Solid-state Bioprocessing Research Articles

Fermented quinoa flour: Implications of fungal solid-state bioprocessing and drying on nutritional and antioxidant properties

Quinoa (Chenopodium quinoa Willd) is a pseudocereal rich in protein and excellent source of vitamins, antioxidants, and minerals. Developing new quinoa-based products would be a strategy to extend its consumption. The aim of this study is to evaluate the impact of solid-state fermentation with Pleurotus ostreatus and drying (hot-air at 50, 60 and 70 °C and lyophilization) on the nutritional and functional properties of fermented white and black quinoa flours. Changes in proximal composition revealed that white variety was more suitable for solid-state fermentation (SSF), according to the higher biomass production, higher increase of protein (2.11%) and insoluble fiber (7.82%) content. SSF was highly effective in phytic acid reduction (90%) regardless the variety. However, after fermentation, quinoa seeds presented reduced total phenolic content (TPC) and antioxidant capacity, especially on black variety. Nonetheless, hot-air drying improved the antioxidant properties (increased TPC and antioxidant capacity) and promoted changes in the phenolic profile. These changes were characterized by a release of gallic acid together with a notable reduction of vanillic, caffeic and ferulic acids, quercetin-3-glucoside and/or quercetin. A notable increase of the angiotensin converting enzyme-I inhibitory activity was found on the fermented flours mainly due to SSF.

Valorization of agricultural residues using Myceliophthora thermophila as a platform for production of lignocellulolytic enzymes for cellulose saccharification

The use of lignocellulosic agricultural residues is emerging as a low-cost, sustainable, and eco-friendly approach for the production of microbial enzymes and other chemicals. In this study, the production of enzymes with lignocellulolytic potential and the secretome profile of the thermophilic fungus Myceliophthora thermophila were evaluated in solid-state cultivation on agricultural residues such as rice straw, soy straw, and wheat bran. In addition, the saccharification potential of the extracts was evaluated. The analysis of enzymatic activities (peptidase, lipase, amylase, CMCase, FPase, β-glucosidase, xylanase, pectinase, and laccase) and the 66 proteins identified in the secretome reinforced the hydrolytic and oxidative properties of the enzymatic lignocellulolytic arsenal produced by M. thermophila. The extracellular extract grown on rice straw obtained after 72 h of solid-state bioprocess achieved 46% saccharification of wheat bran substrate. Furthermore, the overall performance of the wheat bran extract (24 h) indicates the relevance of the CMCase, FPase and xylanase activities in saccharification processes. These results highlight the potential of M. thermophila as a platform for the production of enzymes with biotechnological potential, especially for lignocellulose degradation and cellulose saccharification. Solid-state bioprocess allows exploring agro-industrial residues, as a matrix and source of nutrients, for the enzyme production and emphasizes the possibility of adding value to agricultural waste. Moreover, the use of M. thermophila in solid-state bioprocesses can still be widely explored and optimized as a platform to scale up the production of cheapest fungal enzymes.

Solid-state bioprocessing of sugarcane bagasse with Auricularia fuscosuccinea for phenolic compounds extraction

Sugarcane bagasse is a natural source of phenolic compounds. However, these compounds are bound to lignocellulose components, reducing their ability to function as good antioxidants. These linkages are hydrolyzed by enzymes like β-glucosidases, increasing free phenolics. Auricularia is a food-grade genus capable of producing β-glucosidases. The aim of this work was (I) to determine naturally occurring species of Auricularia and (II) to obtain phenolic compounds through the solid-state bioprocessing of sugarcane bagasse. We have successfully isolated five strains that were assigned to the taxon A. fuscosuccinea. We determined β-glucosidase activity by fluorescence plate assay of the five isolated strains and adjusted an optimal temperature for mycelial growth at 30 °C. A. fuscosuccinea LBM 243 was chosen for solid-state bioprocessing of sugarcane bagasse. β-glucosidase activity (12.2 ± 0.62 U l−1) and protein content (51.58 ± 6.26 mg l−1) were highest on day 20 of culture. The maximum value of total phenolic content (507.5 ± 9.05 mg l−1) was obtained at day 20 and antioxidant capacity (34.44% ± 11.20) was highest at day 10, both in ethanolic extracts. The best performance of ethanol against methanol extraction in this work is highlighted considering ethanol to be a safe, efficient, and low-cost solvent.

OPTIMIZATION OF POMEGRANATE PEEL EXTRACTS FOR THE BIOCONVERSION OF THE ELLAGITANNINS TO ELLAGIC ACID USING ASPERGILLUS NIGER, RHIZOPUS ORYZAE AND MIXED CULTURE

Objective: The present study is aimed at optimization of concentration of substrate (pomegranate peel extract) for the production of Ellagic acid using Aspergillus niger and Rhizopus oryzae. Methods: Test organisms were isolated and identified using standard microbiological techniques. Collected pomegranate peels were dried, grained and used as the substrate for solid-state fermentation. Prepared spore suspension of the test organisms was inoculated and incubated at room temperature. At regular intervals of 24 h, samples were drawn and subjected to analysis of reducing sugar, soluble proteins, hydrolysable tannins and production of extracellular tannase using standard methods. Results: The results of the present investigation demonstrated the optimum substrate concentration for the active conversion of ellagitannins to ellagic was 15g of A. niger, 20g for R. oryzae and 15g for mixed culture. Conclusion: From the current work, it was concluded that solid-state bioprocessing of fruit substrates and fruit wastes using fungi has shown to enrich phenolic antioxidants and improve phytochemical consistency.

Fungal-Mediated Biotransformation of Soybean Supplemented with Different Cereal Grains into a Functional Compound with Antioxidant, Anti-Inflammatory and Antitumoral Activities

The aim of this study was to select fungal strains and alternative substrates to increase the production of bioactive compounds by solid-state bioprocessing using soybeans. Initially, from a total of 17 fungi strains, R. oligosporus NRRL 3267, R. oligosporus NRRL 2710, and R. arrhizus NRRL 2582 were pre-selected for presenting the greatest antioxidant activities during soybean fermentation. The three Rhizopus strains were cultured in soybeans supplemented with different cereal grains (brown rice, wheat, corn, and oat) aiming to achieve a higher antioxidant activity. Soybean supplementation with brown rice increased the concentration of phenolic compounds (0.697 to 6.447 mg GAE g-1) significantly compared with the only use of soybean (1.792 to 3.10 mg GAE g-1), using R. oligosporus NRRL 2710. The improved fungal-mediated biotransformation process (R. oligosporus NRRL 2710 with soybeans and brown rice) generated an isoflavone aglycone-rich product, containing different antioxidant compounds, such as trans-cinnamic acid, gallic acid, myricetin, quercetin, and kaempferol. The fermented substrate also showed great potential to inhibit hyaluronidase enzyme (anti-inflammatory activity) and against CaCo-2 tumor cells growth (antitumor activity). The resulting compound could serve as a value-added food and/or feed ingredient and a rich source of bioactive compounds.

Changes in biochemical composition of cassava and beet residues during solid state bioprocess with Pleurotus ostreatus

The development of the Pleurotus ostreatus in cassava ( Manihot esculenta ) and beet ( Beta vulgaris ) residues via solid state bioprocess (SSB) was evaluated in terms of variation of proximate composition, phenolic compounds, minerals, in vitro digestibility, fungal growth and lipase activity. The substrates were cultured at 30 °C for 35 days and sampled every 5 days. The amount of protein, and fibers of cassava residues, increased by 125.7%, and 253.2%, respectively. The beet residue showed a protein increment of 57.6%, and fibers of 232.7%. In beet residues, the lipid increase was of 261.7% while that for cassava residues was of 136.2%. The cassava residue showed a reduction in phenolic compounds from 18.07 mg of acid galic equivalent (AGE)/g to 4.17 mg AGE/g and beet from 30.96 mg to 3.50 mg. In vitro digestibility increased by 5.24% in cassava residues and 29.38% in beet residues. P. ostreatus reached 8.63 CFU/g (15 days) in cassava residue and 6.08 CFU/g (25 days) in beet residue. Maximum lipase activity was 0.71 U/g (35 days) in beet residues and 0.46 U/g (20 days) in cassava residues. Both beet and cassava residues presented nutritional enrichment (carbohydrate reduction and increase of other compounds) and increased in vitro digestibility with P. ostreatus via SSB. This application presents great biotechnological potentiality, e.g. the utilization as a low-cost nutrient source for animal feed.

Biotransformation of fruit residues via solid state bioprocess using Lichtheimia ramosa

Lichtheimia ramosa is a promising candidate fungus for solid state bioprocesses (SSB) due its rapid colonization. Fruit wastes present ideal conditions for fungal growth and biotransformation, which can promote the release of products of biotechnological interest. Thus, the aim of this work was to evaluate the mycelial growth and the enzymatic activities of L. ramosa in pineapple (Ananas comosus), orange (Citrus sinensis), mango (Mangifera indica), passion fruit (Passiflora edulis) and grape (Vitis vinifera) fruit wastes via SSB, and their influence in the generation of molecules with potential use. The SSB was evaluated in terms of capacity of biotransformation of the substrate (composition, protein enrichment, and fatty acid profile) and production of amylase, carboxymethylcellulase (CMCase), xylanase and lipase enzymes. Main cultivations were carried out at 30 °C for 30 days and every 5 days samples were taken and analyzed for microbiological content, proximal composition and enzymatic profile. Fatty acids were determined at day 0 (baseline) and at the end of the cultivations. L. ramosa grew well in all substrates up to the 25th day, except for on the orange residue, upon which development was slightly lower at this time. Protein enrichment was found in all substrates as follows: passion fruit (309.54%), pineapple (294.89%), mango (263.45%), orange (65.60%) and grapes (19.17%). Regarding enzymes, lipase was not synthetized in any of the substrates, though amylase, CMCase and xylanase were observed at different levels. The fatty acid profile varied from raw to cultivated substrates, indicating that L. ramosa can act in the synthesis and conversion of these acids. It was concluded that L. ramosa and the studied substrates are viable for SSB.

Production of Fungal Laccase under Solid State Bioprocessing of Agroindustrial Waste and Its Application in Decolourization of Synthetic Dyes

Fungal laccases are preferred due to high redox potentials and low substrate specificity to xenobiotics including synthetic dyes. For large-scale applications, low enzyme yield and high cost of production has remained the challenge. Agroindustrial waste such as saw-dust of Terminalia superba abounds locally and was utilized as low-cost alternative substrate for laccase production in Solid State Bioprocessing (SSB) using Trametes sp. isolate G31. The study optimized laccase production using various parameters. Optimal production of laccase was at pH 5.0 - 7.0 with 2356 U/mL - 2369 U/mL and 25°C (2336 U/mL). Among the sources of nitrogen and carbon tested, laccase production in ammonium sulphate and sucrose supplemented media were higher. The effect of activators on laccases production showed that Cu2+ and Ca2+induced high titres of laccase at 4 -5 mM and 2 - 4 mM respectively, while production of laccase by Mn2+ was significantly high at 40 mM. The effect of 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), guaiacol and varatryl alcohol on laccase production was significantly different especially in glycerol. Optimum production for laccase was on day 14 with 2356 U/mL followed by steady declined up to day 34. The purified laccase had a specific activity of 5008 µmol/min/mg, purification factor of 3.85, and a molecular mass of ~40 kDa using N-PAGE. The potential of crude laccase to decolourize diverse dyes was tested. Phenol red achieved 40% decolourization for 1hr, while RBBR (93%), Crystal violet (60%), Methylene blue (53%) and Congo red (51%) for 24 hr, 72 hr, 48 hr and 120 hr respectively. Methyl red and Malachite green attained 42% (72 hr) and 32% (48 hr) decolourization. The enzyme ability to oxidize Phenol red and other synthetic dyes without mediators made it eco-friendly in treating dye wastewaters.

Role of carbohydrate-cleaving enzymes in phenolic mobilization of guava leaves tea during solid state bio-processing with Monascus anka and Bacillus sp

• Solid-state bio-processing (SSB) increased soluble phenolic contents (SPC) of guava leaves tea (GLT). • Relationship between SPC and carbohydrate-cleaving enzyme was investigated during SSB. • High levels of α -amylase and total cellulase activities were observed during SSB. • Good correlations between SPC and α -amylase, cellulase and β -glucosidase activity. • Low correlation on between SPC and xylanase activity. The purpose of this study was to investigate the effect of carbohydrate-cleaving enzymes produced by Monascus anka and Bacillus sp. in the phenolic mobilization during guava leaves tea (GLT) bio-processing. The results showed solid state bio-processing (SSB) greatly increased the contents of soluble phenolic and flavonoid aglycone in GLT. During SSB, the activities of α -amylase (83.05 U/g) and total cellulase (31.32 U/g) were high, while those of xylanase (5.27 U/g) and β -glucosidase (0.19 U/g) were relatively lower. There was a good correlation between the content of soluble phenolic released from GLT and the activities of α -amylase, cellulase and β -glucosidase, while a low correlation between the content of soluble phenolic and the activity of xylanase. Moreover, the contents of soluble phenolic, especially quercetin and kaempferol, in GLT treated with mixture of pure enzymes significantly increased. The study provides theoretical basis for the phenolic mobilization of plant matrices during SSB and lays a solid foundation for seeking more efficient methods for enhancing the efficacy of tea infusions.

Cultivation of Medicinal Mushroom Biomass by Solid-State Bioprocessing in Bioreactors.

Basidiomycetes of various species and their wide range of pharmaceutically interesting products in the last decades represent one of the most attractive groups of natural products in Asia and North America. Production of fungal fruit bodies using farming technology is hardly covering the market. Comprehensive solid-state technologies and bioreactors are the most promising part for fast and large amount of cultivation of medicinal fungi biomass and its pharmaceutically active products. Wood, agriculture, and food industry wastes represent the main substrates that are in this process delignified and enriched in proteins and highly valuable pharmaceutically active compounds. Research in physiology, basic and applied studies in fungal metabolism, process engineering aspects, and clinical studies in the last two decades represent large contribution to the development of these potentials that initiate the development of new drugs and some of the most attractive over-the-counter human and veterinary remedies. Present article is an overview of the achievements in solid-state technology of the most relevant medicinal mushroom species production in bioreactors. Graphical Abstract.

Liberation and recovery of phenolic antioxidants and lipids in chokeberry (Aronia melanocarpa) pomace by solid-state bioprocessing using Aspergillus niger and Rhizopus oligosporus strains

The present study focused on the changes in polyphenols, antioxidant activities and lipid compositions from chokeberry pomace during solid-state fermentation (SSF) with Aspergillus niger and Rhizopus oligosporus. The extractable phenolics increased more than 1.7- fold during both fermentation processes. A similar trend was observed for total flavonoids. The obtained results also indicated that a longer fermentation period resulted in a greater loss of anthocyanins. The free radical scavenging ability of phenolic extracts, evaluated by DPPH and TEAC assays, were significantly enhanced during the SSFs. HPLC-MS analysis of phenolic compounds showed that the amounts of flavonols and cinnamic acids increased while the concentrations of glycosylated anthocyanins decreased substantially. The SSF of chokeberry pomace also resulted in a significant increase in lipids with high linoleic acid content (57–61% of the total fatty acids). The present investigation demonstrated that SSF enriched the chokeberry pomace with phenolic antioxidants and lipids with better nutritional-quality characteristics.

Delignification of Lignocellulose Biomasses by Alginate–Chitosan Immobilized Laccase Produced from Trametes versicolor IBL-04

An extracellular laccase was produced from a lignin-degrading white-rot fungus Trametes versicolor IBL-04 using the solid-state bioprocessing of corncobs. The enzyme thus produced was effectively immobilized on alginate–chitosan beads. Immobilized laccase furnished the highest immobilization yield (81.43 ± 2.98) under optimized working conditions of 3.0% (w/v) sodium alginate concentration, 2.4% CaCl2, 0.25% chitosan concentration following an immobilization time of 5 h. The immobilization process altered the optimum pH from 5.0 to 4.0 (an acidic shift), whereas the temperature optima were displaced from 40 to 50 °C (towards higher temperature). The Michaelis–Menten constant (K m) and the maximum reaction rate (V max) of the free and immobilized laccase were 0.14 mM, and 780 µmol/mL and 0.10 mM and 814 µmol/mL, respectively. The alginate–chitosan immobilized laccase was more stable and tolerant against the activity inhibition caused by the denaturants tested, concerning its soluble laccase. Delignification results revealed that the enzyme led to a marked reduction in lignin content of all the lignocellulosic wastes with the highest delignification of 78.29% in sugarcane bagasse, followed by wheat bran and corn stover after 15 h. It is demonstrated that the immobilized laccase could be a promising option as a green catalyst for the efficient delignification of lignocellulosic biomass residues.

Induction of fungal laccase production under solid state bioprocessing of new agroindustrial waste and its application on dye decolorization

Lignocellulosic wastes are generally produced in huge amounts worldwide. Peach waste of these obtained from fruit juice industry was utilized as the substrate for laccase production by Pleurotus eryngii under solid state bioprocessing (SSB). Its chemical composition was determined and this bioprocess was carried out under stationary conditions at 28 °C. The effects of different compounds; copper, iron, Tween 80, ammonium nitrate and manganese, and their variable concentrations on laccase production were investigated in detail. The optimum production of laccase (43,761.33 ± 3845 U L−1) was achieved on the day of 20 by employing peach waste of 5.0 g and 70 µM Cu2+, 18 µM Fe2+, 0.025% (v/v) Tween 80, 4.0 g L−1 ammonium nitrate, 750 µM Mn2+ as the inducers. The dye decolorization also researched to determine the degrading capability of laccase produced from peach culture under the above-mentioned conditions. Within this scope of the study, methyl orange, tartrazine, reactive red 2 and reactive black dyes were treated with this enzyme. The highest decolorization was performed with methyl orange as 43 ± 2.8% after 5 min of treatment when compared to other dyes. Up to now, this is the first report on the induction of laccase production by P. eryngii under SSB using peach waste as the substrate.

Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The “primed” side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S′1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P′1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.

Production of a generic microbial feedstock for lignocellulose biorefineries through sequential bioprocessing

Lignocellulosic materials, mostly from agricultural and forestry residues, provide a potential renewable resource for sustainable biorefineries. Reducing sugars can be produced only after a pre-treatment stage, which normally involves chemicals but can be biological. In this case, two steps are usually necessary: solid-state cultivation of fungi for deconstruction, followed by enzymatic hydrolysis using cellulolytic enzymes. In this research, the utilisation of solid-state bioprocessing using the fungus Trichoderma longibrachiatum was implemented as a simultaneous microbial pretreatment and in-situ enzyme production method for fungal autolysis and further enzyme hydrolysis of fermented solids. Suspending the fermented solids in water at 50°C led to the highest hydrolysis yields of 226mg/g reducing sugar and 7.7mg/g free amino nitrogen (FAN). The resultant feedstock was shown to be suitable for the production of various products including ethanol.

Kinetic characterization, thermo-stability and Reactive Red 195A dye detoxifying properties of manganese peroxidase-coupled gelatin hydrogel.

An indigenous and industrially important manganese peroxidase (MnP) was isolated from solid-state bio-processing of wheat bran by white-rot fungal strain Ganoderma lucidum IBL-05 under pre-optimized growth conditions. Crude MnP extract was partially purified (2.34-fold) to apparent homogeneity by ammonium sulphate precipitation and dialysis. The homogeneous enzyme preparation was encapsulated on gelatin matrix using glutaraldehyde as a cross-linking agent. Optimal conditions for highest immobilization (82.5%) were: gelatin 20% (w/v), glutaraldehyde 0.25% (v/v) and 2 h activation time using 0.6 mg/mL of protein concentration. Gelatin-encapsulated MnP presented its maximum activity at pH 6.0 and 60 °C. Thermo-stability was considerably improved after immobilization. The optimally active MnP fraction was tested against MnSO4 as a substrate to calculate kinetic parameters. More than 90% decolorization of Sandal-fix Red C4BLN (Reactive Red 195A) dye was achieved with immobilized MnP in 5 h. It also preserved more than 50% of its original activity after the sixth reusability cycle. The water quality parameters (pH, chemical oxygen demand, total organic carbon) and cytotoxicity (brine shrimp and Daphnia magna) studies revealed the non-toxic nature of the bio-treated dye sample. A lower Km, higher Vmax, greater acidic and thermal-resistant up to 60 °C were the improved catalytic features of immobilized MnP suggesting its suitability for a variety of biotechnological applications.

Optimization of lipase production by solid state bioprocess using packed bed bioreactor

Optimization of lipase production by solid state bioprocess using packed bed bioreactor

Extending shelf life of wheat based animal feed using solid state bioprocessing

The recent expansion in the number of biorefineries, due in particular to the advent of the biofuels industry, has led to a massive production of agro-industrial solid residues as by-products. Solid state bioprocessing (SSB) has been targeted as a high-priority technology for the treatment of this type of residues. In this research, the utilisation of SSB technologies using the edible fungus Rhizopus oryzae as a potential method for increasing the shelf life of animal feed, produced from the residues of a wheat bioethanol plant, has been investigated.Experimental results showed that R. oryzae is able to prevent the growth of undesired microorganisms for at least 20 days. Samples that were not inoculated with R. oryzae showed growth after just 2 days and samples that were dosed with a commercial preservative experienced growth of pathogens after 7 days. Currently, preservatives (organic acids) are added to the solid wastes to avoid biodegradation but clearly increase the production costs. It was estimated that for a bioethanol plant producing 180,000tpa of animal feed the substitution of organic acids for the proposed method using SSB would represent savings of up to 500,000 USD/year.

Peptidase with Keratinolytic Activity Secreted by Aspergillus terreus During Solid-State Fermentation

The aim of this study was to evaluate peptidase production by Aspergillus terreus in solid-state bioprocess and evaluate its parameters. The best conditions were 5.0 g of wheat bran as substrate, incubation temperature 30°C, inoculum 2.0x105spores/g and 75% saline volume, with production reaching 677 U/mL (5400 U/g culture medium) after 72 h of fermentation. Biochemical characterization of the crude enzymatic extract showed the optimum pH and temperature of 6.5 and 55°C, respectively. The stability at different temperatures and pH values showed that the extract could endure different pH. The evaluation of the ions influence and inhibitors proved that the enzyme required an ion for better activity, which was corroborated with the inhibition of EDTA and PMSF, characterizing serine and/or metallo peptidase. The extract was also tested for specific activities and showed promising results for keratinolytic and collagenolytic activities (0.252 and 0.165 OD/mL, respectively).