高活力糖化酶发酵技术的研究

本文对糖化酶产生菌ＡＮ１４９的诱变育种方法、发酵产酶工艺、摇瓶培养基配方及小型自动发酵罐条件等进行了系统的研究。实验结果表明，菌种自然分离、紫外线诱变以及ＮＴＧ处理等的配合作用可显著提高产酶量和转化率，出发菌株的产酶活力从８５００ｕ／ｍｌ提高到１５０００ｕ／ｍｌ。按最佳的培养基配方和发酵工艺条件，采用ＷＮＣ－１５突变菌株发酵１６０ｈ，酶活力可达３００００ｕ／ｍｌ加以上，对提高我国的糖化酶活力具有重要的现实意义。     

Proteome analysis of the fungus Aspergillus carbonarius under ochratoxin A producing conditions

Aspergillus carbonarius is an important ochratoxin A producing fungus that is responsible for mycotoxin contamination of grapes and wine. In this study, the proteomes of highly (W04-40) and weakly (W04-46) OTA-producing A. carbonarius strains were compared to identify proteins that may be involved in OTA biosynthesis. Protein samples were extracted from two biological replicates and subjected to two dimensional gel electrophoresis analysis and mass spectrometry. Expression profile comparison (PDQuest software), revealed 21 differential spots that were statistically significant and showed a two-fold change in expression, or greater. Among these, nine protein spots were identified by MALDI-MS/MS and MASCOT database and twelve remain unidentified. Of the identified proteins, seven showed a higher expression in strain W04-40 (high OTA producer) and two in strain W04-46 (low OTA producer). Some of the identified amino acid sequences shared homology with proteins involved in regulation, amino acid metabolism, oxidative stress and sporulation. It is worth noting the presence of a protein with 126.5 fold higher abundance in strain W04-40 showing homology with protein CipC, a protein with unknown function related with pathogenesis and mycotoxin production by some authors. Variations in protein expression were also further investigated at the mRNA level by real-time PCR analysis. The mRNA expression levels from three identified proteins including CipC showed correlation with protein expression levels. This study represents the first proteomic analysis for a comparison of two A. carbonarius strains with different OTA production and will contribute to a better understanding of the molecular events involved in OTA biosynthesis.     

一株黄曲霉拮抗细菌F1的筛选、鉴定及其抑菌特性

为获得能有效抑制食品原料中黄曲霉生长的生防细菌,本研究以乳酸菌发酵制品和香辛料为筛菌出发样品,通过菌株分离、初筛和复筛,从黑胡椒中筛选到一株对黄曲霉有强烈抑制作用的菌株F1。依据菌株的形态特征、培养特征、生理生化特征结合16S rDNA基因序列分析、系统发育树构建来鉴定菌株,最终确定菌株F1为解淀粉芽孢杆菌Bacillus amyloliquefaciens(保藏编号:CGMCC No.10942),与已商品化的生防菌Bacillus amyloliquefaciens FZB42的亲缘关系最为接近。菌株F1的发酵上清液经过超滤处理以及蛋白酶酶解试验和稳定性试验,结果显示,菌株F1的抑菌成分为分子量30 ku~100 ku的蛋白类物质;在100℃处理30 min后仍能保持87.43%的抑菌活性;在pH值3.0~10.0范围内其相对抗菌活性均在70%以上。研究表明,从黑胡椒中分离到的解淀粉芽孢杆菌F1能够通过代谢产生稳定性较强的抗菌蛋白而达到拮抗黄曲霉的目的,在食品领域具有一定的应用前景。     

Improving 2‐phenylethanol and 6‐pentyl‐α‐pyrone production with fungi by microparticle‐enhanced cultivation (MPEC)

Trichoderma atroviride IMI 206040 synthesizes the coconut lactone 6‐pentyl‐α‐pyrone (6‐PAP) de novo and Aspergillus niger DSM 821 produces the rose‐like flavour compound 2‐phenylethanol (2‐PE) from the precursor l ‐phenylalanine. Here, microparticles of different chemical composition and nominal particle diameter in the range 5–250 µm were added to shake‐flask cultures of both fungi to investigate the particles' effect on product formation. Maximum 2‐PE concentration increased by a factor of 1.3 to 1430 mg/l with the addition of 2% w/v talc (40 µm diameter). Maximum 6‐PAP concentration increased by a factor of 2 to 40 mg/l with the addition of 2% w/v iron (II, III) oxide. The influence of ions leaching out of the particles was investigated by cultivating the fungi in leached particle medium. For the first time, the positive effect of the microparticle‐enhanced cultivation (MPEC) technique on the microbial production of volatile metabolites, here flavour compounds from submerged fungal cultures, is demonstrated. The effect is strain‐ and particle‐specific. Copyright © 2014 John Wiley & Sons, Ltd.     

曲霉发酵甘油生产1,3-丙二醇的研究

从实验室常规霉菌米曲霉和黑曲霉出发,通过唯一碳源法筛选出2株可发酵甘油生产1,3-丙二醇的菌株,并建立了气相色谱检测甘油和1,3-丙二醇的方法,用于测定实际发酵液中1,3-丙二醇的含量。目前有关微生物法生产1,3-丙二醇的研究主要集中在几种条件致病菌上,采用霉菌既考虑了安全问题,又丰富了1,3-丙二醇发酵法生产的可用菌株,为进一步的产业化打下基础。     

Effect of contamination of foods by Aspergillus flavus on the nutritive value of protein

The effect of aflatoxin was measured on the protein quality of peanut meal (PNM) and fish meal (FM) Total protein efficiency, protein efficiency ratio, net protein utilisation, examination of the histopathology of the liver, ileal digesion of amino acids and plasma amino acid concentration were used as bioassays together with chemical score (CS), dye binding capacity (DBC), essential amino acid index (EAAI), and discriminant computered PER (DC-PER) as chemical methods. In trial 1, aflatoxin-free PNM was compared with infected PNM at graded levels of toxin when fed to chickens and ducklings. In trial 2, various mixtures of PNM and FM at a constant aflatoxin level (280 μg kg^?1) were fed to compare the effects of aflatoxin on proteins of differing quality. Ducks were more sensitive to the toxin than chickens, as indicated by deterioration of protein quality, and the effects on growth and the histological appearance of the liver were magnified on diets of low quality (PNM), but not of high quality (FM). Contamination of PNM resulted in progressive increase in DBC and, to a lesser extent, in DC-PER, while EAAI and CS were not affected. The importance of these findings lies in the problems of mould contamination of animal feedstuffs in humid, tropical conditions, which may affect the more sensitive animals, and may not be detected by chemical methods of measuring protein quality, nor by bioassay on chickens, if the levels of contamination are low.     

Studies on the Biosynthesis of the Stephacidin and Notoamide Natural Products: A Stereochemical and Genetic Conundrum

The stephacidin and notoamide natural products belong to a group of prenylated indole alkaloids containing a bicyclo[2.2.2]diazaoctane core. Biosynthetically, this bicyclic core is believed to be the product of an intermolecular Diels–Alder (IMDA) cycloaddition of an achiral azadiene. Since all of the natural products in this family have been isolated in enantiomerically pure form to date, it is believed that an elusive Diels–Alderase enzyme mediates the IMDA reaction. Adding further intrigue to this biosynthetic puzzle is the fact that several related Aspergillus fungi produce a number of metabolites with the opposite absolute configuration, implying that these fungi have evolved enantiomerically distinct Diels–Alderases. We have undertaken a program to identify every step in the biogenesis of the stephacidins and notoamides, and by combining the techniques of chemical synthesis and biochemical analysis we have been able to identify the two prenyltransferases involved in the early stages of the stephacidin and notoamide biosyntheses. This has allowed us to propose a modified biosynthesis for stephacidin A, and has brought us closer to our goal of finding evidence for, or against, the presence of a Diels–Alderase in this biosynthetic pathway.     

The effect of pH on bioremediation potential for the removal of direct violet textile dye by Aspergillus niger

Alkaline and acidic media have been used in the textile dye industry, depending on the fabric nature. The bioremoval of textile direct violet dye by Aspergillus niger fungal strain was studied. The effect of pH on dye bioremoval was investigated at a pH range from 2 to 11. The direct violet dye bioremoval reached maximum with 92.4%, 64.0%, 91.4%, and 62.3 % at pH values of 2, 3, 8, and 9, respectively, at 24 h of incubation. The percentages of removal rate after 72 h incubation were 98.9, 97.3, 94.0, 95.0, 97.0, and 97.3 at pH 2, 3, 6, 7, 8, and 9, respectively. The optimum pH values were 2, 3, 8, and 9 for direct dye removal. At the end of the experiments, the treatment with fungal strain could reduce COD value of synthetic dye solution by 76-91%. Pseudo first and second order kinetic models were applied to evaluate differences in the biosorption rates and uptakes of textile dye. Pre-equilibrium biosorption of direct violet dye onto fungus under different dye concentrations followed a pseudo second order kinetic model with a high degree of correlation coefficients (R² > 0.99), and the calculated values of q_e nearly matched the experimental values of textile dye during the biotreatment process.     

Aflatoxin B1 and aflatoxins in ground red chilli pepper after drying

In this study, 180 red chilli pepper (RCP) berry samples were obtained from two different croplands of Gaziantep and Kahramanmara? (Turkey) in August, September and October. RCP berry samples were dried under sunlight and grinded. Ground red chilli pepper (GRCP) samples were analysed for aflatoxins (AFs, sum of B₁, B₂, G₁ and G₂) and AFB₁ contamination. According to the results, in 49 of 180 samples, AFB₁ and in 37 samples, AFs were higher than legal limits. The lowest amounts of AFs and AFB₁ were obtained in August and the highest amounts in October. χ² analysis showed that there were no significant differences (p > 0.05) between cities among 3 months according to number of samples with AFs and AFB₁ above legal limits. According to the Duncan multiple-range test, there was no significant difference between all months. Strict measures are necessary to produce high-quality GRCP. RCP berry must be treated to reduce moulds before production of GRCP.