Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products

Penicillium expansum is known for its destructive rot and patulin production in apple juice. According to the literature, P. expansum can, among other compounds, produce citrinin, ochratoxin A, patulin, penitrem A, and rubratoxin B. In this study the qualitative production of metabolites was examined using TLC (260 isolates), HPLC (85 isolates), and MS (22 isolates). The results showed that none of the 260 isolates produced ochratoxin A, penitrem A, or rubratoxin B. However, chaetoglobosin A and communesin B were produced consistently by all 260 isolates. Patulin and roquefortine C were produced by 98% of the isolates. Expansolides A/B and citrinin were detected in 91 and 85% of the isolates, respectively. Chaetoglobosins and communesins were detected in naturally infected juices and potato pulp, whereas neither patulin nor citrinin was found. Because most P. expansum isolates produce patulin, citrinin, chaetoglobosins, communesins, roquefortine C, and expansolides A and B, foods contaminated with this fungus should ideally be examined for chaetoglobosin A as well as patulin.     

体外法添加苹果酸与不饱和脂肪酸对瘤胃发酵及瘤胃功能菌群的影响

本研究采用体外法在油酸（Oleic acid,OA）、亚油酸（Linoleic acid,LA）、亚麻酸（Linolenic acid,LNA）的基础上添加不同水平苹果酸(Malic acid, MA),研究苹果酸和不饱和脂肪酸对瘤胃发酵模式与瘤胃主要功能菌群的影响。结果表明添加不饱和脂肪酸和苹果酸后,丙酸浓度随着脂肪酸不饱和度的增加显著升高（P<0.01）,总产气量和CH4产量显著降低（P<0.01）,其它挥发性脂肪酸均随着脂肪酸不饱和度的增加显著降低（P<0.01）。添加不饱和脂肪酸LA与LNA的甲酸甲烷杆菌数量显著增加（P<0.01）;白色瘤胃细菌与黄色瘤胃细菌数量比CK组显著增加（P<0.05）;而脂解厌氧弧杆菌和溶纤维丁酸弧菌数量比CK组却显著下降（P<0.05）。联合添加苹果酸和不饱和脂肪酸组中,只有在添加10 mmol/L苹果酸的OA组的甲酸甲烷杆菌,数量明显减少（P<0.05）。结论是不饱和脂肪酸和苹果酸能够改善瘤胃发酵模式,但二者对体外CH4抑制作用之间没有直接的联系。并且联合添加二者对于瘤胃主要功能菌群没有显著影响。     

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.     

Micellar electrokinetic capillary electrophoresis for rapid analysis of patulin in apple cider

A micellar electrokinetic capillary chromatography (MECC) mode was applied to a capillary electrophoresis (CE) method, which was developed for detection and quantitation of patulin in apple ciders. This method used a small sample amount (2 mL) and consumed minimal organic solvent compared to the most commonly used HPLC methods. The sample preparation procedure of the CE method was also simpler than other chromatographic techniques developed for patulin analysis. Patulin was detected with a photodiode array detector at 273 nm. The standard curve was linear (r(2) = 0.9984) from 75 microgram/L to 121 microgram/mL with patulin working solutions corresponding to 3.8 microgram/L to 6.1 microgram/mL patulin in the sample. The linearity was better in a narrower range of concentrations (r(2) = 0.9999) from 75 microgram/L to 24.1 microgram/mL. The limit of detection of the method was 3.8 microgram/L. Patulin recoveries at 4 levels in spiked samples (10-121 microgram/L) ranged from 95.2 to 105.4%. The recoveries were 96. 9% and 99.2% for 2 levels (22.3 and 223 microgram/L, respectively) of patulin in infected apple samples. This method represents a unique alternative method for rapid and sensitive analysis of patulin in apple ciders.     

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.     

Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of (13)C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.     

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.     

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%.     

Ruminal fermentation of propylene glycol and glycerol

Bovine rumen fluid was fermented anaerobically with 25 mM R-propylene glycol, S-propylene glycol, or glycerol added. After 24 h, all of the propylene glycol enantiomers and approximately 80% of the glycerol were metabolized. Acetate, propionate, butyrate, valerate, and caproate concentrations, in decreasing order, all increased with incubation time. Addition of any of the three substrates somewhat decreased acetate formation, while addition of either propylene glycol increased propionate formation but decreased that of butyrate. R- and S-propylene glycol did not differ significantly in either their rates of disappearance or the products formed when they were added to the fermentation medium. Fermentations of rumen fluid containing propylene glycol emitted the sulfur-containing gases 1-propanethiol, 1-(methylthio)propane, methylthiirane, 2,4-dimethylthiophene, 1-(methylthio)-1-propanethiol, dipropyl disulfide, 1-(propylthio)-1-propanethiol, dipropyl trisulfide, 3,5-diethyl-1,2,4-trithiolane, 2-ethyl-1,3-dithiane, and 2,4,6-triethyl-1,3,5-trithiane. Metabolic pathways that yield each of these gases are proposed. The sulfur-containing gases produced during propylene glycol fermentation in the rumen may contribute to the toxic effects seen in cattle when high doses are administered for therapeutic purposes.     

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.     

Fermentation of cottonseed and other feedstuffs in cattle rumen fluid

Bovine rumen fluid was fermented anaerobically over 48 h with cottonseed, corn, alfalfa, or a mixture of these substrates in anaerobic mineral buffer. Samples taken at different incubation times were derivatized with n-butanol and subjected to gas chromatography and mass spectroscopy. No unusual fermentation end-products from the cottonseed substrate were detected. Cottonseed supported rumen fermentation at levels comparable to those of the other substrates. Major components were usually found in the decreasing order of acetate, propionate, butyrate, and valerate, although acetate and propionate concentrations decreased late in the alfalfa and mixed-feed fermentations, eventually allowing butyrate concentrations to exceed those of propionate. As expected, lactate was produced in high concentrations when corn was fermented. The minor components 2-methylpropionate, 2- and 3-methylbutyrate, phenylacetate, phenylpropionate, and caproate also accumulated, with their relative concentrations varying with the substrate. Succinate was produced in substantial amounts only when corn and alfalfa were fermented; it did not accumulate when cottonseed was the substrate. Samples containing cottonseed were derivatized and subjected to reversed-phase high-performance liquid chromatography, revealing that gossypol concentrations did not change during fermentation.     

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.     

High-pressure liquid chromatographic method for the determination of patulin in apple butter.

Patulin is extracted from apple butter samples with ethyl acetate and the extract is cleaned up on a silica gel column, using benzene-ethyl acetate (75+25) as the eluant. High-pressure liquid chromatography, using a 25 cm ZorbaxSil column, isooctane-ethyl ether-acetic acid (750+250+0.5) as the mobile solvent, and a 254 nm ultraviolet detector, is used for the determinative step. Under these conditions, patulin is eluted before 5-hydroxymethylfurfural, a component of apple butter which interferes with other liquid chromatographic and thin layer chromatographic methods. Recoveries of patulin added at levels of 34.6, 138.4, and 276.8 mug/kg ranged from 89.0 to 112.1%.     

瘤胃液对全株甜高粱青贮质量的影响

甜高粱是一种重要的能源作物,为实现长时间贮存并提升糖化效率,该研究分析了瘤胃液不同添加量对全株甜高粱青贮品质和酶解糖化效果的影响。设置R1、R3、R5和R7共4个瘤胃液处理组(添加量依序分别为1、3、5和7 mL/100 g原料)和1个对照组(CK,等量蒸馏水),考察了瘤胃液不同添加量对全株甜高粱青贮过程中有机组分、发酵品质和酶解性能等质量指标的动态影响,并跟踪解析青贮期间微生物菌群的动态演绎。结果表明,添加瘤胃液能明显减少青贮甜高粱中的干物质、水溶性碳水化合物、粗蛋白以及木质纤维组分含量,使青贮pH和氨氮含量显著下降(P0.05),并与瘤胃液添加量呈负相关。青贮中的乳酸、乙酸含量随瘤胃液添加量和青贮发酵时间延长而明显增加(P0.05),瘤胃液强化了青贮发酵并有助于减少干物质损失,尤其在较高添加量时,青贮60d时的甜高粱综纤维素含量反而有所增加。4种瘤胃液处理组的门水平优势细菌主要为厚壁菌和变形菌,厚壁菌相对丰度随时间延长和瘤胃液添加量的增加而逐渐增加,变形菌门丰度则逐渐下降;属水平主要以乳酸杆菌、泛菌和醋酸杆菌为主,乳酸杆菌丰度与时间、瘤胃液添加量呈正相关,而泛菌则呈减少趋势。瘤胃液强化青贮后的甜高粱还原糖得率显著提升,尤其瘤胃液添加量为7 mL/100 g的R7处理组的糖得率较原料分别提高了11.06%(30 d)和19.28%(60 d)。添加瘤胃液能有效改善青贮甜高粱的发酵质量和生物降解性能,起到生物强化的预处理作用,为甜高粱的乙醇化利用奠定了基础。     

Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad (Decapterus maruadsi)

Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).     

Effect of addition of various carbon substrates on denitrification in a vertic Mollisol

Summary Glucose, acetate, malate, and citrate were added to an agricultural soil. The pe values (-log e^-; calculated from the redox potential) obtained 30 min after the addition of C were not correlated with the theoretical reducing power nor with the theoretical total energy of the C compounds. By contrast the number of electron (e^-) equivalents was correlated with pe⁷, indicating that the proton number affected the redox potential (Eh) measurement. After 24 h of incubation, denitrification rates followed the order citrate>malate>glucose and control. No N₂O production was detected with acetate. Denitrification was not correlated with the theoretical reducing power of the added C compounds but was correlated with pe+pH. Similar numbers of e^- equivalents were measured with all treatments. After 72 h of incubation, the order of the denitrification rates was malate>citrate >acetate>glucose and control. The Eh values (lower than after 24h) did not differ with treatment while the number of e^- equivalents was influenced by the quality of the C source. This also demonstrates that the proton number affected the measured Eh. Our results suggest that the different C substrates did not directly influence the soil physicochemical and biological conditions through their degree of oxidation. Any effects appeared to be indirect, arising from the ability of the substrates to generate new metabolites, and consequently initiate different metabolic pathways that modified the soil physicochemical conditions, reducing power and microbial activity.     

The impact of saponins or saponin-containing plant materials on ruminant production--a review

Saponins are steroid or triterpene glycoside compounds found in a variety of plants. Some saponin-containing plants, mainly legumes, have been used as animal feed, but others are toxic. Several studies on the effect of saponins on ruminant production have also been reported. Some in vitro and in vivo experiments that demonstrate the beneficial effects of saponin such as defaunation of the rumen and manipulation of the end products of fermentation are described. Defaunation is the selective removal of protozoa from the rumen microbial ecosystem by a cell membrane cholesterol-saponin interaction, which causes cell rupture. Because protozoa in the rumen cause protein turnover by predating on bacteria, defaunation increases the nitrogen utilization of the ruminant and may lead to an increase in growth, milk, or wool production. The growth-promoting effect was evident in the high roughage diet suggesting that the application of saponins or saponin-containing plant materials may be beneficial for the subsistence farmers in developing countries. Saponins are deglycosylated by rumen microbes. Some sapogenins have been detected in the digestive tract of ruminants; however, the direct action of these compounds on the host animal is still unclear. No information on the effects of saponin on ruminant reproduction is available. There is an urgent need for a systematic evaluation of the most active structural components of the saponins, and their interaction with the microbial community, the host animal, and the diet. Along with these studies, the direct effects of saponins or their microbial degradation products on the host must be examined in order to get the full understanding of the metabolism and beneficial effects of saponins on animals.     

Tannins in tropical browses: effects on in vitro microbial fermentation and microbial protein synthesis in media containing different amounts of nitrogen

Four species of browses (Acacia angustissima, Acacia salicina, Calliandra calothyrsus, andDichrostachys cinerea) were used to study the effect of tannins on microbial fermentation and microbial protein synthesis in incubation media containing high nitrogen (HN) and low nitrogen (LN) in the presence and absence of polyethylene glycol (PEG, MW 6000). The additional nitrogen in HN medium was supplied through ammonium bicarbonate. The use of HN medium significantly (P < 0.05) increased the in vitro gas and short-chain fatty acid (SCFA) production and microbial protein synthesis compared to the LN medium. Incubation of tannin-containing browses alone produced significantly (P < 0.05) lower gas and SCFA compared to in the presence of PEG in both HN and LN media. Inclusion of PEG in tannin-containing browses significantly (P < 0.05) reduced the molar proportion of propionate compared to in its absence. Higher N in the media resulted in 10.4 and 9.9% increases in in vitro gas and SCFA production, respectively, whereas inclusion of PEG to tannin-containing feed to remove the effect of tannins increased the in vitro gas and SCFA production by 186 and 195%, respectively, indicating that the low fermentation of tannin-containing browses could be due to the depressive effects of tannins on microbial activity and only partially accounted for by unavailability of N for rumen microbes. Incubation of browses with straw significantly (P < 0.05) decreased ammonia nitrogen concentration but increased the in vitro gas and SCFA production and microbial protein synthesis compared to straw alone.     

Enzymic release of reducing sugars from oat hulls by cellulase,as influenced by Aspergillus ferulic acid esterase and trichoderma xylanase

Hydroxycinnamic acids, mainly ferulic and p-coumaric acids, are believed to be inhibitory to ruminal biodegradability of complex cell wall materials such as oat hulls. Previous studies have shown that a novel enzyme, Aspergillus ferulic acid esterase, and Trichoderma xylanase act synergistically to break the ester linkage between ferulic acid and the attached sugar of feruloyl polysaccharides, releasing ferulic acid from oat hulls. In this paper, we examined the enzymic release of reducing sugars from oat hulls by the actions of individual enzymes (Aspergillus ferulic acid esterase at 13 mU, 6.4 U, and 4678.4 U/assay; cellulase at 20 levels, ranging from 7.8 mU to 2772.7 U/assay; Trichoderma xylanase at 20 levels, ranging from 7.8 mU to 4096 U/assay) and by the combined action of cellulase at six levels (62.5 mU, 2 U, 16 U, 128 U, 1024 U, and 2772.7 U/assay), Aspergillus ferulic acid esterase at 13 mU/assay, and Trichoderma xylanase at two levels (1 U and 256 U/assay). The amount of total acid-extractable reducing sugars in the oat hulls used in this study was 793.8 +/- 8.0 microg/mg. The results show that after a 24-h incubation with Aspergillus ferulic acid esterase alone, no reducing sugars were observed to be released from oat hulls. With cellulase as the sole enzyme, as the concentration increased from 7.8 mU to 2772.7 U/assay, the release of reducing sugars increased (P < 0.01) from 0 to 39% of the total present, with the highest release at 512 U/assay. With Trichoderma xylanase alone, as the concentration increased from 7.8 mU to 4096 U/assay, the release of reducing sugars increased (P < 0.01) from 4.9 to 33%, with the highest release at 2048 U/assay. When incubated together with Trichoderma xylanase (1 U or 256 U/assay) and Aspergillus ferulic acid esterase (13 mU/assay), cellulase at all six levels (62.5 mU, 2 U, 16 U, 128 U, 1024 U and 2772.7 U/assay) significantly increased the release of reducing sugars (P < 0.01) from 8 to 69%. These results indicate that the synergistic interaction between Aspergillus ferulic acid esterase and Trichoderma xylanase on the release of ferulic acid from feruloyl polysaccharides of oat hulls makes the remainder of the polysaccharides open for further hydrolytic attack and facilitates the accessibility of the main chain of polysaccharides to cellulase. This action extends the cell wall hydrolysis, thus releasing a higher yield of reducing sugars. Such enzymic pretreatment of oat hulls may provide a unique advantage to rumen microorganisms for the biodegradation of the complex cell walls of byproduct feeds such as oat hulls.