Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production.

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways - hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained - one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in ß-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

Selection of Non-Mycotoxigenic Inulinase Producers in the Group of Black Aspergilli for Use in Food Processing.

Research background: Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach: Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions: The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution: The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

Antioxidative Responses of Duckweed (Lemna minor L.) to Phenol and Rhizosphere-Associated Bacterial Strain Hafnia paralvei C32-106/3.

Duckweed (L. minor) is a cosmopolitan aquatic plant of simplified morphology and rapid vegetative reproduction. In this study, an H. paralvei bacterial strain and its influence on the antioxidative response of the duckweeds to phenol, a recalcitrant environmental pollutant, were investigated. Sterile duckweed cultures were inoculated with H. paralvei in vitro and cultivated in the presence or absence of phenol (500 mg L-1), in order to investigate bacterial effects on plant oxidative stress during 5 days. Total soluble proteins, guaiacol peroxidase expression, concentration of hydrogen peroxide and malondialdehyde as well as the total ascorbic acid of the plants were monitored. Moreover, bacterial production of indole-3-acetic acid (IAA) was measured in order to investigate H. paralvei's influence on plant growth. In general, the addition of phenol elevated all biochemical parameters in L. minor except AsA and total soluble proteins. Phenol as well as bacteria influenced the expression of guaiacol peroxidase. Different isoforms were associated with phenol compared to isoforms expressed in phenol-free medium. Considering that duckweeds showed increased antioxidative parameters in the presence of phenol, it can be assumed that the measured parameters might be involved in the plant's defense system. H. paralvei is an IAA producer and its presence in the rhizosphere of duckweeds decreased the oxidative stress of the plants, which can be taken as evidence that this bacterial strain acts protectively on the plants during phenol exposure.

Superior cellulolytic activity of Trichoderma guizhouense on raw wheat straw.

Lignocellulosic plant biomass is the world's most abundant carbon source and has consequently attracted attention as a renewable resource for production of biofuels and commodity chemicals that could replace fossil resources. Due to its recalcitrant nature, it must be pretreated by chemical, physical or biological means prior to hydrolysis, introducing additional costs. In this paper, we tested the hypothesis that fungi which thrive on lignocellulosic material (straw, bark or soil) would be efficient in degrading untreated lignocellulose. Wheat straw was used as a model. We developed a fast and simple screening method for cellulase producers and tested one hundred Trichoderma strains isolated from wheat straw. The most potent strain-UB483FTG2/ TUCIM 4455, was isolated from substrate used for mushroom cultivation and was identified as T. guizhouense. After optimization of growth medium, high cellulase activity was already achieved after 72 h of fermentation on raw wheat straw, while the model cellulase overproducing strain T. reesei QM 9414 took 170 h and reached only 45% of the cellulase activity secreted by T. guizhouense. Maximum production levels were 1.1 U/mL (measured with CMC as cellulase substrate) and 0.7 U/mL (ß-glucosidase assay). The T. guizhouense cellulase cocktail hydrolyzed raw wheat straw within 35 h. Our study shows that screening for fungi that successfully compete for special substrates in nature will lead to the isolation of strains with qualitatively and quantitatively superior enzymes needed for their digestion which could be used for industrial purposes.

Application of new insoluble dietary fibres from triticale as supplement in yoghurt - effects on physico-chemical, rheological and quality properties.

BACKGROUND: The need to increase the daily intake of dietary fibres opens a new chapter in the research of functional foods enriched with fibres. The potential application of an innovative product - insoluble dietary fibres from triticale in yoghurts - was deployed by characterising their food application and evaluating physico-chemical, rheological and sensory properties and was the aim of this research. RESULTS: Detailed characterisations of these fibres are presented for the first time and showed very good hydration properties, optimal pH (slightly acidic), optimal chemical composition, high antioxidant capacity which was proven by phenolics contents. Besides, these fibres showed negligible calorific value, with no phytates and high antioxidant capacity, mainly from ferulic acid. Therefore they could be successfully added to yoghurt. Enrichment of yoghurt having different milk fat content (1.5 and 2.8% w/w) with triticale insoluble fibre (1.5% and 3.0% w/w) significantly influenced the syneresis level, its apparent viscosity, yield stress and thixotropic behaviour. The overall sensory quality scores indicated that yoghurt enriched with 1.5% triticale insoluble fibres was recognised as 'excellent' and had enhanced antioxidant activity. CONCLUSIONS: Insoluble triticale fibre could therefore be used as a supplement to produce functional yoghurt. © 2017 Society of Chemical Industry.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-ß-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates.

Expression and distribution of cellulase, amylase and peptidase isoforms along the midgut of Morimus funereus L. (Coleoptera: Cerambycidae) larvae is dependent on nutrient substrate composition.

The influence of diet composition--two substrates, wheat bran and sawdust--on isoform expression of digestive enzymes (cellulase, amylase and peptidase) in the midgut of Morimus funereus larvae was examined. Their impact on larval development was demonstrated by measuring the increase of larval weight during development and by analysis of digestive enzymes zymographic profiles, where the expression of cellulase isoforms from M. funereus larvae midgut has been examined for the first time in this study. Larvae reared on wheat bran had higher body weight between day 60 and day 100 than larvae reared on sawdust; however, both groups achieved similar body weight after day 110. Wheat bran as substrate induced different cellulase and amylase isoforms. Oak sawdust in substrate acted as inducer of peptidases. The highest cellulase activity and the greatest isoform variability were detected in the midgut extracts of larvae reared on wheat bran. From our results it can be assumed that M. funereus endocellulase, amylase and peptidase are secreted in the anterior midgut, and their concentration gradually decreases towards the hindgut.

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis.

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation.

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized ß-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.

Immobilization of cell wall invertase modified with glutaraldehyde for continuous production of invert sugar.

Yeast cell wall invertase (CWI) was modified with dimethyl suberimidate, glutaraldehyde, formaldehyde, and sodium periodate. Retained activity after modification was 45% for CWI modified with formaldehyde, 77% for CWI modified with sodium periodate, 80% for CWI modified with glutaraldehyde, and 115% for CWI modified with dimethyl suberimidate. Chemically modified and native CWIs showed significantly broad pH stability (pH 3-11), whereas after incubations at 50, 60, and 70 °C, CWI modified with glutaraldehyde showed the highest thermostability. Optimum pH for CWI modified with glutaraldehyde was between 4 and 5, whereas optimum temperature was at 60 °C. Comparison to CWI modified with glutaraldehyde after immobilization within alginate beads showed broader pH optimum (4.0-5.5) as well as broader temperature optimum (55-70 °C). Column bed reactor packed with the immobilized CWI modified with glutaraldehyde was successfully used for the 95% inversion of 60% (w/w) sucrose at the flow rate of 3 bed volumes per hour, pH 4.9, and 45 °C. A 1 month productivity of 3844 kg of inverted sugar/kg of the immobilisate was obtained.

Purification and properties of midgut alpha-amylase isolated from Morimus funereus (Coleoptera: Cerambycidae) larvae.

Using soluble starch as a substrate five isoforms of alpha-amylase were identified in a crude extract of Morimus funereus larvae. The main alpha-amylase (termed AMF-3) was purified by gel filtration chromatography and anion exchange chromatography to obtain a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Its enzymatic purity was confirmed by an in-gel activity assay after SDS-PAGE. The purity of AMF-3 was increased 112-fold with a 15.4% yield. AMF-3 had apparent molecular masses of 33 and 31 kDa when analysed using SDS-PAGE and Superdex 75 FPLC gel filtration chromatography, respectively and a calculated isoelectric point of 3.2. Purified AMF-3 showed maximal activity at pH 5.2 and had an optimum activity temperature of 45 degrees C. AMF-3 retained over 90% of its maximum activity at temperatures from 45 to 60 degrees C. AMF-3 exhibited a high affinity towards soluble starch with a K(m) value of 0.43 mg/mL. Maximal AMF-3 activity was achieved in the presence of 0.1 mM CaCl(2), while at higher concentrations its activity decreased. AMF-3 activity increased with increasing NaCl concentration. AMF-3 activity was significantly inhibited by alpha-amylase wheat inhibitor. Using a number of raw starch substrates maximum AMF-3 activity was achieved with horse-radish starch, in contrast to undetectable activity towards potato starch.

Preparation of combined extract of cell wall and cytosol antigens of Candida albicans for immunoblot analysis.

Immunoblot analysis is not in wide use for diagnosis of invasive candidiasis, mostly because the procedure is not standardized and hence not reliable. This work describes a standardized method for C. albicans antigen extract preparation and immunochemical detection. The major improvement of the method is the preparation of combined antigen extract -- consisting of the cell wall and cytosol antigens. The fungal cells and lysis buffer were mixed at a 1:3 ratio and disintegrated by ultrasound for six cycles of one minute each. After centrifugation, cytosol antigens were obtained in the supernatant and cell wall antigens were in the precipitate. Precipitate was dissolved in lysis buffer with 2% sodium dodecyl sulfate (SDS) and boiled at 100 degrees C for 2 min. After centrifugation, the supernatant was combined with the previous one, so the extract of the combined antigens was obtained in the mixture. With those combined antigen extracts and with sera of three different groups of patients, immunoblot analysis showed sensitivity of 90.2%, specificity of 84.4%, and accuracy of 88.0%.