Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).     

Analysis of some breakfast cereals on the French market for their contents of ochratoxin A,citrinin and fumonisin B1: development of a method for simultaneous extraction of ochratoxin A and citrinin

Crops may be contaminated by mycotoxins which can persist in the final products. Forty-five breakfast cereals were collected in French supermarkets. Ochratoxin A (OTA) and citrinin (CIT) were simultaneously extracted by a new method based on solvent partition validated in-house. The recoveries were over 80% for CIT and OTA. Fumonisin B₁ (FB₁) was analysed by an IUPAC method. The recoveries for FB₁ ranged from 50% to 70%, depending on the matrix. The losses were located at the step of immunoaffinity clean-up.OTA was detected in 69% of the samples; 20% of them were above the EU limit of 3 μg/kg. Twenty percent contained CIT (1.5–42 μg/kg). FBs were detected, not only in cornflakes, but also in products containing oats or rice, in the range 1–1110 μg/kg. Some samples were contaminated by all three mycotoxins.     

The production of ochratoxin A and citrinin in barley

The production of ochratoxin A by Aspergillus ochraceus and of ochratoxin A and citrinin by Penicillium viridicatum growing on previously sterilised barley for 200 days at 5, 10 and 20°C and a water activity of 0.85 is reported. A. ochraceus did not grow at 5°C, multiplied slowly at 10°C but did not produce toxin. At 20°C the organism multiplied more quickly and produced ochratoxin after 19 days, which slowly disappeared over the next 150 days. P. viridicatum grew slowly at 5°C but did not produce any toxin. It multiplied at 10°C and produced ochratoxin A which was only detectable during the period from 100 to 150 days. At 20°C both ochratoxin A and citrinin were produced. Ochratoxin A was detected after 10 days and was still present after 240 days, whereas citrinin was produced in large quantities between 118 and 129 days and then rapidly disappeared.     

Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia

Mycotoxins are products of moulds that frequently contaminate maize. In this study the presence of mycotoxins fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and ochratoxin A (OTA) was determined in 49 maize grain samples collected in autumn 2002. The most frequent finding was that of FB₁(100%), followed by ZEA (84%) and OTA (39%), while FB₂ was found only in three samples. The co-occurrence of two and three mycotoxins was found in 55 and 37% of samples, respectively. The concentrations (mean ± SD) of FB₁, ZEA and OTA in positive samples were 459.8 ± 310.7, 3.84 ± 6.68 and 1.47 ± 0.38 µg kg^-1, respectively, and the concentrations of FB₂ in three positive samples were 68.4, 109.2 and 3084.0 µg kg^-1. Although such low concentrations of mycotoxins are not a significant source of exposure in countries with a European diet, a few samples with extreme values indicate that thorough control is needed.     

Occurrence of aflatoxin B1, citrinin and ochratoxin A in rice in five provinces of the central region of Vietnam

The possible coexistence of three mycotoxins in rice, including aflatoxin B1 (AFB1), citrinin (CIT) and ochratoxin A (OTA), was investigated. The samples of rice were collected in large markets in five provinces of the central region of Vietnam. These toxins were extracted, purified and finally quantified by HPLC with fluorimetry detection. Contamination of AFB1 was found to be the most, followed by OTA, while contamination of CIT was insignificant. The coexistence of CIT with AFB1/OTA in rice was found in high percentage. Some samples overpassed the authorized limit by Europe in OTA and/or AFB1.     

Natural co-occurrence of aflatoxin B1, fumonisin B1 and ochratoxin A in barley and corn foods from Korea

A survey for aflatoxin B₁ (AFB₁), fumonisin B₁ and ochratoxin A (OTA) was conducted on 127 samples that included 30 food-grade barley, 32 barley foods, 18 food-grade corn and 47 corn foods, randomly collected during 1998-99 in Seoul, Korea. The presence of mycotoxins was analysed by direct competitive enzyme-linked immunosorbent assay (ELISA), and most of the positive samples from ELISA were confirmed using high-performance liquid chromatography (HPLC). Recoveries of AFB₁ and OTA spiked at 10 ng g -1 and FB₁ spiked at 50 ng g^-1 were 106, 87 and 105% by ELISA, whereas those by HPLC were 80, 79 and 84%, respectively. Detection limits by ELISA for AFB₁, FB₁ and OTA were 1, 5 and 5 ng g^-1, and those by HPLC were 0.6, 35 and 1 ng g^-1. Naturally occurring AFB₁, FB₁ and OTA were found in 4/32 (12%), 2/32(6%) and 4/32 (12%) samples of barley foods with an average of 26, 16 and 9 ng g^-1, respectively. AFB₁ and FB₁ in corn foods were detected in 4/47 (8%) and 9/47 (19%) samples with the average being 20 and 74 ng g^-1, while no OTA was found in any corn foods samples. No AFB₁, FB₁ or OTA was detected in any of food-grade barley and corn samples. This is the first report on the natural co-occurrence of AFB₁ and FB₁ in barley and corn foods as well as on surveillance of OTA in Korea.     

Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014–2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (r_s = 0.44) and wheat (r_s = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.     

Occurrence of ochratoxin A and citrinin in Czech cereals and comparison of two HPLC methods for ochratoxin A detection

The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg^?1. Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest? (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg^?1 and PART = 44.74 µg kg^?1. For the second sample (feeding barley) they were VICAM = 48.63 µg kg^?1 and PART = 34.40 µg kg^?1. Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg^?1 and PART = 6.03 µg kg^?1; VICAM = 4.12 µg kg^?1 and PART = 3.95 µg kg^?1). CIT was analysed using the PART method only, and its highest content (93.64 µg kg^?1) was found for the malting barley sample with high OTA content (44.74 µg kg^?1 as analysed using PART).     

2011~2019年吉林省市售食品中单增李斯特菌污染情况分析

目的 了解吉林省市售餐饮食品中单增李斯特菌的污染情况,为食品安全管理、食源性疾病的监测提供科学依据。方法 对2011年-2019年从吉林省9个监测地区采集到的8279件市售食品样本进行单增李斯特菌的分离鉴定。结果 2011年 -2019年从吉林省市售食品中共检测出单增李斯特菌阳性菌株352株,总检出率4.25%；各检测地区间长春市阳性检出率最高(7.63%),其次为四平市(6.03%)；12类市售食品中肉及肉质品阳性检出率最高(9.41%),其次为速冻米面食品(6.72%)；各类采样地点中超市的阳性检出率最高(6.89%),其次为农贸市场(5.64%)和快餐店(5.08%)；散装食品的阳性检出率明显高于预包装食品。结论 2019年吉林省的单增李斯特菌阳性检出率较临近三年呈回升态势；长春市与四平市单增李斯特菌污染情况较其他地区严重；肉与肉制品单增李斯特菌的污染情况最为严重,其次为速冻米面食品；流通环节中单增李斯特菌阳性检出率较高；散装食品的单增李斯特菌污染情况较预包装食品严重。有关部门应针对地区现状、食品类型和包装类型的不同加强管理力度。     

Production and stability of patulin, ochratoxin A, citrinin, and cyclopiazonic acid on dry cured ham

Toxinogenic fungal species can be isolated from dry cured meat products, raising the problem of the direct contamination of these foods by mycotoxins known to be carcinogenic or potent carcinogens. Because the contamination of a food by mycotoxins can be considered a balance between production and degradation, the stability of mycotoxins on dry cured meat was also investigated. This study focused on patulin, ochratoxin A, citrinin, and cyclopiazonic acid that can be produced by fungal species previously isolated from dry cured meat products sold on the French market. We demonstrated that neither patulin nor ochratoxin A was produced on dry meat by toxigenic strains, whereas relatively high amounts of citrinin and cyclopiazonic acid were found after a 16-day incubation period at 20 degrees C (87 and 50 mg/kg, respectively). After direct contamination, the initial content of patulin rapidly decreased to become undetectable after only 6 h of incubation at 20 degrees C. For both citrinin and ochratoxin A, the kinetics of decrease at 20 degrees C was less rapid, and the two toxins presented half-lives of 6 and 120 h, respectively. By contrast, more than 80% of the initial contamination in cyclopiazonic acid was still found on ham after a 192-h incubation period. Toxin stability was not affected by storage at 4 degrees C. These results suggest that growth of toxigenic strains of Penicillium has to be avoided on dry meat products.     

Occurrence of aflatoxins, ochratoxin A, and fumonisins in retail foods in Japan

We conducted a survey of aflatoxin B1, B2, G1, and G2, ochratoxin A, and fumonisin B1, B2, and B3 contamination in various foods on the retail market in Japan in 2004 and 2005. The mycotoxins were analyzed by high-performance liquid chromatography, liquid chromatography-mass spectrometry, or high-performance thin-layer chromatography. Aflatoxins were detected in 10 of 21 peanut butter samples; the highest concentration of aflatoxin B1 was 2.59 microg/kg. Aflatoxin contamination was not found in corn products, corn, peanuts, buckwheat flour, dried buckwheat noodles, rice, or sesame oil. Ochratoxin A was detected in oatmeal, wheat flour, rye, buckwheat flour, green coffee beans, roasted coffee beans, raisins, beer, and wine but not in rice or corn products. Ochratoxin A concentrations in contaminated samples were below 0.8 microg/kg. Fumonisins were detected in popcorn, frozen corn, corn flakes, and corn grits. The highest concentrations of fumonisins B1, B2, and B3 in these samples were 354.0, 94.0, and 64.0 microg/kg, respectively.     

Natural co-occurrence of ochratoxin A,ochratoxin B and aflatoxins in Sicilian red wines

The natural occurrence of ochratoxin A, ochratoxin B, aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂ (OTA, OTB, AFB₁, AFB₂, AFG₁, AFG₂) in red wines was investigated by HPLC/FLD after immunoaffinity column clean-up in 57 market samples produced in Sicily (Italy). The results showed a very low incidence of these mycotoxins in analysed samples, confirming the high degree of quality and safety of Sicilian red wines. The results indicated 71.9% and 64.9% positive samples for OTA and OTB respectively, with an average level of 0.13 μg l^–1, well below the European maximum permitted levels (MLs). The aflatoxin most frequently detected in the samples was AFG₁, present in 57.9% of samples, while the other aflatoxins were rarely present. Recovery experiments were carried out on eight mycotoxin-free red wines spiked with OTA, OTB, AFB₁, AFB₂, AFG₁ and AFG₂ at two different levels. The limits of detection (LODs) in wines were 0.02 µg l^–1 for OTA, 0.04 µg l^–1 for OTB, 0.03 µg l^–1 for AFG₁, AFG₂ and AFB₂, and 0.05 µg l^–1 for AFB₁. A good correlation was found, with good performances in term of precision for the method.     

Mycoflora in commercial pet foods

This article reports on the identification of mycoflora of 21 dry pet foods (12 belonging to dogs and 9 to cats) that corresponded to 8 commercial brands made in Argentina and imported. The isolation frequency and relative density of the prevalent fungal genera are compared too. Ten genera and fungi classified as Mycelia sterilia were identified. The predominant genera were Aspergillus (62%), Rhizopus (48%), and Mucor (38%). The most prevalent among Aspergillus was Aspergillus flavus followed by Aspergillus niger and Aspergillus terreus. The predominant Mucor was Mucor racemosus followed by Mucor plumbeus and Mucor globosus. The moisture content of these foods ranged from 5.6 to 10.0% and from 7.2 to 9.9% for dog and cat foods, respectively. A greater moisture content in food for the senior category (9.5 +/- 0.2) was observed only in comparison to adult and kitten/puppy. If the moisture content can be maintained at these levels, mold growth would be prevented or at least it would remain at an insignificant level. Some genera and species isolated and identified from the foods analyzed are potentially producing toxins, which are known as mycotoxins. This involves a risk for animal health.     

Development of a multiplex real-time PCR to quantify aflatoxin, ochratoxin A and patulin producing molds in foods

The antimicrobial activity of 8 Bacillus spp. and 2 Lysinibacillus spp. representing the predominant aerobic sporeformers during traditional maari fermentations, a traditional fermented baobab seeds product from Burkina Faso, was investigated. The antimicrobial activity was assessed against a total of 31 indicator organisms representing various Gram-negative and positive pathogens. The screening showed that 3 Bacillus subtilis strains (B3, B122 and B222) in particular had antimicrobial activity against some Gram-positive organisms and were selected for further studies. It was found that the antimicrobial substances produced were heat stable, in-sensitive to catalase, sensitive to protease and trypsin but resistant to the proteolytic action of papain and proteinase K and equally active at pH values ranging from 3 to 11. Bacteriocin secretion started in late exponential growth phase and maximum activity was detected during the stationary growth phase. The production of bacteriocin by B. subtilis B3, B122 and B222 was dependent on the aeration conditions. Maximum production of bacteriocin was observed under reduced aeration. Specific primers were used to screen isolates B3, B122 and B222 for genes involved in the synthesis of the bacteriocins subtilosin A, subtilin, sublancin and ericin. Amplicons of the expected sizes were detected for iywB, sboA, sboX, albA and spaS involved in the biosynthesis of subtilosin and subtilin, respectively. The translated nucleotide sequences had 100% identity to the YiwB, SboX and SboA amino acid sequences of the subtilosin A producing B. subtilis subsp. subtilis strain 168. Interestingly there was a 3 amino acid deletion at the N-terminal part of AlbA in B3, B122 and B222 that probably alters the activity of this enzyme. Analysis of the spaS gene sequences of B3, B122 and B222, encoding a subtilin precursor peptide, showed that the translated nucleotide sequence had 98% identity with the corresponding SpaS amino acid sequence of subtilin producing B. subtilis subsp. spizizenii strain ATCC6633.     

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods

ABSTRACT

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC₂₀) of the established method was 0.10 ± 0.02 ng mL^?1, with the limit of detection (IC₁₀) being 0.04 ± 0.007 ng mL^?1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (?0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g^?1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.     

Gluten contamination of cereal foods in Canada

Persons suffering from celiac disease (CD) must avoid foods containing gluten or those contaminated with wheat, barley, or rye. This study was designed to estimate gluten contamination of cereal-based foods available in Canada, whether labelled gluten-free or not. About half of the 148 foods sampled were labelled as gluten-free. According to R5-enzyme-linked immunosorbent assay (ELISA), twenty-three cereal-based foods (or 15%) contained more than 20 mg of gluten per kg, including sixteen regular and seven gluten-free foods, the latter being the least contaminated. When used in combination with R5-ELISA, AOAC-ELISA (not detecting barley) was a simple and efficient tool to roughly estimate the nature of the gluten contamination, which was later confirmed by real-time polymerase chain reaction for barley, and wheat/barley/rice. Rice-, corn- or quinoa-based foods were the safest for celiac patients. In addition to misleading food labelling for both gluten-rich and gluten-free foods, critical issues for persons with CD included foods made with oats or buckwheat (contaminated with wheat and barley gluten) in addition to those, such as breakfast cereals, specifically enriched with barley malt ingredients.     

Preliminary data on the presence of mycotoxins (ochratoxin A, citrinin and aflatoxin B1) in black table olives "Greek style" of Moroccan origin

Many mould strains, in particular Aspergillus and/or Penicillium, are able to develop on olive and produce ochratoxin A (OTA) and/or citrinin (CIT) and/or aflatoxin B (AFB) after harvest, during drying and storage of olives. The development of fungi on olives is responsible for the reduction of nutritional quality of olive because they can disturb the synthesis of the fatty acids. OTA, CIT and AFB are particularly dangerous for health, inducing cancer of urinary tracts or liver carcinoma. In this study, ten olive samples bought at retailer and at supermarket in Morocco were analyzed for their OTA, CIT and AFB contents. These three mycotoxins were extracted simultaneously by a method based on solvent partition validated in-house, then separated by HPLC coupled to a fluorescence detector. All olive samples contain OTA ranging from LOQ to 1.02 microg/kg. Respectively, 50 and 25% from retailer and supermarket samples were contaminated by more than 0.65 microg/kg. In addition, 80% of olive samples contained CIT above LOD, and 100% of olive tested contained AFB above 0.5 microg/kg. As simultaneous presence of these toxins increases toxic risks, it is thus essential to have a good control of the conservation of olives after harvest.     

The method of the simultaneous determination of the citrinin and ochratoxin A content in cereals and cereal products

The simplifyed method of simultaneous determination of the citrinin (CT) and ochratoxin A (OTA) in cereals is described. The extraction of mycotoxins was carried out by small volumes of solvents (water/aceton/hexane) without additional sample clean up and concentration. The extracts were analysed by HPLC using mixture of methanol/ethylacetate/phosphoric acid, pH 2.2 as a mobile phase and fluorescence detection (lamda ex 330 nm, lamda em 495 nm). The reliability and reproducibility of results were improved by usage of internal standard--methyl-derivative of ochratoxin A. Average recovery of CT and OTA was 70%, relative standard deviation 12% and 7%, respectively, limit of detection 0.003 mg/kg.     

成都市武侯区肉类食品大肠杆菌污染状况及毒力基因调查

了解成都市武侯区肉类食品中大肠杆菌污染情况和分离菌株的毒力特征,以预防和控制食品污染,保证食品安全。2011~2013年间采集成都市武侯区超市和菜市场的肉类食品,经选择性培养与分离鉴定,采用PCR方法检测8种相关毒力基因(eae A、stx1A、stx2A、hly A、agg R、est、elt A和bfp A)的流行情况。结果表明生肉类中大肠杆菌的检出率为81.07%,熟肉类中大肠杆菌的检出率为40%;在分离的324株大肠杆菌中有49株检测到一种及以上毒力基因;主要流行的毒力基因是eae A和agg R,未检测到est和bfp A基因。通过本研究发现成都市武侯区肉类食品中大肠杆菌污染比较严重,大肠杆菌携带毒力基因以eae A和agg R为主,该结果反映本市肉类产品的卫生安全状况,可为成都地区食品源大肠杆菌控制及毒力基因流行提供科学数据。