Detection of Extracellular Enzymes Produced by Pleurotus Spp Grown on Coffee Pulp

DETECTION OF EXTRACELLULAR ENZYMES PRODUCED BY PLEUROTUS SPP GROWN ON COFFEE PULP

D. Salmones and G. Mata

Instituto de Ecología, A.C.

Apartado Postal 63, Xalapa, Veracruz 91070 México

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ABSTRACT

Variation in the production of laccase activity, manganese-peroxidase, endoglucanase and xylanase was investigated during the degradation of coffee pulp by strains of Pleurotus djamor, P. ostreatus and P. pulmonarius. Minimum activity of endoglucanase and xylanase was observed during the spawn run period, increasing during the formation of primordia and development of the fruiting bodies. The activities of laccase and manganese peroxidase were detected from the fourth day of incubation onwards, reaching the highest points between 12 and 16 days, then decreasing during the formation and development of the fruiting bodies. The concentration of phenols present in the coffee pulp decreased from the fourth day of incubation, thus confirming the detoxifying activity of laccase. All the strains presented good development on the substrate with P. djamor being the most precocious in the formation of primordia. The biological efficiencies fluctuated between 31.74 to 84.31 % in 36 days of cultivation.

INTRODUCTION

Coffee pulp, one of the main waste products produced during the processing of coffee, is a material rich in carbohydrates, proteins and minerals (Elías 1978). It also contains tannins, caffeine and polyphenols, although in lesser quantities. Due to the antinutritional effects of these substances, the waste has limited commercial use, and the surplus is considered a contaminating agent in the areas near the regions that process the grain (Zuluaga 1989). In Mexico alone more than 100 000 t of coffee pulp are generated during each cycle of the coffee crop. Among the proposed technologies for the use of this residual matter, one of the most potentially interesting is the growth of edible mushrooms, as it represents a direct conversion of a residue of low commercial value into a food item of acceptable protein content. Nevertheless, despite the first studies on the cultivation of Pleurotus spp in coffee pulp where biological efficiencies were frequently reported to be well over 100% (Martínez-Carrera 1987) its present use is minimal. This is mainly due to the low level of competition of Pleurotus spp strains used against the incidence of antagonic competitive moulds so frequent in the cultures.

Recent studies have reported that white rot fungi, among which Pleurotus spp are found, produce oxidative and hydrolytic enzymes capable of unfolding the complex molecule of the lignocellulose in compounds of low molecular weight to later be absorbed by the fungi (Buswell et al. 1993, Kerem and Haddar 1997). In these genera, as in Agaricus spp, laccase and endoglucanase activities have been associated with the colonization and fructification stages of the mushroom (Alarcón 1997, Claydon et al. 1988, Geetha and Sivaprakasam 1998) observing that the spawn running on the substrates is a critical period for the cultures. On the other hand, the laccase has shown to act not only in the biodegradation of lignin, but also in the detoxification of the substrate and defense from antagonic moulds (Bollag et al. 1988, Savoie and Mata.1999). Therefore the strains with high production of this enzyme could have advantages during the colonization and biodegradation of the substrate by the mushroom. All of the above has been reported for Pleurotus spp culture in different

substrates, but information regarding the use of coffee pulp is scarce (Velázquez-Cedeño et al. in press).

The aim of the present study was therefore to cultivate Pleurotus spp on coffee pulp, quantifying the variation in the activities of some oxidative and hydrolytic enzymes during the vegetative and reproductive stages of the fungi.

MATERIALS AND METHODS

Fungal strains

Six strains of Pleurotus spp were studied: P. djamor (Fr ) Boedijn, IE-121 and IE-218, P. ostreatus (Jacq. Fr.)Kumm, IE-38 and IE-49, and P. pulmonarius (Fr.) Quél, IE-137 and IE-225. Three of those, IE-38, IE-49 and IE-137, are under commercial cultivation; strain IE-121 was isolated from a Mexican wild specimen, and strains IE-218 and IE-225 were progeny chosen from our breeding programs. All strains were provided by the Fungi Ceparium of our institution, registered in the World Data Centre for Microorganisms (No. 782).

Culture conditions

The fungal cultures were maintained on plates of malt agar extract (EMA, Bioxon) and incubated for one week at 28°C . Spawn was prepared with sterile and hydrated sorghum grains, inoculated with fungal mycelia from the seven day old cultures grown in EMA and incubated at 28ºC for two weeks.

Coffee pulp was collected from a local coffee processing plant, sun dried and stored at room temperature. For enzyme production, the coffee pulp was rehydrated for 12 h, then the excess moisture was drained (reaching 60±5% approximately) and the samples were prepared with 200 g of wet weight (50 g dry weight) in plastic bags sterilized at 121°C for one hour. Each sample was inoculated with 20 g of spawn (seven replicates by strain) and incubated in a dark room at 28±1°C. Two days later small perforations made on the plastic bags allowed gaseous exchange. The cultures were monitored for 36 days. At the same time, control samples were prepared without mycelia.

Enzymatic activities were monitored every four days during the first 16 days of incubation. Thereafter, the polyethylene bags were withdrawn and the substrate was transferred to the production room. The enzymatic activities were then tested as follows: primordial initiation stage, 1st flush and post-1st flush (one week later).

At the same time, the yield was evaluated in samples containing 2 Kg (500 g dry weight) sterilized coffee pulp inoculated with 100 g of spawn. Ten replicates were prepared by strain. Gaseous exchange and ambiental conditions of running spawn period were similar to enzymatic activity tests. After 16 days of incubation, the plastic covering was taken off and the samples were transferred to the production room, in which they were maintained throughout the rest of the experiment. Yield was expressed as the relationship between the mushrooms’ wet weight and the substrate dry weight as a percent (biological efficiency) (Stamets 1993).

Separation of the crude enzyme extract

Mixtures of 0.7 g powdered substrate and 10 ml of distilled water were stirred for 30 min at 45 rpm. The resulting suspensions were filtered through inert fabric and the filtrates were centrifuged twice at 10060 x g for 15 min. The supernatants of crude enzyme extract were stored at 4°C until analysed for enzymatic activities

Enzyme Assays

Except for laccase (LAC), whose activity appears to be lost during liofilization (Velázquez-Cedeño et al. in press.), all the enzymatic assays were performed on lyophilised samples.

Endoglucanase (CMC) and xylanase (XYL) activities were estimated using the DNS method (Miller 1959), measuring the amount of reducing sugars liberated in reaction mixtures containing 2% carboxymethyl cellulose (Sigma) or 0.5% xylan (Sigma) and supernatant. The incubation times were 30 and 45 mins at 30°C, respectively. Standard curves were obtained with glucose or xylose. The activities were expressed in units defined as the quantity of enzyme required from 1µmol of glucose or xylose per minute.

LAC activity was determined by the increase in the absorbance due to the formation of tetramethoxy-azo-bis-methylenequinone, resulting from the oxidation of syringaldazine (Sigma) as described by Leonowicz A. and K. Grzywnowickz (1981). The activity was assayed in mixed reactions containing supernatant, phosphate citrate buffer (pH 5, 0.1M) and syringaldazine 4.4 mM. in methanol. One unit was defined as the amount of enzyme producing a one unit change in absorbance/min at λ=526 nm (ε = 65000 M-1cm-1). Manganese peroxidase (MnP) activity was assayed by oxidation of 3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (Sigma) in the presence of MnSO4. 1 mM (del Pilar-Castillo et al. 1994). The reaction was initiated by addition of hydrogen peroxidase (0.18 mM) and the increase in absorbance measured at 590 nm during 2 mins (Mata and Savoie 1998). One unit of MnP was defined as the amount of enzyme required to form 1 µmol of Mn3+/min using an Σ590 value for Mn3+ of 3.29 x 104/mol/cm. All enzyme assays were carried out in triplicate.

Concentration of phenolic compounds was determined with Folin and Ciocalteu’s reagent as described by Box (1983). Results were expressed in mmol of water soluble phenols per gram of lyophilised substrate.

RESULTS AND DISCUSSION

The enzymatic activities were grouped into two behavioural patterns. One of the groups corresponded to cellulolytic and hemicellulolytic enzymes. This group had low activity during the vegetative stages of the fungi, increased activity in the presence of primordia and development of fruiting bodies, and decreased activity later during the post 1st flush period. The other group included oxidative enzymes, and displayed activity during the vegetative stage of fungi, which decreased in the reproductive stage and increased again in the post 1st flush period.

Figure 1 shows low enzymatic activities during the first 16 days of incubation and minimal fluctuations thereafter in the CMC. There was no outstanding point, as previously reported for cultures of Pleurotus spp in other substrates (Kannan et al. 1990, Alarcón 1997). This was probably due to the low content of cellulose present in the coffee pulp (nearly 20%) (Elías 1978), compared to the contents of other lignocellulosic materials traditionally used for mushroom production, such as straws, where the contents of cellulose are higher than 40% (Dunlap and Chiang 1980). On the other hand, the high activity observed on the reproductive stage of the fungi coincides with Geetha and Sivaprakasam (1998) who suggested a relation between the morphogenesis of the fungi and its cellulolytic activity; since it is known that the function of CMC along with other cellulolytic enzymes is to provide the mycelium with the necessary energy for the formation of fruiting bodies. The decrease in cellulose content during the fructification of Pleurotus spp grown on the coffee pulp, reported by Apráez Guerrero (1989), confirms this pattern.

The XYL activity (Figure 1) showed a similar increase to the CMC, displaying a higher activity from the 12th day of incubation. The strains of P. pulmonarius (IE-137 and IE-225) presented the highest increases in their activities during the formation and development of the fruiting bodies compared with the rest of the strains, even though this may be considered as a characteristic of the species. The results coincide with previous works on Pleurotus spp and Agaricus spp, where maximum activity was reached when the basidiomes were ready for harvesting (Savoie et al. 2000).

IE-38 IE-49 IE-121 IE-137 IE-218 IE-225

Figure 1. Changes in endoglucanase (CMC) and xylanase (XYL) activities during the running spawn (16 days) and fructification stages of six Pleurotus spp strains on coffee pulp. PI= primordia initiation, F=1st flush PF=1st post-harvest flush.

LAC activity was observed from the 4th day of incubation (Figure 2). Maximum activity was observed at the 12th day of incubation, except for IE-121 strain, which reached maximum activity on the 16th day of incubation. Laccase activity diminished drastically during the formation and development of the fruiting bodies. Similar results were observed by Velázquez-Cedeño et al. (in press) in a previous experiment on Pleurotus spp cultivation with coffee pulp. P djamor strains presented the higher fluctuation for the laccase’s maximum activity (8.08 y 33.7 μmol/g/min). For the other strains, the concentrations fluctuated between 28.3-29.2 μmol/g/min.

*The meanings of the symbols used in these figures are specified in Figure 1.

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Variations in MnP activity are shown in Figure 3. The enzyme was detected from, the 4th day of incubation and reached maximum values between the 8th and 16th day. The higher activities were seen for the strains P. pulmonarius (IE 137 and IE-225) and P. ostreatus (IE-38).

An inverse relationship was observed between the concentrations of soluble phenols and LAC and MnP activities. The concentration of phenol diminished from the 4th day of incubation and stayed as low as 5μmol/L. Bollag et al. (1988) related these enzymatic activities to the removal of the phenolic compunds by LAC through oxidative coupling and polymerisation in the substrate. The high oxidative enzymes production during the substrate colonization support previous results with in Agaricus spp and Lentinula spp (Savoie 1998, Mata and Savoie 1998).These results show that LAC and MnP activities influence, at least in an indirect way, mushroom nutrition during the vegetative growth giving it the nitrogen and mineral sources necessary for triggering the reproductive stage.

As for the yield (Table 1), all the strains showed a rapid adaptation to the substrate during the incubation stage, observing samples completely colonized in the first week of culture. Even though the strains of P. djamor developed a less dense mycelial growth they were the most precocious, presenting primordial initiation between 13 and 15 days of incubation. The biological efficiencies fluctuated between 37.74±9.3 to 84.31±12.8%, lower than the ones previously reported (Martínez-Carrera et al. 2000), even though this could be because in the present study the biological efficiencies correspond only to 1 to 2 harvests, and that represents only 80 to 90% of the total expected yield.

Table 1. Mycelial growth and biological efficiencies of Pleurotus spp strains in coffee pulp during 36 days of culture cycles.

*Values are shown with standard deviation and they represent the media of 10 replicates

Relating the results of yields and enzymatic activity of LAC and MnP In der Wiesche et al. (2000) suggest that the measurement of the enzymes during the spawn-run could help in estimating the biomass of the mushroom in the substrate, and in selecting strains with a high capacity for the production of fruiting bodies. Even through our results are not favourable to the proposal, it was observed that the strains with less activity of MnP, corresponding to the specie P. djamor, presented low biological efficiencies in two crops, which corroborates a high relation between the amount of biomass of the fungi and the production of these enzyme.

CONCLUSION

More needs to be known about the functions of extracellular enzymes produced by Pleurotus spp cultivated on coffee pulp to optimalize development of the mushroom. The studies done so far show that laccase plays an important role in the colonization of the substrate and transformation of the phenolic compounds. For a second stage of the study, experiments could be designed to induce the production of this oxidase in the first days of incubation to improve fungal development and

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consequently increase its competitivity with the presence of antagonic moulds in the cultures. Strain IE-225, in parallel studies performed within this project, significantly increased its defenses against Trichoderma ressei and T. viride when it was sown with the supplemented inoculum with lignolytic materials (data not shown) with interesting results in the search to improve the Pleurotus spp adaptation for growing in coffee pulp, which could increase the use of this substrate among the mushroom growers.

ACKNOWLEDGEMENTS

The authors are thankful to CONACyT (Project No. 28530-B) and the authorities of the Institute of Ecology for the financial support given for this research, and to Angélica Velázquez-Cedeño for technical support.

REFERENCES

Apráez Guerrero, J.E. 1989. Evaluación de la digestibilidad y paredes celulares de la pulpa de café inoculada con el hongo Pleurotus ostreatus. MSc. Thesis, Universidad Nacional Autónoma de México, Mexico, D.F.

Alarcón Gutiérrez, E. 1997 Cambio de actividad de celulasa y lacasa y obtención de carpóforos de hongos comestibles (Pleurotus spp) durante su crecimiento sobre algunos desechos lignocelulósicos. MSc. Thesis. Instituto Tecnológico de Veracruz. Veracruz, México.

Bollag J.M., K.L. Shuttleworth, D.H. Anderson. 1988 Laccase mediated detoxification of phenolic compound. Appl. Envinron. Microbiol. 54: 3086-3091.

Box, J.D. 1983 Investigation of the Folin Ciocalteau reagent for the determination of polyphenolic substances in natural waters. Water Res. 17: 511-525.

Buswell, J.A., Y.J. Cai, S.T. Chang.1993. Fungal-and substrate-associated factors affecting the ability of individual mushroom species to utilise different lignocelullosic growth substrates. In: Proceed. of 1st Internat. Conf. Mush. Biol. and Mush. Prod. The Chinese University Press, Hong Kong. 141-150.

Claydon, N., M. Allan, D. Wood. 1988. Fruit body biomass regulated production of extracellular endocellulase during periodic fruiting by Agaricus bisporus . Trans. of the British Myc. Soc. 90: 85-90.

del Pilar-Castillo, M., J. Strenström , P. Ander. 1994. Determination of manganese preoxidase activity with 3-methyl-2-benzothiazolinone by drazone and 3-(dimethylamino)benzoic acid. Anal Bioch. 218: 399-404.

Dunlap, C.E., L. C. Chiang. 1980. Cellulose degradation: a common link. In: S.L. Shulelr (ed.). Utilization and recycle of agricultural wastes and residues. CRC Press, Boca Ratón. 19-65.

Elías, L.G. 1978. Composición química de la pulpa de café y otros subproductos. In: J.E. Braham and R. Bressani (eds). Pulpa de café: composición, tecnología y utilización. INCAP, Guatemala. 19-29.

Geetha, D., K. Sivaprakasam. 1998. Enzyme and sporophore production potential of oyster mushroom (Pleurotus spp). Mush. Res. 7: 39-42

In der Wiesche, C., M. Molter, F. Zadrazil, S. Aksu 2000. Activities of ligninolytic enzymes as a means for monitoring the colonization of straw substrate pretreated at different temperatures by Pleurotus ostreatus. Mush. Sci. 15: 391-398.

Kannan, K., G. Oblisami, B.G. Loganathan. 1990. Enzymology of ligno-cellulose degradation by Pleurotus sajor-caju during growth on paper-mill sludge. Biol. Wastes 33:1-8.

Kerem, Z., Y. Hadar. 1997. The role of manganese in enhanced lignin degradation by Pleurotus ostreatus. In: Biological Sciences Symposium. Tappi Press, Atlanta. 29-33.

Leonowicz A., K. Grzywnowickz. 1981. Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as a substrate. Enzyme Microb. Technol. 3:55-58

Martínez-Carrera, D. 1987. Design of a mushroom farm for growing Pleurotus on coffee pulp. Mush. J. for the Trop. 7 :13-23.

Martínez-Carrera, D. A. Aguilar, W. Martínez, M. Bonilla, P. Morales, M Sobal. 2000.Mushroom cultivation on coffee pulp. In: Proceed. of the 3rd Internat. Seminar on Biotechnology in the coffee agro-industry. Kluywer Academic Pub, Dordrecht. 471-488.

Mata, G. J.M. Savoie. 1998. Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw. World J. of Microb.. and Biotechnol. 14: 1-7.

Miller, G.L. 1959. Use of dinitrosalicylic acid and reagent for determination of reducing sugars. Anal. Chem .31: 426-428.

Savoie, J.M. 1998.Changes in enzyme activities during early growth of the edible mushroom, Agaricus bisporus, in compost. Mycol. Res. 102: 1113-1118.

Savoie, J.M. G. Mata 1999. The antagonistic action of Trichoderma sp. hyphae to Lentinula edodes hyphae changes lignocellulolytic activities during cultivation in wheat straw. World J. of Microb. and Biotechnol. 15: 369-373.