葡萄酒中赭曲霉毒素A的研究
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摘要
赭曲霉素A(以下简称OTA)是一种由真菌(包括疣孢青霉菌,赭曲霉,以及炭黑曲霉)产生的生物毒素,其常见于谷物,谷物产品,水果,咖啡,葡萄干,葡萄酒和啤酒中。由于OTA对人体构成了一种潜在的危害,国际癌症研究机构(IARC)已将其定义为group 2B类致癌物。欧洲整体膳食评估中对食品中OTA进行了分析,结果发现欧洲人从谷物中摄取到的OTA含量占到总摄取量的50%,而葡萄酒以13%仅次于谷物,排在第二位。欧盟(EU)、葡萄与葡萄酒组织(OIV)等国际性组织对葡萄酒中赭曲霉毒素A进行了限量,其限量标准为2μg/L。我国还没有相关限量标准,国内也缺乏相关的研究。
论文主要的研究结果如下：
(1)高效液相-荧光法测定葡萄酒中赭曲霉素A方法的建立
采用HPLC-FLD法测定葡萄酒中OTA时,线性良好,R2为0.9997；经固相萃取小柱浓缩后,测定方法检出限可达到0.002μg／L；回收率为85.7%-102.6%；RSD(n=5)为4.5%。通过对比发现HPLC-FLD方法相对于UPLC-MS/MS具有更好灵敏度。
(2)葡萄酒、葡萄酒生产过程中赭曲霉素A的测定与跟踪
采用建立的测定方法对葡萄酒样进行测定,实验发现干红葡萄酒中OTA含量明显高于干白葡萄酒。对于过程样测定结果发现,葡萄酒酿造过程中OTA的含量变化存在一定规律。
(3)葡萄园中赭曲霉素A产生菌的分离与鉴定
通过麦芽汁浸提物培养基(含氯霉素)与孟加拉红氯霉素培养基对葡萄园中的真菌进行分离,并对其中的曲霉与青霉进行了初步鉴定。共分离出29株真菌。
(4)赭曲霉素A产生菌产毒素能力评估
通过培养基对菌株进行纯培养,比较不同菌株的产OTA能力。炭黑曲霉产OTA能力明显高于赭曲霉。通过比较实验室筛选菌株的产OTA能力后发现,黑曲霉(r-t-7与r-p-5)产OTA能力最为显著,与赭曲霉产OTA能力相当。
(5)赭曲霉素A产生菌产毒素条件评估
通过正交实验评估,发现三种曲霉的产OTA最佳条件均为温度30℃,酒精度5%,糖度300g／L,发酵时间10d。各因素对三种曲霉的影响有所差异,糖度对炭黑曲霉、黑曲霉影响明显,酒精度对赭曲霉影响显著。
Ochratoxin A (OTA) is a kind of mycotoxin produced by several fungal species (include Penicillium verrusosum, Aspergillus ochraceus, Aspergillus carbonarius). Contamination of food commodities, including cereals and cereal products, fruit, coffee, dry vine fruits, wine and beer, has been reported from all over the world. OTA is a potential harm for human. The International Agency for Research on Cancer (IARC) evaluated Ochratoxin A, and classified it as possibly carcinogenic to humans (group 2B). In assessment of dietary intake of Ochratoxin A by the population of EU Member States, it reported cereals was the first contributor (50% of total OTA intake), wine was the second(15% of total OTA intake), The European Union and the International Organization of Vine and Wine (OIV) have established a maximum level for ochratoxin in wine-2μg/L.However, there are less maximum level and research about OTA in China.
The main results were as follow:
1. Establishment of a method for determination OTA in wine by HPLC
OTA detection in red was validated checking the linearity of the method (R2=0.9997). Utilizing concentration, the detection limit of OTA in wine were 0.004μg/L; recovery (85.7-102.6%), and precision (RSD=4.5%, n=5).
2. The determination of OTA from wine and wine fermentation
The results showed that the content of OTA in dried red wine was higher than dried white wine. In the process of making wine, the content of OTA changed regularity.
3. The identification and isolation fungi from vineyard and grape
At first, we isolated fungus from vineyard by two different media, and so identified various species of isolation fungi. The number of isolation was 29 in all.
4. Assessment of the content of OTA produced by fungi
Compared the content of OTA in two positive strain, the content of OTA produced by Aspergillus carbonarius was higher than Aspergillus Ochraceu. Aspergillus niger (r-t-7 and r-p-5) were the highest protector in the identification fungi.
5. Assessment of the factor influenced fungi to product OTA
The result showed three strains had the same optimum condition (temperature-30℃; alcoholic strength-5%; sugar degree-300g/L; fermentation days-10).And different factors influenced different fungi, sugar degree was important to Aspergillus niger, fermentation days and alcoholic strength were important to Aspergillus Ochraceu.
论文主要的研究结果如下：
(1)高效液相-荧光法测定葡萄酒中赭曲霉素A方法的建立
采用HPLC-FLD法测定葡萄酒中OTA时,线性良好,R2为0.9997；经固相萃取小柱浓缩后,测定方法检出限可达到0.002μg／L；回收率为85.7%-102.6%；RSD(n=5)为4.5%。通过对比发现HPLC-FLD方法相对于UPLC-MS/MS具有更好灵敏度。
(2)葡萄酒、葡萄酒生产过程中赭曲霉素A的测定与跟踪
采用建立的测定方法对葡萄酒样进行测定,实验发现干红葡萄酒中OTA含量明显高于干白葡萄酒。对于过程样测定结果发现,葡萄酒酿造过程中OTA的含量变化存在一定规律。
(3)葡萄园中赭曲霉素A产生菌的分离与鉴定
通过麦芽汁浸提物培养基(含氯霉素)与孟加拉红氯霉素培养基对葡萄园中的真菌进行分离,并对其中的曲霉与青霉进行了初步鉴定。共分离出29株真菌。
(4)赭曲霉素A产生菌产毒素能力评估
通过培养基对菌株进行纯培养,比较不同菌株的产OTA能力。炭黑曲霉产OTA能力明显高于赭曲霉。通过比较实验室筛选菌株的产OTA能力后发现,黑曲霉(r-t-7与r-p-5)产OTA能力最为显著,与赭曲霉产OTA能力相当。
(5)赭曲霉素A产生菌产毒素条件评估
通过正交实验评估,发现三种曲霉的产OTA最佳条件均为温度30℃,酒精度5%,糖度300g／L,发酵时间10d。各因素对三种曲霉的影响有所差异,糖度对炭黑曲霉、黑曲霉影响明显,酒精度对赭曲霉影响显著。
Ochratoxin A (OTA) is a kind of mycotoxin produced by several fungal species (include Penicillium verrusosum, Aspergillus ochraceus, Aspergillus carbonarius). Contamination of food commodities, including cereals and cereal products, fruit, coffee, dry vine fruits, wine and beer, has been reported from all over the world. OTA is a potential harm for human. The International Agency for Research on Cancer (IARC) evaluated Ochratoxin A, and classified it as possibly carcinogenic to humans (group 2B). In assessment of dietary intake of Ochratoxin A by the population of EU Member States, it reported cereals was the first contributor (50% of total OTA intake), wine was the second(15% of total OTA intake), The European Union and the International Organization of Vine and Wine (OIV) have established a maximum level for ochratoxin in wine-2μg/L.However, there are less maximum level and research about OTA in China.
The main results were as follow:
1. Establishment of a method for determination OTA in wine by HPLC
OTA detection in red was validated checking the linearity of the method (R2=0.9997). Utilizing concentration, the detection limit of OTA in wine were 0.004μg/L; recovery (85.7-102.6%), and precision (RSD=4.5%, n=5).
2. The determination of OTA from wine and wine fermentation
The results showed that the content of OTA in dried red wine was higher than dried white wine. In the process of making wine, the content of OTA changed regularity.
3. The identification and isolation fungi from vineyard and grape
At first, we isolated fungus from vineyard by two different media, and so identified various species of isolation fungi. The number of isolation was 29 in all.
4. Assessment of the content of OTA produced by fungi
Compared the content of OTA in two positive strain, the content of OTA produced by Aspergillus carbonarius was higher than Aspergillus Ochraceu. Aspergillus niger (r-t-7 and r-p-5) were the highest protector in the identification fungi.
5. Assessment of the factor influenced fungi to product OTA
The result showed three strains had the same optimum condition (temperature-30℃; alcoholic strength-5%; sugar degree-300g/L; fermentation days-10).And different factors influenced different fungi, sugar degree was important to Aspergillus niger, fermentation days and alcoholic strength were important to Aspergillus Ochraceu.
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