The wood-decay fungi are able to bioconvert a wide variety of lignocellulosic residues due to the secretion of extracellular enzymes. The use of agricultural wastes as substrate for mushroom cultivation or enzymes production can help to solve environmental problems caused by inadequate discharge in the nature. The production of hydrolytic and oxidative enzymes by Pleurotus pulmonarius developed in solid state system using orange waste as substrate was evaluated in this work. Among the hydrolytic enzymes, pectinase was the main enzyme produced by the fungus, presenting the highest enzymatic activity of 9.4 U/mL after 35 days of cultivation. Considering the oxidative enzymes, laccase was the main enzyme produced with maximal activity of 12.2 U/mL obtained after 20 days of cultivation. Low enzyme levels of manganese peroxidase, β-glucosidase and β-xy-losidase were detected with activity peaks at the end of the cultivation. The enzymatic levels of amylase, carboxymethyl cellulase and xylanase were similar and less than 1.5 U/mL. No aryl-alcohol oxidase activity was detected. NDF, ADF and cellulose values increased during 45 days of cultivation. There was no lignin degradation during the study period and the fungus culture in orange solid waste caused protein enrichment in the substrate. Our results demonstrate that P. pulmonarius was an efficient producer of two important industrial enzymes, pectinase and laccase in a cheap solid state system using orange waste as substrate.
The orange fruit is one of the widely produced and consumed fruits in the world. Its production exceeds 80 million tons a year. Around 34% of orange production is usually made into juice, however in countries where orange is widely produced (Brazil and the United States) this number rises to 96%, generating a huge amount of citric residue (approximately 50% of the total fruit weight), which may be potentially applied to biotechnology [
There is a variety of white-rot fungi, most of which are basidiomycetes. The lignocellulolytic basidiomycetes are not only important decomposers, but also the main organisms responsible for recycling carbon in the ecosystems.
They degrade the wood components, such as cellulose, hemicellulose and lignin, into low molecular weight components that can be assimilated for their growing and reproduction [
Many of the enzymes produced by the fungi are importantly applied to several industrial areas. Some of these applications are in the production of detergent, starch, beverages, food, textile industry, animal feed, pulp and paper, leather, chemical and biomedical products [
The genus Pleurotus includes species of white-rot fungi that have ability to degrade lignin, cellulose and hemicellulose of wood [
P. pulmonarius (Fr.) Quél.?CCB20 was obtained from the Culture Collection of the Botany Institute of São Paulo. The stock culture of the fungus was maintained at 4˚C through periodical transfer in Petri dish containing wheat bran (20 g/L), agar (18 g/L) and mineral medium [
The cultivation was carried out in Erlenmeyers flasks (125 mL) containing 4 g of orange waste and sufficient amounts of mineral media to obtain initial humidity of 85%. Two mycelial plugs of agar plates containing 7 day- mycelium, (15 mm in diameter) were used as inoculum after the instruments sterilization. The flasks were incubated at 28˚C in the dark. Cultivation was interrupted at determined periods with the addition of 20 mL of distilled water, followed by agitation for 20 min at 8˚C, vacuum filtration and centrifugation (10 min; 10,000 g; 4˚C). The supernatant was used as crude enzymatic solution. The material retained on the filter paper (Whatman N˚ 1) was dried at 40˚C and milled to obtain 1 mm particles and stored to determine the fibers, crude protein, lignin and cellulose.
The xylanase (EC 3.2.1.8), α-amylase (EC 3.2.1.1), pectinase (EC 3.2.1.5) and carboxymethyl cellulase (CMCase) (EC 3.2.1.4) activities were determined from the hydrolysis of their substrates at 1% (w/v): birchwood xylan, starch, pectin and carboxymethyl cellulose ( [
The β-xylosidase (EC 3.2.1.37) and β-glucosidase (EC 3.2.1.21) activities were determined by the synthetic substrates ρ-nitrophenyl-β-D-xylopyranoside and ρ-nitrophenyl-β-D-glucopyranoside. The reaction was interrupted by the addition of a Na2B4O7∙10H2 saturated solution [
The laccase (EC 1.10.3.2) activity was determined spectrophotometrically with 2,2’-Azino-bis[3-ethylbenz- thiazoline-6-sulfonic] (ABTS) at 10 mM as a substrate using acetate buffer 50 mM (pH 5.0), at 40˚C for 5 min. Oxidation of ABTS was monitored by the absorbance increase (A420 = 36,000 M−1∙cm−1) [
The enzymatic activities were expressed in international unities per milliliter (U/mL). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of substrate per minute. The reagents used were from Sigma-Aldrich brand, USA.
Total soluble phenolics in the cell free culture broth were colorimetrically estimated by the Folin-Ciocalteu method [
Neutral detergent fiber (NDF: cellulose, hemicellulose and lignin), acid detergent fiber (ADF: lignocellulose), lignin and cellulose were determined by the Van Soest method from the fermentation residual material [
All the analysis was carried out in duplicate. The data was expressed as the means ± standard deviations. Differences of 5% (p < 0.05) among the means were considered significant according to Dunnett test. The results were analyzed using the Graph-Pad Prism 3.0 (San Diego, EUA) software.
The isolate of P. pulmonarius CCB20 was selected for the present study due to its capability to grow in orange residue. In previous experiments, the fungus was able to completely colonize orange waste at 85% initial moisture content after 8 days of cultivation at 28˚C. The time course of enzymes production by P. pulmonarius was followed for up to 45 days (
The pectinase activity maintained stable until the 20th cultivation day, with a mean of 6.2 ± 0.60 U/mL. After declining, the production increased on the 35th day, with a maximum value of 9.4 ± 0.40 U/mL (
There are two categories of enzymes which degrade hemicellulose: the endo-β-xylanases (EC 3.2.1.8.), which attack the xylan main chain, and the β-xylosidase (EC 3.2.1.3.7.), which hydrolyze xylooligosaccharides into D-xylose. The maximum xylanase and β-xylosidase acitivities were 0.7 ± 0.02 U/mL and 0.3 ± 0.03 U/mL, respectively (
The maximum CMCase and amylase activities were 0.6 ± 0.05 U/mL and 1.1 ± 0.06 U/mL, respectively, on the fifth cultivation day (
Laccase activity was high between the 12th to the 20th day, presenting the highest value of 12.2 ± 0.15 U/mL on the 20th cultivation day (
however it reached its peak when the reducing carbohydrates decreased (
Low MnP activities were found along the cultivation, with a maximum value of 0.07 ± 0.00 U/mL on the last day (
Usually, dry orange waste contains up to 12% of crude fiber (including pectin and cellulose), 6% of protein, 7% of ash, 63% of nitrogen, 19% of total sugar and 9% of reducing sugar [
In this study, NDF, ADF and cellulose values increased (data not show), until the last cultivation day, suggesting the absence of consumption of these polymers by the fungus. There was no lignin degradation during the study period.
The fungi culture in orange solid waste caused protein enrichment in the lignocellulosic substrate. A significant increase (p < 0.01) in the substrate protein content was observed from the 7th cultivation day, resulting in an addition of 4% until the last point of the growth curve.
In the present study there was a detected decrease in antioxidant activity of the crude extract, from 100% to 39%, on the last cultivation day (
Byproducts obtained from citrus juice production are rich in soluble and insoluble carbohydrates, making them an attractive raw material with great potential for biologic conversion to value-added products [
fungi has been scarcely reported, recent studies demonstrate that pectinase may be produced by these fungi in lignocellulosic substrates, including citrus pulp [
The enzymatic secretion responsible for carbohydrates cleavage, for example, pectinase and β-glucosidade, have been associated with lignin degradation [
Studies have demonstrated that when Pleurotus spp. are cultivated under SSF in lignocellulosic substrates they are capable of producing cellulases, xylanases [
Makkar et al. [
The efficient lignin degradation carried out by the white-rot fungi mainly occurs through laccase, LiP and MnP secretion. Oyster mushrooms are well-known laccase and MnP producers, but no typical LiP [
The cellulose in the cultivation of P. sajor-caju increased whereas a linear decrease was observed for NDF but not for ADF content [
The ligninolytic system of Pleurotus spp. has not been fully defined yet, however, there is evidence that it is different from the other white-rot fungi [
Many researchers have demonstrated that the antioxidant activity of plants and mushrooms extracts is associated with the phenolic compounds extracted from these materials [
Thus, the orange waste, besides demonstrating to be capable of inducing the production of pectinase and laccase by P. pulmonarius, also shows that its solid fermentation residual substrate has high potential to be used in the development of animal feed, enriched in its protein content lowering production costs as well as valorizing the residue from the citric industry.
Our results support the use of orange waste as substrate for production of important industrial enzymes. P. pulmonarius CCB20 was able to produce hydrolytic enzymes and laccase was the main oxidative enzyme produced. The isolated used in this work is a mushroom with positive characteristics such as easy cultivation and excellent adaptation. Orange waste is a cheap alternative for cultivation of mushrooms and the fungal growth caused protein enrichment of substrate. In conclusion, the cultivation of this fungus offers a way to bioconversion of lignocellulosic substrates.
This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-proc. 473181/2007-0). F.D. Inácio and R.O. Ferreira are fellowship holders of the Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and R.M. Peralta and C.A.V. de Araujo are fellowship holders of the CNPq.