Isolation and Identification of Yeasts in Orange Juice and Their Rapid Molecular Detection Techniques
|Keywords||Yeast orange juice Isolation and identification Molecular detection Melting curveanalysis|
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.