Veratryl alcohol stimulates fruiting body formation in the oystermushroom, Pleurotus ostreatus

Helio H. Suguimoto a;1, Aneli M. Barbosa b;*, Robert F.H. Dekker a;2,Raul J.H. Castro-Gomez a

a Biotechnology Program, CCA - Departamento de Tecnologia de Alimentos e Medicamentos, Universidade Estadual de Londrina, CxP 6001,CEP 86051-990 Londrina, Parana, Brazil

b CCE - Departamento de Bioqu|mica, Universidade Estadual de Londrina, CxP 6001, CEP 86051-990 Londrina, Parana, Brazil

Abstract

The oyster mushroom, Pleurotus ostreatus, cultivated in solid state on sugarcane bagasse^wheat bran (5:1) medium in the presence ofveratryl alcohol resulted in an increased production of the fruiting body at earlier times compared to when the fungus was grown in theabsence of veratryl alcohol. The results indicate a new physiological role for veratryl alcohol in stimulating fruiting body formation.Veratryl alcohol also stimulated laccase production during the mycelial growth stage. Evidence is also presented that laccases were involvedin the physiological development of the fruiting body. 2001 Federation of European Microbiological Societies.

Keywords: Pleurotus ostreatus ; Physiological role of veratryl alcohol ; Laccase; Fruiting body stimulation; Sugarcane bagasse^wheat bran media

1. Introduction

The mushroom industry is a global expanding industrywith world production greater than 2 million tonnes an-nually [1]. The chief mushroom varieties produced are thechampignon (Agaricus bisporus), shiitake (Lentinus edodes)and the oyster mushroom (Pleurotus ostreatus). Pleurotusspp. alone account for more than 800 000 tonnes of mush-room produced world-wide annually [1]. These mush-rooms are cultivated on lignocellulosic substrates whichcan comprise sawn wood logs, or comminuted residuessuch as wood sawdust and agricultural wastes (cerealstraws, sugarcane bagasse). After the substrates arespawned, the mushroom colonises the substrate and aftera period its mycelium produces a fruiting body (basidio-carp). Fruiting body induction (fructication) is performed

by subjecting the mycelium to external stimuli (light, lowertemperature, humidity). It is not clearly understood whattriggers the dierentiation of the basidiocarp, but severalcompounds, e.g. cerebrosides, cyclic AMP and AMP [1],and surfactants such as saponin [2] and sucrose fatty acidesters [3], have been reported to stimulate fruiting bodyformation in basidiomycetes.

Aromatic alcohols such as veratryl alcohol are second-ary metabolites synthesised de novo from glucose by sev-eral fungi, but mainly the basidiomycetes [4], where theyhave been ascribed to play a specic role as a physiologicalcofactor of lignin peroxidase (LiP) [5], a key enzyme in thedepolymerisation of lignin. The chief function of veratrylalcohol is as a redox mediator between LiP and lignindegradation [6], and in protecting LiP during the redoxcycle from metabolically produced H2O2 [7]. It can alsoact as an inducer of laccases in basidiomycetes [8] andascomycetes [9], and hence promote lignocellulose degra-dation.

In this paper we report on a new physiological role forveratryl alcohol, and demonstrate for the rst time thatveratryl alcohol is involved in the initiation of fruitingbody formation in the oyster mushroom, P. ostreatus,and that it induces the production of laccases during thedierent stages of mushroom development.

* Corresponding author. Fax: +55 (43) 328 4440;E-mail : aneli@sercomtel.com.br

1 Present address: Departamento de Engenharia Alimentos, UNOPAR,Avenida Paris 675, CEP 86051-140, Londrina, Parana, Brazil.

2 CNPq-Visiting Research Professor.

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2. Materials and methods

2.1. Microorganism and solid-state cultivation

P. ostreatus (Fr. Kummer), a commercial strain used inBrazil, was obtained from Universidade Estadual de Lon-drina culture collection (Dr. R. Castro-Gomez), and cul-tured with periodic transfer on potato^dextrose agar(PDA). Inoculum was prepared from mycelia taken fromcultures grown on PDA plates for 6 days. Agar plugs of5-mm diameter were taken from the edge of the growingcolony and used to inoculate media.

Mycelial growth by solid-state cultivation was con-ducted in petri dishes (100U15 mm) containing a mixtureof 5 g (dw) sugarcane bagasse (sieved to pass a 3-mmscreen), 1 g (dw) wheat bran (farelo de trigo, Vitao, Bra-zil) and 16 ml distilled water. The solid medium was au-toclaved at 121C for 30 min, and upon cooling inoculatedwith a single agar plug placed inverted on top of the sub-strate in the centre of the dish, and incubated at 28 1C.Veratryl alcohol (Aldrich) dissolved in 4 ml sterile waterwas added to the surface of the solid media after 4 daysfungal growth at the levels 4, 8, 12, 16 and 20 Wmol g31

medium. Radial growth of the fungus was measured atfour equidistant points from the centre of the growingcolony, and the rate of mycelial growth (mm/day) deter-mined over 24-h intervals. Experiments were carried out induplicate and results are presented as mean values S.E.M.

2.2. Fruiting body production

P. ostreatus was cultivated in stoppered jars (8.5U17cm) containing 60 g bagasse, 12 g wheat bran and 210ml deionised water, which were autoclaved for 1 h at121C. Each jar was inoculated with a single agar pluginserted into the interior of the solid medium, and left togrow at 28 1C. Veratryl alcohol, diluted in 30 ml sterilewater, was added to the solid media at nal concentrationsof 4 and 8 Wmol g31 solid substrate after 20 days followinginoculation. At day 30 of continuous growth, conditionsfor fructication were promoted by opening the jars, andplacing them in a growth chamber at 20C and 90% rela-

tive humidity. Primordia appeared after a further 10 daysof growth, and basidiocarps were harvested 5 days later(total time from inoculation to harvesting: 45 days), andweighed (fresh weight). In another set of experiments, cul-tures were subjected to fructication at day 25 with theobjective of evaluating the eect of veratryl alcohol onreducing the time for basidiocarp development. These cul-tures were harvested after a growing period of 40 days.Experiments were carried out in triplicate and results pre-sented as mean values S.E.M.

2.3. Analytical procedures

Following the growth and removal of mushrooms, thespent-solids medium was extracted by a standardised pro-cedure: a representative sample of the solid medium (10.5g wet weight) was removed from the thoroughly mixedspent material, and transferred to an Erlenmeyer askcontaining 60 ml deionised water and stirred for 15 min(200 rpm) at 20C. The ltrate recovered was used to assayenzyme activity and sugars. Extractions were made onspent material at 10 days (mycelial growth experimentsin petri dishes), at 25 and 30 days (fructication), at thetime when the primordia rst appeared (35 and 40 days),and at the time of harvesting the rst crop of mushrooms(40 and 45 days, respectively).

Reducing sugars were measured by the Somogyi methodusing D-glucose as the standard [10]. The cellulose, hemi-cellulose and lignin contents in the spent-solid media weredetermined by the method of Bailey [11] after successivetreatments with water, 80% aqueous alcohol, 1 N H2SO4and 72% H2SO4, on a 0.2 g (dw) sample. The residuerecovered was ashed at 550C, and the dierence in weightreported as the amount of lignin in the sample. All assayswere performed in duplicate.

2.4. Enzyme assays

Laccase activity (PPO-I and PPO-II) was assayedagainst 2,2P-azino-bis(3-ethyl-benzthiazoline-6-sulfonicacid) (ABTS; Sigma) and 2,6-dimethoxyphenol (Fluka),respectively, as substrates at 50C as previously described[9,12], but in this case at pH 4.0 and 5.5, respectively.

Table 1The eect of veratryl alcohol concentration on mycelial growth rate and laccase production by P. ostreatus in solid-state cultivation on sugarcane bag-asse^wheat bran medium

Veratryl alcohol concentration (Wmol g31) Mycelial growth (mm day31) Laccase activitya (U g31)

0 13.10 0.014b 0.518 0.00414 11.157 0.243 1.251 0.01558 9.786 0.100 1.495 0.1025

12 5.615 0.385 1.989 0.001616 3.643 0.500 2.203 0.046320 1.900 0.557 3.147 0.0854

aPPO-I (activity against ABTS at pH 3.0).bMean S.E.M.

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Laccase activity was also assayed against catechol (Sigma)at pH 5.0 as described elsewhere [13]. Enzyme activitieswere expressed in units (U) as Wmol product formedmin31, and are reported as U g31 solid medium. Enzymeassays were performed in duplicate.

3. Results and discussion

3.1. Mycelial growth on solid media in the presence ofveratryl alcohol

Vegetative (mycelial) growth of P. ostreatus (Fr.Kummer) on sugarcane bagasse^wheat bran mixture wasoptimal at 28C and pH 4.8. After growth the solid me-dium was extracted and assayed for laccases and residualsugars. It was found that 90% of the total laccases (mea-sured as PPO-I), and 85% of reducing sugars could beextracted in one extraction, so subsequent work involveda single extraction.

P. ostreatus grew on bagasse^wheat bran media contain-ing veratryl alcohol, but radial growth was severely limitedat concentrations s 12 Wmol g31 substrate over a periodof 9 days (Table 1). The inhibitory nature of veratryl al-cohol was probably associated with toxicity at high con-centrations [9]. A similar nding was observed when P.ostreatus strain Florida was grown in submerged cultureon glucose media containing veratryl alcohol (unpublishedresults). Enzyme extraction of the solid medium and assayof laccase PPO-I activity showed that laccase productionincreased as the concentration of veratryl alcohol in thesolid media increased up to 20 Wmol g31 substrate (Table1). Several basidiomycetes are known to produce veratrylalcohol, albeit at low levels [4], which induced laccaseproduction [8]. Pleurotus spp. produced extracellular ve-ratryl alcohol oxidases for which veratryl alcohol was asubstrate [14], and intracellular NADPH-dependent arylalcohol dehydrogenases that can use the veratryl alcohol-oxidised product, veratraldehyde, as substrate [15]. To-gether these enzymes constitute a redox system that en-sured a supply of H2O2 for Mn-dependent peroxidase in-

volvement in lignin depolymerisation, and a suitableendogenous source of veratryl alcohol, which in P. ostrea-tus, functioned to induce laccases for subsequent ligninol-ysis. Although there were no reports on endogenous levelsof veratryl alcohol for Pleurotus spp., up to 4 mM veratrylalcohol was reported to be produced in another basidio-mycete, Bjerkandera sp. when grown on lignin-relatedcompounds [16]. If this represented physiological levels,then the veratryl alcohol levels examined in this workfor P. ostreatus were within the physiological range.

3.2. Fruiting body production in the presence of veratrylalcohol

Fructication was induced by lowering the temperatureto 20C at day 30, and basidiocarps harvested at 45 days.These times and temperatures for Pleurotus spp. werewithin the ranges reported [1,17]. Sugarcane bagasse sup-plemented with wheat bran in the ratio 5:1 and watergiving a moisture content of 76% was found to be agood composting mixture for cultivating P. ostreatus formushroom production resulting in a biological eciency

Fig. 1. Fruiting body production by P. ostreatus in solid-state cultiva-tion on sugarcane bagasse^wheat bran medium in the presence of ve-ratryl alcohol. Fructication was induced at 25 and 30 days, and mush-rooms harvested at 40 and 45 days, respectively.

Table 2The eect of veratryl alcohol concentration on laccase (PPO-I) production during dierent phases of growth by P. ostreatus in solid-state cultivation onsugarcane bagasse^wheat bran medium

Veratryl alcohol concentration (Wmol g31) Stages of growtha

fructication primordia harvest

laccase activity at the following times (U g31)b

25 days 30 days 35 days 40 days 40 days 45 days

0 1.640 0.006c 3.426 0.021 2.030 0.057 3.364 0.049 0.230 0.009 0.260 0.0524 3.480 0.099 4.120 0.061 2.190 0.005 3.690 0.006 0.390 0.007 0.361 0.0678 4.220 0.057 4.600 0.036 2.680 0.021 5.140 0.074 0.480 0.033 0.380 0.011

aStages of growth: enzyme levels at the time of initiation of fructication, at the appearance of primordia, and at maturation of the basidiocarps (har-vest), for cultures in which fructication was induced at 25 and 30 days.bAgainst ABTS as substrate (PPO-I).cMean S.E.M.

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(fresh fruit body weight/dried substrate weightU100) of upto 86%, and conrmed earlier work (Suguimoto, unpub-lished work) on the suitability of bagasse for cultivatingoyster mushroom. Veratryl alcohol promoted the biologi-cal eciencies from 79 to 87% when fructication wasinduced at 25 days after inoculation.

The eect of veratryl alcohol on promoting mushroomformation and yield is shown in Fig. 1. When fructicationwas initiated at 25 days after inoculation, the fresh weightof mushrooms produced in the absence of veratryl alcoholwas lower than when fructication was performed at 30days. However, in the presence of veratryl alcohol, theyield of mushrooms at 25 days was signicantly greaterat both veratryl alcohol levels studied, than for similarexperiments, in which fruiting body was induced at 30days. These results demonstrate unequivocally that veratr-yl alcohol not only increased the yields of mushrooms, butalso promoted their earlier production, and therefore wasinvolved in stimulating fruiting body formation. It wasconcluded from these results that a new role for veratrylalcohol was in fruiting body development in P. ostreatus.

During the course of 45 days growth, the weight loss inthe spent solid media was about 17%, with the lignin con-tent decreasing from 18.5 to 15% at harvesting. Theamount of cellulose and hemicellulose in the solid mediaduring this period decreased by 3 and 6%, respectively, butthe reducing sugar levels remained between 0.5 and 1.0%(w/w), which was sucient to sustain active growth by thefungus. Veratryl alcohol did not appear to aect the levelsof free sugars produced during the growth period.

3.3. Laccase production during mushroom development

A study of laccase production in the presence of veratrylalcohol at dierent stages of mushroom growth for thetwo sets of experiments conducted, revealed that the pro-les of enzyme levels for the three types of laccase activ-ities were similar. Only the results for PPO-I activity arepresented (Table 2). The results demonstrated that laccaseswere actively produced during formation of primordia andbasidiocarps, and that veratryl alcohol signicantly in-creased all three types of laccase activities during thephases of fungal dierentiation. Most notably, however,was the signicant drop in all three laccase activities by thetime the mushroom matured (at harvest). The conclusionsdrawn from this observation were that laccases and, ve-ratryl alcohol too, played a role in the physiological devel-opment of the basidiocarp. A role for laccases during fruitbody formation has also been observed in A. bisporus [18].

Acknowledgements

This work was supported by funds from CAPES andCNPq, Brazil.

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