不产桔霉素的红曲霉菌种深层发酵生产莫纳可林K

对三株在YES培养基中不产桔霉素的红曲霉菌种,在摇瓶中研究了它们液体发酵生产莫纳可林K的情况。在大米粉培养基中,红色红曲霉不产莫纳可林K,但是紫色红曲霉和烟灰色红曲霉均能产莫纳可林K,前者产量高于后者。在葡萄糖．甘油培养基中,后两者的产量均很低,但是如果在该培养基中添加酵母膏,紫色红曲霉能产生较为可观的莫纳可林K。在2L的发酵罐中,利用添加了酵母膏的葡萄糖-甘油培养基,紫色红曲霉在第13d的莫纳可林K产量可达104mg/L,培养过程中无桔霉素产生。     

基于适体技术的桔青毒素检测方法的建立

桔青毒素（citrinin,CTN）是以玉米、谷物、奶酪等为主要成分的食品和动物饲料中常见的、由桔青霉菌产生的酮类真菌毒素,可引起人和动物的慢性中毒或癌症,一直缺少灵敏的快速检测方法。本文通过指数富集适体系统进化技术（简称SELEX）对可能与CTN结合的特异性适体进行了筛选,经过15轮循环,得到17条适体。通过二级结构分析、亲和力检测发现适体13（Apt-13）对CTN有较好的亲和度,解离常数Kd为0.06μmol/L。进一步利用非荧光标记染料PicoGreen,利用其与双链DNA结合的原理,建立了桔青毒素非标记荧光检测方法,30min完成检测,最低检测限达到国家标准（50ppb）且与其他毒素无交叉反应。本研究建立的基于适体的桔青毒素检测技术成本低,可以替代传统的基于抗体的检测方法,为霉菌毒素的精准检测技术的开发提供了实验证据。     

Quantum chemical study of the structure and properties of citrinin

Detailed structures and electronic properties of three tautomeric forms of the toxin citrinin were investigated using several quantum calculation methods. Energetic preference of the predominant p- and o-quinone methide tautomeric forms is dependent on the method of calculation. A previously unstudied carboxylic acid enol tautomer was calculated to be surprisingly stable in vacuo, being within 2.5 kcal mol^? 1 at the B3LYP/6-311++G(2d,2p) level of theory. Despite differences in bond nature and connectivity of tautomers, the natural bond orbital analysis revealed that tautomeric forms share similar natural charges and natural electron configurations. Calculated bond lengths corresponded with experimentally observed values and assignments for the calculated infrared vibrational frequencies are reported.     

高产莫纳可林K低产桔霉素红曲霉菌株的筛选和发酵条件初步优化

采集全国各地红曲霉制品中的红曲霉资源,经分离获278株红曲霉菌株。高效液相色谱(HPLC)法测定各菌株发酵液中莫纳可林K(Monacolin K)和桔霉素含量,筛选获1株Monacolin K产量较高、桔霉素含量较低的红曲霉菌株编号为M-22。依据形态特征和ITS基因序列,参照红曲霉属分类检索表,鉴定M-22菌株为紫色红曲霉(Monascus purpureus)。通过摇瓶发酵对温度、初始pH、碳源、氮源、碳氮比(C/N)等因素进行优化,确定M-22菌株摇瓶发酵产Monacolin K适宜条件为发酵温度26℃、初始pH 5.0、转速160 r/min,甘油为碳源、蛋白胨为氮源、碳氮比(C/N)为5:1,Monacolin K产量显著提高,最高为107.16 mg/L。以优化的发酵条件对M-22菌株进行5 L发酵罐发酵,发酵液中Monacolin K产量最高为189.83 mg/L,桔霉素含量32.53μg/L,红曲色素色价为16.38 U/m L。     

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose–response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC₂₀ were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less–than–additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Development of a method for the determination of citrinin in barley,rye and wheat by solid phase extraction on aminopropyl columns and HPLC-FLD

A method for the determination of the mycotoxin citrinin (CT) in rye, wheat and barley is described. The proposed method is based on ethyl acetate extraction, solid phase clean-up (SPE) on aminopropyl columns and reversed phase high performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). The limits of detection and quantification of CT amounted to 0.6–0.9 μg/kg and 1.7– 3.3 μg/kg with mean recovery rates in the range of 77–92% (RSD 4.8–5.5%). This method can also be used for the determination of CT in red-fermented rice. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

高特异性抗桔霉素单克隆抗体的制备及鉴定

以1, 4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白偶联抗原CIT-BSA,将偶联抗原免疫BALB/C小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,应用有限稀释法进行筛选,经过克隆化后筛选到一株稳定分泌抗桔霉素抗体的杂交瘤细胞株H2-F8．该单克隆抗体经过初步鉴定,抗体类型为IgM类,抗体的相对亲和力为4.17×10⁸ L/mol,单抗与黄曲霉毒素B1、赭曲霉毒素A、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮等毒素的交叉反应率均低于0.1%,与红曲色素中的橙色素和红色素的交叉反应率均低于0.01%．在此基础上建立了间接竞争ELISA检测方法,线性范围为0.05～1.0 μg/L,IC₅₀值为0.3 μg/L．结果为快速检测桔霉素的酶联免疫检测方法的建立和检测试剂盒的研制提供技术依据．     

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.