Trametes versicolor (L.) Lloyd as a source of thermostable serine protease: production and characterization

Proteases are ubiquitously present and are among the largest groups of commercially important enzymes. Here, we investigated a wood-rot basidiomycete Trametes versicolor (L.) Lloyd [Syn. Coriolus versicolor (L.) Quél.; Polyporus versicolor (L.) Fr.] as a source of the enzyme serine protease, its production, and optimized to obtain a higher yield of the enzyme.. The significant variables with optimized values for maximum production of the enzyme were temperature (30?C), incubation time (120 h) and wheat bran (10 g). The yield increased by 30.76% by statistically optimizing the media. The optimized temperature and pH for the maximum protease activity was 50?C and pH 7.0, respectively. The enzyme was purified through ion exchange (using DEAE cellulose 52 resin) and gel filtration chromatography (using Superdex 200 column). The purified enzyme had a retention time of 7 min in RP-HPLC. The enzyme was stable at a broad range of temperature (30-60?C) and pH (5.0-8.0) with a half-life of 58.72 min, Vmax of 37.17 ?M min/mL and Km of 0.657 mg/mL. Its activity was enhanced by Na+, Ca2+, Mg2+ ions and SDS surfactant. These properties make this enzyme a valuable candidate for industrial applications

Production of Fungal Pectin Lyase and Polygalacturonase from Fruit Wastes by Solid State Fermentation

Polygalacturonase (PG or PGase) and Pectin lyase (PL) are depolymerase enzymes that split the ?-1, 4-glycosidic linkages in the backbone of homogalacturonans. They are produced by microorganisms degrading pectin-containing substrates. PG and PL were produced in the solid-state fermentation (SSF) of beans testa (BT), mango peels (MP), Plantain peels (PP), and BT: MP: PP (1:1:1). Substrates were seeded with individual fungal strains namely Aspergillus tamarii, Aspergillus terreus, Aspergillus piperis, Aspergillus parasiticus and Mucor piriformis. PG production ranged from 0.0377 - 141.0095 U/g with A. tamarii, A. terreus and A. piperis producing the highest on mango peels. PL production ranged from 50.50 - 10,852.50 U/g with Mucor piriformis producing the highest on plantain peels. The pH of the fermentation medium changed during growth, metabolism, and pectinase production. The best pH for pectinase production falls within the acidic range. Unconventional substrates such as PP are viable for pectinase production, and PL yield in SSF is improved by compositing substrates.

Emericella nidulans (4DP5), Cladosporium herbarum (7DF12) and Bacillus subtilis improve the nutritional value of palm kernel cake (PKC) through solid-state fermentation (SSF)

Aims@#Palm kernel cake (PKC) is a high-protein, high-energy food that is widely utilized in the animal feed business. However, the high fibre and limited amino acid content of untreated PKC were the main issues for it to be used as animal feed, particularly in non-ruminants. To improve the quality of PKC, this study combined the use of solid-state fermentation (SSF) and consortia of fungi and bacteria to treat the PKC.@*Methodology and results@#Two fungi, Emericella nidulans (4DP5) and Cladosporium herbarum (7DF12) and three strains of bacteria, Bacillus subtilis, which were active mannanase producers, were used in different combinations to reduce the hemicellulose content and improve the crude protein content of PKC in a lab-scale solid-state fermentation. PKC inoculated separately with five types of mixed culture treatments were allowed to ferment. The fermentation conditions were 20% inoculum (w/v), 85-92% humidity, pH 7.0 and PKC particle size 0.8 mm. PKC treatments with two fungi, E. nidulans (4DP5) and C. herbarum (7DF12), as well as a fungus-bacterium combination, E. nidulans (4DP5) and B. subtilis, outperformed the other three treatments. The crude protein levels were increased by 3.34% and 1.86%, respectively, due to these treatments. Furthermore, the level of aflatoxins produced increased marginally but remained within the permissible limits.@*Conclusion, significance and impact of study@#The treated PKC has more sugar and crude protein and less than 20 parts per billion (ppb) of aflatoxin, making it appropriate for animal consumption. The SSF technique of combining fungi and Bacilli enhanced the nutritional and market value of PKC substantially, which can be upscaled.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Recovery of Red Pigments from Monascus purpureus FTC 5357 by Extraction of Fermented Solids: Operational Conditions and Kinetics

Abstract Solvent extraction of red pigments from fermented solids is reported. The pigments were produced by solid-state fermentation of oil palm frond (OPF) biomass with the food-safe fungus Monascus purpureus FTC 5357. The effects of extraction solvent and other operational conditions (pH, temperature, agitation rate, contact time) on the recovery of pigments are discussed. The recovery was maximized using aqueous ethanol (60% ethanol by vol) as solvent at pH 6, 30 °C, with an extraction time of 16 h and an agitation rate of 180 rpm. A fermented solids dry mass of 1 g was used for each 160 mL of solvent during extraction. The kinetics of extraction were assessed by fitting the experimental data to different models. Peleg's model proved to be the best for describing solid-liquid extraction of the pigments under the above specified conditions. The highest extraction yield of red pigments under the above specified optimal conditions was 207(6.08 AU g(1 dry fermented solids.

Amylase from Bacillus sp. Produced by Solid State Fermentation Using Cassava Bagasse as Starch Source

Abstract Amylases are enzymes involved in starch hydrolysis, generating the most diverse products, such as maltose, glucose and dextrins. This work aimed the study of the production of amylolytic enzymes via solid-state fermentation (SSF) using "crueira", an essentially starchy cassava residue, as substrate-support and Bacillus sp. as microorganism. For the implementation of the experimental part, a Central Composite Design (CCD) with three variables (initial moisture, pH and temperature) was made. Each test was examined at 24, 48 and 72 hours by the method of starch dextrinizing activity. The optimum production conditions were 60% initial moisture, pH 6 and 37 °C. The maximum yield was 437.76 U/g in 72 hours of fermentation. The optimum temperature of enzyme performance was 65 °C. The pH optimum range was 4 to 6. The Co2 +, Ca2 + and K+ ions positively influenced the activity of enzymes and the Fe2+ ion had no effect on enzymatic activity. On the other hand, the ions Hg2+, Zn2+, Cu2+, Mn2+ and Mg2+ adversely influenced enzymatic activity. Therefore, producing amylases from Bacillus sp. and using crueira as a substrate is possible.

Newly Isolated Penicillium sp. for Cellulolytic Enzyme Production in Soybean Hull Residue

Abstract (1) Background: The aim of this study was to evaluate the production and partial characterization of xylanase and avicelase by a newly isolated Penicillium sp. in solid-state fermentation, using soybean hulls as substrate. (2) Methods: Temperature, time, number of spores, and substrate moisture on xylanase and avicelase bioproduction were evaluated, maximizing activity with 30°C, 1x106 spores/g substrate, 14 and 7 days of fermentation with 70 and 76% substrate moisture contents, for xylanase and avicelase, respectively. (3) Results: Different solvents, temperatures, and agitation in the enzymatic extraction were evaluated, obtaining higher activities, 430.77 and 26.77 U/g for xylanase and avicelase using 30 min extraction and 0.05 M citrate buffer solution (pH 4.5 ), respectively at 60°C and 175 rpm and 50°C and 125 rpm. The optimum pH and temperature for enzymatic activity determination were 5.3 and 50°C. Enzyme extract stability was evaluated, obtaining higher stability with pH between 4.5 and 5.5, higher temperature of up to 40°C. The kinetic thermal denaturation (Kd), half-life time, D-value, and Z-value were similar for both enzymes. The xylanase Ed value (89.1 kJ/mol) was slightly lower than the avicelase one (96.7 kJ/mol), indicating higher thermostability for avicelase. (4) Conclusion: In this way, the production of cellulases using alternative substrates is a way to reduce production costs, since they represent about 10% of the world demand of enzymes, with application in animal feed processing, food production and breweries, textile processing, detergent and laundry production, pulp manufacturing and the production of biofuels.

Solid state fermentation to obtain vegetable products bio-enriched with isoflavone aglycones using lactic cultures / Fermentación en sustrato sólido utilizando cultivos lácticos para la obtención de productos vegetales bioenriquecidos con isoflavonas agliconas

The consumption of soybean isoflavones (IS) is associated with several beneficial properties on human health. Some lactic acid bacteria possess ß-glucosidase enzyme, that allows to obtain the active form of IS (aglycone). The solid state fermentation (SSF) has received great attention in the last years in order to obtain several valuable compounds. SSF, using soybean as substrate and Lactobacillus rhamnosus CRL 981 as starter, was studied in the present work. Sucrose was added into soybean paste to study the effect on the behavior of the selected strain. The development of L. rhamnosus CRL 981 through pH and recount measures, sugar intake, organic acid production, ß-glucosidase activity and IS conversion were analyzed. No significant differences in growth and acidity were observed between soybean pastes with and without sucrose added, but the production of lactic acid was higher in the latter paste. The ß-glucosidase activity was detected in both pastes and the complete hydrolysis of IS at 12 h of fermentation was observed. Also, this strain was able to increase the free amino acids in soybean paste. SSF, using soybean as substrate and L. rhamnosus CRL 981 as starter culture, is an alternative process to obtain a soybean product bio-enriched in active IS with attractive nutritional characteristics.

El consumo de isoflavonas de soja (IS) está asociado a diversos beneficios para la salud humana. Ciertas bacterias lácticas poseen la enzima ß-glucosidasa, que permite obtener la forma bioactiva (agliconas) de las IS. La fermentación en sustrato sólido (FSS) ha recibido gran atención en los últimos anos debido a sus numerosas ventajas, y permite la obtención de productos con valor agregado. En el presente trabajo se estudió la FSS utilizando soja como sustrato y Lactobacillus rhamnosus CRL981 como cultivo iniciador. Con el fin de estudiar el efecto de una fuente de carbono externa sobre el comportamiento de la cepa seleccionada, se adicionó sacarosa a la pasta de soja. Se evaluó el crecimiento de L. rhamnosus CRL 981 a través de medidas de pH y recuento en placa. Además, se analizó el consumo de azúcares, producción de ácidos orgánicos, actividad ß-glucosidasa y conversión de IS. No se observaron diferencias significativas en el crecimiento y acidez entre las pastas de soja sin adición de sacarosa y con ella, sin embargo, la producción de ácido láctico fue mayor en esta última. La actividad de ß-glucosidasa se detectó en ambas pastas y se observó la hidrólisis completa de IS a las 12 h de fermentación. Además, esta cepa fue capaz de aumentar los aminoácidos libres en la pasta de soja. La FSS, utilizando soja como sustrato y L. rhamnosus CRL 981 como cultivo iniciador, es un proceso alternativo para obtener un producto de soja bioenriquecido en IS bioactivas con características nutricionales atractivas.

Screening, Production and Partial Characterization of a Thermostable Laccase from Trametes sp. Isolate B7 with Biotechnological Potentials

The search for efficient and green oxidation technologies has increased interest in utilization of laccases in non conventional methods. Laccases catalyze a wide range of substrates due to low substrate specificity and strong oxidative potentials. Challenges to large-scale enzyme utilization include, low enzyme activity and instability which restrict use in many areas of biotechnology. In the study, 59 fungi comprising Aspergillus niger (40%), Trichoderma harzianum (31%), Aspergillus flavus (9.0%), Trichoderma viride (5.0%), Fusarium oxysporum (5.0%), Rhizopus stolonifer (5.0%), Trametes sp. (3.0%) and Aspergillus nidulans (2.0%) were isolated and screened for laccase production. Plate screening test showed 57.5%, 34.0% and 8.5% of fungi were laccase-positive on ABTS, Guaiacol, and α-naphthol agar respectively. Isolates were further screened in liquid cultures, and the highest laccase producer identified molecularly. Trametes sp isolate B7 was selected for solid state fermentation (SSF). Laccase production in SSF was highest at pH 5.0 (2356 U/mL). The purified laccase showed high activity (pH 3.0 - 6.0) and stability (pH 3.0 - 8.5) using ABTS. It was active (20 - 80°C) and thermostable (30 - 80°C) with optimum stability at 70°C (100% for 1 hour). The percentage decolourization of Phenol red were 28% and 36% using 1000 U/mL and 2000 U/mL crude laccases respectively. Similarly, RBBR (100%), Congo red (75%) and Malachite green (62%), and 77.4%, 64% and 28% were decolourized using 1000 U/mL and 2000 U/mL crude laccases respectively. ABTS agar was very reliable in large-scale screening for laccase which possessed thermostable property and degraded synthetic dyes without use of enzyme mediators. These attribute made the enzyme suitable for application in industry and biotechnology.

Laccase production by Myrothecium gramineum and its optimization under solid state fermentation using cowpea pod as substrate

The present study highlights the utilization of wastes such as cowpea outer pod generated from agro industries forlaccase production using Myrothecium gramineum LCJ177 under solid-state fermentation. Conventional methodswere used to optimize the process parameters. The classical one-factor-at-a-time method showed that the optimalstarch concentration was 1 g/L, peptone concentration was 0.5 g/L, copper sulfate concentration was 0.6 mM, andpyrogallol concentration was 0.8 mM. Likewise, the suitable physical conditions were an initial pH of five of theculture medium, the temperature of 30°C and moisture content of 60%. Utilization of dried cowpea outer pod as asubstrate reduces the pollution levels by converting agro-wastes as useful by-products.

Effects of phytohormones and lignin on growth of Antrodia cinnamomea mycelium / 中草药

Objective: To investigate the effects of phytohormones and lignin on the growth of Antrodia cinnamomea mycelium, and to determine the content of crude polysaccharide and crude triterpenoid and antioxidant activity of cultured products. Methods The product cultured by solid-state fermentation in petri dish was ultrasonically extracted. The phenol-sulfuric acid method was selected to determine the content of crude polysaccharide of the extract, and the vanillin-glacial acetic acid method was used to determine the content of crude triterpenoids of the extract. The half-clearing concentrations (IC50) of DPPH free radicals and ABTS free radicals were indexes for evaluating the anti-oxidant activity of the culture product. Results Sampling near the outer edge of the mycelium layer after 20 d of culture was performed by the activation method to obtain inoculated raw materials with better growth activity; The basal medium with good growth, high content of crude polysaccharide and crude triterpenoids was modified PCA medium; On the basis of this medium, the mycelia with 0.5 g/L lignin added into the medium grew fastest, and the diameter of mycelium layer increased to 1.19 times of the control group; When the concentration of IBA was 0.5 mg/L, the dry weight of mycelium was increased by 89.51% compared with the control group, and the yield of crude polysaccharide was increased by 130.57% compared with the control group, which was much higher than other groups. The content and yield of triterpenoids were also increased significantly, 61.31% higher than the control group, and crude triterpenoids yield reached 133.24 mg/L; The content of crude triterpenoids in mycelium was the highest (5.62%) when adding 0.5 g/L powder of Cinnamomum kanehirai, which was 84.26% higher than that of the control group; In addition, the addition of plant hormones and lignin to the culture medium has a certain effect on the anti-oxidant capacity of the Antrodia cinnamomea mycelium, on the whole, the experimental group had good anti-oxidant activity after adding different substances. Conclusion By adding phytohormone and lignin to the modified PCA medium, the growth of the mycelium of Antrodia cinnamomea can be effectively promoted, the content of the active ingredient can be increased, and the anti-oxidant activity can be enhanced.

Optimization of yeast autolysis under solid-state fermentation conditions / 生物工程学报

Yeast autolysis under solid-state fermentation can effectively promote the release of various active substances, thereby improving the quality of yeast products. The optimal process for yeast autolysis under solid-state fermentation was obtained by optimizing the autolysis temperature, autolysis time and the zinc ion concentration. We analyzed the indexes of free amino acid, soluble protein and α-amino nitrogen in the fermentation material, as well as A₂₆₀/A₂₈₀ ratio to determine yeast autolysis process conditions in the solid-state fermentation. On the basis of the obtained data, L₉ (3³) orthogonal test was designed to optimize the solid-state fermentation parameters for yeast autolysis: temperature at 40, 50 and 55 °C; time 12, 18 and 24 h; zinc ion concentration 2, 4 and 8 mg/kg. The optimum process conditions for yeast autolysis were: autolysis temperature 55 °C, time 18 h, zinc ion concentration 2 mg/kg, and soluble protein content reached 9.31 mg/g, free amino acid 14.36 mg/g, α-amino nitrogen 10.16 μg/g and A₂₆₀/A₂₈₀ 1.73. After optimization of the process, the soluble protein, free amino acid and α-amino nitrogen contents of the yeast autolysis production can be significantly increased, thereby obviously improving the quality of the composite culture.

Local strains Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1 as a reducing and detoxifying agents for deoxynivalenol

Aims@#Deoxynivalenol is a type B trichothecene produced by Fusarium graminearum that can cause serious health problems in human and livestock. The present study aimed to reduce and detoxify deoxynivalenol using a local strain Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1. @*Methodology and results@#Corn as solid substrate artificially inoculated with F. graminearum bio 163252 to produce deoxynivalenol. Deoxynivalenol contaminated corn then inoculated with A. oryzae KKB4 and R. oryzae KP1R1. During fermentation, a decrease in deoxynivalenol levels is analyzed including loss of dry matter and glucosamine content. Deoxynivalenol was extracted from the substrate by solid phase extraction and quantified using high-performance liquid chromatography. The reduction of deoxynivalenol by A. oryzae KKB4 and R. oryzae KP1R1 were 65.91% and 56.82%, respectively after ten days of fermentation. Toxicity analysis revealed that residues of deoxynivalenol were not toxic to growth of Saccharomyces cerevisiae cells. @*Conclusion, significance and impact of study@#Local strains A. oryzae KKB4 and R. oryzae KP1R1 were able to reduce and detoxify deoxynivalenol in solid substrates. This study provides supporting data to control mycotoxin that is critical for food and feed safety.

Cellulases production on paper and sawdust using native Trichoderma asperellum / Producción de celulasas en papel y aserrín usando Trichoderma asperellum / Produção de celulases em papel e serragem usando Trichoderma asperellum

Abstract Microbial cellulases are industrially used enzymes that catalyze the cleavage of the glycosidic bonds of cellulose. This hydrolysis yields sugars that can be used in processes such as bioethanol production. These enzymes are mainly produced by fungi belonging to the genus Trichoderma via submerged or solid state fermentation with cellulosic materials as substrates. Recent publications have increasingly demonstrated that alternatives to T. reesei enzymes in the production of second-generation biofuels exist. Here, cellulolytic activities of crude extracts obtained from a native isolate of T. asperellum from coffe pulp and a strain of T. reesei were evaluated. Solid state fermentations were performed using paper and sawdust as substrates. The activities were measured after 12 days of incubation. The extracts obtained from T. reesei showed higher cellulase and endoglucanase activities (6.5 and 5.8 U/g) than those obtained using T. asperellum (5.6 and 4.1 U/g) with paper as substrate. There were no significant differences between isolates when grown on sawdust. It was possible to verify that native T. asperellum was able to produce cellulases on lignocellulosic material such as moistened paper and sawdust without having undergone a chemical pretreatment.

Resumen Las celulasas microbianas son enzimas utilizadas industrialmente, que catalizan la ruptura de enlaces glicosídicos de celulosa. Esta hidrólisis produce azúcares que pueden utilizarse en procesos tales como la producción de bioteanol. Estas enzimas son producidas principalmente por hongos pertenecientes al género Trichoderma vía fermentación en estado sólido o sumergido, con materiales celulósicos como sustratos. Las publicaciones recientes han demostrado de forma creciente que existen alternativas a las enzimas de T. reesei en la producción de biocombustibles de segunda generación. En este estudio se evaluaron las actividades celulolíticas de extractos crudos obtenidos de un aislamiento nativo de T. asperellum de pulpa de café y una cepa de T. reesei. Las fermentaciones en estado sólido se llevaron a cabo usando como sustratos papel y aserrín. Las actividades se midieron después de 12 días de incubación. Los extractos obtenidos de T. reesei mostraron mayor actividad de celulasa y endoglucanasa (6.5 and 5.8 U/g) que los obtenidos usando T. asperellum (5.6 and 4.1 U/g) con papel como sustrato. No hubo diferencias significativas entre los dos aislamientos cuando crecieron en aserrín. Se pudo verificar que T. asperellum nativa fue capaz de producir celulasas en material lignocelulósico, como papel humedecido y aserrín, que no había pasado por un pretratamiento químico.

Resumo As celulases microbianas são enzimas utilizadas industrialmente, que catalisam o rompimento das ligações glicosídicas da celulose. Esta hidrólise produze açúcares que podem ser utilizados em processos como a produção de bioetanol. Estas enzimas são produzidas principalmente por fungos pertencentes ao gênero Trichoderma, via fermentação em estado sólido ou submerso, com materiais celulósicos como substrato. As publicações recentes veem demonstrando de maneira crescente que existem alternativas as enzimas de T. reesei na produção de biocombustíveis de segunda geração. Neste estudo foram avaliadas as atividades celulolíticas de extratos brutos obtidos de um isolamento nativo de T. asperellum da polpa de café e uma cepa de T. reesei. As fermentações em estado sólido se realizaram usando como substrato papel e serragem. As atividades foram medidas depois de 12 dias de incubação. Os extratos obtidos de T. reesei mostraram maiores atividades de celulase e endoglicanase (6.5 e 5.8 U/g) que os obtidos usando T. asperellum (5.6 e 4.1 U/g) com papel como substrato. Não houve diferenças significativas entre os dos isolamentos quando cresceram em serragem. Foi possível verificar que T. asperellum nativa foi capaz de produzir celulases em material lignocelulósico, como papel humedecido e serragem, que não haviam passado por um pré-tratamento químico.

Production of β-Mannanase by Penicillium italicum Subjected to Different Growth Conditions

Aims: The process parameters affecting enzyme production were optimized to ascertain the best optimal conditions for β-mannanase production by Penicillium italicum in solid state fermentation. Study Design: Four stages of experimental processes were designed for this study. The first experiment, samples were withdrawn after 24, 48, 72, 96, 120, 144,168 and 192 h incubation. In second experiment, the fermentation media were incubated at different temperatures. In third experiment, the effect of different pH values on β-mannanase production was evaluated, while the fourth experiment described the supplementation of surfactants in mineral salt solution for β-mannanase production. Place and Duration of Study: Microbiology Research Laboratory, Federal University of Technology, Akure Nigeria between September 2011 and March 2012. Methodology: β-mannanase production was conducted using Locust Bean Gum (LBG) as the sole carbon source; moisten with mineral salt solution, and enzyme activity determined by dinitrosalicylic acid method, while protein content was determined by Lowry method. Results: Maximum enzyme activity (146.389 U/ml) was observed after 72 h of incubation. Different surfactants were supplemented in the basal medium, and Sodium Dodecyl Sulfate (SDS) was observed to give the highest β-mannanase activity of 53.335 U/ml. Initial pH of the culture medium was optimized and a pH of 6.0 was found to support maximum enzyme activity (173.241 U/mg protein). The optimum incubation temperature was achieved at 35°C. Conclusion: The results obtained provide information on optimal process parameters that might improve the yield of β-mannanase by P. italicum for better fish feed formulation, especially in the larval stages of fish fingerlings when the enzyme system is not efficient.

Decolorization of textile industry wastewater in solid state fermentation with Peach-Palm (Bactris gasipaes) residue / Decoloração de águas residuais da indústria têxtil em fermentação de estado sólido com resíduo de Pupunha (Bactris gasipaes)

Abstract In this work we have assessed the decolorization of textile effluents throughout their treatment in a solid-state fermentation (SSF) system. SSF assays were conducted with peach-palm (Bactris gasipaes) residue using the white rot fungus Ganoderma lucidum EF 31. The influence of the dye concentration and of the amounts of peach-palm residue and liquid phase on both the discoloration efficiency and enzyme production was studied. According to our results, independently of experimental conditions employed, laccase was the main ligninolytic enzyme produced by G. lucidum. The highest laccase activity was obtained at very low effluent concentrations, suggesting the existence of an inhibitory effect of higher concentrations on fungal metabolism. The highest percentage of color removal was reached when 10 grams of peach palm residue was moistened with 60 mL of the final effluent. In control tests carried out with the synthetic dye Remazol Brilliant Blue R (RBBR) decolorization efficiencies about 20% higher than that achieved with the industrial effluent were achieved. The adsorption of RBBR on peach-palm residue was also investigated. Equilibrium tests showed that the adsorption of this dye followed both Langmuir and Freundlich isotherms. Hence, our experimental results indicate that peach-palm residue is suitable substrate for both laccase production and color removal in industrial effluents.

Resumo Neste trabalho, avaliamos a descoloração de efluentes têxteis durante seu tratamento em um sistema de fermentação em estado sólido (SSF). Os ensaios foram conduzidos com resíduo de pupunha (Bactris gasipaes) utilizando o fungo de podridão branca Ganoderma lucidum EF 31. A influência da concentração de corante, as quantidades de resíduo e da fase líquida foram estudadas tanto na eficiência de descoloração como na produção de enzima. De acordo com os resultados, independentemente das condições experimentais utilizadas, a lacase foi a principal enzima ligninolítica produzida por G. lucidum. A atividade de lacase mais elevada foi obtida em baixas concentrações de efluentes, sugerindo um efeito inibitório no metabolismo fúngico. A maior remoção de cor foi obtida com 10 gramas de resíduo da pupunha e 60 mL do efluente final. Nos ensaios de controle realizados com o corante sintético RBBR, foram atingidos cerca de 20% mais descoloração do que os obtidos com o efluente industrial. A adsorção de RBBR no resíduo de pupunha também foi investigada. Os testes de equilíbrio mostraram que a adsorção deste corante seguiu as isotermas de Langmuir e Freundlich. Assim, os resultados experimentais indicam que o resíduo de pupunha é um substrato adequado tanto para a produção de lacase quanto para a remoção de cor em efluentes industriais.

α-L- rhamnosidases Produced under Solid State Fermentation by Few Aspergillus Strains

The production of α-L-rhamnosidase from Aspergillus ochraceous MTCC -1810, A. wentii MTCC- 1901, A. sydowii MTCC- 635, A. foetidus MTCC-508 under solid- state fermentation using easily available agro- industrial residues such as corn cob, rice bran, sugarcane bagasse, wheat bran and citrus peel as substrate. Among these, sugarcane bagasse in combination with naringin and sucrose were found to be the best substrate. The α-L-rhamnosidase production was highest after the 4th day of incubation at 30ºC and a substrate to moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused the maximum production of the enzyme. The temperature optima and pH optima of α-L-rhamnosidases were determined in the range of 50-60ºC and 4.0-5.0 respectively. The α-L-rhamnosidases secreted from the above fungal strains is suitable for the debittering of orange fruit juice.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante 3 estrategias de propagación / Production of infectious units of Isaria fumosorosea ( Hypocreales: Cordycipitaceae) from different indigenous isolates of northeastern Mexico using 3 propagation strategies

El objetivo del presente estudio fue evaluar la producción de blastosporas y conidios de diferentes aislados nativos de México del hongo entomopatógeno Isaria fumosorosea y de una cepa de colección mediante diferentes técnicas de propagación. En la producción de blastosporas se utilizaron 2 medios de cultivo líquidos (sumergidos), uno a base de casaminoácidos y el otro a base de peptona de colágeno como fuentes de nitrógeno, con glucosa como fuente de carbono en ambos. Para la producción de conidios, los hongos se cultivaron en agar papa dextrosa, a partir de esos cultivos se prepararon suspensiones de 1 x 10(6) conidios/ml para inocular matraces con caldo dextrosa Sabouraud, para iniciar así la fase líquida del cultivo bifásico, denominado también precultivo. Posteriormente con el precultivo y las suspensiones de conidios se inocularon bolsas con granos de arroz, que se incubaron durante 14 días para el cultivo bifásico y para la fermentación sólida, respectivamente. El aislado HIB-23 fue el que logró la más elevada concentración de blastosporas obtenida en el cultivo sumergido: 4,90 x 10(8) blastosporas/ml en el medio casaminoácidos; y en el medio con peptona de colágeno se obtuvieron 2,15 x 10(8) blastosporas/ml. La máxima producción de conidios en fermentación sólida la logró la cepa Pfr-612 (1,58 x 10(9) conidios/g), mientras que la máxima en cultivo bifásico correspondió al aislado HIB-30 (9,00 x 10(6) conidios/g). La fermentación sólida resultó ser el método más efectivo, con un promedio de 1,09 x 10(9) conidios/g, mientras que el cultivo bifásico fue el menos efectivo, con un promedio de 2,76 x 10(6) conidios/g. Para la producción de blastosporas en los medios sumergidos no se obtuvo diferencia significativa alguna.

The aim of this study was to evaluate the production of blastospores and conidia of different native isolates and a strain of Isaria fumosorosea using different propagation techniques. Two liquid culture media of casamino acids and peptone as nitrogen sources and glucose as carbon source for both media cultures were respectively used in the production of blastospores, while for the production of conidia, the fungi were grown in potato dextrose agar; from these cultures, solutions of conidia to a concentration of 1 x 10(6) per milliliter were prepared to inoculate flasks with Sabouraud dextrose broth for the liquid phase of the biphasic culture, also known as preculture. Subsequently, rice grain bags were inoculated with the preculture and the conidia solutions, which were incubated for 14 days for solid fermentation and biphasic culture, respectively. The HIB-23 isolate recorded a concentration of 4.90 x 10(8) blastospores/ml in the casamino acid medium, while a concentration of 2.15 x 10(8) blastospores/ml was obtained in the peptone collagen medium. For the Pfr-612 strain, the conidia production in solid-state fermentation was 1.58 x 10(9) conidia/g, and for HIB-30 in the biphasic culture of 9.00 x 10(6) conidia/g. Solid-state fermentation proved to be the most effective method with an average of 1.09 x 10(9) conidia/g, whereas the biphasic culture was the least effective method with 2.76 x 10(6) conidia/g; no significant difference was reported for the submerged production media.

Process Optimization for Lipolytic Bacterial Fermentation: Value Addition to the By-products of Oil Seeds

Background: Lipase enzyme has wide application in industries, particularly food and detergent, but high production cost has always limited its use. Extensive studies are underway on production of high quality and low cost lipase enzyme in large amounts, for which microbial sources have been found to be the best. Aim: To estimate the potential of oil cakes for bacterial lipase production. Methodology: By-products of different oil seeds viz. neem, sesame, flax, mustard, coconut, castor, and groundnut were used for the preparation of fermentation media to culture lipolytic Pseudomonas aeruginosa. Optimization of growth condition was done with respect to different parameters such as fermentation time, nitrogen supplements, carbon additives, and lipid sources. Results: A good lipolytic P. aeruginosa JCM5962 (T) strain was isolated from soil of sugarcane field. Results of the study showed that coconut, sesame, neem, flax and mustard oilcakes induced good lipolytic activity from bacteria. Negligible lipase activity was obtained when organism was cultured in castor and groundnut oilcake medium. 1% ammonium nitrate as an additional nitrogen supplement was found to be ideal parameter for improved production. Conclusion: According to present work, lipases could be economically produced by P. aeruginosa using low cost oil cakes as potent substrate for fermentation medium.