术。它具有特异、敏感、快速、简便、重复性好、易自动化突出优点。传统方法检测食源性微生物(有益细菌,腐败菌,病原细菌)的步骤繁琐费时,需经富集培养、分离培养、形态特征观察、生理生化反应、血清学鉴定以及必要的动物试验等过程。并且传统方法无法对那些难以人工培养的微生物进行检测。目前常用的耐热菌检测方法是常规的培养检测法,耗时很长,一般需要4-5日才能出检测报告。而应用PCR技术,只需数小时就可以检测出目的菌。
试验选择了耐热菌的16SrDNA的高变区V2区和V4区某一段作为特异性引物的接合区域,以实现对此种微生物的特异的快速检测。通过试验研究得到以下结论:
1.从本研究结果看出:在苹果汁中生长的酸土环脂酸芽抱杆菌不是单一的菌,而是形态不同,生理生化性质不同,营养利用状况不同的菌群。除此之外,还有别的脂环酸芽孢杆菌菌种存在。
2.通过优化试验,最优PCR反应体系为:模板DNA 5μL,10×buffer KCl 2.5μL,浓度为25mmol/μL的Mg2+ 2.5μL,浓度为2.5mmol/μL的dNTPS 2μL,浓度为10μg/L的正反引物各0.5μL,浓度为5U/μL的Taq DNA聚合酶0.25μL,总体积25μL。反应条件为:94℃预变性4min后进入PCR循环,循环程序是94℃变性30S,58℃30S,72℃30S,扩增35个循环后72℃延伸5min。
3.得出耐热菌DNA最佳提取方法:比较了CTAB法、氯化苄法、水浴煮沸法和微波炉提取法,发现微波炉提取法是最佳DNA模板提取法,其优点是效率高,单管操作,简便快捷,费时最少。
4.优化后的扩增体系下,PCR检测耐热菌低限是3.2×10~2cfu/mL。
5.优化后的PCR扩增体系检测灵敏度不受苹果汁的干扰,在实际的生产检测中,当果汁里含有芽孢浓度为1cfu/10mL～2 cfu/10mL时,在45℃,260r/min的培养条件下,10 h后目的菌可被检测出来。
A.acidoterrestris is a kind of mostly important spoil bacteria in food industry. Its significant characters is that it can grow on a widely range of 20～70℃temperature and pH2.0～6.0,surviving in the high temperature and low acid environment condition. Its spore capability of thermo resistant is respectively 85℃56min、90℃15min、95℃24min,so that pasteurization process can’t kill it. No metabolism and reproduction are found in concentrate juice and its existence will not influence the quality of the juice. However, it can grow and produce the guaiacol and halogen phenol, which decrease apple juice quality by leading to bad flavor and turbidity. European and American make regulation that the amount of the stain in 10g apple juice should be less than 1cfu.So controlling A.acidoterrestris is becoming a technology trade barrier in apple juice import. The research of this bacterial is in initial stage in China .Concerning research mostly focus on isolating method and control. NO research objecting to investigation which species the bacteria existing in apple juice belong to. The research on rapid detection is also lack.
PCR technology is Polymerase Chain Reaction, and it is nucleic acid amplification technology in vitro, developing gradually after 80 era. Its significant advantages are good specificity, high sensitivity、rapid、simple、good repeat quality and automation.
The procedure of traditional method detecting the food source microorganism(useful organism、spoil organism、pathogen organism)is cumbersome and time-consuming. Steps of traditional method include increasing culture, isolating incubation, morphological observation, physiological and biochemical identification, serological identification, even animal experiment and so on. In additional, the traditional method can not detect the microorganisms which are not cultured. As for the researched bacteria here common detection method need 4～5d.However,application of PCR just spend several hours, and through it the only 0.1mgDNA of objects are detected by electrophoresis.
In this paper, two primers were designed from the V2 and V4 region of A.acidoterrestris’16SrDNA sequence. A PCR method for rapid and specific detecting of A.acidoterrestris is investigated.
The results of the studies are listed as follows:
1. Conculding this research results, A.acidoterrestris in apple juice are not simple one strain. They show different morphological、different physiological and biochemical character、different、different ability of using carbonhydrates. Besides for this, other strains of Alicyclobacillus maybe exist in apple juice.
2. Through optimum experiment, the PCR reaction system: template DNA5μL, 10×buffer KCl 2.5μL, 2.5μL of concentration 25mmol/μL Mg2+, 2μL of concentration 2.5mmol/μL dNTPS, respectively 0.5μL of concentration 10μg/L positive and negative primer, total volume 25μL. Reaction condition: pre-denature at 94℃for 4min,35 cycles of 94℃30S、58℃30S、72℃30S, final extent ion at 72℃for 5min.
3. Optimal extraction DNA of A.acidoterrestris method: compared the methods of CTAB、Benzyl chloride、boiling in water bath、boiling by microwave oven. The results find boiling by microwave oven high efficiency、one tube、rapid、simple and saving time. So it is regarded as optimal extraction method in this research. 4. Under optimum amplification system, the lowest limit amount is that it can detect 3.2×10~2cfu/mL of concentration bacteria.
5. The sensitivity is not affected by apple juice. When 1cfu/10mL～2 cfu/10mL of the spore is infected in apple juice artificially, after incubating 10h at 45℃,460r/min condition, the objective can be detected.
PCR技术是聚合酶链反应(Polymerase Chain Reaction , PCR),80年代逐渐发展起来的体外核酸扩增技术。它具有特异、敏感、快速、简便、重复性好、易自动化突出优点。传统方法检测食源性微生物(有益细菌,腐败菌,病原细菌)的步骤繁琐费时,需经富集培养、分离培养、形态特征观察、生理生化反应、血清学鉴定以及必要的动物试验等过程。并且传统方法无法对那些难以人工培养的微生物进行检测。目前常用的耐热菌检测方法是常规的培养检测法,耗时很长,一般需要4-5日才能出检测报告。而应用PCR技术,只需数小时就可以检测出目的菌。
试验选择了耐热菌的16SrDNA的高变区V2区和V4区某一段作为特异性引物的接合区域,以实现对此种微生物的特异的快速检测。通过试验研究得到以下结论:
1.从本研究结果看出:在苹果汁中生长的酸土环脂酸芽抱杆菌不是单一的菌,而是形态不同,生理生化性质不同,营养利用状况不同的菌群。除此之外,还有别的脂环酸芽孢杆菌菌种存在。
2.通过优化试验,最优PCR反应体系为:模板DNA 5μL,10×buffer KCl 2.5μL,浓度为25mmol/μL的Mg2+ 2.5μL,浓度为2.5mmol/μL的dNTPS 2μL,浓度为10μg/L的正反引物各0.5μL,浓度为5U/μL的Taq DNA聚合酶0.25μL,总体积25μL。反应条件为:94℃预变性4min后进入PCR循环,循环程序是94℃变性30S,58℃30S,72℃30S,扩增35个循环后72℃延伸5min。
3.得出耐热菌DNA最佳提取方法:比较了CTAB法、氯化苄法、水浴煮沸法和微波炉提取法,发现微波炉提取法是最佳DNA模板提取法,其优点是效率高,单管操作,简便快捷,费时最少。
4.优化后的扩增体系下,PCR检测耐热菌低限是3.2×10~2cfu/mL。
5.优化后的PCR扩增体系检测灵敏度不受苹果汁的干扰,在实际的生产检测中,当果汁里含有芽孢浓度为1cfu/10mL～2 cfu/10mL时,在45℃,260r/min的培养条件下,10 h后目的菌可被检测出来。
A.acidoterrestris is a kind of mostly important spoil bacteria in food industry. Its significant characters is that it can grow on a widely range of 20～70℃temperature and pH2.0～6.0,surviving in the high temperature and low acid environment condition. Its spore capability of thermo resistant is respectively 85℃56min、90℃15min、95℃24min,so that pasteurization process can’t kill it. No metabolism and reproduction are found in concentrate juice and its existence will not influence the quality of the juice. However, it can grow and produce the guaiacol and halogen phenol, which decrease apple juice quality by leading to bad flavor and turbidity. European and American make regulation that the amount of the stain in 10g apple juice should be less than 1cfu.So controlling A.acidoterrestris is becoming a technology trade barrier in apple juice import. The research of this bacterial is in initial stage in China .Concerning research mostly focus on isolating method and control. NO research objecting to investigation which species the bacteria existing in apple juice belong to. The research on rapid detection is also lack.
PCR technology is Polymerase Chain Reaction, and it is nucleic acid amplification technology in vitro, developing gradually after 80 era. Its significant advantages are good specificity, high sensitivity、rapid、simple、good repeat quality and automation.
The procedure of traditional method detecting the food source microorganism(useful organism、spoil organism、pathogen organism)is cumbersome and time-consuming. Steps of traditional method include increasing culture, isolating incubation, morphological observation, physiological and biochemical identification, serological identification, even animal experiment and so on. In additional, the traditional method can not detect the microorganisms which are not cultured. As for the researched bacteria here common detection method need 4～5d.However,application of PCR just spend several hours, and through it the only 0.1mgDNA of objects are detected by electrophoresis.
In this paper, two primers were designed from the V2 and V4 region of A.acidoterrestris’16SrDNA sequence. A PCR method for rapid and specific detecting of A.acidoterrestris is investigated.
The results of the studies are listed as follows:
1. Conculding this research results, A.acidoterrestris in apple juice are not simple one strain. They show different morphological、different physiological and biochemical character、different、different ability of using carbonhydrates. Besides for this, other strains of Alicyclobacillus maybe exist in apple juice.
2. Through optimum experiment, the PCR reaction system: template DNA5μL, 10×buffer KCl 2.5μL, 2.5μL of concentration 25mmol/μL Mg2+, 2μL of concentration 2.5mmol/μL dNTPS, respectively 0.5μL of concentration 10μg/L positive and negative primer, total volume 25μL. Reaction condition: pre-denature at 94℃for 4min,35 cycles of 94℃30S、58℃30S、72℃30S, final extent ion at 72℃for 5min.
3. Optimal extraction DNA of A.acidoterrestris method: compared the methods of CTAB、Benzyl chloride、boiling in water bath、boiling by microwave oven. The results find boiling by microwave oven high efficiency、one tube、rapid、simple and saving time. So it is regarded as optimal extraction method in this research. 4. Under optimum amplification system, the lowest limit amount is that it can detect 3.2×10~2cfu/mL of concentration bacteria.
5. The sensitivity is not affected by apple juice. When 1cfu/10mL～2 cfu/10mL of the spore is infected in apple juice artificially, after incubating 10h at 45℃,460r/min condition, the objective can be detected.
引文
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