Identification of fungi using DNA sequences: an approach to identify Fusarium species isolated from domestic unpolished rice

Molecular approaches are being developed to provide for the rapid and objective identification of fungi. We attempted the identification of Fusarium species by a genetic analysis to validate practically the utility of a molecular approach for fungal identification and to reveal its limitations, and sequenced three regions, the 5' end of the 28S rRNA gene (D2 region) and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions, in the rRNA genes. The DNA sequences of 38 Fusarium strains isolated from domestic unpolished rice were compared for similarity with entries in the GenBank. Based on this comparison, it was estimated that all these three regions, as a minimum, must be compared with the database to identify Fusaria at the species level. According to the combinations of sequences in the three regions, the 38 isolates were classified into 13 groups. Out of the 13 groups, 6 groups (20 isolates in total) could be identified as definite species based only on the sequence data. For the other 6 groups (17 isolates in total), candidate species were limited on the basis of the sequence similarity, and then the isolates were identified at the species level with the aid of morphology. Only one isolate could not be identified. These results verified that DNA sequence comparison with the GenBank database is useful for the identification of Fusarium species.     

营养糙米面包的研制

对以糙米为原料制作面包的技术进行了研究,通过正交试验确定了营养糙米面包的制作工艺参数和配方,开发出新型营养保健型糙米食品。     

The effect of substrate on mycotoxin production of selected Penicillium strains

Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.     

糙米大豆复合粉加工技术的研究

以糙米粉和大豆粉为原料,制作速溶糙米大豆复合粉。通过单因素和正交实验对复合粉主要影响因素进行了优化,结果表明:将糙米粉和大豆粉分别在100、120℃下熟化30min后用100目细目筛初步筛分,按1∶2比例配比混匀,以20g混料为基准向其中加入水26%、乳糖4g和β-环糊精1.6g,混匀后用细目筛筛分造粒,取80目制得速溶的糙米大豆复合粉,不仅具有大豆和糙米的芳香气味,还具有糙米和大豆的营养价值和生理功效,具有良好的溶解性,色泽均匀剔透,口感细腻。     

速冻面米食品生产许可证审查细则

一、发证产品范围及申证单元 实施食品生产许可证管理的速冻面米食品是指以小麦粉、米、杂粮等粮食为主要原料或同时配以(单一或多种配料)肉、禽、蛋、奶、蔬菜、果料、糖、油、调味品为馅料,经成型、生制或熟制、速冻、包装而成的食品.例如速冻水饺、包子、花卷、馒头、汤圆、八宝饭、棕子、玉米、南瓜饼、脆皮鲜奶等.根据加工方式速冻面米食品可分为生制品、熟制品(包括发酵类及非发酵类).速冻面米食品的申证单元为1个.企业具备了生产熟制品的能力,也可以生产同种产品的生制品.     

国内外真菌毒素检测标准现状及分析

该文介绍目前国内外现有主要真菌毒素检测标准,对国际标准化组织(ISO)、国际分析化学师协会(AOAC)、欧洲标准化委员会(CEN)和我国现有主要真菌毒素检测标准进行了比较和探讨.综述了相关技术在现有标准中的应用,并分析相关技术和标准的发展趋势。     

Factors affecting patulin production by Penicillium expansum

Patulin, a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to human health because exposure can result in severe acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects. In this study, we investigated the effects of Penicillium expansum isolate, apple cultivar, storage temperature and time, and pH on the production of patulin. Patulin was analyzed by a previously developed micellar electrokinetic capillary electrophoresis method. P. expansum isolates originating from across Ontario produced widely differing levels of patulin, ranging from 0 to >6 mg/g by dry mycelial weight. The highest patulin levels were those for isolates displaying aggressive growth (characterized by rapidly increasing acidity) accompanied by profuse mycelial development. Distinct patterns in fungal growth rates and patulin production were evident among isolates grown in McIntosh, Empire, and Mutsu ciders. Extensive fungal growth and higher patulin levels (538 to 1,822 microg/ml on day 14) in apple ciders were associated with incubation at room temperature (25 degrees C), although potentially toxic patulin levels (75 to 396 microg/ml on day 24) were also found in refrigerated ciders (4 degrees C) inoculated with P. expansum.     

Determination of ten sulfonyl urea herbicides in unpolished rice by solid-phase extraction cleanup and LC-diode array detection

A liquid chromatographic (LC) method with diode array detection (DAD) was developed for screening of 10 sulfonyl urea herbicide residues in unpolished rice. The investigated herbicides were azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, ethoxysulfuron, flazasulfuron, imazosulfuron, metsulfuron-methyl, pyrazosulfuron-ethyl and tribenuron-methyl. Acetonitrile-water (2:1) extracts of rice samples were cleaned up with solid-phase extraction cartridges (octadecylsilane-bonded silica (ODS) and graphitized carbon black (GCB)). Three fractions of the GCB eluate were taken for analysis using 3 separate injections in order to avoid interference in LC-DAD analysis and to reduce analyte coelution problems. Recoveries from rice samples fortified with the 10 herbicides at 0.05 and 0.2 microgram/g ranged from 46.6 to 119.6%, and coefficients of variation were 3.1-12.6%. The quantitation limits were 0.01-0.02 microgram/g.     

Growth and metabolites production by Penicillium brevicompactum in yoghurt

Penicillium brevicompactum, commonly encountered in the indoor air, is known to produce a mycotoxin, mycophenolic acid (MPA). This mould has been isolated from a wide range of foods; considering that we had previously isolated this species from contaminated yoghurt, in this study we have evaluated its growth in yoghurt sweetened with sucrose, fructose and fructose added with fruit pieces. Fungal growth was evaluated monitoring CO(2) production in the headspace during yoghurt storage at 4+/-1, 8+/-1 and 10+/-1 degrees C throughout 21 days. P. brevicompactum grew well in the samples sweetened with fructose at 8 and 10 degrees C. The addition of sucrose influenced the growth negatively, particularly at 4 degrees C. Volatile Organic Compounds (VOC) and MPA production was determined at 8 degrees C in inoculated and uninoculated yoghurt, as well as in liquid malt extract. Differences in VOC profiles and in MPA production were correlated with the age of the fungus and with the growth medium. This study points out for the first time the early qualitative changes in volatile production patterns of a common indoor mould, grown in yoghurt, as well as the production of MPA during storage at refrigeration temperatures.     

Trichothecene and beauvericin mycotoxin production and genetic variability in Fusarium poae isolated from wheat kernels from northern Italy

The importance and widespread incidence of Fusarium poae as a natural contaminant of wheat in different climatic areas warrants investigation into the genetic diversity and toxin profile of a northern Italy population. Eighty-one strains of F. poae isolated from durum wheat kernels, identified by species-specific polymerase chain reaction and translation elongation factor-1α gene sequence analysis, were genetically characterized by the amplified fragment length polymorphism (AFLP) technique and analysed by high-pressure liquid chromatography for their ability to produce the beauvericin (BEA) and trichothecene mycotoxins. A high level of variability was observed by using AFLP analyses, with the lowest level of genetic similarity among the strains being approximately 61%. Most of the strains, 95%, produced BEA at <2655 µg g^?1; 88% produced the trichothecene nivalenol at <865 µg g^?1 and 76% produced the trichothecene fusarenon-X at <167 µg g^?1. These data show that F. poae can produce high amounts of BEA together with trichothecenes, and can represent a high potential mycotoxin risk in Italy for wheat colonized by this species.     

Effects of substrate, water activity, and temperature on growth and verrucosidin production by Penicillium polonicum isolated from dry-cured ham

Penicillium polonicum, a common mold on dry-cured meat products, is able to produce verrucosidin, a potent neurotoxin. The ability of P. polonicum isolated from dry-cured ham to grow and produce verrucosidin from 4 to 40 degrees C at water activities (a(w)) of 0.99, 0.97, and 0.95 on malt extract agar (MEA) and a medium made up with meat extract, peptone, and agar (MPA) was evaluated. Verrucosidin was quantified by high-pressure liquid chromatography and mass spectrometry. P. polonicum was able to grow on MEA and MPA at all the a(w) values tested from 4 to 37 degrees C but not at 40 degrees C. The optimal environmental conditions for growth were 20 degrees C, 0.99 a(w) on MEA and 20 to 25 degrees C, 0.97 a(w) on MPA, but the highest amount of verrucosidin was obtained at 25 degrees C, 0.99 a(w) in both media. No direct correlation between extension of mold growth and verrucosidin production was found. Temperature appears to be the most important factor ruling mycelial growth, whereas verrucosidin accumulation is mostly influenced by a(w). However, analysis of variance of the data showed that there was a complex interaction among all the environmental factors (medium, temperature, and a(w)) that significantly (P < 0.0001) affected growth and verrucosidin production. The reduction of a(w) to intermediates values of 0.95 has a stronger effect on growth on MEA than on MPA. Given that the meat-based medium proved to be an appropriate substrate for the biosynthesis of verrucosidin by P. polonicum, the ability of this mold to produce the toxin on meat products should be established.     

Mycotoxin production capability of Penicillium roqueforti in strains isolated from mould-ripened traditional Turkish civil cheese

Mould-ripened civil is a traditional cheese produced mainly in eastern Turkey. The cheese is produced with a mixture of civil and whey curd cheeses (lor). This mixture is pressed into goat skins or plastic bags and is ripened for more than three months. Naturally occurring moulds grow on the surface and inside of the cheese during ripening. In this research, 140 Penicillium roqueforti strains were isolated from 41 samples of mould-ripened civil cheese collected from Erzurum and around towns in eastern Turkey. All strains were capable of mycotoxin production and were analysed using an HPLC method. It was established that all the strains (albeit at very low levels) produced roquefortine C, penicillic acid, mycophenolic acid and patulin. The amounts of toxins were in the ranges 0.4–47.0, 0.2–43.6, 0.1–23.1 and 0.1–2.3 mg kg^?1, respectively. Patulin levels of the samples were lower than the others. The lowest level and highest total mycotoxin levels were determined as 1.2 and 70.1 mg kg^?1 respectively. The results of this preliminary study may help in the choice of secondary cultures for mould-ripened civil cheese and other mould-ripened cheeses.     

Mycotoxin production by Penicillium expansum on blackcurrant and cherry juice.

The production of mycotoxins and other secondary metabolites by Penicillium expansum on blackcurrant and cherry juice has been studied at 10 degrees C and 25 degrees C under storage imitated conditions. P. expansum was able to synthesize extracellular patulin under all conditions, and together with extracellular chaetoglobosin A when unlimited oxygen was available. Patulin, the chaetoglobosins A and C, the communesins A and B and the expansolides A and B could be detected intracellularly depending on the conditions. The metabolites were detected using thin-layer chromatography and high-performance liquid chromatography with diode array detection by comparison to standards. A method to detect the expansolides A and B by TLC was developed.     

Flavor production from edible oils and their constituents by Penicillium corylophilum

Production of volatile substances from edible oils and their constituents by Penicillium corylophilum was studied to clarify the mechanism of flavor production from a non-stick oil by the organism in a rice cake system. First, edible oils from plant and animal origins were tested for flavor production. Among the oils tested, coconut oil was the only one from which the flavor was produced. Second, triacylglycerols consisting of fatty acids with various lengths of carbon chain (C6 to C13) were studied for flavor production. Among the triacylglycerols tested, flavors were produced from those consisting of fatty acids with carbon chains of C6 to C11. The flavors consisted of methylketones and secondary alcohols, whose carbon chains were one carbon shorter than the precursor fatty acid molecules of the triacylglycerols. Flavors similar to that from the non-stick oil were produced from tricaprylin (C8), trinonanoin (C9), and tridecanoin (C10) among the triacylglycerols tested. Formation of mould spores was more strongly suppressed by triacylglycerols with shorter chain fatty acids. Third, fatty acids with various lengths of carbon chain (C7 to C15) were studied for flavor production. Among the fatty acids tested, flavors were produced from decanoic (C10) and undecanoic (C11) acids only. The flavors also consisted of methylketones and secondary alcohols one carbon shorter than the precursor fatty acids. Fatty acids with short carbon chains (C7 to C9) completely inhibited the mould growth. Our study showed that the range of carbon chain length of fatty acids capable of the flavor production (C10 to C11) was narrower than that of triacylglycerols (C6 to C11). It was also found that the non-stick oil and coconut oil contain tricaprylin and tridecanoin as triacylglycerols and decanoic acid as fatty acid.     

Mould growth on traditional greek sausages and penicillin production by Penicillium isolates

Visible moulds were isolated and identified from traditional Greek sausages from Northern Greece. Penicillium species were isolated from 90.8% of visibly mouldy sausages. Penicillium solitum, P. nalgiovense and P. commune species made up 60.6% of the total number of isolates. The most frequently occurring species was P. solitum (26.1%). P. nalgiovense and P. olsonii were found to be positive to penicillin production in an agar assay and further examination for antibiotic production in liquid culture with complex media designed for penicillin production, confirmed their ability for penicillin biosynthesis. Penicillin production by P. olsonii is reported for the first time in this study.     

Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergillus parasiticus by extracts of Agave species

In this work, the effect of ethanolic, methanolic and aqueous extracts of Agave asperrima and Agave striata on growth and production of aflatoxin (in A&M medium) and cyclopiazonic acid (CPA; in Czpaek-Dox medium) and on growth in corn under storage conditions was determined. Aspergillus strains were inoculated (10(6) conidia per ml of medium or per 6 g of corn), then plant extracts were added and incubated without shaking at 28 degrees C for 8 days (for aflatoxin-producing analysis) or for 12 days (for CPA-producing analysis). Aflatoxin was assayed by HPLC and cyclopiazonic acid by absorbance at 580 nm using the Erlich reagent. The extracts that most effectively inhibited growth were those from the flowers of both plants. These exhibited an MIC from 0.5 to 2 mg/ml in culture media. Extracts from scape showed an MIC from 15 to 30 mg/ml in culture media. The MIC of the flower extracts was higher (>30 mg/g) when examined in corn. However, concentrations lower than the MIC drastically inhibited production of aflatoxins in culture medium or in corn. Half of the MIC inhibited 99% of the production of aflatoxins and 85% of cyclopiazonic acid.     

Evaluation of several culture media for production of patulin by Penicillium species

The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.     

Influence of storage conditions of apples on growth and patulin production by Penicillium expansum

Penicillium expansum causes blue mould rot, a serious post-harvest disease of apples, and is the main producer of the mycotoxin patulin. The present study aimed to determine the influence of storage conditions (i.e. temperature and O(2) level) on growth and patulin production by different P. expansum strains on a simulation medium and on apples. Growth was strongly influenced by the temperature, while the used atmosphere (20, 3, and 1% O(2); <1% CO(2)) had no effect. Optimal growth was observed at 25 degrees C for every strain tested. Patulin production was stimulated when the temperature decreased (from 20 to 10 or 4 degrees C), while a further decrease of the temperature to 1 degrees C caused a reduction in patulin production. The temperature at which the stimulation was changed into suppression was strain dependent. Similar results were observed for the O(2) level. A reduction of the O(2) level from 20 to 3% O(2) could stimulate or suppress patulin production depending on the strain, while a clear decrease of the patulin production was observed when the O(2) level was reduced from 3 to 1%. These results show that the induction of limited stress to the fungus, such as lowering the temperature or lowering the O(2) levels stimulates patulin production. However, the combination of different stress conditions (e.g. low temperature and low O(2)) will result in a reduced formation of the toxin. The combination of stress conditions, at which the transition from stimulation to suppression is observed, is strain dependent. Moreover, patulin production is characterized by a high natural variability. The presented results show that the temperature and O(2) level has to be as low as possible during the storage of apples in order to suppress patulin production and to guarantee food safety.