饲料中圆弧青霉菌的分离培养及其毒素的提取与鉴定
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摘要
青霉酸(Penicillic Acid,PA)主要是由圆弧青霉菌产生的代谢产物,主要污染小麦、大米、玉米等农产品和动物饲料,在饲料中的最高含量可以达到2%。青霉酸是危害养殖业的常见霉菌毒素之一,长期饲喂含青霉酸的饲料,能使动物肝脏肿大,肝细胞变性并能抑制动物细胞DNA合成,严重的可使动物细胞DNA断裂。青霉酸能与其他霉菌毒素产生联合毒性,若与其他霉菌毒素共同作用能产生更大的毒性,对人类和动物的健康威胁十分巨大。因此对饲料中的圆弧青霉菌及其代谢产物的研究对提高畜产品的品质和食品安全有十分重要的意义。
研究目的
本研究旨在通过对饲料中的圆弧青霉菌的分离培养及鉴定和饲料中圆弧青霉菌的代谢产物青霉酸的提取、纯化和鉴定,为饲料中的圆弧青霉菌毒素——青霉酸的毒性研究和青霉酸的快速检测提供了可靠的实验方法及理论依据。
研究方法
本研究首先对可疑霉变饲料进行培养,饲料中的霉菌生长出来以后接着进行单独接种培养,根据单独接种的霉菌生长成熟以后根据大小、颜色、形态初步分类。分类以后将霉菌单独接种于平放在灭菌培养皿中的已滴加液体高盐察氏培养基盖玻片上,置于25℃霉菌培养皿中培养3～5 d,待有孢子长出后即染色在显微镜下观察霉菌孢子和菌丝的形态结构。将圆弧青霉菌孢子接种于饲料中在25℃霉菌培养箱中培养14 d,用氯仿提取饲料中的毒素。氯仿提取液浓缩后过柱,分离组分经过薄层层析鉴定,将用薄层层析鉴定后确定目的组分再通过高效液相色谱——质谱联检分析系统(HPLC-MS)进行液相—质谱检测,进一步确定青霉酸的含量。
研究结果
1.从采集的21份霉变饲料样品中共分离、鉴定出青霉属真菌6株:白色圆弧青霉(P.cyclopium Westling)、桔青霉(P.citrinum Thom)、绳状青霉(P.funiculosum)、扩展青霉(P.expansum)、棒行青霉(P.claviforme)和皮落青霉(P.crustosum)。
2.从经过培养的接种圆弧青霉菌饲料中成功提取并分离出圆弧青霉菌的代谢产物——青霉酸。
研究结论
青霉菌是饲料污染的主要霉菌之一,圆弧青霉菌的主要代谢产物为青霉酸。从湖南各地采集的饲料中已分离、培养、鉴定了6种青霉菌,其中白色圆弧青霉菌为产生青霉酸的霉菌,通过薄层层析和高效液相色谱——质谱联检分析系统已鉴定了分离到的圆弧青霉毒素为青霉酸。
Penicillic Acid(PA),which is mostly producted by Penicillium cyclopium,PA major pollution wheat,rice,corn and other agricultural products and animal feed,in feed can achieve the highest level of 2%.PA is one of the common mycotoxin,which is harmful to the agriculture industry.Long-term feeding with PA feed,make animal hepatomegaly,hepatic cell degeneration and animal cells can inhibit DNA synthesis, serious can make animal cell DNA breakage.PA can produce joint toxicity with other mycotoxin and PA could synergism with other common mycotoxin may have greater toxicity and threat to human and animal health.Therefore,studying the Penicillium cyclopium and PA in feed would make great significance sense to enhance the quality of livestock products and the security of food.
Purpose:
Through the isolated culturing and appreciation of Penicillium cyclopium and the extraction,depuration,and appreciation of the Penicillium cycopium metabolic product-PA,We attempt to establish a method and theory for the toxicity studying and fast-detection of PA in this research.
Method:
The first suspicious mouldy feed to culture,feed mold growth in the future followed by a separate inoculation training,according to a separate vaccination fungal growth under mature beyond the size,color,shape preliminary classification.After the mold separate classification will be inoculated in high salt liquid Czapek's Medium glass coverslips,at 25℃feed mold incubator cultured 3 to5 d,wait until there are spores sprouted after Dyeing is observed under the microscope mold spores and hyphae of the morphological structure.Penicillium cyclopium spores will be inoculated in 25℃feed mold incubator cultured 14 d,chloroform extract toxins in the feed.Chloroform extract concentration after column chromatography,identified the fractions by TLC, determined purpose component through HPLC-MS Spectrometry to further define the content of Penicillium acid.
Result:
1.We isolated 6 kinds of Penicillium from 21 moldy feed,they are P.cyclopium Westling,P.citrinum Thom,P.funiculosum,P.expansum,P.claviforme and P. crustosum.
2.We have extracted and isolated the metabolic product-PA from cultured Penicillium cyclopium.
Conclusion:
Penicillium pollution is the major pollution of feed,and the major metabolic product of Penicillium cyclopium is penicillic acid.Separated and identified 6 kinds of Penicillium from the feed,with collected from Hunan Province.White Penicillium cyclopium metabolite is PA,which identified by thin-layer chromatography and HPLC-MS seized analysis system.
研究目的
本研究旨在通过对饲料中的圆弧青霉菌的分离培养及鉴定和饲料中圆弧青霉菌的代谢产物青霉酸的提取、纯化和鉴定,为饲料中的圆弧青霉菌毒素——青霉酸的毒性研究和青霉酸的快速检测提供了可靠的实验方法及理论依据。
研究方法
本研究首先对可疑霉变饲料进行培养,饲料中的霉菌生长出来以后接着进行单独接种培养,根据单独接种的霉菌生长成熟以后根据大小、颜色、形态初步分类。分类以后将霉菌单独接种于平放在灭菌培养皿中的已滴加液体高盐察氏培养基盖玻片上,置于25℃霉菌培养皿中培养3～5 d,待有孢子长出后即染色在显微镜下观察霉菌孢子和菌丝的形态结构。将圆弧青霉菌孢子接种于饲料中在25℃霉菌培养箱中培养14 d,用氯仿提取饲料中的毒素。氯仿提取液浓缩后过柱,分离组分经过薄层层析鉴定,将用薄层层析鉴定后确定目的组分再通过高效液相色谱——质谱联检分析系统(HPLC-MS)进行液相—质谱检测,进一步确定青霉酸的含量。
研究结果
1.从采集的21份霉变饲料样品中共分离、鉴定出青霉属真菌6株:白色圆弧青霉(P.cyclopium Westling)、桔青霉(P.citrinum Thom)、绳状青霉(P.funiculosum)、扩展青霉(P.expansum)、棒行青霉(P.claviforme)和皮落青霉(P.crustosum)。
2.从经过培养的接种圆弧青霉菌饲料中成功提取并分离出圆弧青霉菌的代谢产物——青霉酸。
研究结论
青霉菌是饲料污染的主要霉菌之一,圆弧青霉菌的主要代谢产物为青霉酸。从湖南各地采集的饲料中已分离、培养、鉴定了6种青霉菌,其中白色圆弧青霉菌为产生青霉酸的霉菌,通过薄层层析和高效液相色谱——质谱联检分析系统已鉴定了分离到的圆弧青霉毒素为青霉酸。
Penicillic Acid(PA),which is mostly producted by Penicillium cyclopium,PA major pollution wheat,rice,corn and other agricultural products and animal feed,in feed can achieve the highest level of 2%.PA is one of the common mycotoxin,which is harmful to the agriculture industry.Long-term feeding with PA feed,make animal hepatomegaly,hepatic cell degeneration and animal cells can inhibit DNA synthesis, serious can make animal cell DNA breakage.PA can produce joint toxicity with other mycotoxin and PA could synergism with other common mycotoxin may have greater toxicity and threat to human and animal health.Therefore,studying the Penicillium cyclopium and PA in feed would make great significance sense to enhance the quality of livestock products and the security of food.
Purpose:
Through the isolated culturing and appreciation of Penicillium cyclopium and the extraction,depuration,and appreciation of the Penicillium cycopium metabolic product-PA,We attempt to establish a method and theory for the toxicity studying and fast-detection of PA in this research.
Method:
The first suspicious mouldy feed to culture,feed mold growth in the future followed by a separate inoculation training,according to a separate vaccination fungal growth under mature beyond the size,color,shape preliminary classification.After the mold separate classification will be inoculated in high salt liquid Czapek's Medium glass coverslips,at 25℃feed mold incubator cultured 3 to5 d,wait until there are spores sprouted after Dyeing is observed under the microscope mold spores and hyphae of the morphological structure.Penicillium cyclopium spores will be inoculated in 25℃feed mold incubator cultured 14 d,chloroform extract toxins in the feed.Chloroform extract concentration after column chromatography,identified the fractions by TLC, determined purpose component through HPLC-MS Spectrometry to further define the content of Penicillium acid.
Result:
1.We isolated 6 kinds of Penicillium from 21 moldy feed,they are P.cyclopium Westling,P.citrinum Thom,P.funiculosum,P.expansum,P.claviforme and P. crustosum.
2.We have extracted and isolated the metabolic product-PA from cultured Penicillium cyclopium.
Conclusion:
Penicillium pollution is the major pollution of feed,and the major metabolic product of Penicillium cyclopium is penicillic acid.Separated and identified 6 kinds of Penicillium from the feed,with collected from Hunan Province.White Penicillium cyclopium metabolite is PA,which identified by thin-layer chromatography and HPLC-MS seized analysis system.
引文
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