Mould Strains Research Articles

Identification of biological characteristics and preliminary research on genetic transformation of the Aspergillus niger TL8 strain isolated in Vietnam

Aspergillus niger is a mold commonly used in industrial production of many enzymes and organic acids. Because this fungus can produce different extracellular enzymes to degrade plant materials, it also causes the damages for some agricultural products at postharvest stages. In this study, we isolated a black mold strain named TL8 from a decayed dragon fruit. Based on morphological characteristics and the rDNA ITS (internal transcribed spacer) sequence, the TL8 strain was identified as A. niger. The A. niger TL8 strain is able to use different carbon sources for the growth and decay the peel of dragon fruits in vitro. In order to establish the basis for future studies on the mechanism of plant material decomposition of the fungus, we have successfully transferred and expressed the GFP reporter gene in this A. niger strain using the Agrobacterium tumefaciens-mediated transformation method and the hygromycin resistance marker.

Thin antimicrobial silver, copper or zinc containing SiOx films on wood polymer composites (WPC) applied by atmospheric pressure plasma chemical vapour deposition (APCVD) and sol–gel technology

Microbial colonization of wood and wood polymer composites (WPC) by bacteria and mould can cause aesthetic damage, negative impact on mechanical properties or even health hazards. This work investigated the structural morphology, elemental composition and antimicrobial properties of thin functional silicon oxide films deposited on WPC using atmospheric pressure chemical vapour deposition (APCVD) and sol–gel technology. Scanning electron microscopy results indicated the creation of flat, thin and closed films on top of the surface of this material. By embedding of active agents such as silver, copper or zinc, the APCVD films became slightly rougher. The corresponding sol–gel-films showed a certain number of cracks. The elemental composition of the films was investigated by X-ray photoelectron spectroscopy and depth profiling indicating the integration of active agents into the created SiOx film matrices. Moreover, the possibility to generate combinations of active agents in one layer could be demonstrated. BacTiter-Glo® tests as a part of antibacterial investigations showed strong bactericidal properties for silver and copper as well as sufficient properties for zinc-containing layers against Escherichia coli. Antimycotic effects on mould strains Aspergillus niger, Paecilomyces variotii, Penicillium funiculosum and Trichoderma viride were low for films containing only one active agent. Significant improvements in activity were achieved by the combination of these active agents.

Analysis of Mold Overall Deformation and Contact Stress During Biomass Cold Compression Molding

Ansys is adopted to analyze mold deformation and stress field distribution rule during the process of compressing biomass under pressure of 20Mpa. By means of unit selection, material property setting, mesh partition, contact pair establishment, load and constraint applying, and solver setting, the stress and strain of overall mold are analyzed; meanwhile, the size as well as distribution of various contact stresses is explored. The mold deformation is small while the displacement of compression bar and compressed fuel is larger. Since displacement is the manifestation of overall deformation situation, the lower part of fuel will definitely be lower than that of upper part with the compaction of fuel, but the displacement of compression bar is the largest. Overall equivalent pressure diagram indicates that, the pressure on compression bar is the largest, followed by compressor; The pressure at the contact place of compressor and base is larger while other places is smaller so that it decreases in the form of semi circle. The contact stresses are various in different places of contact pair and the difference in some is large. However, fuel compression is a repetitive periodic process, so each contact pair will generate alternating stress, under which fatigue phenomenon may quickly occur in mold.

A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry.

The source tracking of fungal contamination in the food industry is an important aspect of food safety. Currently, all available methods are time consuming and require the use of a reference library that may limit the accuracy of the identification. In this study, we report for the first time, a library-independent FTIR spectroscopic approach for MST of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on the genus and species level. Therefore, such an approach possesses great importance for food safety control in food industry.

Microbial Diversity in Daqu during Production of Luzhou Flavored Liquor

Yeast, bacterial, and mold strains in different stages during the daqubased production of Luzhou-flavored liquor were isolated and identified by analyzing the 26S rRNA, 16S rRNA, and internal transcribed spacer region sequences, respectively. In total, 113 yeast, 63 bacteria, and 128 mold strains were identified, with the majority identified to the species level and the remaining strains identified to the genus level. In the final daqu product (day 90), the dominant bacteria, yeast, and mold strains were Bacillus subtilis subsp. inaquosorum, Saccharomycopsis fibuligera, and Lichtheimia ramosa, respectively. Thus, in the finished product, the daqu microflora mainly comprised bacteria and molds, mostly consisting of heat-resistant spore-forming Bacillus spp. and filamentous fungi. Corresponding changes in the enzyme activity patterns during daqu production revealed that Mucor circinelloides f. circinelloides was strongly correlated with protease, whereas saccharifying enzyme activity was mainly correlated with Rhizopus oryzae. A relatively strong correlation was found between saccharifying and liquefied enzyme activities and Staphylococcus saprophyticus subsp. saprophyticus, Bacillus tequilensis, Weissella cibaria, and Enterobacter mori. These results indicate the benefits of molecular methods for detailed microbial isolation and identification and provide basic information for subsequent studies to reveal the functional microorganisms of daqu during the production of Luzhou-flavored liquor.

IMPACT OF DISPLAY AND HANDLING ON THE CHEMICAL AND MICROBIAL CONTENT OF BEEF FROZEN LIVER

A total of 60 beef frozen liver samples from different market retail from different origins in Kafr-El-sheikh Governorate were collected then transferred immediately to the laboratory under aseptic conditions. These collected samples were subjected to chemical, bacteriological and mycological analysis. The obtained results revealed that the mean values of pH and TVN-B (Total Volatile Nitrogen) in examined frozen liver samples were 5.54±0.01 and 6.99±0.28mg/100gm, respectively. While in microbial analysis the Staphylococcus aureus was isolated from 44 samples (66.7%), and there were two strains have the ability to produce enterotoxins (A), only one strain can produce enterotoxine (B), one strain can produce enterotoxine (B&D) and one strain produce enterotoxine (A&D). The percentage of other Staphylococci were Staph saprophyticus (12.5%), Staph epidermidis (23.8%), Staph intermedius (7.5%) and Staph capitis (2.5%). Also the mold were isolated from 80% from the examined frozen liver samples and the isolated mold strains were identified as 24 A.niger (26.1%) , 10 A. flavus (10.9%), 8 A. fumigatus (8.7%), 10 Penicillium (10.9%), 6 Fusarium Spp . (6.5%), 6 Rhizopus Spp . (6.5%), 6 Mucor Spp . (6.5% ), 6 Thamnidium Spp . (6.5%), 4 Nigrospora Spp .(4.3%) and 2 Trichothecium Spp . (2.2%). Multiplex PCR was done to detect the virulence of isolated strains of A.flavus and found that the Lane(1) was Positive A. flavus strain for nor-1 and omtA genes, Lane(2) was Positive A. flavus strain for nor-1, ver-1, omtA and aflR genes, Lane(3) was Positive A. flavus strain for nor-1 gene, Lane(4) was Positive A. flavus strain for nor-1, ver-1 and aflR genes and Lane(5) was Positive A. flavus strain for nor-1, ver-1 and omtA genes. In conclusion, the obtained results indicate that consumption of frozen liver may cause a public health hazard to the consumer. Measures to control the quality of beef frozen liver during display and handling should be taken.

Characterization of Aspergillus sojae Isolated from Meju, Korean Traditional Fermented Soybean Brick.

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and α-amylase with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.

Screening of Microorganisms and Raw Materials for Lipase Production by Solid-State Fermentation

The production of biodiesel from vegetable oils using eco-friendly processes is a hot topic actually. These processes are based on enzymatic biocatalysts, namely lipases, and present many advantages over classical processes i.e. they do not require the use of sodium hydroxide, nor huge quantities of water. Lipases are widespread in nature, being produced by many microorganisms. However, fungal lipases have benefits over bacterial lipases due to their low cost of production, thermal and pH stability, substrate specificity and activity in organic solvents. These low cost production processes rely, most of the time, on solid-state fermentation (SSF). The aim of this research was to select microorganisms for their ability to secrete lipolytic enzymes and to grow on a solid support compatible with SSF. Thirty-five yeast and mold strains were tested in term of growth rate and extracellular lipase production. Different solid support such as vermiculite, crushed wheat husk, cacao seed-husk and carbon sources such as soy oil, sunflower oil, olive oil or sucrose were also tested for their ability to support cell growth and lipase production.

Dominant Selectable Markers for Penicillium spp. Transformation and Gene Function Studies

Penicillium spp. has been genetically manipulated and gene function studies have utilized single gene deletion strains for phenotypic analysis. Fungal transformation experiments have relied on hygromycin and hygromycin phosphotransferase (hph) as the main dominant selectable marker (DSM) system in Penicillium spp. This poses a limitation on the number of loci that can be analyzed and complemented in reverse genetic studies. Additionally, many economically important Penicillium spp. have not been evaluated to determine the utility of additional chemicals that can serve as DSMs. Therefore, six compounds were examined for 15 blue mold strains and their Minimum Inhibitory Concentrations (MICs) determined. Phleomycin, neomycin and G418 were deemed ineffective, as Penicillium spp. growth was observed on media amended with 1000 μg/ml of each compound. The efficacy of bialophos to inhibit fungal growth was intermediate, with MICs ranging from 250 to 1000 μg/ml and was species-dependent. However, chlorimuron ethyl and benlate had the lowest MIC values and minimal variation in efficacy within and between species. Therefore, benlate and chlorimuron ethyl are good candidates for use as since corresponding fungal resistance genes have been cloned, characterized and are available from a variety of public and academic sources.

Nadzor nad mikrobiološkim statusom zraka u zdravstvenoj ustanovi u prostorima za sterilizaciju

SUMMARY: Microorganisms in the air are part of the bioaerosol which is composed of different particles of biological substances, dust particles, bacteria cells and their portions, mould spores and a number of by-products of their metabolism. This research focuses on analysing the primary microclimate indicators (temperature and relative humidity) and the presence of microorganisms in the work environment (air). Measurements were taken after the process of sterilisation of infective waste over three months with the objective to determine the total number of aerobic mesophilic bacteria and the total number of moulds. The microclimate indicators were measured with an attested device. The study monitored the microbiological purity of the air in a given place using the sedimentation method, with samples subsequently processed in a microbiology lab in keeping with the prescribed standards. After incubation and following the counting of developed colonies on microbiological substrates (Tryptic soy agar and Sabouraud dextrose agar), different strains of bacteria and moulds were identified. The most common were Gram (+) sporogenes bacteria Micrococcus spp and Staphylococcus spp., and as for fungi the most commonly found were Aspergillus spp and Penicillium spp. It was found that temperature and relative humidity affect the total number of aerobic mesophilic bacteria and fungi which was confirmed using the Wilcoxon equivalent pairs test at the level of statistic significance p<0.05.

Engineering Aspergillus niger for galactaric acid production: elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9.

Background meso-Galactaric acid is a dicarboxylic acid that can be produced by the oxidation of d-galacturonic acid, the main constituent of pectin. Mould strains can be engineered to perform this oxidation by expressing the bacterial enzyme uronate dehydrogenase. In addition, the endogenous pathway for d-galacturonic acid catabolism has to be inactivated. The filamentous fungus Aspergillus niger would be a suitable strain for galactaric acid production since it is efficient in pectin hydrolysis, however, it is catabolizing the resulting galactaric acid via an unknown catabolic pathway.ResultsIn this study, a transcriptomics approach was used to identify genes involved in galactaric acid catabolism. Several genes were deleted using CRISPR/Cas9 together with in vitro synthesized sgRNA. As a result, galactaric acid catabolism was disrupted. An engineered A. niger strain combining the disrupted galactaric and d-galacturonic acid catabolism with an expression of a heterologous uronate dehydrogenase produced galactaric acid from d-galacturonic acid. The resulting strain was also converting pectin-rich biomass to galactaric acid in a consolidated bioprocess.ConclusionsIn the present study, we demonstrated the use of CRISPR/Cas9 mediated gene deletion technology in A. niger in an metabolic engineering application. As a result, a strain for the efficient production of galactaric acid from d-galacturonic acid was generated. The present study highlights the usefulness of CRISPR/Cas9 technology in the metabolic engineering of filamentous fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0613-5) contains supplementary material, which is available to authorized users.

Infections in the tissue material and their impact on the loss of transplants in the Laboratory of in vitro Cell and Tissue Culture with Tissue Bank in the years 2011-2015.

Radiation sterilization eliminates microbiological infections but causes the degradation of the cell factor. The negative result of microbiological examination for tissue transplants is one of the conditions for approval for distribution in patients. The study attempts to verify impact of the presence of microbes onto material for transplant loss. In the 2011-2015 period, we analyzed 293 donors of skin and amnion. Microbiological sampling was performed. The total of 21 strains of bacteria, molds and fungi was identified in collected tissue. The widest spectrum of strains was found in skin (17), followed by amnia (8). The total number of positive findings was 147 and was again highest in skin (129), while the number of positive findings in amnia was 18 only. The general percentage of fungal infections was very low. The presence of fungal strains was only observed in allogeneic skin (2%). Large number of microorganisms isolated from the skin before sterilization was observed, so it seems impossible to use allogeneic intravital skin. However, the intravital application of allogeneic amnion obtained from cesarean section remains to be considered.

Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production

Speck is a meat product obtained from the deboned leg of pork that is salted, smoked and seasoned for four to six months. During speck seasoning, Eurotium rubrum and Penicillium solitum grow on the surface and collaborate with other moulds and tissue enzymes to produce the typical aroma. Both of these strains usually predominate over other moulds. However, moulds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can also co-grow on speck and produce ochratoxin A (OTA). Consequently, speck could represent a potential health risk for consumers. Because A. ochraceus and P. nordicum could represent a problem for artisanal speck production, the aim of this study was to inhibit these mould strains using Debaryomyces hansenii and Saccharomycopsis fibuligera. Six D. hansenii and six S. fibuligera strains were tested in vitro to inhibit A. ochraceus and P. nordicum. The D. hansenii DIAL 1 and S. fibuligera DIAL 3 strains demonstrated the highest inhibitory activity and were selected for in vivo tests. The strains were co-inoculated on fresh meat cuts for speck production with both of the OTA-producing moulds prior to drying and seasoning. At the end of seasoning (six months), OTA was not detected in the speck treated with both yeast strains. Because the yeasts did not adversely affect the speck odour or flavour, the strains are proposed as starters for the inhibition of ochratoxigenic moulds.

Effects of different drying treatments on fungal population and ochratoxin A occurrence in sultana type grapes

ABSTRACTThis study aimed to determine the changes in mould and ochratoxin A (OTA) occurrence in sultanas under three different conventional drying conditions. Five different vineyards were chosen, and the three different treatments were applied to these grapes while drying. At the end of the drying process, total mould and black aspergilli (BA) populations in the samples varied from 2.45 to 5.61 log colony-forming units (CFU) g–1 and from 0 to 4.92 log CFU g–1, respectively. Significant increases (p < 0.05) occurred in mould loads depending on the extending drying period. However, independent of vineyard location, all the samples treated with cold dipping solution showed the lowest fungal loads. These results indicate that dipping solution treatment was the most effective drying method to minimise fungal infection of grapes. The expected results could not be achieved by drying grapes artificially contaminated with ochratoxigenic Aspergillus carbonarius spores. Seventy-one of 96 isolates (73.95%) obtained during drying were Aspergillus spp., and the remaining (n = 25, 26.05%) belonged to other genera, such as Penicillium, Trichoderma and Cladosporium. Grape juice-based agar medium was used to determine the realistic OTA production capacities of the isolated mould strains. The highest OTA production capacities were 809.70 ± 9.19, 87.58 ± 16.89 and 45.44 ± 18.78 ng g–1 in 50% grape juice agar (GJ50), all five of which were from A. niger isolates. OTA was not present in any sample during the drying period; however, OTA was detected in two samples at 0.32 ± 0.15 and 0.52 ± 0.36 µg kg–1 after the end of the drying process. The limit of detection (LOD) and limit of quantitation (LOQ) of the method used for detecting OTA in samples were 0.1 and 0.3 µg kg–1, respectively.

Identification and control of moulds responsible for black spot spoilage in dry-cured ham

The aims of this work were to identify moulds responsible for black spot spoilage in the drying and cellar stages of dry-cured ham processing and evaluate the effectiveness of preventive actions for controlling this alteration. Four mould strains isolated from spoiled hams were identified by morphological characteristics and the ITS and β-tubulin sequencing. Two of them were Cladosporium oxysporum, one was C. cladosporioides and the remaining one was C. herbarum. These spoiling strains reproduced the black spots on dry-cured ham-based media and ham slices. Additionally, the effect of water activity (aw) conditions reached throughout dry-cured ham ripening and the activity of the protective culture Penicillium chrysogenum CECT 20922 against the spoiling moulds were evaluated. In the dry-cured ham model system the growth of the Cladosporium strains was minimised when the aw approaches 0.84 or in P. chrysogenum CECT 20922 inoculated dry-cured ham slices. Therefore such combination could be used to avoid the black spot formation in dry-cured ham.

Nghiên cứu chế tạo sản phẩm chống nấm mốc cho giày da quân nhu từ nguyên liệu quế

This paper shows the process of preparation of an anti-mold product containing α-brom cinnamaldehyde transformed from Vietnam cinnamon oil. The obtained α-brom cinnamaldehyde in the product is confirmed by modern chemical physic methods such as: IR, 1H-NMR, 13C-NMR. 6 strains of mould on military leather shoes are isolated and identified by morphology characters. The obtained product is examined by acceleration test method. The result indicates strong inhibitory effect of α-brominated cinnamaldehyde on the development of the mould destroying military leather shoes.

Characterization and amylolytic activity of yeast and mold strains from Thai sweet rice

Characterization and amylolytic activity of yeast and mold strains from Thai sweet rice

Fungal infection of cystic fibrosis patients - single center experience.

Cystic fibrosis (CF) is the most common monogenetic autosomal recessive disease in the human population. This systemic disease is characterized by changes in multiple organs, mainly in the lung tissue and digestive tract. More than 59% of CF patients become sensitized to fungal spores, mostly Aspergillus fumigatus. 5-15% of CF patients develop allergic bronchopulmonary aspergillosis. The aim of the study was to analyse the occurrence of yeast and filamentous fungi of the respiratory infections in CF patients and evaluation of drug resistance. Between 2006 and 2014, mycological evaluation of 42 patients hospitalized at the National Institute of Tuberculosis and Lung Diseases was carried out. 217 specimens from pulmonary tract were collected from 42 patients with cystic fibrosis. 205 (68%) strains of yeast and 96 (32%) filamentous fungi strains were cultured. The most common mould strain was A. fumigatus - 22,2% (67 species). All isolates of filamentous fungi were in vitro 100% susceptible to itraconazole, voriconazole, posaconazole and amphotericin B. A. fumigatus and C. albicans were the most common etiological agents of fungal respiratory pathogens associated with CF patients. A. fumigatus strains were in vitro 100% susceptible to azole and amphotericin B. Two strains of C. albicans and one strain of C. tropicalis were non-susceptible to azole (fluconazole, itraconazole and voriconazole). Scedosporium apiospermum was resistant to amphotericin B (MIC > 32 mg/l) and susceptible to voriconazole (MIC 0.094 mg/l).

High-throughput screening of high Monascus pigment-producing strain based on digital image processing.

This work proposed a new method which applied image processing and support vector machine (SVM) for screening of mold strains. Taking Monascus as example, morphological characteristics of Monascus colony were quantified by image processing. And the association between the characteristics and pigment production capability was determined by SVM. On this basis, a highly automated screening strategy was achieved. The accuracy of the proposed strategy is 80.6 %, which is compatible with the existing methods (81.1 % for microplate and 85.4 % for flask). Meanwhile, the screening of 500 colonies only takes 20-30 min, which is the highest rate among all published results. By applying this automated method, 13 strains with high-predicted production were obtained and the best one produced as 2.8-fold (226 U/mL) of pigment and 1.9-fold (51 mg/L) of lovastatin compared with the parent strain. The current study provides us with an effective and promising method for strain improvement.

The Wine: Typicality or Mere Diversity? The Effect of Spontaneous Fermentations and Biotic Factors on the Characteristics of Wine

Abstract Wine is probably one of the main fermented beverages for which the recognition of the “territoriality” is fundamental for its appreciation. The sensory profile of wine is significantly affected by microbial activities, and indigenous microorganisms may significantly contribute to the expression of wine typicality. The microbial ecology of wines is complex and includes several species and strains of yeasts, bacteria and molds. Several works showed the positive effects of spontaneous fermentations on the quality of wine as a consequence of the growth of different species and/or strains together at high levels. Furthermore, a new style of “natural” winemaking is gaining importance, since the resulting wines are obtained thanks to the action of spontaneous autochthonous agents and the use of chemical addition is not allowed. In this contest, natural winemaking could provide enhanced opportunities for products with unique characters and popularly recognized as typical. The present work reports on microbial ecology and molecular profile characterizing natural large-scale vinifications, and an innovative procedure, named “fortified pied de cuve”, to accelerate the alcoholic fermentation performed spontaneously is also reported. Furthermore, this work reports on how the biotic factors, such as migratory birds, contribute in disseminating of wine-related yeasts over long distances, opening up new fields of research that will allow to unravel connection between wine and environmental factors.