产黄曲霉毒素B1降解酶菌株的筛选鉴定及发酵条件优化

以香豆素为唯一碳源筛选到27株能高效降解黄曲霉毒素B1(AFB1)的微生物菌株.用高效液相色谱检测AFB1含量的方法进行AFB1降解酶活力测定.以不同菌株发酵上清液中AFB1降解酶活力高低为复筛条件,筛选到AFB1降解酶活力最高的一株菌并命名为HSD8.该菌株经形态学、生理生化及系统发育学方法鉴定为Sinomonas sp..筛选所得最优菌株的发酵上清液中酶活达443 U/mL,通过单因素试验对其产酶发酵条件进行优化,以提高酶活.优化所得最佳发酵条件为:装液量50 mL/250 mL,发酵周期48 h,初始pH 5.0,接种量8％,发酵温度37℃,摇床转速160r/min.在最佳发酵条件下,该菌株发酵上清液中酶活可达548 U/mL,比优化前提高23.7％.优选菌株HSD8在生物降解黄曲霉毒素B1方面具有应用潜力,值得进一步研究开发.     

八种菊科中草药抗霉菌及饲料霉变的研究

从我国南方霉变的粮食和饲料中分离得到黄曲霉、黑曲霉等10种霉菌并作为供试菌;采用抑菌圈及二倍稀释法研究了野菊花、艾叶等8种菊科中草药的提取物拮抗此10种供试霉菌的活性．结果表明,蒲公英、虾须草等6种中草药及8种中草药的等比混合品抗霉菌活性最强,对各种供试菌的MIC值均不超过12．5g/L,其中中草药混合品和蒲公英显示了MIC的最小值0．391g／L．对此8种中草药提取物及其混合品抗饲料霉变及黄曲霉毒素的产生做了进一步研究,发现中草药混合品的拮抗霉菌生长及黄曲霉毒素产生的效力很强,对黄曲霉毒素的抑制率可超过95%,显示了广阔的应用前景．     

Different media and methodologies for the detection of aflatoxin production by Aspergillus flavus strains isolated from trout feed

We have studied the aflatoxin producing capacity of 41 Aspergillus flavus strains isolated from the mycoflora present of natural media (wheat, rice and mixed feed) synthetic medium (Aflatoxin Producing Ability Medium) and semisynthetic media (Coconut Agar Medium and Glucose Yeast Extract Agar) were compared. Aflatoxins were analysed on days 4 and 8 post-inoculation under an incubation temperature of 28 °C. A total of 30 strains (75.7%) were producers on natural media as detected by Thin Layer Chromatography: 23 strains on wheat, 27 on rice and 12 on mixed feed. The results by qualitative flourescence tests on synthetic and semisynthetic media were: 3 strains positive on Coconut Agar Medium (CAM) 1 on Glucose Yeast Extract Agar (GY + Agar) and none on Aflatoxin Producing Ability Medium (APA).     

Aflatoxin biosynthesis gene clusters and flanking regions

AIMS: To compare the biosynthetic gene cluster sequences of the main aflatoxin (AF)-producing Aspergillus species. METHODS AND RESULTS: Sequencing was on fosmid clones selected by homology to Aspergillus parasiticus sequence. Alignments revealed that gene order is conserved among AF gene clusters of Aspergillus nomius, A. parasiticus, two sclerotial morphotypes of Aspergillus flavus, and an unnamed Aspergillus sp. Phylogenetic relationships were established using the maximum likelihood method implemented in PAUP. Based on the Eurotiomycete/Sordariomycete divergence time, the A. flavus-type cluster has been maintained for at least 25 million years. Such conservation of the genes and gene order reflects strong selective constraints on rearrangement. Phylogenetic comparison of individual genes in the cluster indicated that ver-1, which has homology to a melanin biosynthesis gene, experienced selective forces distinct from the other pathway genes. Sequences upstream of the polyketide synthase-encoding gene vary among the species, but a four-gene sugar utilization cluster at the distal end is conserved, indicating a functional relationship between the two adjacent clusters. CONCLUSIONS: The high conservation of cluster components needed for AF production suggests there is an adaptive value for AFs in character-shaping niches important to those taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first comparison of the complete nucleotide sequences of gene clusters harbouring the AF biosynthesis genes of the main AF-producing species. Such a comparison will aid in understanding how AF biosynthesis is regulated in experimental and natural environments.     

Sequential Pathology of Experimental Aflatoxicosis in Quail and the Effect of Selenium Supplementation in Modifying the Disease Process

Feeding of aflatoxin B1 @ 1 ppm to 2-week old Japanese quail for a period of 8 weeks produced gross and microscopic changes in the liver, skeletal muscles, heart and bursa of Fabricius. These included fatty changes, bile duct hyperplasia and lymphoid aggregation in liver; haemorrhages in thigh, breast muscles and myocardium; mild depletion of lymphocytes, cystic degeneration and fibrous tissue proliferation in bursa of Fabricius. More or less similar lesions were seen in quail chicks fed on aflatoxin with sodium selenite @ 5 ppm but these were of lesser intensity and appeared at later stages of the experiment thereby indicating that supplementation of selenium had some protective action against the toxic effect of aflatoxin B1 in Japanese quail.     

Influence of dietary culture material containing aflatoxin and T<Subscript>2</Subscript> toxin on certain serum biochemical constituents in Japanese quail

Forty newly hatched unsexed Japanese quail (Coturnix coturnix japonica) chicks were fed diets containing 3 ppm aflatoxin (AF) and 4 ppm T₂ toxin either singly and in combination for 35 days. Sera samples were collected from six birds in each group at the end of the trial to study the effect of certain serum biochemical parameters. Highly significant (P < 0.01) differences were observed for serum total protein, albumin, globulin, glucose, cholesterol, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) between control and toxin treated groups. Toxin treated groups did not reveal any significant difference for serum urea nitrogen, creatinine, uric acid, calcium, phosphorus, sodium and potassium. There was a reduction in the levels of serum total protein, albumin, globulin, glucose, cholesterol, ALT and elevation of AST and GGT and variable ALP levels observed in toxin treated groups. Forms part of M.V.Sc. thesis of first author approved by the Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, India.     

Electrochemical monitoring of aflatoxin M1 in milk samples using silver nanoparticles dispersed on α‐cyclodextrin‐GQDs nanocomposite

Aflatoxins are potential food pollutants produced by fungi. One of important toxins is aflatoxin M1 (AFM1). A great deal of concern is associated with AFM1 toxicity. In the present study, an innovative electrochemical interface for quantitation of AFM1 based on ternary signal amplification strategy was fabricated. In this work, silver nanoparticles was electrodeposited onto green and biocompatible nanocomposite containing α‐cyclodextrin as conductive matrix and graphene quantum dots as amplification element. Therefore, a multilayer film based on α‐cyclodextrin, graphene quantum dots, and silver nanoparticles was exploited to develop a highly sensitive electrochemical sensor for detection of AFM1. Fully electrochemical methodology was used to prepare a transducer on a glassy carbon electrode, which provided a high surface area toward sensitive detection of AFM1. The surface morphology of electrode surface was characterized by high‐resolution field emission scanning electron microscope. The proposed sensing platform provides a simple tool for AFM1 detection. Under optimized condition, the calibration curve for AFM1 concentration was linear in 0.015mM to 25mM with low limit of quantification of 2μM. The practical analytical utility of the modified electrode was illustrated by determination of AFM1 in unprocessed milk samples.     

A versiconal hemiacetal acetate converting enzyme in aflatoxin biosynthesis

Conversion of the aflatoxin biosynthetic intermediate versiconal hemiacetal acetate (VHA) in a cell free extract ofAspergillus parasiticus ATCC 15517 is investigated. The enzymatic reaction is monitored by a method using high performance liquid chromatography (HPLC). The major product of the enzymatic reaction is a water soluble compound not chloroform-extractable at pH 7.5. The product becomes chloroform extractable upon acidification of the reaction medium and is separated and quantitated by reversed-phase HPLC. It is tentatively identified as versiconal hemiacetal alcohol, which is converted to versicolorin C (VC) upon acid treatment.