| Experiment 1:The aim of this research was to analyze and evaluate the contamination situation of aflatoxin(AF)in ingredients and complete feeds in Beijing region.Results showed that the detection rates of AFB1 in corn,bran,soybean meal,DDGS,and complete feeds were 45.8%,52.2%,40.9%,86.5%and 72.0%,respectively;the over standard rates were 8.3%,0.0%,22.7%,8.7%and 4.0%,respectively;and the average contents were 5.48,0.25,87.15,10.52 and 2.14 ?g/kg,respectively.These results indicated that the average contents of aflatoxin in complete feeds,corn and DDGS were the highest.Aflatoxin contamination was widely found in both feeds and feedstuffs.Experiment 2:The aim of this research was to clone gene of Myxobacteria aflatoxin degradation enzyme(MADE)from Myxococcus fulvus.Using amino acid sequence of peptide to design degenerate primers,part of MADE gene was obtained via degenerate PCR,and the length was 865 bp.And then using RACE combined with nested PCR technology to amplify the 5' end and 3' end sequences.The full length of MADE DNA sequence was 1659 bp,encoding 552 amino acids,the molecular weight was about 57.0 kDa,and isoelectric point was about 4.74.The amino acid sequence deduced from MADE gene had a putative signal peptide of 24 amino acids,36 putative phosphorylation sites and one putative N-glycosylation site.Experiment 3:The aim of the research was to express MADE gene in E.coli BL21.The MADE gene and expression vector pET30a were both cleaved by BamH I and Sal I,and then were used to construct the expression plasmid pET30a-made.The expression plasmid was transformed into E.coli BL21.By IPTG inducing,the recombinant protein rMADE was expressed successfully.And 1 000 mg rMADE was obtained from 1 L fermentation culture,having a total activity of 100 000 U and a specific activity of 100 U/mg to degrade A FB1.Experiment 4:The aim of this research was to express MADE gene in Pichia Pastoris GS115.The optimal MADE gene and expression vector pPICZaA were both cleaved by Xhol I and Not I,and then were used to construct the expression plasmid pPICZaA-made-opt.The expression plasmid.was transformed into Pichia Pastoris GS115 competent cell after linearized with restriction enzyme Sac I.The optimal expression conditions have been determined as follows,the pH value of BMMY was 4.0,methanol concentration was 1.5%,temperature was 28? and fermentation time was 5 d.And 2 000 mg rMADE was obtained from 1 L fermentation culture,having a total activity of 300 000 U and a specific activity of 150 U/mg to degrade AFB1.Experiment 5:The aim of this research was to investigate the protective effect of B.subtilis ANSB060 on broilers fed moldy peanut meal naturally contaminated with aflatoxins.A total of 360 one-week-old male broilers(Ross 308)were assigned to six dietary treatments for six weeks.The treatment diets were:CO(basal diet);C1.0(CO + 1.0 g B.subtilis ANSB060/kg diet);MO(basal diet formulated with moldy peanut meal);M0.5,M1.0 and M2.0(M0 + 0.5,1.0 and 2.0 g B.subtilis ANSB060/kg diet,respectively).The results showed that aflatoxins increased(P<0.05)serum aspartate transaminase activity,decreased(P<0.05)serum glutathione peroxidase activity,and enhanced(P<0.05)malondialdehyde contents in both the serum and liver.Aflatoxins also caused gross and histological changes in liver tissues,such as bile duct epithelium hyperplasia,vacuolar degeneration and lymphocyte infiltration.The supplementation of ANSB060 reduced aflatoxin levels in the duodenum and liver,and counteracted the negative effects of aflatoxins,leading to the conclusion that ANSB060 has a protective effect against aflatoxicosis and this protection is dose-related. |
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