The effect of royal jelly and propolis alone and in combination on inhibition of Aspergillus parasiticus growth,aflatoxin production,and aflR gene expression

The objective of this study was to determine the inhibitory effect of royal jelly (RJ) and propolis on growth, aflatoxin production and aflR gene expression in Aspergillus parasiticus. Inhibitory effect of RJ and propolis against a standard strain of A. parasiticus(ATCC 15517) was determined alone and in combination in accordance with the CLSI M38-A2 and checkerboard methods, respectively. The aflatoxin concentrations in the control and treated media were determined by HPLC. Also, the quantitative changes in the aflR gene expression were analyzed. The minimum inhibitory concentrations (MIC) of RJ and propolis alone were 3,200 and 100μg/ml, respectively. Also, the MICs of RJ and propolis in combination were 200 and 25μg/ml, respectively. When combined, a synergistic interaction was observed with a FICI of 0.312. Total levels of aflatoxin decreased from 386.1ppm to 8.72, 3.01 and 1.75ppm at 1,600μg/ml of RJ, 50μg/ml of propolis and 100+12.5μg/ml of RJ and propolis, respectively. In addition, the level of afIR gene expression was significantly decreased after treatment with RJ and propolis extracts alone and with their combination. The findings reveal that RJ and propolis extracts, either alone or in combination, have a significant inhibitory effect on aflR gene expression in aflatoxin production.     

Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare

Mutants of Aspergillus parasiticus highly resistant to phenylpyrroles were isolated at a high mutation frequency, after UV-mutagenesis and selection on media containing fludioxonil. Studies on the effect of mutation(s) on the aflatoxin production resulted in the identification of two fludioxonil-resistant phenotypes: aflatoxigenic (FLD(afl)(+)) and non-aflatoxigenic (FLD(afl)(-)) mutant strains. Most of the FLD(afl)(+) mutant strains produced the aflatoxin B(1) at similar or even higher (up to 2.5-fold) concentrations than the wild-type parent strain on yeast extract sucrose medium. Interestingly, in most of these mutant strains the aflatoxigenic ability significantly increased (up to 4-fold) when the mutants were grown on fungicide-amended medium. However, a significant reduction in the aflatoxin production was observed in wheat grains by all FLD(afl)(+) mutant strains. Tests on the response of mutant strains to high osmotic pressure showed that most fludioxonil-resistant mutants were more sensitive to high osmolarity than the wild-type parent strain. Study of other fitness determining parameters showed that the mutation(s) for resistance to phenylpyrroles may or may not affect the mycelial growth rate, sporulation and conidial germination. However, in a number of aflatoxigenic-mutant strains these fitness parameters were unaffected or only slightly affected. Cross resistance studies with fungicides from different chemical groups showed that the mutation(s) for resistance to fludioxonil also highly reduced the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides. No effect of phenylpyrroles resistance mutation(s) on fungitoxicity of triazoles, benzimidazoles, anilinopyrimidines, phenylpyridinamines, strobilurin-type fungicides and to the non site-specific inhibitors chlorothalonil and maneb was observed. The above mentioned data indicate, for the first time, the potential risk of increased aflatoxin contamination of agricultural products by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to aromatic hydrocarbon, dicarboximide and phenylpyrrole fungicides.     

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.     

一株具有抑制麦芽糖酶活性的乳酸菌的筛选与鉴定

以抑制麦芽糖酶为目的,采用α-葡萄糖苷酶抑制剂的体外筛选模型,筛选出具有较好麦芽糖酶抑制效果的乳酸菌菌株JH28。菌株JH28的MRS发酵上清及无细胞提取物均对麦芽糖酶有抑制作用。将该菌株的无细胞提取物经透析膜（截留分子量1000Da,去离子水为透析液）透析后取透析液冻干,测定不同浓度冻干物对麦芽糖酶的抑制效果,最高抑制率可达67.19%,经计算其IC为4.12mg/mL。菌株经16SrDNA鉴定为干酪乳杆菌,具有潜在的工业应用价值。      

Effect of anilinopyrimidine resistance on aflatoxin production and fitness parameters in Aspergillus parasiticus Speare

Mutants of Aspergillus parasiticus resistant to the anilinopyrimidine fungicides were isolated at a high mutation frequency after UV-mutagenesis and selection on media containing cyprodinil. In vitro fungitoxicity tests resulted in the identification of two predominant resistant phenotypes that were highly (R₁-phenotype) and moderately (R₂-phenotype) resistant to the anilinopyrimidines cyprodinil, pyrimethanil and mepanipyrim. Cross-resistance studies with fungicides from other chemical groups showed that the highly resistance mutation(s) did not affect the sensitivity of R₁-mutant strains to fungicides affecting other cellular pathways. Contrary to that, a reduction in the sensitivity to the triazoles epoxiconazole and flusilazole, the benzimidazole carbendazim, the phenylpyrrole fludioxonil, the dicarboximide iprodione and to the strobilurin-type fungicide pyraclostrobin was observed in R₂-mutant strains. Study of fitness parameters of anilinopyrimidine-resistant strains of both phenotypic classes showed that all R₁ mutant strains had mycelial growth rate, sporulation and conidial germination similar to or even higher than the wild-type parent strain, while these fitness parameters were negatively affected in R₂ mutant strains. Analysis of the aflatoxin production showed that most R₁ mutant strains produced aflatoxins at concentrations markedly higher than the wild-type parent strain. A considerable reduction in the aflatoxin production was observed on cultured medium and on wheat grains by all R₂ mutant strains, indicating a possible correlation between fitness penalties and aflatoxigenic ability of A. parasiticus. The potential risk of increased aflatoxin contamination of agricultural products and their byproducts by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to the anilinopyrimidines is discussed.     

Inhibition of growth and aflatoxin production of Aspergillus parasiticus by citrus oils

Summary Aspergillus parasiticus was inoculated into grapefruit juice and a glucose-yeast extract medium; both contained 500–7000 ppm of citrus oils that were incorporated into the media by sonication. Orange and lemon oil were more inhibitory to mold growth and aflatoxin production than was d-limonene, the main constituent of the two peel oils. After 7 days at 28° C, 2000 ppm of lemon and 3000 ppm of orange oil in grapefruit juice afforded maximum suppression of mold growth and toxin formation. When the glucose-yeast extract medium was used, 3000 ppm of either oil were needed to achieve the same result. After 4 days at 28° C, orange oil at 3500 ppm in either medium markedly inhibited mold growth (as evidenced by dry weight of mold mycelium) and aflatoxin production (only 14 and 1% of the amount normally produced in the juice and artificial medium, respectively). Higher concentrations of orange oil further reduced mold growth and aflatoxin production and also delayed the onset of sporulation, if it occurred. Although aflatoxin was detected in all samples, only 0.2 to 0.5% of the amount found in controls (without the citrus oil) was present when the medium contained 7000 ppm orange oil. The mold consistently grew, albeit very poorly, on the glass at the liquid-atmosphere interface even when the substrate contained a large amount of citrus oil.

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Hemmung des Wachstums und der Aflatoxinproduktion von Aspergillus parasiticus durch Orangenöl, Citronenöl und d-Limonen

Zusammenfassung Pampelmusensaft und ein Glucose-Hefeextrakt-Medium wurden beide mit 500–7000 ppm Orangenöl, Citronenöl oder d-Limonen angereichert und dann mitAspergillus parasiticus beimpft. Wachstum und Aflatoxinproduktion des Pilzes wurden stärker durch die Öle als durch d-Limonen gehemmt, obwohl dieser der Hauptbestandteil der beiden Öle ist. 2000 ppm Citronenöl bzw. 3000 ppm Orangenöl in Pampelmusensaft genügten zur starken Hemmung der Wachstums- and der Aflatoxinproduktion vonA. parasiticus während 7 Tage bei 28° C. Wenn Glucose-Hefeextrakt als Nährboden diente, dann wiesen bei 3000 ppm beide Öle gleiche Hemmung auf. Wenn beide Nahrboden nur 4 Tage bei 28° C gehalten wurden, dann waren 3500 ppm Orangendl notwendig, um Wachstum and Aflatoxinproduktion zu hemmen. Pampelmusensaft mit einem Orangenöl-Gehalt von 3500 ppm enthielt nur 14% der Aflatoxin-Menge des beimpften Saftes ohne Öl. Das Medium mit Glucose, Hefeextrakt und Orangendl hatte nur 1 % des Aflatoxin-Gehaltes der Kontrolle. Höhere Konzentrationen von Orangenöl hemmten noch stärker und verzögerten den Beginn der Konidienbildung. Wenn das Medium 7000 ppm Orangenöl enthielt, dann konnte nur geringes Pilzwachstum und Aflatoxinproduktion (0,2–0,5% der Kontrolle) beobachtet wurden; das minimale Wachstum des Pilzes geschah an der Grenzfläche Nährboden und Atmosphare.

     

Inhibitory effect of phenolics extracted from sorghum genotypes on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production

The inhibitory activity of bioactive polyphenols present in six sorghum genotypes—two red (AON 486 and IS 620), two yellow (LPJ and IS 17779) and two white (SPV 86 and SPV 462) varieties—on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production was evaluated. In the first experiment the production of aflatoxins in the six sorghum genotypes after removal of surface phenolics by acidic methanol treatment was studied and compared with that in untreated grains. Aflatoxin production was found to be fourfold higher in treated grains. The total phenols and bioactive polyphenols extracted by acidic methanol were quantified using the Folin–Denis method and the bovine serum albumin–benzidine conjugate procedure respectively. In the second experiment the effect of extracted sorghum phenolics under in vitro conditions on fungal growth and aflatoxin production was studied at two concentrations (0.01% and 0.1%) of phenolics. Extracted phenolics added to yeast extract sucrose (YES) medium at 0.1% concentration showed an inhibitory effect on aflatoxin production. At 0.01% phenolic concentration, aflatoxin production was minimal on day 3 after infection. At other time points the aflatoxin content was similar to that in the control. At 9 days after infection the fungal biomass in IS 620 was significantly lower than that in the control. At 0.1% phenolic concentration, aflatoxin production was minimal and the red genotype IS 620 showed maximum resistance. Fungal biomass was lowest at all growth stages in IS 620 as compared with the control. Polyphenol oxidase (PPO) activity was not detected in A. parasiticus grown on YES medium (control). PPO activity was not induced in A. parasiticus by the addition of phenolics to the liquid culture medium (no PPO activity was detected in the culture medium). The inhibitory activity of bioactive polyphenols could be attributed to the lack of PPO enzyme in this fungus. Copyright © 2007 Society of Chemical Industry     

Ethylene inhibits aflatoxin biosynthesis in Aspergillus parasiticus grown on peanuts

The filamentous fungi Aspergillus parasiticus and Aspergillus flavus synthesize aflatoxins when they grow on a variety of susceptible food and feed crops. These mycotoxins are among the most carcinogenic naturally occurring compounds known and they pose significant health risks to humans and animals. We previously demonstrated that ethylene and CO2 act alone and together to reduce aflatoxin synthesis by A. parasiticus grown on laboratory media. To demonstrate the potential efficacy of treatment of stored seeds and grains with these gases, we tested ethylene and CO2 for ability to inhibit aflatoxin accumulation on Georgia Green peanuts stored for up to 5 days. We demonstrated an inverse relationship between A. parasiticus spore inoculum size and the level of toxin accumulation. We showed that ethylene inhibits aflatoxin synthesis in a dose-dependent manner on peanuts; CO2 also inhibits aflatoxin synthesis over a narrow dose range. Treatments had no discernable effect on mold growth. These observations support further exploration of this technology to reduce aflatoxin contamination of susceptible crops in the field and during storage.     

具有ACE抑制活性乳酸菌的特性研究

选择了3株Lactobacillus helveticus(H—1，H—2，LH)和Lactobacillus casei(C，LC)为实验对象，分别制作发酵乳，并测定其蛋白水解活性、生长特性及ACE抑制活性(ACEI)。结果表明，发酵乳中脯氨酸及含有脯氨酸的多肽越多，其ACE抑制活性越强；而且具有较大滴定酸度的菌种很可能具有较高的ACEI活性。经筛选，H—1的A—CEI活性最高。该菌经30℃培养18h后具有最大约ACE抑制活性。     

Novel techniques for controlling the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts

The effects of modified atmosphere packaging involving oxygen absorbents, storage temperature and packaging film barrier characteristics on the growth of and aflatoxin production by Aspergillus parasiticus in packaged peanuts was investigated. Mold growth was barely visible in air-packaged peanuts using a high gas barrier film (ASI) while extensive mold growth was observed in peanuts packaged under similar gaseous conditions using a low barrier film (ASIII). Incorporation of an oxygen absorbent (Ageless type S) inhibited mold growth in peanuts packaged in film ASI, while mold growth occurred in peanuts packaged with an absorbent/carbon dioxide generator (Ageless type G) in film ASI and in all absorbent-packaged peanuts in film ASIII. Aflatoxin B₁ production was detected at levels greater than the regulatory limit of 20 ng g^-1 in air-packaged peanuts using film ASI at 20°C and 25°C with the maximum level of aflatoxin (52·95 ng g^-1) being detected in air-packaged peanuts using film ASIII. However, aflatoxin production in all absorbent-packaged peanut samples was less than the regulatory level of 20 ng g^-1 irrespective of the barrier characteristics of the packaging film. Discoloration was more intensive in air-packaged peanuts in film ASIII especially at 25°C and 30°C than those packaged under similar or modified gaseous conditions using film ASI. This study has shown that oxygen absorbent technology is a simple and effective means of controlling the growth of and aflatoxin production by A. parasiticus. However, the effectiveness of these absorbents is dependent on the gas barrier properties of the packaging film surrounding the product.     

浅谈乳酸菌在黄酒生产中的作用

为了全面地了解乳酸茵兼在黄酒生产全过程中的作用，介绍了黄酒米浆水中乳酸茵的应用、黄酒深层酿造兼氧浸米中乳酸菌的作用和乳酸菌在黄酒发酵过程中的作用，同时也介绍了乳酸茵代谢产物乳酸和乳酸乙酯在黄酒中的含量和作用。     

Inhibitory effects of Satureja hortensis L. essential oil on growth and aflatoxin production by Aspergillus parasiticus

In an effort to screen the essential oils of some Iranian medicinal plants for novel aflatoxin (AF) inhibitors, Satureja hortensis L. was found as a potent inhibitor of aflatoxins B1 (AFB1) and G1(AFG1) production by Aspergillus parasiticus NRRL 2999. Fungal growth was also inhibited in a dose-dependent manner. Separation of the plant inhibitory substance(s) was achieved using initial fractionation of its effective part (leaf essential oil; LEO) by silica gel column chromatography and further separation by reverse phase-high performance liquid chromatography (RP-HPLC). These substances were finally identified as carvacrol and thymol, based on the interpretation of 1H and 13C NMR spectra. Microbioassay (MBA) on cell culture microplates contained potato-dextrose broth (PDB) medium (4 days at 28 degrees C) and subsequent analysis of cultures with HPLC technique revealed that both carvacrol and thymol were able to effectively inhibit fungal growth, AFB1 and AFG1 production in a dose-dependent manner at all two-fold concentrations from 0.041 to 1.32 mM. The IC50 values for growth inhibition were calculated as 0.79 and 0.86 mM for carvacrol and thymol, while for AFB1 and AFG1, it was reported as 0.50 and 0.06 mM for carvacrol and 0.69 and 0.55 mM for thymol. The results obtained in this study clearly show a new biological activity for S. hortensis L. as strong inhibition of aflatoxin production by A. parasiticus. Carvacrol and thymol, the effective constituents of S. hortensis L., may be useful to control aflatoxin contamination of susceptible crops in the field.     

The effect of zinc and phytic acid on the incorporation of 1-14C-acetate into aflatoxin by resting mycelia of Aspergillus parasiticus.

The effect of zinc and phytic acid on [1-14C]-acetate incorporation into aflatoxins by resting mycelium was studied. When different levels of ZnSO4 were used to study its effect on the incorporation of [1-14C]-acetate into aflatoxins, it was found that the specific radioactivity incorporation into aflatoxins was maximum at the level of 10 mM-ZnSO4. At this concentration the change in the specific radioactivities of aflatoxins B1 + B2 and aflatoxins G1 + G2 were +74.61% and +29.66%, respectively. On the other hand, phytic acid had an inhibitory effect on the incorporation of [1-14C]-acetate. These observations have been correlated in order to find out why soyabean is unable to produce aflatoxins by Aspergillus parasiticus.     

乳酸菌蛋白水解体系及相关基因表达的研究进展

乳酸菌的蛋白水解体系包括胞外酶、转运系统和多种胞内酶.它们将外源蛋白质逐步水解成能被乳酸菌直接利用的游离氨基酸,弥补了因乳酸菌自身不能直接利用外源无机氯和蛋白质的缺陷,对于乳酸菌的正常生长具有非常重要的意义.目前,国内外正在研究利用现代分子技术,从基因表达层面检测不同菌株之间或菌株经过不同处理前后关键蛋白水解酶的表达水平差异,筛选出蛋白酶高表达量的菌株,从而得到蛋白水解能力强的优质乳酸菌发酵剂菌株.本文将对乳酸菌的蛋白水解体系及近几年乳酸菌蛋白水解体系中相关基因表达情况的研究进展进行综述,以期为乳酸菌蛋白质代谢的进一步研究提供参考.     

乳酸菌蛋白水解体系及相关基因表达的研究进展

乳酸菌的蛋白水解体系包括胞外酶、转运系统和多种胞内酶。它们将外源蛋白质逐步水解成能被乳酸菌直接利用的游离氨基酸,弥补了因乳酸菌自身不能直接利用外源无机氮和蛋白质的缺陷,对于乳酸菌的正常生长具有非常重要的意义。目前,国内外正在研究利用现代分子技术,从基因表达层面检测不同菌株之间或菌株经过不同处理前后关键蛋白水解酶的表达水平差异,筛选出蛋白酶高表达量的菌株,从而得到蛋白水解能力强的优质乳酸菌发酵剂菌株。本文将对乳酸菌的蛋白水解体系及近几年乳酸菌蛋白水解体系中相关基因表达情况的研究进展进行综述,以期为乳酸菌蛋白质代谢的进一步研究提供参考。