利用抑制性差减杂交技术鉴定谢瓦氏曲霉间型变种产孢相关的基因

谢瓦氏曲霉间型变种Aspergillus chevalieri var.intermedius俗称"金花菌",是茯砖茶发酵中的优势菌种,该菌在不同的渗透压胁迫下产生不同类型的孢子。采用抑制性差减杂交技术(suppression subtractive hybridization,SSH),以低渗条件下产生的子囊孢子和高渗条件下产生的分生孢子为材料分别与营养菌丝体构建两个正反向文库。每个库随机挑选400个阳性克隆进行测序。然后将所得到的基因片段在GenBank进行BLASTx和BLASTn同源比对,筛选出的差异表达的基因涉及蛋白质代谢、细胞代谢、转录调控等多个方面,其中可能参与有性产孢调控的基因有13条,可能参与无性产孢调控的基因有10条。     

Microbiological Safety Evaluation of an Industrial Refuse Incinerator

An industrial refuse incinerator was tested to determine minimal operating temperatures required to prevent release of viable microorganisms into the atmosphere. A liquid suspension of Bacillus subtilis var. niger spores was disseminated into the firebox as an aerosol, and dry spores mixed with animal bedding were dumped into the firebox. The minimal requirement for wet spores was 575 F (302 C) for the firebox air temperature and 385 F (196 C) for the firebrick refractory lining. When dry spores were used, these temperatures were 700 and 385 F (371 and 196 C), respectively.     

Eurotium spp. and echinulin in feed refused by swine.

Feed refused by swine contained a high-propagule density of Eurotium chevalieri Mangin (anamorph, Aspergillus chevalieri (Mangin) Thom and Church), Eurotium amstelodami Mangin (anamorph, Aspergillus amstelodami (Mangin Thom and Church), and Aspergillus candidus Link. Echinulin (8 micrograms/g of feed) was detected in the feed. Isolates of E. chevalieri and E. amstelodami but not A. candidus produced echinulin on rice or cracked corn. Mice refused to drink water containing 90 micrograms of echinulin per ml. This is the first report of the alkaloid echinulin in feed refused by swine.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

Interrelationship of Heat and Relative Humidity in the Destruction of Clostridium botulinum Type E Spores on Whitefish Chubs

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Incidence of Clostridium botulinum in honey of various origins

By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one. Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C. Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F). The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%). It was estimated that most positive samples contained spores in one per gram or lower concentrations. One sample contained 4 type A spores per gram and another 36-60 type F spores per gram. No distinct biochemical properties were found with the honey isolates.     

Differential adaptation of membranes of two osmotolerant fungi,Aspergillus chevalieri and Penicillium expansum to high sucrose concentrations

Aspergillus chevalieri and Penicillium expansum were able to tolerate sucrose concentrations in the growth media up to 80% (w/v). At 50% sucrose the growth rate is approximately 1.4 and 1.2 times, respectively, higher than in the control. While at 80% sucrose it drops to 35% and 45% of the control level for both fungi. Lipids and proteins in plasma membranes increased with increasing sucrose concentrations in the growth medium. Phospholipid content in membranes of both organisms being also increased, phosphatidyl glycerol was the major detected phospholipid and represented the highest increase. The fatty acid composition of fraction enriched plasma membrane of both fungi changed when they were grown in high sucrose concentrations. Some fatty acids which had not been detected in control cultures were present and the proportions of other fatty acids changed. At 50% sucrose the unsaturation index of membranes decreased by 20-25% in both fungi, indicating that the plasma membrane is less fluid at this concentration. At 80% sucrose a similar trend was observed for P. expansum but for A. chevalieri the unsaturation index was little changed compared with the control. The fluorescence polarization values of 1,6-diphenyl 1,3,5-hexatriene (DPH) in membranes of both fungi grown at 80% sucrose increased, indicating a decrease in membrane fluidity. At 50% sucrose the increase in saturation of membrane fatty acids would tend to reduce membrane fluidity but in A. chevalieri at 80% sucrose fatty acids did not become more saturated. In this case the marked increase in sterols at this sucrose concentration may be responsible for low membrane fluidity.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chemical composition and antibacterial activities of the essential oils of Lippia chevalieri and Lippia multiflora from Burkina Faso

The chemical composition of the essential oils of Lippia chevalieri and Lippia multiflora obtained from the air-dried leaves by hydrodistillation were analysed using GC and GC-MS. L. chevalieri and L. multiflora belonged to thymol/p-cymene/2-phenyl ethyl propionate and thymol/p-cymene/thymyl acetate chemotypes, respectively. The essential oils were also tested against 09 strains using a broth microdilution method. The Gram-negative bacteria were the most sensitive. The essential oil of L. multiflora was the most active.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

A Note on the Nature and Sequence of Yeasts during Fermentation of Apples grown in India

Six morphologically different groups of yeasts comprising Kloeckera apiculata. Candida krusei, Candida valida, Candida sorbosa, Metschnikowia pulcherrima and Saccharomyces chevalieri were isolated from fresh, fermenting and fermented apple juice. This is the first report on the isolation of C. valida, C. sorbosa and S. chevalieri from fermenting and fermented apple juice.     

Effect of volatile and gaseous metabolites of germinating pea seeds on micromycetes

Differences in the effect of volatile and gaseous metabolites of germinating pea seeds on the germination of spores of Mucor racemosus and macroconidia of Fusarium oxysporum are described. Germination of spores of M. racemosus was inhibited by seed metabolites whereas germination of macroconidia of F. oxysporum was stimulated during the first two days and inhibition occurred only after further two days of germination of the seeds. A pronounced inhibition of germination of spores of both micromycetes took place due to absorption of CO2 from volatile and gaseous metabolites. Absorption of some components of seed metabolites in a KMnO4 solution led to a decrease of the inhibitory effect on germination of spores of M. racemosus and stimulatory effect on germination of macroconidia of F. oxysporum.     

Effect of volatile substances fromOriganum majorana andOcimum basilicum on spore respiration and germination of some soil fungi

Oxygen uptake by the spores ofFusarium moniliforme, F. oxysporum, F. semitectum, F. solani, Mucor racemosus andTrichoderma viride was increased in the presence of volatile substances extracted, fromOriganum majorana andOcimum basilicum. This increase was greater in the presence of volatile substances fromO. basilicum thanO. majorana, except in the case ofF. semitectum where the reverse was true. A drop in the RQ of all the germinating spores was observed in the presence of these substances. Volatile substances fromO. majorana reduced the spore germination ofM. racemosus whereas the spores ofT. viride were stimulated to germinate. Volatile substances fromO. basilicum stimulated the spore germination ofM. racemosus whereasT. viride spores were not affected.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7˙2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of a_w during heating. Spore resistance to heat increased in the order: buffer ⋖ commercial oil < olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low a_w (0˙479 and 0˙492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values ( ca 28°C in oils and 10°-12°C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Toxicity to Chicks of Aspergillus and Penicillium Species Isolated from Moldy Pecans

Isolates of Aspergillus chevalieri, A. flavus, A. ochraceus, A. repens, and Penicillium funiculosum and complexes of P. citrinum-P. implicatum isolated from moldy pecan meats were toxic to chicks.     

Use of a novel method to characterize the response of spores of non-proteolytic Clostridium botulinum types B, E and F to a wide range of germinants and conditions

AIMS: Limited information is available on the germination triggers for spores of non-proteolytic Clostridium botulinum. An automated system was used to study the effect of a large number of potential germinants, of temperature and pH, and aerobic and anaerobic conditions, on germination of spores of non-proteolytic Cl. botulinum types B, E and F. METHODS AND RESULTS: A Bioscreen analyser was used to measure germination by decrease in optical density. Results were confirmed by phase-contrast light microscopy. Spores of strains producing type B, E and F toxin gave similar results. Optimum germination occurred in L-alanine/L-lactate, L-cysteine/L-lactate and L-serine/L-lactate (50 mmol l(-1) of each). A further 12 combinations of factors induced germination. Sodium bicarbonate, sodium thioglycollate and heat shock each enhanced germination, but were not essential. Germination was similar in aerobic and anaerobic conditions. The optimum pH range was 5.5-8.0, germination occurred at 1-40 degrees C, but not at 50 degrees C, and was optimal at 20-25 degrees C. CONCLUSIONS: The automated system enabled a systematic study of germination requirements, and provided an insight into germination in spores of non-proteolytic Cl. botulinum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results extend understanding of germination of non-proteolytic Cl. botulinum spores, and provide a basis for improving detection of viable spores.     

Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

Pure yeast culture fermentation of cocoa (Theobroma cacao L): effect on yield of sweatings and cocoa bean quality

Cocoa sweatings, the pale yellowish liquid that drains off during cocoa fermentation, is the breakdown product of the mucilage surrounding the fresh cocoa bean, and constitutes about 10% of the weight of the cocoa fruit. On average, about 1.9 million l of sweatings are produced annually in Ghana during the cocoa harvesting season. It has been shown to be a suitable medium for the production of wines, alcohol, marmalade, jam and syrup. Its rapid collection in high yields and quality is the first step to its utilization on a commercial scale. Thus pure yeast culture fermentation of cocoa under controlled temperature conditions and its effect on yield of sweatings and final cocoa bean quality was investigated. Cocoa fermentations employing Saccharomyces chevalieri or Kluyveromyces fragilis alone gave significantly higher yields of sweatings (p 0.05) than controls. The initial rates of sweating by the two strains were also very high but dropped to a constant minimum value after 12h of fermentation. In contrast, fermentations employing Torulopsis candida or Candida norvengensis alone as well as different combinations of all the yeast strains did not give any significant difference in yield compared to controls (p 0.05). Fermentations using S. chevalieri alone or other combinations in which S. chevalieri was present gave beans with acceptable quality based on different quality indices used for grading cocoa beans commercially.