春草莓表面霉菌鉴定及其致腐能力研究

目的调查春草莓表面霉菌的总体状况,对主要致腐真菌进行致腐能力和产毒素研究,为草莓的保鲜及食用安全评价提供理论依据。方法对60份春草莓样品进行霉菌检测,分离霉菌采用ITS序列测序方法进行鉴定;利用高效液相色谱法及酶联免疫法对腐烂草莓及真菌发酵液中赭曲霉毒素A(ochratoxin A,OTA)和展青霉素(patulin,PAT)进行检测,对出现率较高的霉菌进行草莓复接试验,判断其致腐能力。结果健康草莓表面的霉菌计数对数值3.5~5.5的占样品总数的83%;毛霉菌属、枝孢菌属和青霉菌属在草莓中出现率较高;由这3类霉菌致草莓腐烂样品和其发酵液均检测不出真菌毒素OTA和PAT,复接试验中毛霉属真菌在36 h在草莓表面形成明显菌斑,72 h可形成明显菌斑而更大面积软腐。结论草莓表面存在大量且多种霉菌,霉菌侵染草莓是导致其腐烂主要诱因,毛霉属霉菌占主要生态位但青霉菌属、枝孢菌属霉菌更易导致其软腐,草莓表面霉菌不会产生真菌毒素而对草莓进行二次污染。     

臭氧降解玉米中赭曲霉毒素A的效果及对玉米脂肪酸的影响

针对粮食贮藏过程中真菌污染造成的食用安全性问题,探讨臭氧在玉米储藏中的应用可行性。以玉米中易污染的赭曲霉毒素A(OTA)为研究对象,在不同质量浓度臭氧,不同处理时间下,考察臭氧对OTA的降解效果,以及对玉米脂肪酸的影响。结果表明：30g/m3臭氧处理120min或者60g/m3臭氧处理90min能将80μg/L的OTA标准品几乎降解完全;利用60g/m3臭氧处理10h能有效的将玉米中污染80μg/kg OTA降解到安全范围(5μg/kg)以下,并且臭氧处理对玉米脂肪酸无显著影响。说明臭氧可有效降解玉米储藏中污染的OTA,并且不会破坏其中的不饱和脂肪酸,初步证明臭氧在玉米储藏中应用的可行性。     

中国南方稻谷主产区黑曲霉菌群的流行特征及产毒能力研究

本研究从我国南方十省份代表性粮库中采集稻谷样品,根据形态学特征筛选初步鉴别出黑曲霉群(Aspergillus niger aggregate)菌株,进一步依据ITS和CaM序列特征在种水平上进行鉴定;采用ELISA、HPLC-FLD方法,结合OTA生物合成基因鉴定方法联合确证黑曲霉群中分离株的产OTA能力。结果表明,从稻谷样品中共筛选出107株,划分为4个种：黑曲霉(A.niger)、百岁兰曲霉(A.welwitschiae)、塔宾曲霉(A.tubingensis)和新黑曲霉(A.neoniger)。其中百岁兰曲霉和黑曲霉在广西和广东地区流行率较高,塔宾曲霉的流行率随纬度的升高而升高,说明地理环境因素对我国南方地区仓储稻谷中黑曲霉群流行特征的影响较大。虽然黑曲霉群菌株产OTA测定中ELISA检测出7株菌假阳性,但进一步采用HPLC-FLD结合OTA生物合成基因鉴定方法确证107株黑曲霉群菌株均不产生OTA。本研究为我国稻谷的安全储藏和霉菌针对性防控提供了数据支撑。     

新疆酿酒葡萄中赭曲霉毒素A来源菌的筛选及其产毒条件研究

为筛选酿酒葡萄及根系土壤中赭曲霉毒素A(ochratoxin A,OTA)来源菌及其产毒条件,以新疆四大产区(焉耆盆地、吐哈盆地、天山北麓、伊犁河谷)酿酒葡萄和根系土壤为样品,进行微生物分离纯化和分子生物学鉴定,利用荧光紫外法初筛和酶联免疫法(enzyme-linked immunoassay,ELISA)复筛选择OTA来源菌,并通过单因素试验对其产OTA条件进行探究。筛选结果表明,样品中共分离出霉菌12株,其中,M2(黑曲霉Aspergillus niger)、M7(炭黑曲霉Aspergillus carbonarius)为OTA来源菌,M7产OTA含量较高;单因素结果表明,接种量、温度、酿酒葡萄有无损伤对炭黑曲霉产OTA能力的影响较为明显。该研究为构建葡萄酒OTA污染的预警机制提供了理论依据。     

黑曲霉产赭曲霉毒素A合成培养基的设计及产毒优化

针对目前没有适合黑曲霉产赭曲霉毒素A（OTA）全合成培养基的现状,进行黑曲霉产OTA合成培养基的设计,并优化高产OTA培养条件,以期为黑曲霉产OTA代谢和产毒机制的研究提供基础。通过碳源、氮源的筛选确定黑曲霉产OTA合成培养基的成分,利用高效液相色谱-荧光检测法检测此培养基在不同时间、pH、温度条件下OTA的产量,确定产OTA最佳培养条件。结果表明,培养基成分为FeSO4·7H2O 0.01 g/L,MgSO4·7H2O 0.5 g/L,Na2HPO4·2H2O 0.5 g/L,KCl 1.0 g/L,葡萄糖100 g/L,苯丙氨酸0.3 g/L。培养条件为初始pH 值为7,培养温度25 ℃,培养时间8 d。在此条件下黑曲霉产OTA产量最高,为4.19 μg/g,菌体生物量为3.4 g。     

葡萄赭曲霉毒素污染及其产毒素菌株的筛选方法研究进展

赭曲霉毒素A(ochratoxin,OTA)是由曲霉属和青霉属等真菌产生的一类真菌毒素,其毒性很强,分布广泛,对人类和动植物的健康有着巨大的影响。葡萄及其制品是食品中OTA的主要来源之一,从病害的葡萄表面筛选分离产生赭曲霉毒素的菌株是最常用的研究产毒素菌株的方法。由于产毒素菌株主要分布在葡萄果实的表面,葡萄组织受损后会极大提高赭曲霉素的污染程度,研究产毒菌株分布及产毒素能力对控制赭曲霉素污染及寻找一种有效地生物防治方法提供参考。本文综述了葡萄赭曲霉毒素的污染情况及其产毒素菌株的筛选方法,为控制葡萄及其产品中的OTA污染提供依据。      

葡萄中碳黑曲霉的分离及其产生赭曲霉毒素A的研究

碳黑曲霉(Aspergillus carbonarius)是葡萄中产生赭曲霉毒素A(Ochratoxin A,OTA)的重要菌株.采集烟台赤霞珠(Cabernet sauvignon)葡萄,接种于孟加拉红培养基,从中分离到7株黑曲霉群(Aspergillus section black group)真菌,其中3株鉴定属于碳黑曲霉种,占黑色曲霉的43.8%.此3菌株分别接种在粮粒培养基上,静置培养,全部产生OTA,最高浓度达到1300μg/kg,而且,其中2株碳黑曲霉菌株在可可浆培养基上产生荧光,而从葡萄样品中未检出OTA.     

不同条件对炭黑曲霉产赭曲霉毒素A能力的影响

为了解一株分离自新疆葡萄园的炭黑曲霉菌株在不同培养条件下产生赭曲霉毒素A(OTA)的能力,试验采用孟加拉红培养基,利用高效液相-荧光法对OTA进行定量检测,在单因素试验的基础上,采用响应面法优化产毒条件。结果表明,对该菌株产生OTA的条件影响从大到小依次为葡萄糖质量浓度、乙醇体积分数、SO2质量浓度。最优产毒条件:在温度28℃条件下,葡萄糖质量浓度92.26g/L、SO2质量浓度8.26mg/L、乙醇体积分数0.05%。在此条件下,OTA产量为7.427ng/mL。     

赭曲霉毒素A人工抗原的合成与鉴定

采用N-羟琥珀酰亚胺酯（N-hydroxysuccinimide NHS）法将赭曲霉毒素A（OTA）与牛血清白蛋白（BSA）偶联制备了免疫抗原OTA-BSA,采用1-乙基-3（3-二甲基氨基丙基）碳二亚胺（EDC）法将赭曲霉毒素A（OTA）与卵清白蛋白（OVA）偶联制备了包被抗原OTA-OVA。紫外扫描和SDS-PAGE凝胶电泳结果初步表明偶联成功。用OTA-BSA分10μg/只和50μg/只两个剂量分别免疫BALB/C小鼠,获得了高效价的特异性多克隆抗血清,效价可达1∶104,抑制效价达14.7ng,证明偶联成功,并显示低剂量免疫可以提高小鼠pAb的敏感性。本研究为OTA单抗的制备及其免疫学分析方法的建立奠定了基础。      

赭曲霉毒素A人工抗原的合成与鉴定

采用N-羟琥珀酰亚胺酯(N-hydroxysuccinimide NHS)法将赭曲霉毒素A(OTA)与牛血清白蛋白(BSA)偶联制备了免疫抗原OTA-BSA,采用1-乙基-3(3-二甲基氨基丙基)碳二亚胺(EDC)法将赭曲霉毒素A(OTA)与卵清白蛋白(OVA)偶联制备了包被抗原OTA-OVA。紫外扫描和SDS-PAGE凝胶电泳结果初步表明偶联成功。用OTA-BSA分10μg/只和50μg/只两个剂量分别免疫BALB/C小鼠,获得了高效价的特异性多克隆抗血清,效价可达1∶104,抑制效价达14.7ng,证明偶联成功,并显示低剂量免疫可以提高小鼠pAb的敏感性。本研究为OTA单抗的制备及其免疫学分析方法的建立奠定了基础。     

Incidence of toxigenic fungi and ochratoxin A in dried fruits sold in Brazil

A total of 117 dried fruit samples (black sultanas, white sultanas, dates, dried plums, dried figs and apricots) from different origins were analysed both for toxigenic fungi and for the presence of ochratoxin A. Amongst the fungi found, Aspergillus niger was predominant, with 406 isolates, of which 15% were ochratoxin A producers. They were followed by A. ochraceus, with 15 isolates and 87% ochratoxigenics, and A. carbonarius, with only five isolates of which 60% were ochratoxin A producers. The average infection rates for A. niger in black sultanas, plums, figs, dates and white sultanas were 22.0, 8.0, 4.0, 1.5 and 0.5%, respectively. The apricot samples were not contaminated by any fungi or ochratoxin A. Black sultana and dried figs contained the highest contamination with ochratoxin A, with 33 and 26.3% of the samples containing more than 5 µg kg^-1 respectively, while all the white sultanas, dates and plums had no sample that exceeded this limit.     

Effect of operating conditions on ochratoxin A extraction from roasted coffee

Operating conditions affect ochratoxin A (OTA) extraction from roasted coffee. The OTA content found in the beverage can thus be greater than that found in the roasted coffee used to prepare it. Three extraction parameters were studied for roasted coffee: type of extraction solvent (alkaline, neutral, acid), temperature (ambient temperature/23°C, 60°C and 85°C), and extraction time (5, 20, 30, 40, 50, 60 and 80 min). The alkaline solvent used in the method recommended by the European Union extracted OTA better, but a maximum content was obtained at 60°C after 50 min. At least a 100% improvement in extraction was obtained when compared with the European Union usual quantification method that is carried out at ambient temperature. It turned out that the OTA extraction parameters for roasted coffee, as defined by that method, were not optimum and needed to be modified. These results were verified in double-extraction experiments showing that OTA is not completely extracted by this method. Confirmation was obtained by comparison of extraction methods on several commercial samples of roasted coffee.     

降解赭曲霉毒素A菌株的筛选及其鉴定

为了提供筛选生物降解赭曲霉毒素A（ochratoxin A,OTA）的便利条件,优化水系中OTA的测定方法。通过在水系中添加甲醇或乙醇的方法,使得水系中OTA回收率超过了90%,此外通过对比,当甲醇或乙醇与样液体积比为1∶1时,足以准确测定水系中OTA含量。利用以上方法对降解OTA的菌株进行筛选,发现细菌B-1和酵母Y-2均能够降解OTA,且细菌B-1在2 d时能降解87%的OTA,3 d将OTA全部降解,而酵母Y-2在2 d时就能够降解84%的OTA,在后续的培养中维持降解率不变。此外,利用液质联用法进一步确定了菌株B-1和Y-2的降解作用,并分析了降解产物。最后,通过分别分析细菌B-1和酵母Y-2的16S rDNA和ITS rDNA序列,菌株B-1鉴定为泡囊短波单胞菌,菌株Y-2鉴定为耶罗维亚酵母。     

抗赭曲霉毒素A单克隆抗体的制备及免疫学鉴定

通过碳化二亚胺法(EDC法)制备免疫原OTA-BSA,并免疫小鼠,经杂交瘤技术建立产抗赭曲霉毒素A(OTA)单克隆抗体的杂交瘤细胞株1株。通过体内诱生腹水法制备腹水,纯化后对其亚型、效价、灵敏性、亲和性、特异性等免疫学特性进行鉴定;单抗亚类为IgG1型,间接ELISA效价1:6.4×105,IC50为112.8pg/mL,亲和常数为9.74×1011L/mol,与赭曲霉毒素B交叉反应率为5.6%,与其他常见真菌产物交叉小于0.003%;本实验制备的抗OTA单克隆抗体具有高效、敏感、特异等特点,适合OTA的免疫学快速检测。     

Occurrence and Safety Evaluation of Ochratoxin A in Cereal‐based Baby Foods Collected from Iranian Retail Market

Contamination of agricultural commodities with ochratoxin A (OTA) is a worldwide concern in recent decades. Consumption of OTA‐contaminated baby foods exerts health implications especially in children as the most vulnerable subpopulations. In the current study, for the first time in Iran, 64 baby foods (rice, wheat, and multigrain) samples from five different brands available in the Iranian market were analyzed to determine OTA level, using a HPLC with fluorescence detector. Overall, OTA was observed in 41% of analyzed samples with a mean and maximum level of 0.42 ± 0.27 and 1.1 μg/kg, respectively. OTA levels in five of 64 samples (7.8 %) were higher than the permissible limit recommended by European Commission (permissible limit: 0.5 μg/kg) and OTA levels in two of 64 samples (3.1%) were higher than the standard set by Iranian standard organization (1 μg/kg). The highest OTA contamination was observed in rice‐based baby food cereals (1.1 μg/kg; 57% of the samples), followed by wheat‐based (23%) and multigrain (20%) samples. OTA intake in infants (≥9 months old) was more than established provisional tolerable weekly intake by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA) (100 and 120 ng OTA per kg of body weight, respectively). OTA content in baby food and cereals, as well as other raw foodstuff should be investigated comprehensively to reduce the exposure rate of young children to OTA.     

FTIR-ATR infrared spectroscopy for the detection of ochratoxin A in dried vine fruit

A method of screening sultanas for ochratoxin A (OTA) contamination, using mid-infrared spectroscopy/Golden Gate single-reflection ATR (attenuated total reflection), is described. The main spectral characteristics of sultanas from different sources were identified in a preliminary acquisition and spectral analysis study. Principal component analysis (PCA) showed that samples of various origins had different spectral characteristics, especially in water content and the fingerprint region. A lack of reproducibility was observed in the spectra acquired on different days. However, spectral repeatability was greatly improved when water activity of the sample was set at 0.62. A calibration curve of OTA was constructed in the range 10-40 µg OTA kg^-1. Samples with OTA levels higher than 20 µg kg^-1 were separated from samples contaminated with a lower concentration (10 µg OTA kg^-1) and from uncontaminated samples. The reported methodology is a reliable and simple technique for screening dried vine fruit for OTA.     

Co‐occurrence of citrinin and ochratoxin A in rice in Asia and its implications for human health

Citrinin (CIT) and ochratoxin A (OTA) are nephrotoxic mycotoxins, produced by several Aspergillus and Penicillium species and their co‐occurrence in rice may cause health effects in humans. Rice is an important food crop worldwide and is a major staple food in Asia which may be invaded by CIT and OTA producing fungal spores in the field, during harvest and storage. Humans are exposed to these mycotoxins through ingestion of contaminated rice and other food commodities. Yet, data on the combined presence to these food contaminants are still insufficient to estimate human exposure in Asia. This review describes the prevalence of CIT and OTA in rice in Asia and its implications on human health, which may help in establishing and carrying out proper management strategies against mould development on rice. From the health point of view, combined exposition of CIT and OTA should be a public concern as both are nephrotoxic and long‐term exposure can pose detrimental health effects. Thus, it is necessary for local farmers and food factories to implement strict measures and to improve methods for rice preservation during the distribution to consumers, particularly in the markets. Moreover, regular surveys for CIT and OTA occurrence in rice and human biomonitoring are recommended to reduce the health effects in Asian population. © 2017 Society of Chemical Industry     

Ochratoxigenic Black Aspergilli Isolated from Dried Agricultural Products in Yogyakarta, Indonesia

Black aspergilli, the potential ochratoxin A （OTA） producers, were predominant fungi in fermented cocoa bean, coffee bean and dried cassava in Yogyakarta. Identification of black aspergilli at species level will be useful to make clear link between OTA contamination on food product and the toxin producer. The objective of this study was to identify the species of the black aspergilli producing OTA which contaminated dried agriculture products. In this study, 16 isolates were obtained, and four isolates among of them （YAC-9, YAK-6, YAK-12 and YAG-2） were found as OTA producing-strains, with the highest OTA found on Yeast Extract Sucrose （YES） solid medium of 57.68 ppb. Based on morphological characters, 16 isolates can be grouped into four species, after confirmation by molecular data based on PCR method, the groups were identified as A. carbonarius, A. niger, A. tubingensis and A. aculeatus. OTA producing-strains were identified as A. carbonarius and A. niger, meanwhile, A. tubingensis and A. aculeatus were found as non-OTA producing-strains.