Effects of Fungal Solid-State Fermentation on the Profile of Phenolic Compounds and on the Nutritional Properties of Grape Pomace.

Grape pomace (GP) is considered a natural source of bioactive compounds. To improve the extractability of bioactive compounds, in this work, GP was biologically treated for 15 days with the white-rot fungus Trametes versicolor in laboratory jars and a tray bioreactor under solid-state fermentation (SSF) conditions. During SSF, the activity of lignolytic (laccase and manganese peroxidase) and hydrolytic (xylanase, cellulase, ß-glucosidase, and invertase) enzymes was measured, with the activities of laccase (2.66 U/gdb in jars and 0.96 U/gdb in the bioreactor) and xylanase (346.04 U/gdb in jars and 200.65 U/gdb in the bioreactor) being the highest. The effect of the complex enzyme system was reflected in the changes in the chemical composition of GP with increasing ash, crude protein, and free fat content: 28%, 10%, and 17% in the laboratory jars, and 29%, 11%, and 7% in the bioreactor, respectively. In addition, the biological treatment improved the extractability of 13 individual phenolic compounds. Therefore, the applied SSF technique represents an effective strategy to improve the profile of phenolic compounds and the nutritional composition of GP, promoting their valorization and opening the door for potential applications in the food industry and other sectors.

Fabrication of Ciprofloxacin-Loaded Sodium Alginate Nanobeads Coated with Thiol-Anchored Chitosan Using B-390 Encapsulator Following Optimization by DoE.

Most infectious diseases of the gastrointestinal tract can easily be treated by exploiting the already available antibiotics with the change in administration approach and delivery system. Ciprofloxacin (CIP) is used as a drug of choice for many bacterial infections; however, long-term therapy and off-site drug accumulation lead to an increased risk of tendinitis and peripheral neuropathy. To overcome this issue, nanotechnology is being exploited to encapsulate antibiotics within polymeric structures, which not only facilitates dose maintenance at the infection site but also limits off-site side effects. Here, sodium alginate (SA) and thiol-anchored chitosan (TC) were used to encapsulate CIP via a calcium chloride (CaCl2) cross-linker. For this purpose, the B-390 encapsulator was employed in the preparation of nanobeads using a simple technique. The hydrogel-like sample was then freeze-dried, using trehalose or mannitol as a lyoprotectant, to obtain a fine dry powder. Design of Experiment (DoE) was utilized to optimize the nanobead production, in which the influence of different independent variables was studied for their outcome on the polydispersity index (PDI), particle size, zeta potential, and percentage encapsulation efficiency (% EE). In vitro dissolution studies were performed in simulated saliva fluid, simulated gastric fluid, and simulated intestinal fluid. Antibacterial and anti-inflammatory studies were also performed along with cytotoxicity profiling. By and large, the study presented positive outcomes, proving the advantage of using nanotechnology in fabricating new delivery approaches using already available antibiotics.

Microencapsulation of Grape Pomace Extracts with Alginate-Based Coatings by Freeze-Drying: Release Kinetics and In Vitro Bioaccessibility Assessment of Phenolic Compounds.

The phenols from grape pomace have remarkable beneficial effects on health prevention due to their biological activity, but these are often limited by their bioaccessibility in the gastrointestinal tract. Encapsulation could protect the phenolics during digestion and influence the controlled release in such an intestine where their potential absorption occurs. The influence of freeze-drying encapsulation with sodium alginate (SA) and its combination with gum Arabic (SA-GA) and gelatin (SA-GEL) on the encapsulation efficiency (EE) of phenol-rich grape pomace extract and the bioaccessibility index (BI) of phenolics during simulated digestion in vitro was investigated. The addition of a second coating to SA improved the EE, and the highest EE was obtained with SA-GEL (97.02-98.30%). The release of phenolics followed Fick's law of diffusion and the Korsmeyer-Peppas model best fitted the experimental data. The highest BI was found for the total phenolics (66.2-123.2%) and individual phenolics (epicatechin gallate 958.9%, gallocatechin gallate 987.3%) using the SA-GEL coating were used. This study shows that freeze-dried encapsulated extracts have the potential to be used for the preparation of various formulations containing natural phenolic compounds with the aim of increasing their bioaccessibility compared to formulations containing non-encapsulated extracts.

Characterization of Polyphenol Composition and Starch and Protein Structure in Brown Rice Flour, Black Rice Flour and Their Mixtures.

The study investigates the structural and chemical properties of brown rice flour (WRF), black rice flour (BRF) and their mixtures in ratios of 25%, 50% and 75% to provide reference information for the gluten-free bakery industry. BRF contains higher concentrations of proteins, lipids, total minerals, crude fiber, total polyphenols, proanthocyanidins and flavonoids than WRF. A higher amylose content in BRF than in WRF resulted in flour mixtures with slower starch digestion and a lower glycemic response depending on the BRF ratio added. Differences in the chemical composition of WRF and BRF led to improved composition of the flour mixtures depending on the BRF ratio. The presence of anthocyanidins and phenolic acids in higher concentrations in the BRF resulted in a red-blue color shift within the flour mixtures. The deconvoluted FTIR spectra showed a higher proportion of α-helixes in the amide I band of BRF proteins, indicating their tighter folding. An analysis of the FTIR spectra revealed a more compact starch structure in BRF than in WRF. By processing reflection spectra, nine optically active compound groups were distinguished in rice flour, the proportion in BRF being 83.02% higher than in WRF. Due to co-pigmentation, the bathochromic shift to higher wavelengths was expressed by the proanthocyanins and phenolic acids associated with the wavelengths 380 nm to 590 nm and at 695 nm. Anthocyanins, protein-tannin complexes, methylated anthocyanins and acylated anthocyanins, associated with wavelengths 619, 644 and 668 nm, exhibited a hypsochromic effect by shifting the wavelengths to lower values. This research represents a first step in the development of rice-based products with increased nutritional value and a lower glycemic index.

Whole Black Rice Flour Improves the Physicochemical, Glycemic, and Sensory Properties of Cracker Snacks.

The present study describes the enhancement of the nutritional values of gluten-free rice crackers by adding whole black rice grain flour. The crackers were prepared by combining whole brown rice flour (WRF) and whole black rice flour (BRF) in ratios of 0% (WRC), 25% (25-BRC), 50% (50-BRC), 75% (75-BRC), and 100% (BRC). The resulting samples underwent in-vivo effects on postprandial blood glucose levels as well as physicochemical and sensory analysis. In comparison to WRC, the samples containing 100% added black rice flour presented higher nutritional qualities in terms of protein, by 16.61%, 8.64% for lipids, 5.61% for ash, 36.94% for crude fiber, 58.04% for total polyphenols, 95.49% for proanthocyanidins, and 88.07% for flavonoids. The addition of BRF had a suppressing effect on lightness (L*) and yellowness (b*), while redness (a*) increased. The results of the glycemic measurements confirmed that consumption of crackers made from brown or black whole-grain rice grain flour does not generate glycemic peaks above the limit of 30 mg/dL in baseline blood glucose levels. The results of developing rice crackers from black and brown flour blends showed promising physicochemical and nutritional properties and could provide a good alternative to wheat flour as a gluten-free product.

Characterization of Grape Pomace Extract Microcapsules: The Influence of Carbohydrate Co-Coating on the Stabilization of Goat Whey Protein as a Primary Coating.

Both grape pomace and whey are waste products from the food industry that are rich in valuable ingredients. The utilization of these two by-products is becoming increasingly possible as consumer awareness of upcycling increases. The biological activities of grape pomace extract (GPE) are diverse and depend on its bioavailability, which is influenced by processes in the digestive system. In this work, goat whey protein (GW) was used as the primary coating to protect the phenolic compounds of GPE during the spray drying process. In addition, trehalose (T), sucrose (S), xylose (X), and maltodextrin (MD) were added to the goat whey proteins as co-coatings and protein stabilizers. All spray drying experiments resulted in microcapsules (MC) with a high encapsulation efficiency (77.6-95.5%) and yield (91.5-99.0%) and almost 100% recovery of phenolic compounds during the release test. For o-coumaric acid, the GW-coated microcapsules (MC) showed a bioavailability index of up to 731.23%. A semi-crystalline structure and hydrophilicity were characteristics of the MC coated with 10% T, S, X, or 5% MD. GW alone or in combination with T, S, MD, or X proved to be a promising carrier for polyphenols from grape pomace extract and ensured good bioavailability of these natural antioxidants.

The Release of Grape Pomace Phenolics from Alginate-Based Microbeads during Simulated Digestion In Vitro: The Influence of Coatings and Drying Method.

Grape pomace is a byproduct of wineries and a sustainable source of bioactive phenolic compounds. Encapsulation of phenolics with a well-chosen coating may be a promising means of delivering them to the intestine, where they can then be absorbed and exert their health-promoting properties, including antioxidant, anti-inflammatory, anticancer, cardioprotective, and antimicrobial effects. Ionic gelation of grape pomace extract with natural coatings (sodium alginate and its combination with maltodextrins, gelatin, chitosan, gums Tragacanth and Arabic) was performed, and the resulting hydrogel microbeads were then air-, vacuum-, and freeze-dried to prevent spoilage. Freeze-drying showed advantages in preserving the geometrical parameters and morphology of the microbeads compared to other drying techniques. A good relationship was found between the physicochemical properties of the dried microbeads and the in vitro release of phenolics. Freeze-dried microbeads showed the highest cumulative release of phenols in the intestinal phase (23.65-43.27 mgGAE/gMB), while the most suitable release dynamics in vitro were observed for alginate-based microbeads in combination with gelatin, gum Arabic, and 1.5% (w/v) chitosan. The results highlight the importance of developing encapsulated formulations containing a natural source of bioactive compounds that can be used in various functional foods and pharmaceutical products.

In Vitro Bioaccessibility Assessment of Phenolic Compounds from Encapsulated Grape Pomace Extract by Ionic Gelation.

Grape pomace is a by-product of winemaking characterized by a rich chemical composition from which phenolics stand out. Phenolics are health-promoting agents, and their beneficial effects depend on their bioaccessibility, which is influenced by gastrointestinal digestion. The effect of encapsulating phenol-rich grape pomace extract (PRE) with sodium alginate (SA), a mixture of SA with gelatin (SA-GEL), and SA with chitosan (SA-CHIT) on the bioaccessibility index (BI) of phenolics during simulated digestion in vitro was studied. A total of 27 individual phenolic compounds (IPCs) were quantified by UHPLC. The addition of a second coating to SA improved the encapsulation efficiency (EE), and the highest EE was obtained for SA-CHIT microbeads (56.25%). Encapsulation affected the physicochemical properties (size, shape and texture, morphology, crystallinity) of the produced microbeads, which influenced the delivery of phenolics to the intestine and their BI. Thus, SA-GEL microbeads had the largest size parameters, as confirmed by scanning electron microscopy (SEM), and the highest BI for total phenolic compounds and IPCs (gallic acid, 3,4-dihydroxybenzoic acid and o-coumaric acid, epicatechin, and gallocatechin gallate) ranged from 96.20 to 1011.3%. The results suggest that encapsulated PRE has great potential to be used as a functional ingredient in products for oral administration.

Bioconversion of Grape Pomace with Rhizopus oryzae under Solid-State Conditions: Changes in the Chemical Composition and Profile of Phenolic Compounds.

Grape pomace is a sustainable source of bioactive phenolic compounds used in various industries. The recovery of phenolic compounds could be improved by biological pretreatment of grape pomace, as they are released from the lignocellulose structure by the activity of the enzymes produced. The influence of grape pomace pretreatment with Rhizopus oryzae under solid-state conditions (SSF) on the phenolic profile and chemical composition changes was studied. SSF was performed in laboratory jars and in a tray bioreactor for 15 days. Biological pretreatment of grape pomace resulted in an increase in the content of 11 individual phenolic compounds (from 1.1 to 2.5-fold). During SSF, changes in the chemical composition of the grape pomace were observed, including a decrease in ash, protein, and sugar content, and an increase in fat, cellulose, and lignin content. A positive correlation (r > 0.9) was observed between lignolytic enzymes and the hydrolytic enzyme's xylanase and stilbene content. Finally, after 15 days of SSF, a weight loss of GP of 17.6% was observed. The results indicate that SSF under experimental conditions is a sustainable bioprocess for the recovery of phenolic compounds and contributes to the zero-waste concept by reducing waste.

Physicochemical Characterization and Evaluation of Gastrointestinal In Vitro Behavior of Alginate-Based Microbeads with Encapsulated Grape Pomace Extracts.

Grape pomace is a byproduct of wineries and a rich source of phenolic compounds that can exert multiple pharmacological effects when consumed and enter the intestine where they can then be absorbed. Phenolic compounds are susceptible to degradation and interaction with other food constituents during digestion, and encapsulation may be a useful technique for protecting phenolic bioactivity and controlling its release. Therefore, the behavior of phenolic-rich grape pomace extracts encapsulated by the ionic gelation method, using a natural coating (sodium alginate, gum arabic, gelatin, and chitosan), was observed during simulated digestion in vitro. The best encapsulation efficiency (69.27%) was obtained with alginate hydrogels. The physicochemical properties of the microbeads were influenced by the coatings used. Scanning electron microscopy showed that drying had the least effect on the surface area of the chitosan-coated microbeads. A structural analysis showed that the structure of the extract changed from crystalline to amorphous after encapsulation. The phenolic compounds were released from the microbeads by Fickian diffusion, which is best described by the Korsmeyer-Peppas model among the four models tested. The obtained results can be used as a predictive tool for the preparation of microbeads containing natural bioactive compounds that could be useful for the development of food supplements.

Antioxidant and antiproliferative potentials of phenolic-rich extracts from biotransformed grape pomace in colorectal Cancer.

BACKGROUND: Colorectal carcinoma is one of the most commonly diagnosed malignancies worldwide. Consumption of dietary supplements and nutraceuticals such as phenolic compounds may help combat colorectal carcinoma. The effect of two phenolic-rich extracts prepared from biotransformed grape pomace on the antioxidant properties and antiproliferative activity against two colorectal cancer cell lines (Caco-2 and SW620) were investigated. METHODS: A 15-day solid-state fermentation with the white-rot fungi Phanerochaete chrysosporium and Trametes gibbosa was used to biotransform grape pomace. Solid-liquid extraction was then performed to extract bioactive compounds. The extract was analyzed for the determination of phenolic compounds by ultra-high performance liquid chromatography and in vitro assays of biological activities (antioxidant activity, antiproliferative activity, cell cycle analysis). RESULTS: The 4 days of solid-state fermentation proved to be the optimal period to obtain the maximum yield of phenolic compounds. The tested extracts showed significant antioxidant and antiproliferative activities. Grape pomace treated with P. chrysosporium and T. gibbosa reduced cancer cell growth by more than 60% at concentrations (solid/liquid ratio) of 1.75 mg/mL and of 2.5 mg/mL, respectively. The cell cycle perturbations induced by the grape pomace extracts resulted in a significant increase in the number of cells in the S (9.8%) and G2/M (6.8%) phases of SW620 exposed to T. gibbosa after 48 hours, while P. chrysosporium increased the percentage of cells in the G1 phase by 7.7%. The effect of grape pomace extracts on Caco-2 was less pronounced. CONCLUSIONS: The obtained results suggest the presence of bioactive compounds in biotransformed grape pomace as a residue from winemaking, which could be used to prevent colon cancer.

A Comparative Study of the Influence of Various Fungal-Based Pretreatments of Grape Pomace on Phenolic Compounds Recovery.

Wineries produce considerable amounts of grape pomace, which is a readily available natural source of bioactive phenolic compounds. In this study, grape pomace was used as a substrate for the cultivation of eleven filamentous fungi (Trametes versicolor TV6, Trametes versicolor TV8, Trametes versicolor AG613, Trametes gibbosa, Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Pleurotus eryngii, Ganoderma lucidum, Ganoderma resinaceum, Humicola grisea, and Rhizopus oryzae) under solid-state conditions (SSF) for 15 days with the aim of improving the recovery of the individual phenolic compounds. Twenty-one phenolic compounds were quantified and the recovery of seventeen of them (gallic acid, ellagic acid, p-hydroxybenzoic acid, syringic acid, vanillic acid, 3,4-dihydroxybenzoic acid, ferulic acid, o-coumaric acid, p-coumaric acid, epicatechin gallate, galocatechin gallate, quercetin, kaempferol, procyanidin B1, procyanidin B2, resveratrol, and ε-viniferin) were positively affected by SSF. Ellagic acid is the most recovered compound, whose content increased 8.8-fold after 15 days of biological treatment with Ceriporiopsis subvermispora compared to the untreated initial sample. Among the microorganisms tested, the fungi Pleurotus eryngii and Rhizopus oryzae proved to be the most effective in increasing the recovery of most phenolic compounds (1.1-4.5-fold). In addition, the nutrient composition (proteins, ash, fats) of grape pomace was positively affected by the biological treatments.

Dual Role of Phenyl Amides from Hempseed on BACE 1, PPARγ, and PGC-1α in N2a-APP Cells.

In Alzheimer's disease (AD) the accumulation of amyloid ß (Aß) plaques in the brain leads to neuroinflammation, neuronal cell dysfunction, and progressive memory loss. Therefore, blocking the formation of Aß plaques has emerged as one of the most promising strategies to develop AD treatments. Hempseed is widely used as a food, and recently its compounds have shown beneficial effects on neuroinflammation. The objective of this study was to investigate whether a fraction rich in phenyl amide compounds, N-trans-caffeoyltyramine (CAFT) and N-trans-coumaroyltyramine (CUMT), can affect gene expression: ß-site amyloid-precursor-protein-cleaving enzyme 1 (BACE 1), peroxisome proliferator-activated receptor gamma (PPAR γ), and PPARγ-coactivator-1α (PGC-1α) in N2a-APP cells. The mRNA levels were measured using RT-qPCR. The ethyl acetate fraction and CAFT were found to reduce BACE1 gene expression and are promissory PPARγ and PGC-1α natural agonists. The results show that hempseed compounds can inhibit the expression of BACE 1, which is involved in the accumulation of Aß plaques and positively affect transcription factors involved in complex and diverse biological functions.

A Comprehensive Review on Valorization of Agro-Food Industrial Residues by Solid-State Fermentation.

Agro-food industrial residues (AFIRs) are generated in large quantities all over the world. The vast majority of these wastes are lignocellulosic wastes that are a source of value-added products. Technologies such as solid-state fermentation (SSF) for bioconversion of lignocellulosic waste, based on the production of a wide range of bioproducts, offer both economic and environmental benefits. The versatility of application and interest in applying the principles of the circular bioeconomy make SSF one of the valorization strategies for AFIRs that can have a significant impact on the environment of the wider community. Important criteria for SSF are the selection of the appropriate and compatible substrate and microorganism, as well as the selection of the optimal process parameters for the growth of the microorganism and the production of the desired metabolites. This review provides an overview of the management of AFIRs by SSF: the current application, classification, and chemical composition of AFIRs; the catalytic function and potential application of enzymes produced by various microorganisms during SSF cultivation on AFIRs; the production of phenolic compounds by SSF; and a brief insight into the role of SSF treatment of AFIRs for feed improvement and biofuel production.

Trametes versicolor in lignocellulose-based bioeconomy: State of the art, challenges and opportunities.

Although Trametes versicolor is one of the most investigated white-rot fungi, the industrial application of this fungus and its metabolites is still far from reaching its full potential. This review aims to highlight the opportunities and challenges for the industrial use of T. versicolor according to the principles of circular bioeconomy. The use of this fungus can contribute significantly to the success of efforts to valorize lignocellulosic waste biomass and industrial lignocellulosic byproducts. Various techniques of T. versicolor cultivation for enzyme production, food and feed production, wastewater treatment, and biofuel production are listed and critically evaluated, highlighting bottlenecks and future perspectives. Applications of T. versicolor crude laccase extracts in wastewater treatment, removal of lignin from lignocellulose, and in various biotransformations are analyzed separately.

Transesterification of Sunflower Oil over Waste Chicken Eggshell-Based Catalyst in a Microreactor: An Optimization Study.

The statistical experimental design (DoE) and optimization (Response Surface Methodology combined with Box-Behnken design) of sunflower oil transesterification catalyzed by waste chicken eggshell-based catalyst were conducted in a custom-made microreactor at 60 °C. The catalyst was synthesized by the hydration-dehydration method and subsequent calcination at 600 °C. Comprehensive characterization of the obtained catalyst was conducted using: X-ray powder diffractometry (XRD), X-ray fluorescence (XRF), Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), N2 physisorption, and Hg-porosimetry. Structural, morphological, and textural results showed that the obtained catalyst exhibited high porosity and regular dispersity of plate-like CaO as an active species. The obtained optimal residence time, catalyst concentration, and methanol/oil volume ratio for the continuous reaction in microreactor were 10 min, 0.1 g g-1, and 3:1, respectively. The analysis of variance (ANOVA) showed that the obtained reduced quadratic model was adequate for experimental results fitting. The reaction in the microreactor was significantly intensified compared to a conventional batch reactor, as seen through the fatty acid methyl esters (FAMEs) content after 10 min, which was 51.2% and 18.6%, respectively.

Role of the Encapsulation in Bioavailability of Phenolic Compounds.

Plant-derived phenolic compounds have multiple positive health effects for humans attributed to their antioxidative, anti-inflammatory, and antitumor properties, etc. These effects strongly depend on their bioavailability in the organism. Bioaccessibility, and consequently bioavailability of phenolic compounds significantly depend on the structure and form in which they are introduced into the organism, e.g., through a complex food matrix or as purified isolates. Furthermore, phenolic compounds interact with other macromolecules (proteins, lipids, dietary fibers, polysaccharides) in food or during digestion, which significantly influences their bioaccessibility in the organism, but due to the complexity of the mechanisms through which phenolic compounds act in the organism this area has still not been examined sufficiently. Simulated gastrointestinal digestion is one of the commonly used in vitro test for the assessment of phenolic compounds bioaccessibility. Encapsulation is a method that can positively affect bioaccessibility and bioavailability as it ensures the coating of the active component and its targeted delivery to a specific part of the digestive tract and controlled release. This comprehensive review aims to present the role of encapsulation in bioavailability of phenolic compounds as well as recent advances in coating materials used in encapsulation processes. The review is based on 258 recent literature references.

Enhancement of the anti-inflammatory properties of grape pomace treated by Trametes versicolor.

The application of solid-state fermentation for the production of value-added products from the agro- and food-industry residues has been recently investigated greatly. The white-rot basidiomycete Trametes versicolor is a widely used fungi for the degradation lignocellulosic material in solid-state conditions. Grape pomace constitutes the major by-product of Vitis vinifera L. and is a source of compounds with recognized health benefits. In this study, a process for treating grape pomace with Trametes versicolor for 15 days under solid-state conditions was developed, and the phenolic profile and anti-inflammatory potential of the grape pomace extracts before and after treatment was studied. The anti-inflammatory potential of the grape pomace extracts was studied via tests based on the inhibition of 5-lipoxygenase and hyaluronidase, two key enzymes in inflammatory processes. A total of 24 phenolic compounds were identified and quantified by HPLC methods. With the exception of anthocyanins, an increase in phenolic acids, flavan-3-ols and the flavonol rutin was observed after a treatment period of 1-4 days with T. versicolor. Moreover, the increase in the phenolic content was accompanied by an enhancement in the anti-inflammatory activity of the grape pomace extracts, which was confirmed by the strong correlation between them. This is the first study providing evidence of the benefits of the application of fungal-based solid-state fermentation as an environmentally friendly process for the enhancement of the phenolic composition and anti-inflammatory potential of grape pomace, increasing the possibility of profiting from the great waste produced by the grape-processing industry.

Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by Trametes versicolor.

Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm3), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm3). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45-70 °C with the maximal activity at pH = 4.5.