Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Production of milk-coagulating protease by fungus Pleurotus djamor through solid state fermentation using wheat bran as the low-cost substrate.

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus Pleurotus djamor PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

Improving the extraction yield of essential oil from Pimenta dioica (L.) Merr. using Aspergillus niger ATCC 1004 enzyme blend.

The objective of this work was to optimize the application of an enzymatic blend produced by Aspergillus niger ATCC 1004 on the Pimenta dioica fruits for essential oil extraction. The enzyme blend was obtained from the fermentation of cocoa bean shells, an agro-industrial residue. The effects of the enzymatic pre-treatment on the extraction yield, the chemical composition of the oil through gas chromatography, and the fruit structure through scanning electron microscopy (SEM) were assessed. A Doehlert design was used to optimize the process conditions, resulting in an extraction with 117 mL of enzyme during 77 min, which increased the extraction yield by 387.5%. The chemical composition was not altered, which proves that the enzyme blend preserves the quality of the essential oil extracted. The content of eugenol (70%), the major compound in the P. dioica essential oil, had a great increase in its concentration (560%). The enzyme activity analyses showed the presence of endoglucanase (0.4 U/mL), exoglucanase (0.25 U/mL), ß-glucosidase (0.19 U/mL), and invertase (135.08 U/mL). The microscopy analyses revealed changes in the morphology of fruit surface due to the enzymatic action. These results demonstrate the great potential of using enzyme blends produced by filamentous fungi from agro-industrial residues for the essential oils extraction of interest for the pharmaceutical and food industries.

Production, purification and characterization of phytase from Pichia kudriavevii FSMP-Y17and its application in layers feed.

INTRODUCTION: Phytase, recognized for its ability to enhance the nutritional value of phytate-rich foods, has has gained significant prominence. The production of this enzyme has been significantly boosted while preserving economic efficiency by utilizing natural substrates and optimizing essential factors. This study focuses on optimizing phytase production through solid-state fermentation and evaluating its effectiveness in enhancing nutrient utilization in chicken diets. OBJECTIVE: The objective is to optimize phytase production via solid-state fermentation, characterize purified phytase properties, and assess its impact on nutrient utilization in chicken diets. Through these objectives, we aim to deepen understanding of phytase's role in poultry nutrition and contribute to more efficient feed formulations for improved agricultural outcomes. METHODOLOGY: We utilized solid-state fermentation with Pichia kudriavzevii FSMP-Y17 yeast on orange peel substrate, optimizing variables like temperature, pH, incubation time, and supplementing with glucose and ammonium sulfate. Following fermentation, we purified the phytase enzyme using standard techniques, characterizing its properties, including molecular weight, optimal temperature and pH, substrate affinity, and kinetic parameters. RESULTS: The optimized conditions yielded a remarkable phytase yield of 7.0 U/gds. Following purification, the enzyme exhibited a molecular weight of 64 kDa and displayed optimal activity at 55 °C and pH 5.5, with kinetic parameters (Km = 3.39 × 10-3 M and a Vmax of 7.092 mM/min) indicating efficient substrate affinity. CONCLUSION: The addition of purified phytase to chicken diets resulted in significant improvements in nutrient utilization and overall performance, including increased feed intake, improved feed conversion ratio, enhanced bird growth, better phosphorus retention, and improved egg production and quality. By addressing challenges associated with phytate-rich diets, such as reduced nutrient availability and environmental pollution, phytase utilization promotes animal welfare and sustainability in poultry production.

Nutritional attributes and microbial metagenomic profile during solid-state fermentation of soybean meal inoculated with Lactobacillus acidophilus under non-sterile conditions.

BACKGROUND: Soybean meal (SBM) is used widely in animal feed but it contains anti-nutritional factors (ANFs) such as protease inhibitors - immunogenic proteins that limit its utilization. Fermentative processes could help to reduce these ANFs. The aim of this study was to evaluate the nutritional attributes, bacterial community dynamics, and microbial metagenomic profile during the solid-state fermentation of SBM using a strain of the bacterium Lactobacillus acidophilus with or without pre-autoclaving treatment. RESULTS: Following fermentation, there was a reduction in the pH and a concurrent increase in the population of lactic acid bacteria. Fermentation also resulted in an increase in both crude and soluble protein levels. Trypsin inhibitor levels decreased after fermentation, particularly in fermented SBM that had not been pre-autoclaved, with an inactivation rate higher than 90%. Moreover, high-molecular-weight peptides (44-158 kDa), specifically some polypeptides from the soybean immunogen glycinin and ß-conglycinin, underwent degradation during the fermentation process. Bacterial community analysis revealed the dominance of the Lactobacillus genus in all samples, regardless of the treatments applied. Metagenomic profiling identified L. acidophilus as the dominant species in inoculated SBM, irrespective of whether pre-autoclaving was conducted or not. CONCLUSION: This study demonstrates the feasibility of solid-state fermentation with L. acidophilus under non-sterile conditions to inactivate trypsin inhibitor and increase protein concentration and hydrolysate immunogen proteins into low-molecular-weight peptides in SBM. Lactobacillus acidophilus inoculum also inhibited the growth of undesirable bacteria. This knowledge contributes to our understanding of the potential applications of solid-state fermentation with L. acidophilus in improving the nutritional quality of SBM. © 2024 Society of Chemical Industry.

Efecto de la fermentación en medio sólido sobre el contenido de fenoles totales y la capacidad antioxidante del maíz / Effect of solid medium fermentation on total phenol content and antioxidant capacity of maize

Resumen: El maíz contiene un gran número de compuestos antioxidantes, muchos de ellos unidos a componentes de la pared celular, por lo que requieren tratamientos para liberarlos, como el uso de enzimas o procesos de fermentación. La fermentación en medio sólido (FMS) con Rhizopus oryzae se ha aplicado para aumentar la capacidad antioxidante (CA) y el contenido fenólico en cereales y leguminosas. El objetivo del presente trabajo fue evaluar el efecto de la FMS con R. oryzae sobre la CA y el contenido de fenoles totales (CFT) del maíz. La FMS se realizó en bolsas zip-lock (25 cm2) a 30 °C/72 h, con un inóculo de 1 x 106 esporas/g. Se tomaron muestras cada 12 h, el extracto se recuperó con etanol al 80 % y se utilizó para determinar el CFT y la CA (ensayo ABTS+, DPPH y FRAP). Los valores más altos se obtuvieron a las 60 h de cultivo, con un CFT de 1.92 mg/ gramos de materia seca (gms) y una CA de 1.47 mg de equivalentes Trolox por gramo de materia seca (mg ET/gms), 1.27 mg ET/gms y 5.8 mg Fe+2/gms para los ensayos de ABTS+, DPPH y FRAP, respectivamente. El uso de FMS permitió aumentar hasta 0.83 y 1.25 veces el CFT y la CA del maíz, con respecto al tiempo 0 h. El maíz fermentado con R. oryzae mostró potencial para ser empleado como materia prima para el desarrollo de alimentos funciona les, al incrementar su CA a través de un bioproceso.

Abstract: Maize contains a large number of antioxidant compounds. However, many of them are not in free form, as they are bound to components of the cell wall of maize kernels. For this reason, the use of treatments is required to release them, such as the use of enzymes or fermentation processes. Fermentation in solid medium (FMS) with Rhizopus oryzae has been applied to increase the antioxidant capacity (AC) and phenolic content in cereals and legumes. The objective of the present work was to evaluate the effect of FMS with R. oryzae on AC and total phenolic content (TPC) of maize. Fermentation on solid medium was carried out in zip-lock bags (25 cm2) at 30 °C for 72 h, with an inoculum of 1 x 106 spores/g. Samples were taken every 12 h, the extract was recovered with 80% ethanol, and used to determine TPC and AC (ABTS+, DPPH and FRAP essay). The highest values were obtained at 60 h of culture, with a TPC of 1.92 mg/gram dry metter (gdm) and an AC of 1.47 mg TE/gmd, 1.27 mg TE/gdm and 5.8 mg Fe+2/gdm for the ABTS+, DPPH and FRAP assays, respectively. The use of FMS allowed to increase up to 0.83 and 1.25 times the CFT and CA of corn, with respect to time zero. Corn fermented with R. oryzae showed potential to be used as a raw material for the development of functional foods, by increase its AC through a bioprocess.

Lignocellulosic materials valorization in second generation biorefineries: an opportunity to produce fungal biopigments.

Second generation biorefineries play an important role in the production of renewable energy and fuels, utilizing forest and agro-industrial residues and by-products as raw materials. The integration of novel bioproducts, such as: xylitol, ß-carotene, xylooligosaccharides, and biopigments into the biorefinery's portfolio can offer economic benefits in the valorization of lignocellulosic materials, particularly cellulosic and hemicellulosic fractions. Fungal biopigments, known for their additional antioxidant and antimicrobial properties, are appealing to consumers and can have applications in various industrial sectors, including food and pharmaceuticals. The use of lignocellulosic materials as carbon and nutrient sources for the growth medium helps to reduce production costs, increasing the competitiveness of fungal biopigments in the market. In addition, the implementation of biopigment production in biorefineries allows the utilization of underutilized fractions, such as hemicellulose, for value-added bioproducts. This study deals with the potential of fungal biopigments production in second generation biorefineries in order to diversify the produced biomolecules together with energy generation. A comprehensive and critical review of the recent literature on this topic has been conducted, covering the major possible raw materials, general aspects of second generation biorefineries, the fungal biopigments and their potential for incorporation into biorefineries.

Performance evaluation of Trichoderma reseei in tolerance and biodegradation of diuron herbicide in agar plate, liquid culture and solid-state fermentation.

The present study evaluated the performance of the fungus Trichoderma reesei to tolerate and biodegrade the herbicide diuron in its agrochemical presentation in agar plates, liquid culture, and solid-state fermentation. The tolerance of T. reesei to diuron was characterized through a non-competitive inhibition model of the fungal radial growth on the PDA agar plate and growth in liquid culture with glucose and ammonium nitrate, showing a higher tolerance to diuron on the PDA agar plate (inhibition constant 98.63 mg L-1) than in liquid culture (inhibition constant 39.4 mg L-1). Diuron biodegradation by T. reesei was characterized through model inhibition by the substrate on agar plate and liquid culture. In liquid culture, the fungus biotransformed diuron into 3,4-dichloroaniline using the amide group from the diuron structure as a carbon and nitrogen source, yielding 0.154 mg of biomass per mg of diuron. A mixture of barley straw and agrolite was used as the support and substrate for solid-state fermentation. The diuron removal percentage in solid-state fermentation was fitted by non-multiple linear regression to a parabolic surface response model and reached the higher removal (97.26%) with a specific aeration rate of 1.0 vkgm and inoculum of 2.6 × 108 spores g-1. The diuron removal in solid-state fermentation by sorption on barley straw and agrolite was discarded compared to the removal magnitude of the biosorption and biodegradation mechanisms of Trichoderma reesei. The findings in this work about the tolerance and capability of Trichoderma reesei to remove diuron in liquid and solid culture media demonstrate the potential of the fungus to be implemented in bioremediation technologies of herbicide-polluted sites.

Fermentation of Plant-Based Feeds with Lactobacillus acidophilus Improves the Survival and Intestinal Health of Juvenile Nile Tilapia (Oreochromis niloticus) Reared in a Biofloc System.

This study evaluated the effect of fermentation with Lactobacillus acidophilus on the biochemical and nutritional compositions of a plant-based diet and its effects on the productive performance and intestinal health of juvenile Nile tilapia (Oreochromis niloticus) reared in a biofloc technology (BFT) system. The in vitro kinetics of feed fermentation were studied to determine the L. acidophilus growth and acidification curve through counting the colony-forming units (CFUs) mL-1 and measuring the pH. Physicochemical and bromatological analyses of the feed were also performed. Based on the microbial growth kinetics results, vegetable-based Nile tilapia feeds fermented for 6 (FPB6) and 18 (FPB18) h were evaluated for 60 days. Fermented diets were compared with a positive control diet containing fishmeal (CFM) and a negative control diet without animal protein (CPB). Fermentation with L. acidophilus increased lactic acid bacteria (LAB) count and the soluble protein concentration of the plant-based feed, as well as decreasing the pH (p < 0.05). FPB treatments improved fish survival compared with CPB (p < 0.05). Fermentation increased feed intake but worsened feed efficiency (p < 0.05). The use of fermented feeds increased the LAB count and reduced pathogenic bacteria both in the BFT system's water and in the animals' intestines (p < 0.05). Fermented plant-based feeds showed greater villi (FPB6; FPB18) and higher goblet cell (FPB6) counts relative to the non-fermented plant-based feed, which may indicate improved intestinal health. The results obtained in this study are promising and show the sustainable potential of using fermented plant-based feeds in fish feeding rather than animal protein and, in particular, fishmeal.

Production of Gibberellic Acid by Solid-State Fermentation Using Wastes from Rice Processing and Brewing Industry.

Gibberellic acid (GA3) is a natural hormone present in some plants used in agricultural formulations as a growth regulator. Currently, its production on an industrial scale is performed by submerged fermentation using the fungus Gibberella fujikuroi, which is associated with low yields, leaving the purification stages with high costs. An alternative is solid-state fermentation (SSF), which makes it possible to obtain higher concentrations of product using low-cost substrates, such as agroindustrial by-products. This research investigated the use of raw rice bran (RRB) and barley malt residue (BMR) as substrates for GA3 production by the fungus Gibberella fujikuroi. Through two statistical designs, the effect of moisture (50 to 70 wt.%) and medium composition (RRB content between 30 and 70 wt.% to a mass ratio between RRB and BMR) was first evaluated. Using the best conditions previously obtained, the effect of adding glucose (carbon source, between 0 and 80 g·L-1) and ammonium nitrate-NH4NO3-(nitrogen source, between 0 and 5 g·L-1) on GA3 productivity was analyzed. The best yield was obtained using 30 wt.% RRB and 70 wt.% BMR for a medium with 70 wt.% of moisture after 7 days of process. It was also found that higher concentrations of NH4NO3 favor the GA3 formation for intermediate values of glucose content (40 g·L-1). Finally, a kinetic investigation showed an increasing behavior in the GA3 production (10.1 g·kg of substrate-1 was obtained), with a peak on the seventh day and subsequent tendency to stabilization.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola / Produção de protease e caracterização molecular de um gene de protease - DPAP (dipeptidil-aminopeptidase) de diferentes cepas de Sordaria fimicola

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

A pesquisa atual foi projetada para alcançar o potencial de produção de protease extracelular em diferentes cepas de Sordaria fimicola que foram previamente obtidas do Dr. Lamb (Imperial College, Londres) de North Facing Slope e South Facing Slope de Evolution Canyon. Após a triagem inicial e secundária, duas cepas hiperprodutoras S2 e N6 foram selecionadas para fermentação submersa e condições culturais, incluindo temperatura, pH, período de incubação, tamanho do inóculo, concentração de substrato, e diferentes fontes de carbono e nitrogênio foram otimizadas para produção de enzima. A cepa S2 apresentou produção máxima de protease de 3,291 U/mL após 14 dias de incubação a 30 °C com pH 7, concentração de substrato de 1% e inóculo de 1 mL, enquanto a cepa N6 apresentou produção máxima de protease de 1,929 U/mL em condições otimizadas de fermentação. Outro objetivo da presente pesquisa foi sustentar a biodiversidade da genética e modificações pós-tradicionais (PTMs) da protease DPAP (peptidil-aminopeptidase) em Sordaria fimicola. Cinco sítios polimórficos foram observados na sequência de aminoácidos de cepas de S. fimicola com referência a Neurospora crassa. A previsão de PTMs a partir de ferramentas de bioinformática previu 38 locais de fosforilação em resíduos de serina para protease peptidil-aminopeptidase na cepa S1 de S. fimicola, enquanto 45 locais de fosforilação em serina na cepa N7 e 47 modificações de fosforilação de serina foram previstas em N. crassa. A pesquisa atual deu uma ideia de que a mudança na composição genética afetou os PTMs que, em última análise, afetaram a produção da enzima protease em diferentes cepas do mesmo organismo (S. fimicola). A produção e os dados moleculares da pesquisa revelaram que o estresse ambiental tem fortes efeitos sobre genes específicos por meio de mutações que podem causar diversidade genética. S. fimicola é um fungo não patogênico e tem um ciclo de vida curto. Esse fungo pode ser escolhido para produzir enzima protease em escala comercial.

Solid-State Fermented Pineapple Peel: A Novel Food Ingredient with Antioxidant and Anti-Inflammatory Properties.

It has been reported that pineapple (Ananas comosus) contains healthy nutrients and phytochemicals associated with antioxidant and anti-inflammatory capacities. However, a substantial amount of pineapple residue is produced due to a lack of valorization applications at the industrial scale, resulting in the loss of valuable nutrients. Solid-state fermentation (SSF) is proposed as an innovative strategy to enhance the release of bound phenolics from pineapple residues. In this work, the effects of SSF of pineapple peels with Lactobacillus plantarum, Lactobacillus rhamnosus, and Aspergillus oryzae on the release of phenolic compounds and their antioxidant and anti-inflammatory activities were evaluated, respectively. Pineapple peel extracts after SSF showed an increase in the release of phenolic compounds (248.11% with L. plantarum, 182% with A. oryzae, and 180.10% with L. rhamnosus), which led to an increase in the cellular antioxidant (81.94% with L. rhamnosus) and anti-inflammatory potential (nitric oxide inhibition of 62% with L. rhamnosus) compared to non-fermented extracts. Therefore, SSF of pineapple peels with L. plantarum, L. rhamnosus, and A. oryzae thrives as a new approach for the production of secondary metabolites with remarkable biological benefits, which can be the precursors for novel biofortified and nutraceutical-enriched foods that meet the needs of the most demanding and health-conscious consumers.

Enhanced production of cordycepin under solid-state fermentation of Cordyceps militaris by using combinations of grains/substrates.

This manuscript deals with cordycepin, an interesting secondary compound produced from entomopathogenic fungus, Cordyceps. It has attracted commercial interest due to its immense pharmacological importance beneficial to human health. In this study, the contents of cordycepin and its derivatives, like adenine and adenosine, were evaluated through solid-state fermentation using combinations of various grains as substrate. Treatment with grain combination numbers 2, 7, 8, and 9 exhibited higher cordycepin content (1.621, 1.929, 1.895, and 1.996 mg/g cordycepin, respectively) than control (rice). The grain combination number 7 exhibited significantly higher adenine content (700 mg/g) than the control and all other combinations. Treatments with grain combination numbers 2, 5, and 7 exhibited higher adenosine content (2.719, 2.938, and 3.392 mg/g, respectively); however, no significant increase in adenosine content was noted in any treatments. The biomass including fresh mycelium and fruit body was found higher in grain combination numbers 7 and 9, leading to enhanced cordycepin content. Overall, the increase in the fresh biomass significantly enhanced cordycepin accumulation. The level of cordycepin was recorded as higher than that of its derivatives, adenosine and adenine. The grain combination of rice, wheat, jowar, bajra, and sugarcane bagasse added to basal medium exhibited the highest cordycepin content and was found suitable for solid-state fermentation of Cordyceps militaris. To our understanding, the present study is the first to use combinations of cereals for the production of cordycepin from C. militaris.

Chicken viscera meal as substrate for the simultaneous production of antioxidant compounds and proteases by Aspergillus oryzae.

The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01-26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < - 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes.

Enhanced laccase activity in Trametes versicolor (L.: Fr.) Pilát by host substrate and copper.

Laccases are appealing biocatalysts for various industrial utilizations. The fungus Trametes versicolor (L.: Fr.) Pilát causes white rot in wood and has been identified as an important fungal laccase producer. To investigate laccase production and activity in T. versicolor, the native isolate was collected from the host (Quercus castaneifolia) in the forests of Guilan province, northern Iran, and then purified and identified using the molecular marker. Its ability to produce laccase enzyme in the presence of different plant substrates including sawdust and wood chips of oak, poplar, and pine was evaluated. Also, the effect of copper as an enzyme inducer was investigated in vitro. The results showed that adding the wood to the culture medium increased laccase production, and among these, oak sawdust had the greatest effect, a 1.7-fold increase from that in the control (4.8 u/l vs. 2.8 u/l). Also, the enzyme extraction time effect on the optimal recovery yield showed that the 5-h enzyme extraction cycle resulted in the highest yield of the enzyme (18.97 u/l). Moreover, adding different concentrations of copper to the fungal culture medium increased the production of laccase, and the highest amount of enzyme (92.04 u/l) was obtained with 3.5 mM of CuSO4 along with oak sawdust. Based on the results, the addition of host wood sawdust ("oak" in this work) and copper particles together stimulates the fungal growth and the laccase production during submerged cultivation of T. versicolor. Therefore, it would be a safe and cheap strategy for the commercial production of laccase by filamentous fungi.

Probiotic growth-stimulating capacity and antimicrobial activities of aqueous extracts of Lentinus crinitus (L.) Fr (polyporales, basidiomycota).

Lentinus crinitus (L.) Fr is a wild macrofungus that is popular as antimicrobial and various biological activities. This study aims to determine the capacity growth stimulation of Lactobacillus paracasei and antimicrobial activity of aqueous extracts of L. crinitus obtained from wild basidiomata, mycelial biomass by liquid fermentation and spent mushroom substrate obtained by solid-state fermentation. The antimicrobial activity was investigated against bacterial and fungal pathogens and growth stimulation L. paracasei probiotic bacterium. The total carbohydrate and ß-glucan contents of the extracts were determined using colorimetric analysis. The aqueous extracts obtained showed inhibition against Fusarium oxysporum., Penicillium sp., Rhizopus oryzae, Aspergillus niger, Escherichia coli and Salmonella typhimurium. The aqueous extract obtained from wild basidiomata, and mycelial biomass showed the highest percentage of stimulation of L. paracasei growth in 48 h. The extracts obtained from L. crinitus have antimicrobial potential and stimulating capacity of the probiotic Lactobacillus paracasei. Additionally, different biotechnological techniques such as liquid and solid-state fermentation can be used to obtain aqueous extracts.

Sustainable use of giant reed to produce industrialized enzymes.

The giant reed (Arundo donax) is a fast-growing plant adapted to different climatic and soil conditions; although its origin is Asian, the species has spread throughout the world. During its development, it consumes three times more water than typical native vegetation and is responsible for changing the landscape of riparian areas; the high biomass productivity and the annual harvest period make this crop an alternative to produce and/or extract industrial bioproducts. The main objective of this research was to evaluate the feasibility of using giant reed in a bioprocess that produces enzymes by a solid-state fermentation experiment, four fungal species were tested (Aspergillus niger GH1, Aspergillus niger PSH, Trichoderma harzianum, and Rhizopus oryzae); enzyme activities were performed using reported methodologies varying only reaction volumes. The A. niger GH1 and PSH strains were the best adapted to the plant material, A. niger GH1 was capable to produce 4 of the 5 evaluated enzymes (cellulase-endoglucanase (174.39 ± 19.62 U/L), xylanase (1313.31 ± 39.25 U/L), invertase (642.22 ± 23.55 U/L), and polyphenol oxidase (6094.01 ± 306.54) while A. niger PSH was able to produce 3 of the 5 evaluated enzymes (cellulase-endoglucanase (147.09 ± 13.88 U/L), xylanase (1307.76 ± 31.40 U/L), and invertase (603.92 ± 3.14 U/L).

Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of ß-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Bati Butter as a Potential Substrate for Lipase Production by Aspergillus terreus NRRL-255.

This study evaluated bati butter (Ouratea parviflora) as a substrate for lipase production by solid-state fermentation (SSF) using Aspergillus terreus NRRL-255. A gas chromatograph with a flame ionization detector determined the bati butter fatty acid profile. Lipase production and spore count were optimized using a 32 experimental design and evaluated using the response surface methodology. Moreover, the crude enzyme extract was evaluated against different pH, temperature, and activating and inhibitors reagents. Regarding the fatty acids identified, long-chain accounted for 78.60% of the total lipids. The highest lipase production was obtained at 35 °C and 120 h of fermentation, yielding 216.9 U g-1. Crude enzyme extract presented more significant activity at 37 °C and pH 9. ß-Mercaptoethanol increased the enzyme activity (113.80%), while sodium dodecyl sulfate inactivated the enzyme. Therefore, bati butter proved to be a potential substrate capable of inducing lipase production by solid-state fermentation.

Production of a Fungal Punicalagin-Degrading Enzyme by Solid-State Fermentation: Studies of Purification and Characterization.

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.