Patulin in Italian commercial apple products

Patulin is a mycotoxin produced by microscopic fungi belonging to the Penicillium and Aspergillus genera. The natural occurrence of patulin in four apple products marketed in Italy and purchased from the supermarket, herbalist, and retail shops was studied. Thirty-three samples of the four products had no detectable patulin contamination. The 11 positive samples had a concentration ranging between 1.4 and 74.2 microg/L with a mean of 26.7 microg/L. All vinegar samples were negative for patulin; of 10 apple-based baby foods, two samples were contaminated with 17.7 and 13.1 microg/L and both were labeled as "organic food". Comparing organic and conventional agricultural practices, no significant differences were found. Finally, optimization of extraction protocol more general and useful for juices, clarified juices, baby foods, vinegars, and purees was performed. The low incidence of the patulin level in Italian apple products is a clear parameter to judge the quality of the fruit, and the process is of a high standard.     

Prediction of Penicillium expansum spoilage and patulin concentration in apples used for apple juice production by electronic nose analysis

Classification models for Penicillium expansum spoilage of apples and prediction models for patulin concentration in apples usable for apple juice production were made on the basis of electronic nose (e-nose) analysis correlated to HPLC quantification of patulin. A total of 15 Golden Delicious and 4 Jonagold apples were surface sterilized and divided into three groups per variety. The Golden Delicious group consisted of five apples each. Group 1 was untreated control, group 2 was surface inoculated with P. expansum, and group 3 was inoculated in the core with P. expansum. The apples were incubated at 25 degrees C for 10 days. E-nose analysis was performed daily. At day 10 the Golden Delicious apples were individually processed for apple juice production. During apple juice production the mash and juice were analyzed by e-nose, and samples were taken for patulin analysis by HPLC. The volatile metabolite profile was obtained by collection of volatile metabolites, on tubes containing Tenax TA, overnight between the 9th and 10th days of incubation and subsequent analysis of the collected compounds by GC-MS. Prediction models using partial least-squares, with high correlation, for prediction of patulin concentration in shredded apples as well as apple juice were successfully created. It was also shown that it is possible to classify P. expansum spoilage in apples correctly on the basis of soft independent modeling of class analogy classification of e-nose analysis data. To the authors' knowledge this is the first report of a regression model between e-nose data and mycotoxin content in which actual concentrations are reported. This implies that it is possible to predict mycotoxin production and concentration by e-nose analysis.     

Prevention of patulin toxicity on rumen microbial fermentation by SH-containing reducing agents

Patulin, a toxic fungal metabolite, negatively affects rumen fermentation. This mycotoxin has also been associated with intoxication cases in cattle. This study investigates the use of SH-containing reducing compounds to prevent patulin's negative effects on the rumen microbial ecosystem. The effect of 50 microg/mL patulin on the fermentation of alfalfa hay was measured in batch cultures with and without reducing agents. Sulfhydryl-containing cysteine and glutathione prevented the negative effects of the toxin on dry matter degradation, gas, and volatile fatty acid production (P < 0.01). However, non-sulfhydryl-containing ascorbic and ferulic acids did not protect against patulin's toxicity (P > 0.01). Patulin was unstable in buffered rumen fluid as the concentration decreased by half after 4 h of incubation. In the presence of sulfhydryl groups, the toxin disappeared rapidly and was not detected after 1 h of incubation. The utilization of sulfhydryl-containing compounds such as cysteine to avert patulin toxicity could have practical implications in ruminant nutrition.     

Volatility of patulin in apple juice

Patulin is a mycotoxin produced by certain fungi, such as those found commonly on apples. The patulin content of apple juice is a regulatory concern because patulin is a suspected carcinogen and mutagen. A simple model of the apple juice concentration process was carried out to examine the possible contamination of patulin in apple aroma, a distillate produced commercially in the concentration of apple juice. The results show no evidence for patulin volatility, and document a reduction in patulin content by at least a factor of 250 in the apple distillate obtained from apple juice. Furthermore, a survey of several commercial apple aroma samples found no evidence of patulin content.     

Rapid liquid chromatographic determination of patulin in apple juice

A rapid method is described for the quantitative determination of patulin in apple juice. The mycotoxin is extracted from the sample with ethyl acetate and the extract is cleaned up by extraction with a sodium carbonate solution. Patulin is determined by reverse phase liquid chromatography using a muBondapak C18 column and a 254 nm ultraviolet detector. The lower detection limit in patulin standard solution is 0.32 ng and recovery is greater than 75%.     

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.     

Quantification of the mycotoxin patulin by a stable isotope dilution assay.

Two stable isotope dilution assays for the quantification of patulin [4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one] in foods were developed using (13)C-labeled patulin as the internal standard. One method was performed by means of LC/MS in negative electrospray ionization mode without derivatization; the other used HRGC/HRMS after trimethylsilylation of the patulin isotopomers. In comparison with previously reported methods based on high-performance liquid chromatography with UV detection, HRGC/HRMS of the derivatized samples showed better repeatability, higher recovery rates (96% at a spike level of 200 ng/L), and a 100 times lower detection limit (12 ng/L). In contrast, LC/MS showed a much lower performance as compared to HPLC/UV or HRGC/HRMS. Using HRGC/HRMS, the mycotoxin was quantified in many different fruit products and in molded wheat bread.     

酵母菌对果蔬采后病害防治的研究进展

该文对酵母菌对果蔬采后病害防治的研究现状进行了综述，其中包括防治效果、防治机理、防治的优势及最新研究成果。许多酵母菌对果蔬采后病害具有明显的抑制作用，酵母菌抑制霉菌的机理主要在于营养与空间的竞争、对病原菌的直接寄生作用及诱导寄主产生抗病性。酵母菌与其它生防微生物相比，具有拮抗效果好、不产生毒素、可以和化学杀菌剂共同使用等优点。使用生物工程技术改造拮抗酵母菌从而提高其生防效果，是酵母菌对果蔬采后生物防治的最新研究领域。该文还指出了酵母菌对果蔬采后生物防治存在的问题，并对未来的研究提出了新的思路。     

Analysis of patulin in apple juice by diphasic dialysis extraction with in situ acylation and mass spectrometric determination.

A procedure combining diphasic dialysis extraction with in situ acylation and gas chromatography/mass spectrometry (GC/MS) determination was developed for detection and quantification of the mycotoxin patulin in apple juice. Apple juice samples spiked with 4-N,N-dimethylaminopyridine were dialyzed using methane chloride and acetic anhydride inside dialysis tubing. Patulin was derivatized into its acetate and collected in the tubing after diphasic dialysis and was directly determined using GC/MS with the selective ion monitoring mode without further concentration and cleanup steps. Quantification was carried out by a calibration curve with an internal standard of correlation. The appropriate parameters of both dialysis and derivatization were examined. The linear range of the calibration curve was found to be 10-250 microg/L for patulin, and the limit of quantification was 10 microg/L. Levels of patulin ranging from 0 to 107.2 microg/L with 77-109% recovery were found in 10 apple samples. The technique combining diphasic dialysis extraction and acylation was demonstrated and showed potential for other applications.     

Stability of patulin to sulfur dioxide and to yeast fermentation.

The affinity of patulin for sulfur dioxide (SO2) is much less than was previously reported and is of little significance at the SO2 concentrations (below 200 ppm) used in the processing of apple juice and cider. However, at concentrations of 2000 ppm SO2 and 15 ppm patulin, combination was 90% complete in 2 days. Removal of SO2 liberated only part of the patulin, which suggests that 2 mechanisms are involved: one reversible (opening the hemiacetal ring) and one irreversible (SO2 addition at the double bond). Test with 2 yeasts used in English commercial cider making confirmed that patulin is effectively removed during yeast fermentation.     

Biodegradation of the fungicide iprodione by Zygosaccharomyces rouxii strain DBVPG 6399

Iprodione is a contact fungicide used to control several pathogens such as Botrytis cinerea, Monilia, and Sclerotinia. This paper reports the ability of an iprodione-resistant strain of Zygosaccharomyces rouxii to degrade iprodione at a concentration of 1 mg L(-1). The yeast Z. rouxii was chosen also for its ability to grow at high osmolarity. Also of note is that in bioremediation situations and in the food industry such resistance could be important. The kinetic and metabolic behaviors of the fungicide in the media are described. The results show a new transformation pathway of iprodione by the yeast leading to the formation of N-(3,5-dichlorophenyl)-2,4-dioxoimidazoline, 3-isopropylhydantoin, and 3,5-dichloroaniline. These compounds were identified by 1H NMR, 13C NMR, and GC-MS analyses. This study provides a basis to employ yeast strains in biodegradation studies in relation to their ability in the disappearance and degradation of xenobiotics into simpler molecules.     

采用Biolog, DGGE 和PLFA三种方法研究利用方式对土壤微生物群落结构的影响

Biolog, 16S rRNA gene denaturing gradient gel electrophoresis （DGGE）, and phospholipid fatty acid （PLFA） analyses were used to assess soil microbial community characteristics in a chronosequence of tea garden systems （8-, 50-, and 90- year-old tea gardens）, an adjacent wasteland, and a 90-year-old forest. Biolog analysis showed that the average well color development （AWCD） of all carbon sources and the functional diversity based on the Shannon index decreased （P 〈 0.05） in the following order： wasteland 〉 forest 〉 tea garden. For the DCCE analysis, the genetic diversity based on the Shannon index was significantly lower in the tea garden soils than in the wasteland. However, compared to the 90-year-old forest, the tea garden soils showed significantly higher genetic diversity. PLFA analysis showed that the ratio of Gram positive bacteria to Cram negative bacteria was significantly higher in the tea garden soils than in the wasteland, and the highest value was found in the 90-year-old forest. Both the fungal PLFA and the ratio of fungi to bacteria were significantly higher in the three tea garden soils than in the wasteland and forest, indicating that fungal PLFA was significantly affected by land-use change. Based on cluster analysis of the soil microbial community structure, all three analytical methods showed that land-use change had a greater effect on soil microbial community structure than tea garden age.     

Transformation of [14C] and [35S]labeled lignosulfonates during soil incubation

[¹⁴C] and [³⁵S]labeled lignosulfonates (LS) or [¹⁴C]labeled coniferyl alcohol dehydropolymer (DHP) were aerobically incubated in soil for 17 weeks. Respiratory ¹⁴CO₂ was compared with that from DHP or that from [U¹⁴C]cellulose. Less CO₂ was released from ring and side chain carbons of LS than from DHP, though similar amounts of CO₂ were released from the methoxyl groups of both compounds. After incubation, the soil samples were exhaustively extracted with water and then with a sodium pyrophosphate-NaOH solution. The water solubility of the originally completely-soluble LS carbons was greatly decreased by incubation, and a large portion of the extracted ³⁵S was detected as sulfate. The pyrophosphate extract was separated into humic and fulvie acids. The humic acid from soils incubated with LS contained low ³⁵S activity and a similar ¹⁴C activity to that from soils incubated with DHP. The fulvic acid from the soils incubated with LS contained higher amounts of ¹⁴C (and ³⁵S) than that of the soils incubated with DHP. More side chain ¹⁴C activity than other ¹⁴C activity was found in both, the water extract and the fulvic acid from soils incubated with LS. The high ³⁵S together with the high side chain ¹⁴C activity probably indicates an elimination of the side chain carbons together with sulfonic acid groups. Anaerobic incubation of soil with LS or DHP promoted breakdown and incorporation of LS and DHP into humus much less than aerobic incubation. The possible reduction in potential pollution from lignosulfonates due to the observed transformations in soil are discussed.     

Soil extracts and co-culture assist biodegradation of 2,4,6-tribromophenol in culture and soil by an auxotrophic Achromobacter piechaudii strain TBPZ

Achromobacter piechaudii strain TBPZ is a 2,4,6-tribromophenol (TBP) degrader that was isolated from contaminated desert soil. In the current study we have found that yeast extract has a substantial impact on the debromination activity of non-dividing cells of TBPZ harvested during the stationary phase. The results suggest that yeast extract provides a factor that is necessary for activity and that is missing in stationary cells. Nevertheless, strain TBPZ from the stationary phase did survive and degrade TBP in soils with different degrees of contamination without yeast extract supplements. Experiments with soil extracts showed that a soluble factors is responsible for this phenomenon in a similar way to yeast extract. To test whether other soil bacteria provide these factors, co-culture experiments with strain TBPZ and a bisphenol A (BPA) degrader, Sphingomonas strain WH1, that was isolated from the same contaminated desert soil, were carried out. These experiments clearly demonstrated that growth of strain WH1 on BPA enables strain TBPZ to degrade TBP rapidly, suggesting that a metabolic product from WH1 is involved. In experiments with mixed amino acid blend (casamino acid), slower growth was observed when compared to growth with yeast extract. Providing vitamins and a defined mixture of two amino acids, tryptophan and phenylalanie, caused rapid degradation of TBP without the rapid growth seen in yeast extract treatment, indicating that the amino acids are needed to sustain biodegradation activity. The results of this study highlight that degradation of TBP by strain TBPZ is possibly controlled by micronutrients. In addition, the results suggest that an auxotrophic bacterium able to degrade halo-organic pollutants can survive in a natural environment due to its interaction with natural organic matter or other microorganisms.     

Trimming as a means of removing patulin from fungus-rotted apples.

Penicillium expansum 1071, 1172, NRLL 973, and Penicillium patulum ATCC 24550 were inoculated into Red Delicious, Golden Delicious, and McIntosh apples. The decayed tissue was trimmed from the sound tissue, each fraction was weighed, and the patulin concentration in the juice was assayed by thin layer chromatography. The quantity of patulin in the whole apples and in decayed tissues was calculated and these values were used to determine the percentage of total patulin removed by trimming. The patulin content ranged from 140 to 4880 mug/apple. Trimming removed 93-99% of the total patulin, regardless of incubation temperature, fungus strain, or apple variety. Trimming of defective tissue from fungus-rotted apples could substantially reduce the patulin concentration.     

番茄SlJAZ11的表达分析及互作蛋白的筛选与验证

茉莉酸（JA）参与调节植物生长、发育和防御等多种重要生物过程,其中JAZ蛋白作为抑制子,在茉莉酸介导的生物和非生物胁迫响应过程中起到重要作用。为研究SlJAZ11的功能,本研究分析了水杨酸（SA）、茉莉酸等激素以及病原菌侵染处理后SlJAZ11的表达模式。结果表明,SlJAZ11能够响应水杨酸、茉莉酸及丁香假单胞菌（Pst DC3000）的诱导。通过酵母双杂交筛选番茄cDNA文库,获得了14个SlJAZ11潜在的互作蛋白。进一步采用酵母双杂交点对点验证和双分子荧光互补技术（BiFC）对候选的SlENT、SlOOLG与SlJAZ11间的互作关系进行验证,发现SlENT和SlJAZ11存在互作。对SlENT的表达模式进行分析,发现其表达被SA和Pst DC3000显著抑制,而MeJA能显著诱导其表达,表明SlJAZ11可能通过与SlENT互作来调控JA介导的番茄抗病性。本研究可为阐明SlJAZ11功能与作用机制奠定良好的分子基础。     

New monascus metabolite isolated from red yeast rice (angkak,red koji)

Red yeast rice (angkak, red koji) obtained as cultures of Monascus purpureus on rice was extracted and analyzed by HPLC. In addition to the known red, orange, and yellow pigments and the mycotoxin citrinin, a new Monascus metabolite was detected. It is present in the original red yeast rice and formed in higher amounts when red yeast rice is heated. High-resolution mass spectrometry indicated the molecular formula C(15)H(12)O(4). The chemical structure was elucidated by analysis of NMR data. The new compound, named monascodilone, is characterized by a propenyl group on a pyrone ring, an aromatic ring, and a gamma-lactone group.     

Generation of monochloropropanediols (MCPDs) in model dough systems. 1. Leavened doughs

The effect of dough recipe ingredients and processing on the generation of monochloropropanediol isomers (MCPDs) in leavened wheat doughs has been investigated. Commercial ingredients having no effect on MCPD formation were acetic acid and baking fats (triacylglycerols). Ingredients making a significant contribution to MCPD levels were yeast and flour improver [ascorbic acid, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and soya flour]. The results showed that free glycerol is a key precursor of MCPDs in leavened doughs. This glycerol is primarily generated by the yeast during proving but is also present in the flour, the yeast, and the improver. Under conditions of high dough moisture content (45%), MCPD formation was approximately proportional to glycerol concentration but showed a weaker dependence on chloride level, suggesting that the mechanisms of formation involved at least some reversible stages. MCPD generation increased with decreasing dough moisture to a point where the formation reaction was limited by chloride solubility and competing reactions involving glycerol and key precursor intermediates. These results could be predicted by a kinetic model derived from the experimental data. Glycerol was shown to account for 68% of MCPDs generated in proved full recipe dough.     

13C solid-state NMR assessment of decomposition pattern during co-composting of sewage sludge and green wastes

The fate of organic matter during composting is poorly understood. Therefore, we analysed composts of sewage sludges and green wastes (44 samples representative of 11 stages of biodegradation) by conventional chemical methods: pH, humic (HA) and fulvic acid (FA) content, C, N and organic matter (OM) content, and by ¹³C CPMAS NMR to assess the decomposition process of the organic matter. Chemical changes clearly occurred in two phases: first, decomposition of OM during the first 2 months was characterized by decreased C/N ratios, OM content and increased pH; and second, a humification process with increased HA/FA ratios. NMR spectrum changes confirmed this pattern, with an increase in aromaticity and a decrease in alkyl C. A decrease of syringyl to guaiacyl ratio (S/G), a sign of lignin transformation, also indicated humification during composting. NMR spectroscopic properties of composts were also studied by means of principal components analysis (PCA) and revealed changes according to the degree of compost maturation. The factorial map presents a chronological distribution of composts on the two first principal components. The influences of eight chemical factors on the PCA ordination of composts as monitored by their evolution by NMR were also studied by multivariate analyses. PCA clearly indicated two phases: the rapid decomposition of organic matter followed by the formation of humic‐like substances. The first phase, that is ‘new’ composts, was strongly correlated with OM contents, pH and C/N ratios whereas the second phase, corresponding to ‘old’ compost, was correlated with pH, HA content and HA/FA ratio. These results confirm that knowledge of the formation of humic substances is indispensable to suitable monitoring of the composting process.