Synthesis of Citrinin Artificial Antigen and Its Coupling Ratio Determination

[Objective] This study was to establish a method for the rapid and accurate detection of the coupling ratio of artificial antigen. [Method] Artificial synthetic antigen of citrinin （CIT） was first conjugated with vector protein bovine serum albumin （BSA） and then identified by SDS-PAGE electrophoresis,and its coupling ratio was measured by UV absorption and mass spectrometry. [Result] The identification result showed that artificial antigen of CIT was successfully coupled,and the coupling ratio was 8.4 by UV absorption while 6.0 by mass spectrometry. [Conclusion] The comparison experiment showed that mass spectrometry could rapidly identify the artificial antigen and accurately detect its coupling ratio. This study provided basis for the preparation of CIT antigen as well as the establishment of an for enzyme-linked immunosorbent assay （ELISA） for citrinin determination.

红曲制品的桔霉素(Citrinin)问题及应对措施

对于红曲中桔霉素的文章已有一些报道,但专门探讨解决桔霉素问题的文章尚未见,本文在论述桔霉素问题的由来及合成途径的基础上,结合当前国际最新研究进展和作者的实践,提出了多种切实可行的措施,以期抛砖引玉,尽早解决桔霉素问题,为我国红曲制品的出口铺平道路。

Preparation of Artificial Antigen and Egg Yolk-derived Immunoglobulin(IgY) of Citrinin for Enzyme-Linked Immunosorbent Assay

Objective To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin （IgY） to build an enzyme-linked immunosorbent assay （ELISA） for citrinin （CTN）. Methods CTN was conjugated with bovine serum albumin （BSA）, ovalbumin （OVA） with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet （UV） spectrometry and Infrared （IR） spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied. Results UV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng·mL^-1, with a good linearity ranging 20-640 ng·mL^-1. Conclusion Artificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.

Cytotoxic and antibacterial substances against multi-drug resistant pathogens from marine sponge symbiont:Citrinin,a secondary metabolite of Penicillium sp.

Objective:To Isolate,purify,characterize,and evaluate the bioaclive compounds from the sponge-derived fungus Penicillium sp.FF001 and to elucidate its structure.Methods:The fungal strain FF001 with an interesting bioactivity profile was isolated from a marine Fijian sponge Melophlus sp.Based on conidiophores aggregation,conidia development and mycelia morphological characteristics,the isolate FF001 was classically identified as a Penicillium sp.The bioactive compound was identified using various spectral analysis of UV,high resolution electrospray ionization mass spectra,1H and 13C NMR spectral data.Further minimum inhibitory concentrations(MICs)assay and brine shrimp cytotoxicity assay were also carried out to evaluate the biological properties of the purified compound.Results:Bioassay guided fractionation of the EtOAc extract of a static culture of this Penicillium sp.by different chromatographic methods led the isolation of an antibacterial,anticryptococcal and cytotoxic active compound,which was identified as citrinin(1).Further,citrinin(1)is reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus(S.aureus),rifampicin-resistant 5.aureus,wild type S.aureus and vancomycin-resistant Enterococcus faecium showed MICs of 3.90,0.97,1.95 and7.81μg/mL,respectively.Further citrinin(1)displayed significant activity against the pathogenic yeast Cryptococcus neoformans(MIC 3.90μg/mL),and exhibited cytotoxicity against brine shrimp larvae LD_(50)of 96μg/mL.Conclusions:Citrinin(1)is reported from sponge associated Penicillium sp.from this study and for its strong antibacterial activity against multi-drug resistant human pathogens including cytotoxicity against brine shrimp larvae,which indicated that sponge associated Penicillium spp.are promising sources of natural bioactive metabolites.

Cloning and Sequence Analysis of the Full-length cDNA of a Novel yp05 Gene Associated With Citrinin Production in Monascus aurantiacus

Objective To obtain the full-length cDNA of a novel gene （named yp05） associated with citrinin production-related genes in Monascus aurantiacus. Methods Total RNA was extracted from mycelium, 3＇ and 5＇ cDNA end of yp05 gene was amplified using smartTM trace cDNA amplification kit, and the full-length cDNA of a novel gene （named yp05） was obtained from the electronic assembly of 3＇-RACE and 5＇- RACE products. Results This yp05 gene was 787 bp including a 597 bp open reading frame （ORF） and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription ofyp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced. Conclusion The transcription of yp05 gene belongs to differential expression genes of citfinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

Serial Analysis of Gene Expression in Monascus aurantiacus Producing Citrinin

To construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture. Methods Total RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method. Results Five hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaⅢ enzyme in inserts. There were seven repeated clones. Conclusion With the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags≥10) and 143 unique tags to moderately expressed genes (repeat tags≥2).

Peculiarities of feed contamination with citrinin and ochratoxin A

Occurrence of citrinin and ochratoxin A in different feed ingredients and compound feeds was screened by accredited methods based on the indirect competitive enzyme-linked immunosorbent assay. High frequency co-occurrence of both toxins was found in wheat grain and processed sunflower seeds. Citrinin levels exceeded those of ochratoxin A in the majority of co-contaminated feed samples, and the ratio of (1.1 - 10):1 proved to be the most frequent. A possible role of Aspergillus and Penicillium fungi in separate and simultaneous OTA and CIT occurrence in feeds is also discussed.

基于分子对接及动力学模拟的红曲橘霉素合成小分子抑制剂的高通量筛选

通过计算机模拟技术,对红曲橘霉素合成关键酶CitE的抑制剂进行高通量筛选。首先,采用Alphafold 2对CitE蛋白进行同源建模,并与相似序列比对以预测配体结合区域;随后,在Arthor数据库中检索天然配体和黄酮骨架类似物,构建了一个包含20000个化合物的配体库;之后,通过Maestro对CitE蛋白和小分子化合物库进行分子对接,筛选出潜在的CitE配体;并采用Gromacs动力学模拟评估结合稳定性,通过MM/PBSA计算结合自由能,之后对蛋白-配体的结合模式进行分析,进一步确定了槲皮素、木犀草素、芹菜素、染料木素、5,4’-二羟基黄酮、漆黄素6个小分子配体具有较强的CitE结合性能。固态及液态发酵结果表明,上述化合物具有显著的橘霉素抑制活性。其中,固态发酵(20mg/28g干物料)添加上述化合物分别使橘霉素产量降低42.52%、48.81%、32.54%、32.57%、21.02%和13.67%,液态发酵添加(0.1 g/L)上述化合物分别使橘霉素产量降低33.77%、15.58%、33.33%、62.34%、58.87%、50.22%。本研究对建立高效、安全的红曲发酵体系具有借鉴意义。

光调控红色红曲菌M7及MrVeA基因相关突变株的功能分析

为了探讨光调控对红色红曲菌(Monascus ruber)M7、MrVeA基因相关突变株ΔMrVeA、OEMrVeA功能的影响,采用MrVeA基因敲除、过表达技术分别构建基因缺失菌株(ΔMrVeA)和过表达菌株(OEMrVeA)。以避光(黑暗)为对照,分析红光和白光对红色红曲菌M7、MrVeA基因突变株ΔMrVeA、OEMrVeA生长发育、红曲色素(Mps)和桔霉素(CIT)产生的影响。结果表明,黑暗条件下,突变株OEMrVeA闭囊壳和分生孢子的产量增加,菌丝发育、菌落颜色、生物量及色素含量与红色红曲菌(M.ruber)M7相比均无显著性差异,但产桔霉素能力比菌株M7降低7.03%,说明MrVeA基因的过表达会促进菌株M7的有性/无性繁殖,抑制桔霉素的生成。红光有利于突变株OEMrVeA的生长发育、有性/无性繁殖、红曲色素的生成,不利于突变株OEMrVeA生成桔霉素;白光不利于突变株OEMrVeA的生长发育、有性/无性繁殖、红曲色素的生成。研究结果可为低产甚至不产桔霉素的红曲菌工业化应用提供参考。

高产色素低产桔青霉素红曲霉菌筛选及发酵工艺优化研究

该研究通过扩大菌株分离源,选育优质红曲霉菌M7-5。以该菌株为发酵菌株,采用单因素试验研究大米糊化水量、发酵温度、碳源种类及添加量、氮源种类及添加量对红曲发酵色价及桔青霉素含量的影响,在此基础上,利用响应面试验优化最佳控制参数。结果表明,红曲最佳发酵条件为大米糊化水量13%、甘油添加量8%、硝酸钠添加量1.85%,在此条件下,色价与桔青霉素含量的比值(p/c值)达到22158.2。该研究可为传统红曲发酵工艺的改进和工业化应用提供参考。

红曲菌主要代谢产物生产工艺研究进展

红曲是我国传统的谷物发酵产品,广泛应用于发酵食品、医药、化妆品等行业,其主要次级代谢产物有红曲色素(Monascus pigments,MPs)、莫纳可林K(Monacolin K,MK)、桔霉素(citrinin,CIT)等。为了更好地开发红曲,文章论述了红曲中主要代谢产物特性、不同发酵工艺对产量的影响以及红曲菌的生产与应用,并展望了未来红曲菌的研究前景。

降解桔霉素酵母菌的筛选及降解条件的优化

目的从阿瓦提红葡萄果园土壤以及实验室现有酵母菌株中筛选出一株降解桔霉素(citrinin,CIT)的酵母菌,并对其降解桔霉素的培养条件进行优化。方法以YES为基本培养基,将筛选得到的酵母菌与高产CIT红曲菌M7共培养,挑选对CIT降解率较高的菌株,采用形态学和分子生物学进一步对菌株进行鉴定,采用单因素结合正交试验法优化酵母菌降解CIT的培养条件。结果得到一株降解CIT能力较高的酵母菌株PT,经鉴定为库德毕赤酵母,酵母菌PT降解CIT的最优条件为:葡萄糖添加量为165 g/L,FeSO_(4)添加量为0.50 g/L,pH为3.0,于28℃培养13d,在此培养条件下酵母菌PT对CIT的降解率为76.78%。结论采用生物法降解CIT是可行的途径。本研究结果可为微生物脱除CIT的方法提供参考,同时筛选的酵母菌也可应用于其他发酵食品中。

橙色红曲菌中Rab1与CtnE蛋白的亚细胞定位

红曲菌(Monascus)是我国传统的发酵菌株,在发酵过程中会产生一种具有肾毒性的真菌毒素——桔霉素(Citrinin,CIT)。桔霉素的存在极大地限制了红曲制品的应用与发展。明确红曲菌合成CIT的机制,对于开发抑制CIT合成的方法具有十分重要的意义。小G蛋白Rab1可以介导调控丝状真菌中蛋白的转运,ctnE基因编码红曲菌AS3.4384中CIT合成基因簇中的脱氢酶。然而,目前尚不清楚Rab1蛋白是否参与了CtnE蛋白的转运。通过农杆菌介导转化的方法构建了AS3.4384::Rab1-GFP(绿色荧光蛋白,Green Fluorescent Protein)和AS3.4384::Rab1-GFP::ctnE-mCherry(红色荧光蛋白,monomeric cherry red fluorescent protein)两种红曲菌株,并通过荧光共定位确定了Rab1蛋白和CtnE蛋白在菌体中的分布情况。结果表明,CtnE-mCherry蛋白大多聚集于红曲菌丝体的顶端以及分生孢子中,并在菌丝体内呈均匀弥散状分布。Rab1蛋白和CtnE蛋白在红曲菌体内的分布位点基本一致,表明CIT的合成与囊泡转运密切相关。通过Rab1蛋白与CtnE蛋白的荧光共定位情况进一步解析了CIT合成位点。

贵州黔东南腌鱼中桔青霉素和亚硝酸盐含量的检测及分析

目的:了解贵州省黔东南腌鱼中桔青霉素及亚硝酸盐的含量水平,为腌鱼的食用安全性提供参考。方法:采集贵州省黔东南榕江县、从江县农户自制的腌鱼,采用荧光分光光度法测定样品中桔青霉素含量,盐酸萘乙二胺可见分光光度法测定样品中亚硝酸盐含量。结果:检测腌鱼样品共60份,检测出的桔青霉素平均含量为0.1725μg·kg^(-1),亚硝酸盐平均含量为1.3820 mg·kg^(-1)。对榕江县、从江县两县的桔青霉素、亚硝酸盐检出含量进行比较,差异不具有统计学意义(P>0.05)。结论:腌鱼样品中桔青霉素和亚硝酸盐含量较低,均未超过食品安全国家限量标准要求。

高产开环型monacolin K红曲霉菌株的筛选及其在红曲酒酿造中的应用

该研究采用38株红曲霉菌株固态发酵制备功能红曲米,采用高效液相色谱法测定红曲米中monacolin K和桔霉素含量,筛选高产开环型莫纳可林K(monacolin K)红曲霉菌株,通过形态观察及分子生物学技术对筛选菌株进行鉴定,并将其应用于红曲酒酿造,分析红曲酒的品质。结果表明,筛选得到1株高产开环型monacolin K、不产桔霉素的菌株ABQ2,经鉴定,其为丛毛红曲霉(Monascus pilosus)。采用该菌株发酵制备功能红曲米中的monacolin K含量达到25.10 mg/g,开环率为90.5%,且桔霉素未检出。采用菌株ABQ2酿造的红曲酒酒精度为12%vol,酸度为1.91 g/100 mL,monacolin K含量为0.48 mg/mL,其理化指标均符合相关行标要求;与小曲酒相比,红曲酒中总酯含量(433.65 mg/L)极显著增加(P<0.01),其中乳酸乙酯和乙酸乙酯含量最高,高级醇类含量(145.52 mg/L)略低,主要为正丙醇、异丁醇、正戊醇、β-苯乙醇等。

两种同时检测茶叶中赭曲霉毒素A与桔霉素方法的比对

目的对比两种同时检测茶叶中赭曲霉毒素A(ochratoxin A,OTA)与桔霉素(citrinin,CIT)的方法。方法考察不同提取方法、溶剂、料液比的提取效果,比较适配体结合超滤法与免疫亲和柱纯化效果;对比高效液相色谱-荧光检测器(high performance liquid chromatography-fluorescence detector,HPLC-FLD)与超高效液相色谱-三重四极杆质谱法(ultra performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)测定OTA与CIT的检出限、精密度、准确度等差异,评价方法的适用性。结果采用70%甲醇超声与振荡的提取方法,在料液比为1:3的条件下,OTA、CIT的提取率最佳,回收率分别为99.49%的92.77%;经适配体结合超滤纯化后,CIT与OTA的回收率为82.07%~92.95%,与免疫亲和柱纯化方法效果类似;UPLC-MS/MS测定CIT和OTA的基质效应(20.0%、-20.0%)小于HPLC-FLD测定CIT和OTA的基质效应(48.8%、50.3%),二者测定CIT与OTA均具有良好线性度,相关性系数均不小于0.9989;HPLC-FLD测定CIT与OTA的检出限分别为0.09μg/kg、0.12μg/kg;UPLC-MS/MS测定CIT和OTA的检出限分别为0.12μg/kg、0.09μg/kg,两种方法的加标回收率分别为84.20%~100.70%与89.22%~97.88%,相对标准偏差分别为1.30%~4.31%与0.74%~4.04%。结论适配体结合超滤纯化方法操作简单,成本低廉,免疫亲和柱纯化方法成本较高且低浓度下的准确度高;HPLC-FLD和UPLC-MS/MS均适用于茶叶基质中CIT与OTA的提取与检测,但后者基质效应更低,定性更准确。

红曲中红曲菌的鉴定及优质菌的筛选

红曲由红曲菌发酵而得,为提高红曲质量,需对红曲菌进行分离鉴定并筛选优质菌株,而当前红曲菌的鉴定方法和优质菌的筛选方法仍有不足。为此,本文在构建红曲菌菌株库的基础上,对比形态学鉴定和分子生物学鉴定对红曲菌物种鉴定的异同,通过相关性分析提出合理鉴定方案,同时对不同形态学分型的菌株测定色素和桔霉素产量,结合产量换算方法筛选出优质红曲菌。结果:单个红曲中可能存在多株形态不同的红曲菌,以44个红曲样本为菌源构建了45个不同形态学分型,共计148株红曲菌的菌株库。形态学分析表明多数红曲菌与紫色红曲菌、橙色红曲菌及丛毛红曲菌形态相似,而ITS rDNA测序和18S rDNA测序易将未知红曲菌匹配至紫色红曲菌、丛毛红曲菌和红色红曲菌。相关性分析表明ITS rDNA测序相比于18S rDNA测序更接近于形态学分析结果。由于ITS rDNA测序的基因对比库中可参照菌株的种类与数量较多,在多种鉴定方法存在结果差异时,建议优选ITS rDNA测序结果。最后同时参考色素产量、桔霉素产量、桔霉素产量与色素产量之比值3个指标筛选出高产色素(83.3 U/mL)和低产桔霉素(81.5 ng/mL)的菌株红曲菌MZT27,以黄色素与总色素的比值筛选出高产黄色素的菌株红曲菌MZT39(黄色素占比70.06%)。本文旨在为红曲霉种质资源的挖掘和红曲质量的提升提供借鉴。

桔青霉素诱导小鼠肠道屏障功能障碍的机制研究

旨在探讨内质网应激在桔青霉素诱导肠道屏障功能障碍中的作用。试验选用24只初始体重为(27.8±1.5)g的7周龄昆明小鼠,随机分为4组,分别为对照组(5%乙醇)和桔青霉素低(1.25 mg·kg^(-1))、中(5 mg·kg^(-1))、高(20 mg·kg^(-1))剂量组。适应性饲养1周后,对照组按0.1 mL·10g^(-1)体重灌胃5%乙醇,毒素处理组分别灌胃不同剂量桔青霉素,持续14 d。试验结束后,采血并分离血清,测量小肠长度并收集小鼠空肠样本。HE染色观察肠道病理变化;ELISA检测血清中二胺氧化酶(diamine oxidase,DAO)、D-乳酸(D-lactic acid,D-LA)、内毒素(endotoxin,ET)的含量;检测空肠组织抗氧化酶的活性及活性氧(reactive oxygen species,ROS)的释放。为进一步探究内质网应激在桔青霉素诱导肠道损伤中的作用,选用32只昆明小鼠随机分为4组,分别为对照组(5%乙醇)、桔青霉素中剂量(5 mg·kg^(-1))组、内质网应激抑制剂4-苯基丁酸苯基丁酸(4-Phenylbutyric acid,4-PBA)(240 mg·kg^(-1))组和桔青霉素(5 mg·kg^(-1))+4-PBA(240 mg·kg^(-1))组。4-PBA经腹腔注射预处理小鼠1 h后,用桔青霉素对小鼠进行灌胃处理。14 d后,检测空肠组织病理损伤,氧化损伤,细胞凋亡情况及内质网应激相关蛋白表达,并用Spearman相关性分析确定内质网应激信号通路与肠道氧化应激、肠道屏障功能障碍之间的相关性。结果显示,不同剂量的桔青霉素会造成肠道组织损伤、氧化应激及屏障功能障碍。与桔青霉素中剂量组相比,4-PBA预处理能提高小鼠空肠总超氧化物歧化酶(total superoxide dismutase,T-SOD)、过氧化氢酶(catalase,CAT)的活性和总抗氧化能力(total antioxidant capacity,T-AOC),降低ROS和丙二醛(malondialdehyde,MDA)含量,抑制细胞凋亡及内质网应激,降低血清中DAO、D-LA和ET的含量,升高空肠组织中紧密连接蛋白ZO-1、Occludin和Claudin-1 mRNA表达,缓解桔青霉素诱导的空肠组织损伤,且内质网应激信号通路与肠道氧化应激及肠道屏障功能障碍之间存在显著相关性。综上表明,内质网应激在桔青霉素诱导的空肠屏障功能障碍中具有重要调控作用。

桔霉素分子印迹预组装体系的量子化学模拟及吸附性能研究

为解决桔霉素分子印迹聚合物在构建和应用过程中模板分子泄露和功能单体筛选工作量大的问题,以1-羟基-2-萘甲酸为假模板分子,基于液相色谱法构建桔霉素和1-羟基-2-萘甲酸的同时检测方法,该方法对分子印迹洗脱液中的两种目标物具有良好的回收率(≥95%),也能对功能红曲醇提液中桔霉素进行分离。运用量子化学方法,通过分子构型优化、原子电荷分布计算和结合能计算优选了构建分子印迹聚合物的功能单体2,6-二氨基吡啶。以响应面对其制备工艺进行优化,优化后的聚合物对桔霉素的吸附载量为(195.10±13.21)ng/g,相比优化前提高了0.79倍。以甲基丙烯酸为功能单体构建的桔霉素分子印迹聚合物为对照,对比了优化后的聚合物和对照聚合物的吸附性能,前者对桔霉素的吸附载量为后者的3倍,证实计算机模拟技术在桔霉素分子印迹聚合物构建中的实用性,旨在为分子印迹聚合物的构建和桔霉素富集与检测提供参考。

红曲桔霉素致毒机理的网络药理学分析

为探讨红曲中桔霉素导致毒性的内在机理,通过搜索Swiss Target Prediction、Similarity ensemble approach、Comparative Toxicogenomics Database和GeneCards数据库,利用Metascape、Kobas 3.0、Cytoscape 3.7.2和AutoDock软件,开展了网络药理学分析。结果显示,通过筛选去重获得桔霉素与“liver toxicity”和“kidney toxicity”相关的潜在靶点110个,GO富集分析功能条目496条,其中涉及分子功能、生物过程和细胞组成的条目分别为97、354和45条。KEGG信号通路富集靶点数和显著性均较高的是肿瘤MicroRNA通路、肿瘤通路、乙型肝炎等通路。桔霉素可能通过TP53、CASP3、MAPK3和ALB等关键靶点导致肝肾毒性。分子对接实验证明,桔霉素与靶点可以结合,揭示了桔霉素导致肝肾毒性的潜在作用靶点和信号通路,为进一步研究桔霉素致毒机理和健康防护提供理论参考。