Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Antifungal effect of gaseous nitric oxide on mycelium growth, sporulation and spore germination of the postharvest horticulture pathogens, Aspergillus niger, Monilinia fructicola and Penicillium italicum

Aim:  To evaluate the antifungal activity of nitric oxide (NO) against the growth of the postharvest horticulture pathogens Aspergillus niger , Monilinia fructicola and Penicillium italicum under in vitro conditions.
Methods and Results:  Different volumes of NO gas were injected into the Petri dish headspace to obtain the desired concentrations of 50–500  μ l l⁻¹ . The growth of the fungi was measured for 8 days of incubation in air at 25°C . All concentrations of NO were found to produce an antifungal effect on spore germination, sporulation and mycelial growth of the three fungi, with the most effective concentration for A. niger and P. italicum being 100 and 500  μ l l⁻¹ for M. fructicola .
Conclusions:  Short-term exposure to a low concentration of NO gas was able to inhibit the subsequent growth of A. niger , M. fructicola and P. italicum .
Significance and Impact of the Study:  NO gas has potential use as a natural fungicide to inhibit microbial growth on postharvest fruit and vegetables.     

OTA-producing fungi isolated from stored cocoa beans

Aims:  The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates.
Methods and Results:  Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [−1 culture medium; LOD = 6  μ g kg⁻¹ culture medium].
Conclusions:  No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin.
Significance and Impact of the Study:  Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.     

Metabolism of the soy isoflavones daidzein and genistein by fungi used in the preparation of various fermented soybean foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, (1)H-, and (13)C-NMR spectra.     

Screening of some wild fungal isolates for cellulolytic activities

Six wild fungal strains, Trichoderma viride, T. harzianum, Gliocladium virens, Aspergillus terreus, A. niger and Tiarosporella phaseolina , isolated from decomposed jute stacks and diseased jute stem, were tested for their cellulolytic and hemicellulolytic activities and compared with T. reesei MCG 77. Filter paper cellulase production by all these wild strains were lower than those produced by T. reesei while some strains ( T. viride, T. harzianum and G. virens ) possessed carboxymethyl cellulase, β-glucosidase, xylanase and β-xylosidase activities comparable to T. reesei. A. terreus and A. niger produced 3·2 and 1·2 times respectively, greater β-glucosidase activity compared to T. reesei when grown on microcrystalline cellulose.     

Feeding ecology of the freshwater detritivore Ptychoptera paludosa (Diptera, Nematocera)

1. We examined selected aspects of the nutritional ecology of larval Ptychoptera paludosa and their role in nutrient cycling in the Breitenbach, a first-order stream in Hesse, Germany.
2. Food preference experiments demonstrated significant preference for sediments with a high organic matter content and live bacteria.
3. pH was circumneutral in all sections of the gut.
4. Enzymatic activity (β-glucosidase and amino-peptidase) in different parts of the gut was measured using 4-methylumbelliferyl-β- D -glucopyranoside (MUF-Glc) and leucine-4-methylcoumarinyl-7-amide (Leu-MCA). β-glucosidase activity was highest in the hindgut.
5. The mean larval gut passage time was between 7 and 8 h.
6. The egestion rate of last instar larvae was about 0.35 mg dry weight (DW) faeces per larva h^–1 and about 1.25 mg ash-free dry weight (AFDW) faeces per mg larval AFDW day^–1.
7. Larval faeces contained at least 4–18 times more organic matter than the average in the sediments in which they were feeding, that is, larvae fed selectively, extracting organic matter from sediments.
8. P. paludosa larvae are important in the dynamics of detritus in slow-flowing reaches of the Breitenbach. They gather organic material from the sediment to a depth of 3 cm, and release it as faeces onto the sediment surface. A total of 770 g DW faeces m^–2 yr^–1, comprising about 16% organic matter, was produced by the Ptychoptera population.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

Enzyme productivity from the protoplast regenerants of Aspergillus awamori

Protoplasts from the hydroiase enzyme producer Aspergillus awamori were isolated using Bulgarian enzyme preparations—Trichocease-SU and Xylanase. Among the regenerated colonies from protoplasts two were selected with enhanced productivity of xylanase, endoglucanase and β-glucosidase. The endoglucanase produced by the parent and one of the regenerants differ in their absorptivity, sensitivity to product inhibition and heat stability.     

Isolation of the antioxidant pyranonigrin-A from rice mold starters used in the manufacturing process of fermented foods

Rice mold starters prepared from Aspergillus species are commonly used for the manufacture of koji in the production of oriental fermented foods. Methanol extracts of rice mold starters fermented by the Aspergillus species, A. awamori, A. kawachii, A. oryzae, A. saitoi, and A. sojae, were examined for their antioxidative activity by using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging system. The extracts of A. awamori, A. kawachii, and A. saitoi exhibited higher activity than those of A. oryzae and A. sojae. An antioxidant was isolated from the extract of A. saitoi and identified as pyranonigrin-A by 1H-NMR, 13C-NMR, and FAB-MS analyses. The antioxidative activity of pyranonigrin-A was approximately equivalent to that of ferulic acid, an antioxidant in cereal grain. It was present in rice mold starters prepared by A. awamori, A. kawachii, and A. saitoi, although there was no pyranonigrin-A in the A. oryzae and A. sojae starters. The results suggest that the content of pyranonigrin-A in rice mold starters has a correlation with the antioxidative activity, and that it is induced in rice mold starters at the sporulation stage.     

Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments

Abstract β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β- d -glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity ( V _max) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity( K m)was higher in the suspended sediments. V _maxand K _m both increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. V _max was uninfluenced by whether ground or stream water was perfused through the sediments, but K _m was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.     

Aspergillus niger var. taxi, a new species variant of taxol-producing fungus isolated from Taxus cuspidata in China

Aims:  To characterize and identify a new taxol-producing fungal strain HD86-9 isolated from Taxus cuspidata in China.
Methods and Results:  Taxol extracted from strain HD86-9 was identified by HPLC and MS analyses. Strain HD86-9 was cultured and its morphology and phenotypes were described. HD86-9 displayed morphology most similar to that of Aspergillus niger but presented differences in the shape and size of the conidia. The growth evaluation showed that the maximal tolerable temperature of the new strain was 43°C, higher than that of the model Aspergillus niger . The 18S rDNA and the internal transcribed spacer region including the 5·8S rDNA of HD86-9 were amplified by PCR; molecular analysis of these sequences revealed their high similarity of 98% to those of Aspergillus niger .
Conclusions:  The morphology and molecular analysis identified HD86-9 as a new variant of taxol-producing endophytic fungi, and it was named Aspergillus niger var. taxi D.P. Zhou, K. Zhao and W.X. Ping, var. nov.
Significance and Impact of the Study:  As the first report of a taxol-producing variant of Aspergillus niger species, this study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Aims:  The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants.
Methods and Results:  The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l⁻¹ of FaeB was selected (12-fold more than the A. niger overproducing strain).
Conclusions:  This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days.
Significance and Impact of the Study:  This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.     

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine

Aims:  To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum ) in wine fermentation.
Methods and Results:  Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose⁻ and Melibiose⁺ strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum ). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and β-glucosidase activities were assayed on whole cells during fermentation at 13° and 20°C. Saccharomyces uvarum displayed higher esterase activity at 13°C, while S. cerevisiae displayed higher β-glucosidase activity at both temperatures.
Conclusions:  The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine.
Significance and Impact of the Study:  The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.     

Influence of relative gas humidity on the inactivation efficiency of a low temperature gas plasma

Aims:  To investigate the effect of relative gas humidity on the inactivation efficiency of a cascaded dielectric barrier discharge (CDBD) in air against Aspergillus niger and Bacillus subtilis spores on PET foils.
Methods and Results:  The inactivation kinetics as a function of treatment time were determined using synthetic air with different relative humidity as the process gas. Spores of A. niger and B. subtilis respectively were evenly sprayed on PET foils for use as bioindicators. In the case of A. niger, increased spore mortality was found at a high relative gas humidity of 70% (approx. 2 log₁₀). This effect was more evident at prolonged treatment times. In contrast, B. subtilis showed slightly poorer inactivation at high gas humidity.
Conclusions:  Water molecules in the process gas significantly affect the inactivation efficiency of CDBD in air.
Significance and Impact of the Study:  Modifying simple process parameters such as the relative gas humidity can be used to optimize plasma treatment to improve inactivation of resistant micro-organisms such as conidiospores of A. niger .     

The stability of extracellular β-glucosidase fromAspergillus niger is significantly enhanced by non-covalently attached polysaccharides

The removal of noncovalently bound polysaccharide coating from the extracellular enzymes ofAspergillus niger, by the technique of compartmental electrophoresis, had a very dramatic effect on the stability of β-glucosidase. The polysaccharide-β-glucosidase complex was extremely resistant to proteinases and far more stable against urea and temperature as compared with polysaccharide-free β-glucosidase. The β-glucosidase-polysaccharide complex was 18-, 36-, 40-, and 82-fold more stable against chymotrypsin, 3 mol/L urea, total thermal denaturation and irreversible thermal denaturation, respectively, as compared with polysaccharide-free β-glucosidase. The activation energy of polysaccharide-complexed β-glucosidase (55 kJ/mol) was lower than polysaccharide-free enzyme (61 kJ/mol), indicating a slight activation of the enzyme by the polysaccharide. No significant difference could be detected in the specificity constant (V/K _m) for 4-nitrophenyl β-d-glucopyranoside between polysaccharide-free and polysaccharide-complexed β-glucosidase. We suggest that the function of these polysaccharides secreted by fungi includingA. niger might be to protect the extracellular enzymes from proteolytic degradation, hence increasing their life span.     

Enhancement of β-galactosidase productivity of Aspergillus niger NCIM-616

A series of mutations was carried out with Aspergillus niger NCIM-616 as the parent strain. A mutant strain NG-4 with 28% increased β-galactosidase productivity was produced with N -methyl- N -nitro- N -nitrosoguanidine (NTG) at 1500µg ml^-1 concentration and exposure timeof 60 min. This mutant yielded a third generation u.v.-treated strain, UV—5 with a 117·6% increase in β-galactosidase productivity with respect to the parent strain.     

Thiamine increases β-glucosidase production in the newly isolated strain of Fomitopsis pinicola

Aims:  To isolate a high β-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain.
Methods and Results:  A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l⁻¹) improved BGL production in F. pinicola cultures by 3·7-fold to give a specific activity of 114·4 μmol min⁻¹ mg⁻¹ protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported.
Conclusion:  Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812.
Significance and Impact of the Study:  Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.