Early detection of spoilage moulds in bread using volatile production patterns and quantitative enzyme assays

AIMS: Early detection of spoilage fungi (two Eurotium spp., a Penicillium chrysogenum species) in bread analogues over periods of 72 h at 25 degrees C and 0.95 water activity was evaluated using volatile production patterns, hydrolytic enzyme production, and changes in fungal populations. METHODS AND RESULTS: Using an electronic nose system it was possible to differentiate between uninoculated controls and samples contaminated with P. chrysogenum within 28 h. After 40-48 h it was possible to differentiate between the Eurotium spp., P. chrysogenum and the control using Principal Component Analysis (PCA). Cluster analyses could differentiate between the control, P. chrysogenum and the Eurotium spp. after 40 h. Of seven hydrolytic enzymes examined after 48 h, the specific activities of three were significantly different from uninoculated control bread. There were also differences between the mould species in production of three enzymes. Penicillium chrysogenum populations increased fastest, from about 10(3) cfu g(-1) to 10(6)-10(7) cfu g(-1) after 72 h. For the Eurotium spp. this increase was slower. CONCLUSIONS: Overall, this study suggests, for the first time, that an electronic nose system using a surface polymer sensor array is able to detect qualitative changes in volatile production patterns for the early detection of the activity of spoilage moulds in bakery products. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for application of such systems for microbial quality assurance in intermediate moisture food products.     

Experimental use of a new surface acoustic wave sensor for the rapid identification of bacteria and yeasts

AIMS: Use of an electronic nose (zNose(TM)) to discriminate between volatile organic molecules delivered during bacterial/fungal growth on agar and in broth media. METHODS AND RESULTS: Cultures of bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli) and yeasts (two Candida albicans strains) were grown on agar and in broth media and incubated for 24 h at 37 degrees C. Headspace samples from microbial cultures were analysed by the zNose(TM), a fast gas chromatography-surface acoustic wave detector. Olfactory images of volatile production patterns were observed to be different for the various species tested after 24 h. Moreover, some strains (two K. pneumoniae, two C. albicans) did not show changes in volatile production patterns within our species. CONCLUSIONS: Our experiments demonstrate that the electronic nose system can recognize volatile production patterns of pathogens at species level. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results, although preliminary, promise exciting challenges for microbial diagnostics.     

Early discrimination of fungal species responsible of ochratoxin A contamination of wine and other grape products using an electronic nose

An electronic nose (e-nose) system using an array of metal oxide sensors (Fox 3000, Alpha MOS) was used to detect and discriminate two ochratoxigenic fungal species, Aspergillus carbonarius (Bain.) Thom and A. niger Van Tieghem, that are responsible for the contamination of wine and other wine grape products, using their volatile production patterns. Two well-known ochratoxigenic strains were used in this study: A. carbonarius A941 and A. niger A75. These strains were grown on three culture media, Czapek Dox modified (CDm) agar, yeast extract sucrose (YES) agar and white grape juice (WGJ) agar, and the volatile organic compounds produced in the headspace by these species were evaluated over periods of 48–120 h. The e-nose system was able to differentiate between the two species within 48 h of growth on YES and WGJ agar using principal component analysis (PCA), which accounted for 99.9% and 97.2% of the data respectively, in principal components 1 and 2, based on the qualitative volatile profiles. This differentiation was confirmed by cluster analysis of data. However, it was not possible to separate these species on CDm agar. Our results show that the two closely related ochratoxigenic species responsible for the contamination of wine and other wine grape products can be discriminated by the use of qualitative volatile fingerprints. This approach could have potential for rapid identification of A. carbonarius and A. niger on wine grape samples, thereby significantly reducing the time of detection of these ochratoxin A producing species.     

Use of an electronic nose system for diagnoses of urinary tract infections

The use of volatile production patterns produced by bacterial contaminants in urine samples were examined using electronic nose technology. In two experiments 25 and 45 samples from patients were analysed for specific bacterial contaminants using agar culture techniques and the major UTI bacterial species identified. These samples were also analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. In the first experiment analysis of the data using a neural network (NN) enabled identification of all but one of the samples correctly when compared to the culture information. Four groups could be distinguished, i.e. normal urine, Escherichia coli infected, Proteus spp. and Staphylococcus spp. In the second experiment it was again possible to use NN systems to examine the volatile production patterns and identify 18 of 19 unknown UTI cases. Only one normal patient sample was mis-identified as an E. coli infected sample. Discriminant function analysis also differentiated between normal urine samples, that infected with E. coli and with Staphylococcus spp. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology for the first time. These findings will have implications for the development of rapid systems for use in clinical practice.     

Recognition of anaerobic bacterial isolates in vitro using electronic nose technology

AIMS: Use of an electronic nose (e.nose) system to differentiation between anaerobic bacteria grown in vitro on agar media. METHODS AND RESULTS: Cultures of Clostridium spp. (14 strains) and Bacteroides fragilis (12 strains) were grown on blood agar plates and incubated in sampling bags for 30 min before head space analysis of the volatiles. Qualitative analyses of the volatile production patterns was carried out using an e.nose system with 14 conducting polymer sensors. Using data analysis techniques such as principal components analysis (PCA), genetic algorithms and neural networks it was possible to differentiate between agar blanks and individual species which accounted for all the data. A total of eight unknowns were correctly discriminated into the bacterial groups. CONCLUSIONS: This is the first report of in vitro complex volatile pattern recognition and differentiation of anaerobic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest the potential for application of e.nose technology in early diagnosis of microbial pathogens of medical importance.     

Potential for detection of microorganisms and heavy metals in potable water using electronic nose technology

Studies have been carried out to determine the potential for the detection of different microbial species (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa), alone and in the presence of low concentrations of different heavy metals (As, Cd, Pb and Zn) in bottled, reverse osmosis (RO) and tap water, using an electronic nose. Studies show that it is possible to discriminate control water samples from water contaminated with 0.5 ppm of a mixture of metals. The presence of heavy metals may modify the activity of microorganisms and thus the volatile production patterns. Bacterial species at 10(2)-10(4) colony forming units (CFUs) ml(-1) could be detected after 24 h of incubation. Work is in progress to identify the limits of detection for a range of other microorganisms, including, fungi and cyanobacteria, and chlorinated phenols using electronic nose technology.     

Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. METHODS AND RESULTS: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA. They were heat stable and retained activity even after treatment at 121 degrees C for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.     

The use of gas-sensor arrays to diagnose urinary tract infections

Sensorial analysis based on the utilisation of human senses, is one of the most important and straightforward investigation methods in food and chemical analysis. An electronic nose has been used to detect in vivo Urinary Tract Infections from 45 suspected cases that were sent for analysis in a UK Health Laboratory environment. These samples were analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. An intelligent model consisting of an odour generation mechanism, rapid volatile delivery and recovery system, and a classifier system based on learning techniques has been considered. The implementation of an Extended Normalised Radial Basis Function network with advanced features for determining its size and parameters and the concept of fusion of multiple classifiers dedicated to specific feature parameters has been also adopted in this study. The proposed scheme achieved a very high classification rate of the testing dataset, demonstrating in this way the efficiency of the proposed scheme compared with other approaches. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology.     

Detection and discrimination between ochratoxin producer and non-producer strains of <Emphasis Type="Italic">Penicillium nordicum</Emphasis> on a ham-based medium using an electronic nose

The aim of this work was to evaluate the potential use of qualitative volatile patterns produced by Penicillium nordicum to discriminate between ochratoxin A (OTA) producers and non-producer strains on a ham-based medium. Experiments were carried out on a 3% ham medium at two water activities (a_w ; 0.995, 0.95) inoculated with P. nordicum spores and incubated at 25°C for up to 14 days. Growing colonies were sampled after 1, 2, 3, 7 and 14 days, placed in 30-ml vials, sealed and the head space analysed using a hybrid sensor electronic nose device. The effect of environmental conditions on growth and OTA production was evaluated based on the qualitative response. However, after 7 days, it was possible to discriminate between strains grown at 0.995 a_w, and after 14 days, the OTA producer and non-producer strain and the controls could be discriminated at both a_w levels. This study suggests that volatile patterns produced by P. nordicum strains may differ and be used to predict the presence of toxigenic contaminants in ham. This approach could be utilised in ham production as part of a quality assurance system for preventing OTA contamination.     

Marker enzyme assessment in the liver of cyprinus carpio (L.) exposed to 2,4-D and azinphosmethyl

The potential utility of antioxidant enzymes and lipid peroxidation as indicators of exposure to 2,4-D and azinphosmethyl together with the toxic effects of these compounds in freshwater fish Cyprinus carpio were evaluated. Biochemical parameters were recorded spectrophotometrically in fish liver, which were exposed to a single dose of 2,4-D and azinphosmehtyl (1/3 LC(50)), and their mixture at 1:1 ratio for 24, 48, 72, and 96 h. The most sensitive parameter was glutathione S-transferase (GST) activity, which significantly increased with experimental exposures. Glucose 6-phosphate dehydrogenase activity did not change after 24 and 48 h while there was an elevation after 72 h in all exposure groups. The activity decreased only when these were applied in combination at 96 h. Superoxide dismutase activity increased after azinphosmethyl exposure for 48 and 96 h. 2,4-D decreased the activity after 24 h while the activity remained at the same level with control after 48 h. An elevation was found between 72 and 96 h. Mixture treatment did not changed the activity. Glutathione reductase and catalase enzyme activities, and malondialdehyde levels remained constant in all the treatment groups compared with controls. These results suggest that induction of GST activity may be used as biomarker for the assessment of water pollution in C. carpio.     

Influence of growth conditions on the production of extracellular proteolytic enzymes in Paenibacillus peoriae NRRL BD-62 and Paenibacillus polymyxa SCE2

AIMS: To analyse the extracellular protease profile of two Paenibacillus species, Paenibacillus peoriae and Paenibacillus polymyxa, as well as how different growth media influenced its expression. METHODS AND RESULTS: Both bacteria were cultured in five media [Luria-Bertani broth, glucose broth, thiamine/biotin/nitrogen broth (TBN), trypticase soy broth and a defined medium] for 48 h at 32 degrees C. Our results showed a heterogeneous protease secretion pattern whose expression was dependent on medium composition. However, TBN induced the most quantitative and qualitative protease production on both Paenibacillus. The proteases were detected in neutral-alkaline pH range, being totally inhibited by 1,10-phenanthroline, a zinc-metalloprotease inhibitor. We also analysed the protease expression during the growth and, at least to P. peoriae, the most elevated protease activity was measured at 96 h, in which the highest number of spores and a low concentration of viable cells were observed. CONCLUSIONS: The results presented add P. peoriae and P. polymyxa to the list of neutral-alkaline extracellular protease producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Paenibacillus species are ubiquitous in nature, are capable to form resistant spores and to produce several hydrolytic enzymes, including proteases. However, only few data concerning the production of these enzymes are available. Proteases produced by Paenibacillus strains may represent new sources for biotechnological use.     

取食施硅水稻对褐飞虱成虫体内保护酶和解毒酶活性的影响

【目的】本研究旨在明确褐飞虱Nilaparvata lugens成虫取食施硅水稻对其体内保护酶及解毒酶活性的影响,为硅介导的水稻抗虫性的应用提供证据。【方法】以TN1(感虫品种)为供试水稻品种,采用营养液添加Na₂SiO₃·9H₂O的方法设置112 mg Si/L(Si+)和0 mg Si/L(Si-)2种施硅水平,通过酶活性分析测定取食Si+或Si-水稻植株24, 48, 72和96 h后褐飞虱成虫体内保护酶[过氧化氢酶(catalase, CAT)、过氧化物酶(peroxidase, POD)和超氧化物歧化酶(superoxide, SOD)]以及解毒酶[谷胱甘肽S-转移酶(glutathione-S-transferase, GST)、羧酸酯酶(carboxylesterase, CarE)和多功能氧化酶(mixed-functional oxidase, MFO)]活性的动态变化。【结果】与取食Si-水稻植株的褐飞虱成虫相比,取食Si+水稻植株24和48 h时,褐飞虱成虫POD活性分别显著增加101.2%和55....     

Effect of extender, centrifugation and removal of seminal plasma on cooled-preserved Amiata donkey spermatozoa

In the donkey species, the application of cooled semen artificial insemination could aid the survival of endangered breeds. Fifteen ejaculates collected from three Amiata donkeys were used to evaluate the effect of three extenders on spermatozoal motility characteristics after cooling and preservation for up to 72 h. Semen was diluted at a 1:4 semen:extender ratio in INRA96, INRA82 and INRA82 added of 2% centrifuged egg yolk (INRA82-Y) and motility was evaluated by the computerized analyzer CEROS 12.1 at hours 0, 24, 48 and 72. Total motility, and rapid spermatozoa after 24, 48 and 72 h of preservation were higher in INRA82-Y than in INRA96 or INRA82, as was progressive motility after 72 h. INRA82-Y was thus used in a second study, where the effects of centrifugation and of removal of seminal plasma on cooled donkey semen were evaluated on 12 ejaculates from four males. Rapid spermatozoa after 24 and 72 h, and total motility after 72 h were better preserved in the non-centrifuged samples than when seminal plasma was removed, the contrary was true for the proportion of spermatozoa keeping their progressive motility at hour 48. In conclusion, INRA82-Y kept sperm motility characteristics during cooled storage better than INRA82 or INRA96, and removal of seminal plasma during in vitro preservation did not seemed advantageous. Further studies are needed to better understand the changes in motility patterns of donkey spermatozoa caused by seminal plasma and semen extenders, and their relation to fertility.     

Enzymatic systems involved in decomposition reflects the ecology and taxonomy of saprotrophic fungi

One hundred and eleven strains of Basidiomycota, 39 strains of Ascomycota and 2 strains of Mucoromycotina belonging to wood decomposers that cause white-rot (WR) or brown-rot (BR), other wood associated saprotrophs (WA), litter decomposing cord-forming Basidiomycota (LDF), and saprotrophic microfungi (SA), were screened for the production of hydrolytic enzymes and laccase. The presence of enzyme-encoding genes was also analysed in the published genomes of saprotrophic fungi. Several genes, including those for acidic phosphatase, β-glucosidase and N-acetylglucosaminidase, were common in the genomes with enzyme activity widely displayed by fungi, while other enzymes, such as certain hemicellulases or laccase, were produced less frequently. Enzyme production by saprotrophic fungi was shaped by the combination of their ecophysiology and taxonomy. Basidiomycota exhibited higher activities of all enzymes, except alkaline phosphatase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and α-fucosidase, than Ascomycota. The SA and BR fungi showed distinct enzyme production patterns, while the enzyme production by WR, LDF and WA was similar. Differences among species were typically reflected in the level of enzyme activity rather than in the absence of enzymes. Enzyme screening results showed that in several cases, fungi exhibited enzyme activity without the presence of the corresponding gene and vice versa. This indicates that the use of genome-derived information for the prediction of potential enzyme production has substantial limitations and cannot replace functional screening of fungal cultures.     

Destruxin A production by Metarhizium brunneum strains during transient endophytic colonisation of Solanum tuberosum

Metarhizium spp. are known to produce destruxin A (dtx A) and can act as endophytes. Data regarding the fate and behaviour of secondary metabolites in the environment are necessary for registration. Endophytic colonisation and dtx A production on potato plants were monitored at 24, 48, 72, 96 and 120?h after inoculation with Metarhizium brunneum strains (BIPESCO5 and EAMa 01/58-Su). Both strains were recovered from leaves, stem, tuber and root fragments of fungal-challenged potato plants. Although a similar colonisation was observed for both strains, there were differences in percentages in different parts of the plants, with the higher values occurring in the leaves at 96?h for EAMa 01/58-Su (83.3%) and BIPESCO5 (81.6%), and the lower ones, 10–13.3%, observed in tubers and roots at 72, 96 and 120?h post-inoculation for both strains. For strain EAMa 01/58-Su, dtx A was quantified at 24?h (2.49?±?1.7 and 2.0?±?1.4?µg/kg, respectively), and the same concentration was found in both tuber and root at 96?h (2.5?±?1.7?µg/kg); for BIPESCO5, the concentrations differed in tuber at 24?h and in root at 48?h (6.8?±?4.8 and 2.1?±?1.4?µg/kg, respectively). The concentration of dtx A in plant tissues was very low compared to the colonisation levels, suggesting that dtx A production by the fungus may be temporary and that the compound might degrade rapidly.     

Growth characteristics and glucose oxidase production by mutant Penicillium funiculosum strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.     

南极抗细菌活性菌株的筛选及系统发育分析

分别以大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、青枯假单胞菌、绿脓假单胞菌和苏云金芽孢杆菌为指示菌,采用琼脂扩散法对实验室保存的580株极地细菌进行了抗菌活性菌株的筛选与活性验证,从中筛选出4株对上述指示菌株具有明显抗菌效果的活性菌株,其编号分别为97、Z11、Z18及Z19,并对其生长曲线、抗菌活性曲线和系统发育地位进行研究。结果表明,4株菌均在培养24 h后进入指数生长期,菌株97在培养48 h后达到稳定期,而菌株Z11、Z18及Z19在培养60 h后达到稳定期。抗菌活性分别在培养84、96、72和72 h时达到最高。系统发育分析表明,该4株菌分别属于伦黑墨氏菌属(Rheinheimera)、嗜冷杆菌属(Psychrobacter)、假单胞菌属(Pseudomonas)和嗜冷杆菌属(Psychrobacter)。     

Relationship between macrotetrolide production and specific activity of some hydrolases in a high and a low producing strain of Streptomyces griseus

The activities of five hydrolytic enzymes in the culture filtrate and in cell-free extracts from strains of Streptomyces griseus, differing in macrotetrolide production, have been determined over a fermentation period of 200 h. The specific activities of phosphatase, phosphodiesterase, and adenosine triphosphatase in the medium, and phosphatase and phosphodiesterase in the cell-free extract were lower in the low than in the high producing strain. No significant difference was found between the strains, for adenosine triphosphatase and protease activity in the cell-free extract or protease activity in the medium. The specific activity of esterase was higher in the low than in the high producing strain.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.