橙汁中酵母菌的分离鉴定及其快速分子检测技术研究
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摘要
橙汁是最重要的柑橘加工品,其国际贸易额已占柑橘贸易总额的1/3以上。我国的柑橘种植面积和产量均居世界第一,但橙汁产量只占世界总产量的1%左右。为满足快速增加的橙汁消费量,减少对进口的依赖,我国正逐渐从优化柑橘产业布局、增加汁用甜橙栽培面积和降低原料成本等方面促进橙汁产业发展。伴随着这种发展,我国急需建立和完善橙汁的质量和安全保障体系。由酵母菌污染引起的橙汁腐败是产业长期面临的主要问题之一,更是不添加防腐剂的NFC橙汁生产中亟待解决的关键问题之一。与致病菌的检测相比,国内针对食品中酵母菌检测的研究较少。目前,我国食品和饮料中的酵母菌检测仍采用传统的平板培养法,所需时间长、检测结果滞后,不能及时指导生产。因此,为有效控制酵母菌污染,建立一套快速、有效的酵母菌检测方法对我国橙汁的安全生产和消费都具有重要意义。
本论文采用平板法分离橙汁中的酵母菌,通过形态观察、5.8S rDNA及其ITS间隔区序列RFLP分析和26S rDNA D1/D2区序列测定相结合的方法鉴定分离菌株。并且在改进DNA提取方法、优化PCR扩增体系的基础上建立了酵母菌PCR快速检测体系。主要研究结果如下：
1.以63份腐败橙汁为材料,利用平板法进行微生物分离计数,测得腐败橙汁中酵母菌的浓度在104CFU/mL～107CFU/mL。进一步分析表明,104CFU/mL酵母菌使橙汁呈现出胀瓶和变清等腐败特征,106CFU/mL-107CFU/mL酵母菌能使橙汁产品出现明显的胀瓶现象。
2.对橙汁中主要的酵母菌进行了形态观察。Pichia fermentans在YPD培养基上生长,菌落表面呈现辐射分布的精致的条纹,可用于快速识别该种酵母。对菌株Y17-2和Y19菌落形态进行连续观察,发现酵母菌落形态是动态变化的,可通过特定时期出现的明显特征识别酵母菌。Pichia属的7株酵母菌可根据菌落形态差异分为6组,与核糖体DNA序列聚类分析的结果一致。也对Clavispora lusitaniae、Candida sake、Hanseniaspora uvarum和τCandida parapsilosis等主要酵母污染菌在WL培养基上的菌落特征进行观察,这些菌落在颜色和形态上都有差异。研究结果表明：酵母在菌落特征上的差异与其核糖体DNA序列差异的结果一致。因此,采用菌落特征观察来鉴别橙汁中主要的腐败酵母菌是行之有效的快速鉴定方法之一。
3.以107CFU/mL和104CFU/mL的Saccharomyces cerevisiae菌液为材料,对细胞破碎方式进行比较后选择最佳破壁方法,并对研磨破壁时提取液的用量进行了优化。优化后的DNA提取方法如下：
1.5mL样品在13000rpm离心3min收集菌体；80gL提取液、90mg石英砂研磨3min,加320μL提取液；水浴10min,加250μL6mol/L NaCl溶液和等体积的氯仿：异戊醇(24.1)去除杂质,然后无水乙醇沉淀DNA,最后用50μL ddH2O溶解。
4.以提取自104CFU/mL菌液的DNA为模板优化PCR扩增体系。对PCR各组分进行优化的结果是：‘INTP0.2mmol/U引物浓度各0.4μmol/L, rTaq酶15U/25μL—g2+1.5mmol/L,或者rTaq酶1U/25μL—g2+2.5mmol/L有利于检测低浓度酵母菌。
5.以8株代表酵母菌为材料比较5.8S-ITS区RFLP分析、5.8S-ITS区测序分析和26S rDNAD1/D2区测序分析对酵母菌的鉴定效果。3种鉴定法方法的鉴定结果一致,表明5.8S-ITS区RFLP分析与D1/D2区序列测定结合可快速准确地鉴定酵母菌。从橙汁分离菌中鉴定出分属于17属的26种酵母,主要的酵母菌及其出现的频率如下Clavispora lusitaniae (13%)、Candida sake (11%)、Saccharomyces cerevisiae (11%)、Hanseniaspora uvarum(7%)、Candida parapsilosis (6%)和Meyerozyma guilliermondii (6%)。分离菌中有10种酵母菌在橙汁中少见报道,表明橙汁中酵母菌的种类多,值得进一步分析。
6.以106CFU/mL～101CFU/mL菌液和接种橙汁为材料,用常规PCR、热启动PCR和荧光定量PCR进行检测。常规PCR可检测接种橙汁中104CFU/mL酵母菌,表明检测灵敏度比原有的常规PCR检测106CFU/mL酵母菌提高了100倍,比原有的DNA经纯化后检测105CFU/mL酵母菌的灵敏度提高了10倍；使用热启动PCR可检测103CFU/mL酵母菌,是提高检测灵敏度的方法之一。以真菌通用引物NL1/NL4为引物,荧光定量PCR可检测40fg酵母菌DNA,能检测菌液中101CFU/mL酵母,但只能检测接种橙汁中104CFU/mL的酵母菌,这表明荧光定量PCR检测灵敏度高于常规PCR,但仍受橙汁中的某些物质的抑制而无法检测低浓度的酵母菌。
7.以7种已鉴定的橙汁分离酵母菌为材料,进行5.8S-ITS区熔解曲线分析,以考察能否用通过5.8S-ITS区熔解温度鉴别橙汁中的酵母菌。7种酵母菌5.8S-ITS区的熔解温度如下：Candida parapsilosis (80.10±0.0℃)、Rhodotorula glutinis (80.95±0.05℃)、Debarryomyces hansenii (81.70±0.0℃)、Meyerozyma guilliermondii Y56(82.60±0.0℃)、Candida intermedia (83.80±0.0℃)、Hanseniaspora sp.(84.20±0.0℃)、Pichia kluyvero(84.23±0.047℃),结果表明熔解曲线分析结果准确,可用于区分熔解温度相差0.2℃的酵母菌。
Orange juice is the most important processed citrus product, and its international trade value accounts for one third of the total citrus trade value. Although both the cultivation area and outputs of citrus in China now rank the first in the world, the orange juice production of China occupies only～1%of the world. To meet the increasing domestic demand for orange juice, and to reduce the dependence on imports, the development of orange juice industry in China is promoted gradually by optimizing the cultivation area of citrus, increasing the ratio of processing orange, and reducing the raw material cost.
With the rapid development of China's citrus industry, a quality safety and supervision system for orange juice is badly needed in China. The spoilage caused by yeast is one of the main problems of orange juice industry, especially to the Not from Concentrate Orange Juice (NFCOJ), in which preservative is not allowed. Compared with the detection of disease-causing bacteria, few studies have been carried out for the yeast. Up till now, traditional plate count method is still widely used in detection of yeast in food and beverage in China. Traditional method takes time and can not give in-time information for the production. To effectively control yeast contamination of orange juice, a rapid and effective detection method for detection of yeast in orange juice is of great importance to the consumption and production safety of orange juice in China.
In the present study, plate count method was used to isolate yeasts from orange juice. Then the isolates were identified based on morphology, restriction enzyme digestion analysis of the internal transcribed spacers (ITS) and5.8S rRNA gene, and sequence analysis of the D1/D2domain of26S rRNA gene. A molecular rapid detection system was established for the spoilage yeast in orange juice on the basis of a modification of DNA extraction method and optimization of PCR reaction system. The main results obtained were as follows:
1.63samples of spoiled orange juice were analyzed by plate count method. Yeast in spoiled juice was103CFU/ml-107CFU/ml. Further analysis shows that104CFU/ml yeast concentration caused the phenomenon of "bottle-swelling" and spoilage of the juice, and106CFU/ml-107CFU/ml yeast caused significant "bottle-swelling" phenomenon.
2. The colony morphology of yeast on YPD agar was observed. The surface of Pichia fermentans was characterized by wrinkle with stripes radiated from center to edge, which can be used to identify this strain. The dynamic change of colony morphology of strain Y17-2and Y19was observed. It is found that colony morphology of yeast at different development stages was different, and their morphological characteristics can be used to identify the yeast strains. Based on the morphological data obtained,7isolates of the genus Pichia can be divided into6groups, which was consistent to the result of cluster analysis based on ribosome DNA sequence. The colony morphology of Clavispora lusitaniae, Candida sake, Hanseniaspora uvarum, and Candida parapsilosis on WL medium was also observed. These colonies showed different color and shape. The results indicated that the difference of colony morphology of yeast is consistent with difference of ribosomal DNA sequence. Therefor, the morphology of yeast on WL medium is an effective and rapid method to identify the main spoilage yeasts in orange juice.
3. In order to choose the optimum cell disruption method,5cell wall disruption methods were compared using107and10CFU/ml diluents of S. cerevisiae. The volume of extraction buffer for grinding was optimized. Details of the optimized DNA extraction method is as follows:1.5ml specimen were centrifuged at13000rpm for3min;80μl extraction buffer and80-100mg quartz sand were added for grinding3min with pestle by hand, then320μl extraction buffer was added. The suspension of disrupted cells was incubated at65℃for10min, and purified with250μl6mol/L NaCl solution and equal volume of chloroform-isoamyl alcohol (24:1), followed by alcohol precipitation; finally, DNA was dissolved in50μl ddH2O.
4. The PCR amplification system was optimized for detecting yeast at low concentration using DNA extracts of water containing104CFU/ml yeast cells. The results obtained were as follows:0.2mmol/L dNTP and0.4μmol/L each primer,1.5U/25μl rTaq enzyme with1.5mmol/L Mg2+, or1U/25μl rTaq enzyme with2.5mmol/L Mg2+
5. Three molecular identification methods, RFLP analysis of5.8S-ITS region,5.8S-ITS region sequencing, and26S rDNA D1/D2sequence analysis, were tested using8yeast strains. Consistent results were obtained by these three methods. Our results indicated that RFLP analysis of5.8S-ITS region combined with D1/D2sequence analysis obtained accurate identification.68yeast strains were identified as26yeast species, which belonged to17genera according to D1/D2domain sequencing. The dominant yeasts species are as follows:Clavispora lusitaniae (13%), Candida sake (11%), Saccharomyces cerevisiae (11%), Hanseniaspora uvarum (7%), Candida parapsilosis (6%) and Meyerozyma guilliermondii (6%).10yeast species were firstly reported in this study, therefore, there might be more unknown yeast species in orange juice.10yeast species in this study were firstly detected in orange juice in this study. This result implied that there might be more unknown yeast species existing in orange juice.
6. DNA was extracted from water and orange juice containing106-101CFU/ml yeast cell using the improved DNA extraction method. Conventional PCR, hot-start PCR and real-time fluorescence quantitative PCR were used, respectively, to detect yeast. Conventional PCR could detect104CFU/ml yeast in orange juice, which means that the detection limit was improved100times comparing to the current limit of106CFU/ml yeast and10times comparing to the limit of105CFU/ml yeast with purified DNA. Furthermore, the detection limit of Hot-start PCR reached103CFU/ml, so Hot-start PCR is also one of the methods to improve detection sensitivity.40fg yeast DNA, DNA extracted from101CFU/ml yeast diluent, and DNA from104CFU/ml yeast inoculated orange juice were all able to be detected by real-time PCR using fungi universal primer NL1/NL4. This result suggested that the real-time PCR has higher detection sensitivity than conventional PCR. Unfortunately, this method failed to detect yeast at low concentration in samples due to the inhibition of some substances in orange juice.
7. Melting curve analysis of5.8S-ITS region from7identified yeast isolates from orange juice was performed. Their melting temperature were as follows:Candida parapsilosis (80.10±0.0℃), Rhodotorula glutinis (80.95±0.05℃), Debarryomyces hansenii (81.70±0.0℃), Meyerozyma guilliermondii Y56(82.60±0.0℃), Candida intermedia (83.80±0.0℃), Hanseniaspora sp.(84.20±0.0℃), and Pichia kluyveri (84.23±0.047℃). The Tm value of PCR products from the5.8-ITS region of5yeasts isolates differed by more than0.8℃, thus, allowing the differentiation of them by melting peak analysis.
本论文采用平板法分离橙汁中的酵母菌,通过形态观察、5.8S rDNA及其ITS间隔区序列RFLP分析和26S rDNA D1/D2区序列测定相结合的方法鉴定分离菌株。并且在改进DNA提取方法、优化PCR扩增体系的基础上建立了酵母菌PCR快速检测体系。主要研究结果如下：
1.以63份腐败橙汁为材料,利用平板法进行微生物分离计数,测得腐败橙汁中酵母菌的浓度在104CFU/mL～107CFU/mL。进一步分析表明,104CFU/mL酵母菌使橙汁呈现出胀瓶和变清等腐败特征,106CFU/mL-107CFU/mL酵母菌能使橙汁产品出现明显的胀瓶现象。
2.对橙汁中主要的酵母菌进行了形态观察。Pichia fermentans在YPD培养基上生长,菌落表面呈现辐射分布的精致的条纹,可用于快速识别该种酵母。对菌株Y17-2和Y19菌落形态进行连续观察,发现酵母菌落形态是动态变化的,可通过特定时期出现的明显特征识别酵母菌。Pichia属的7株酵母菌可根据菌落形态差异分为6组,与核糖体DNA序列聚类分析的结果一致。也对Clavispora lusitaniae、Candida sake、Hanseniaspora uvarum和τCandida parapsilosis等主要酵母污染菌在WL培养基上的菌落特征进行观察,这些菌落在颜色和形态上都有差异。研究结果表明：酵母在菌落特征上的差异与其核糖体DNA序列差异的结果一致。因此,采用菌落特征观察来鉴别橙汁中主要的腐败酵母菌是行之有效的快速鉴定方法之一。
3.以107CFU/mL和104CFU/mL的Saccharomyces cerevisiae菌液为材料,对细胞破碎方式进行比较后选择最佳破壁方法,并对研磨破壁时提取液的用量进行了优化。优化后的DNA提取方法如下：
1.5mL样品在13000rpm离心3min收集菌体；80gL提取液、90mg石英砂研磨3min,加320μL提取液；水浴10min,加250μL6mol/L NaCl溶液和等体积的氯仿：异戊醇(24.1)去除杂质,然后无水乙醇沉淀DNA,最后用50μL ddH2O溶解。
4.以提取自104CFU/mL菌液的DNA为模板优化PCR扩增体系。对PCR各组分进行优化的结果是：‘INTP0.2mmol/U引物浓度各0.4μmol/L, rTaq酶15U/25μL—g2+1.5mmol/L,或者rTaq酶1U/25μL—g2+2.5mmol/L有利于检测低浓度酵母菌。
5.以8株代表酵母菌为材料比较5.8S-ITS区RFLP分析、5.8S-ITS区测序分析和26S rDNAD1/D2区测序分析对酵母菌的鉴定效果。3种鉴定法方法的鉴定结果一致,表明5.8S-ITS区RFLP分析与D1/D2区序列测定结合可快速准确地鉴定酵母菌。从橙汁分离菌中鉴定出分属于17属的26种酵母,主要的酵母菌及其出现的频率如下Clavispora lusitaniae (13%)、Candida sake (11%)、Saccharomyces cerevisiae (11%)、Hanseniaspora uvarum(7%)、Candida parapsilosis (6%)和Meyerozyma guilliermondii (6%)。分离菌中有10种酵母菌在橙汁中少见报道,表明橙汁中酵母菌的种类多,值得进一步分析。
6.以106CFU/mL～101CFU/mL菌液和接种橙汁为材料,用常规PCR、热启动PCR和荧光定量PCR进行检测。常规PCR可检测接种橙汁中104CFU/mL酵母菌,表明检测灵敏度比原有的常规PCR检测106CFU/mL酵母菌提高了100倍,比原有的DNA经纯化后检测105CFU/mL酵母菌的灵敏度提高了10倍；使用热启动PCR可检测103CFU/mL酵母菌,是提高检测灵敏度的方法之一。以真菌通用引物NL1/NL4为引物,荧光定量PCR可检测40fg酵母菌DNA,能检测菌液中101CFU/mL酵母,但只能检测接种橙汁中104CFU/mL的酵母菌,这表明荧光定量PCR检测灵敏度高于常规PCR,但仍受橙汁中的某些物质的抑制而无法检测低浓度的酵母菌。
7.以7种已鉴定的橙汁分离酵母菌为材料,进行5.8S-ITS区熔解曲线分析,以考察能否用通过5.8S-ITS区熔解温度鉴别橙汁中的酵母菌。7种酵母菌5.8S-ITS区的熔解温度如下：Candida parapsilosis (80.10±0.0℃)、Rhodotorula glutinis (80.95±0.05℃)、Debarryomyces hansenii (81.70±0.0℃)、Meyerozyma guilliermondii Y56(82.60±0.0℃)、Candida intermedia (83.80±0.0℃)、Hanseniaspora sp.(84.20±0.0℃)、Pichia kluyvero(84.23±0.047℃),结果表明熔解曲线分析结果准确,可用于区分熔解温度相差0.2℃的酵母菌。
Orange juice is the most important processed citrus product, and its international trade value accounts for one third of the total citrus trade value. Although both the cultivation area and outputs of citrus in China now rank the first in the world, the orange juice production of China occupies only～1%of the world. To meet the increasing domestic demand for orange juice, and to reduce the dependence on imports, the development of orange juice industry in China is promoted gradually by optimizing the cultivation area of citrus, increasing the ratio of processing orange, and reducing the raw material cost.
With the rapid development of China's citrus industry, a quality safety and supervision system for orange juice is badly needed in China. The spoilage caused by yeast is one of the main problems of orange juice industry, especially to the Not from Concentrate Orange Juice (NFCOJ), in which preservative is not allowed. Compared with the detection of disease-causing bacteria, few studies have been carried out for the yeast. Up till now, traditional plate count method is still widely used in detection of yeast in food and beverage in China. Traditional method takes time and can not give in-time information for the production. To effectively control yeast contamination of orange juice, a rapid and effective detection method for detection of yeast in orange juice is of great importance to the consumption and production safety of orange juice in China.
In the present study, plate count method was used to isolate yeasts from orange juice. Then the isolates were identified based on morphology, restriction enzyme digestion analysis of the internal transcribed spacers (ITS) and5.8S rRNA gene, and sequence analysis of the D1/D2domain of26S rRNA gene. A molecular rapid detection system was established for the spoilage yeast in orange juice on the basis of a modification of DNA extraction method and optimization of PCR reaction system. The main results obtained were as follows:
1.63samples of spoiled orange juice were analyzed by plate count method. Yeast in spoiled juice was103CFU/ml-107CFU/ml. Further analysis shows that104CFU/ml yeast concentration caused the phenomenon of "bottle-swelling" and spoilage of the juice, and106CFU/ml-107CFU/ml yeast caused significant "bottle-swelling" phenomenon.
2. The colony morphology of yeast on YPD agar was observed. The surface of Pichia fermentans was characterized by wrinkle with stripes radiated from center to edge, which can be used to identify this strain. The dynamic change of colony morphology of strain Y17-2and Y19was observed. It is found that colony morphology of yeast at different development stages was different, and their morphological characteristics can be used to identify the yeast strains. Based on the morphological data obtained,7isolates of the genus Pichia can be divided into6groups, which was consistent to the result of cluster analysis based on ribosome DNA sequence. The colony morphology of Clavispora lusitaniae, Candida sake, Hanseniaspora uvarum, and Candida parapsilosis on WL medium was also observed. These colonies showed different color and shape. The results indicated that the difference of colony morphology of yeast is consistent with difference of ribosomal DNA sequence. Therefor, the morphology of yeast on WL medium is an effective and rapid method to identify the main spoilage yeasts in orange juice.
3. In order to choose the optimum cell disruption method,5cell wall disruption methods were compared using107and10CFU/ml diluents of S. cerevisiae. The volume of extraction buffer for grinding was optimized. Details of the optimized DNA extraction method is as follows:1.5ml specimen were centrifuged at13000rpm for3min;80μl extraction buffer and80-100mg quartz sand were added for grinding3min with pestle by hand, then320μl extraction buffer was added. The suspension of disrupted cells was incubated at65℃for10min, and purified with250μl6mol/L NaCl solution and equal volume of chloroform-isoamyl alcohol (24:1), followed by alcohol precipitation; finally, DNA was dissolved in50μl ddH2O.
4. The PCR amplification system was optimized for detecting yeast at low concentration using DNA extracts of water containing104CFU/ml yeast cells. The results obtained were as follows:0.2mmol/L dNTP and0.4μmol/L each primer,1.5U/25μl rTaq enzyme with1.5mmol/L Mg2+, or1U/25μl rTaq enzyme with2.5mmol/L Mg2+
5. Three molecular identification methods, RFLP analysis of5.8S-ITS region,5.8S-ITS region sequencing, and26S rDNA D1/D2sequence analysis, were tested using8yeast strains. Consistent results were obtained by these three methods. Our results indicated that RFLP analysis of5.8S-ITS region combined with D1/D2sequence analysis obtained accurate identification.68yeast strains were identified as26yeast species, which belonged to17genera according to D1/D2domain sequencing. The dominant yeasts species are as follows:Clavispora lusitaniae (13%), Candida sake (11%), Saccharomyces cerevisiae (11%), Hanseniaspora uvarum (7%), Candida parapsilosis (6%) and Meyerozyma guilliermondii (6%).10yeast species were firstly reported in this study, therefore, there might be more unknown yeast species in orange juice.10yeast species in this study were firstly detected in orange juice in this study. This result implied that there might be more unknown yeast species existing in orange juice.
6. DNA was extracted from water and orange juice containing106-101CFU/ml yeast cell using the improved DNA extraction method. Conventional PCR, hot-start PCR and real-time fluorescence quantitative PCR were used, respectively, to detect yeast. Conventional PCR could detect104CFU/ml yeast in orange juice, which means that the detection limit was improved100times comparing to the current limit of106CFU/ml yeast and10times comparing to the limit of105CFU/ml yeast with purified DNA. Furthermore, the detection limit of Hot-start PCR reached103CFU/ml, so Hot-start PCR is also one of the methods to improve detection sensitivity.40fg yeast DNA, DNA extracted from101CFU/ml yeast diluent, and DNA from104CFU/ml yeast inoculated orange juice were all able to be detected by real-time PCR using fungi universal primer NL1/NL4. This result suggested that the real-time PCR has higher detection sensitivity than conventional PCR. Unfortunately, this method failed to detect yeast at low concentration in samples due to the inhibition of some substances in orange juice.
7. Melting curve analysis of5.8S-ITS region from7identified yeast isolates from orange juice was performed. Their melting temperature were as follows:Candida parapsilosis (80.10±0.0℃), Rhodotorula glutinis (80.95±0.05℃), Debarryomyces hansenii (81.70±0.0℃), Meyerozyma guilliermondii Y56(82.60±0.0℃), Candida intermedia (83.80±0.0℃), Hanseniaspora sp.(84.20±0.0℃), and Pichia kluyveri (84.23±0.047℃). The Tm value of PCR products from the5.8-ITS region of5yeasts isolates differed by more than0.8℃, thus, allowing the differentiation of them by melting peak analysis.
引文
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