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Hydrogen peroxide concentration measured in cultivation substrates during

growth and fruiting of the mushrooms Agaricus bisporus and Pleurotus spp.

Article  in  Journal of the Science of Food and Agriculture · May 2007

DOI: 10.1002/jsfa.2854

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Journal of the Science of Food and Agriculture J Sci Food Agric 87:1337–1344 (2007)

Hydrogen peroxide concentration

measured in cultivation substrates
during growth and fruiting of the mushrooms
Agaricus bisporus and Pleurotus spp.
Jean-Michel Savoie,1 Dulce Salmones2∗ and Gerardo Mata2
1 UPR Mycologie et Sécurité des Aliments, INRA, B.P. 81, 33883 Villenave d’Ornon, France
2 Instituto
de Ecologia, Unidad de Micologia, A. C., Apdo. Postal 63, C.P. 91000, Xalapa, Veracruz, Mexico

Abstract: Hydrogen peroxide is suspected of being highly implicated in mushroom nutrition and in substrate
bleaching during cultivation. The parameters for measuring H2 O2 in compost samples were examined and the
methodology was applied to samples from both compost colonized by cultivars and wild isolates of Agaricus
bisporus, and wheat straw or coffee pulp colonized by Pleurotus spp. Laccase and peroxidase activities were
also measured. H2 O2 concentration measured after heating at 80 ◦ C for inactivating laccases and peroxidases was
probably both H2 O2 pre-existing in the compost and H2 O2 generated from quinones and active oxygen species.
This potential H2 O2 concentration increased during the vegetative growth for all the strains, in agreement with
a direct relationship between H2 O2 concentration and active biomass of A. bisporus or Pleurotus spp. in their
cultivation substrates. Correlations were observed between H2 O2 concentration and manganese peroxidase activity
in cultivation substrates at the stage of primordia formation. At this stage of development, H2 O2 generation via
biotic or abiotic mechanisms should be an important physiological trait of mushrooms.
 2007 Society of Chemical Industry

Keywords: laccase; manganese peroxidase; compost; straw; coffee pulp; mushroom

INTRODUCTION period. Polyphenol oxidases, plant cell wall lytic

Hydrogen peroxide (H2 O2 ) has been implicated
in lignin and cellulose-degrading systems of both
white-rot and brown-rot basidiomycete fungi. H2 O2
acts as co-substrate for ligninases and manganese
peroxidases, but it is also thought to play a role in
the initially rapid and destructive attack of cellulose
by mediating the production of hydroxyl radicals1 and
it prevents the polymerizing activity of laccases during
lignin degradation.2 This molecule could be highly
implicated in mushroom nutrition and in substrate
bleaching during cultivation.
The species of the genus Pleurotus are among the
most cultivated mushrooms after Agaricus bisporus
(Lange) Imbach. and Lentinula edodes (Berk.) Pegler.
Pleurotus species are wood rots and they are
generally cultivated on non-composted lignocellulosic
substrates. Agaricus bisporus, the commercial button
mushroom is a leaf-litter rot. Its cultivation requires
a substrate that is produced through the composting
of wheat straw, straw-based horse manure, poultry
manure, gypsum and varying amounts of additives.
Good compost will promote the initial growth and
colonization of A. bisporus to the exclusion of
competing micro-organisms and will then support
its nutritional requirement throughout the cultivation
enzymes and microbial cell wall lytic enzymes have
been identified in axenic as well as non-axenic
cultures of both commercial strains of A. bisporus3 – 6
and various strains of Pleurotus spp.7,8 During
vegetative growth of A. bisporus in compost, increasing
laccase and manganese peroxidase activities and low
polysaccharidase activities are generally measured
whereas the opposite is observed during formation
and extension of sporocarps.4 – 6,9,10 Pleurotus ostreatus
(Jacq.: Fr.) Kumm and Pleurotus pulmonarius (Fr.)
Quél also produce manganese peroxidases and their
activities increase during the vegetative growth and
decrease during the enlargement of the sporocarps.8
The release of H2 O2 by A. bisporus was demon-
strated during incubation of mycelium grown on cereal
grains (commercial spawn) for 2–6 h in water,11 and
the presence of H2 O2 in compost colonized by A.
bisporus has been reported.12 The measurement of
H2 O2 concentration was proposed as a means to esti-
mate the changes in the mycelial biomass activity
from the beginning to the end of the culture of A.
bisporus in compost. Laccases, whose activity is also
used as a biomass marker in composts,13 are inacti-
vated when sporocarps develop, and the correlation
between mycelial biomass and H2 O2 concentration is

∗
Correspondence to: Dulce Salmones, Instituto de Ecologia, Unidad de Micologia, A. C., Apdo. Postal 63, C.P. 91000, Xalapa, Veracruz, Mexico
E-mail: dulce@ecologia.edu.mx
Contract/grant sponsor: ECOS/ANUIES; contract/grant number: M00-A01
(Received 23 May 2006; revised version received 30 August 2006; accepted 7 September 2006)
Published online 2 April 2007; DOI: 10.1002/jsfa.2854

 2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

J-M Savoie, D Salmones, G Mata
less susceptible to variations in substrate composition
than the correlation between mycelial biomass and
laccase activity.12
In the present work, the parameters of H2 O2
measurement in compost samples were re-examined
and the methodology was applied to determine both
compost colonization by cultivars and wild isolates of
A. bisporus, and wheat straw or coffee pulp colonization
by Pleurotus spp. The aims of this study were to
strengthen the role of H2 O2 and reactive oxygen
species in substrate colonization and utilization, and
to propose a method of measurement of these
compounds which could be used as a parameter
to estimate the efficiency of the colonization of
lignocellulosic substrates by white-rot fungi.
MATERIALS AND METHODS
Mushroom strains
Eight strains of A. bisporus were studied. The strains
C45 (Le Lion) and HU1 (Sylvan) were cultivars
from commercial spawn producers. The other six
strains were field isolates that are maintained in
the INRA/CTC collection (Bordeaux, France). They
were isolated from two distant sites corresponding to
two types of habitat. The site of Dinard (D) was in
western France under Cupressus macrocarpa near the
seashore,14 for the strains Bs256, Bs261, Bs270. The
site of Gradignan (G) in south-west of France was in
a meadow spread with horse stable manure,14 for the
strains Bs78C, Bs78F, Bs78Y.
Six strains of Pleurotus spp. were studied. IE-38, IE-
49 (both of Pleurotus ostreatus) and IE-137 (Pleurotus
pulmonarius) were commercial strains from Europe
and Asia. Pleurotus djamor (Fr.) Boedijn, IE-121, was
isolated from a wild Mexican specimen growing on a
decomposing Bursera simaruba log. IE-218 (P. djamor)
and IE-225 (P. pulmonarius) were obtained by genetic
crossing. All Pleurotus strains were deposited in the
Strain Collection (IE) of The Institute of Ecology
(Xalapa, Mexico).
Cultivation of Agaricus bisporus
Spawning material was prepared by inoculating millet
grains with cultures of the different strains grown on
malt agar. Grain spawn (8 g kg−1 of wet compost)
was mixed with conventional horse manure and wheat
straw-based compost (68% moisture) and 100 g of the
mixture was placed in plastic boxes (15 × 10 × 6 cm).
The spawned compost was incubated in the dark for
15 days at 24 ± 2 ◦ C and 90% relative humidity. The
colonized compost was then covered with a standard
casing layer and the temperature reduced to 16 ◦ C to
provide a suitable environment for fruiting. The casing
material was a 1:1 (v/v) mixture of brown sphagnum
Irish peat and ground limestone. Water was added to
obtain maximum water-holding capacity. Throughout
the cropping cycle, the air temperature was maintained
at 16 ◦ C and the relative air humidity at 88–91%.
1338
For each strain, 18 boxes were filled with spawned
compost and three boxes were collected for analyses
at each sampling time that were: 9 days after spawning
(A1), 15 days after spawning (A2), at the time of
primordia appearance (A3), when the first fruit bodies
were harvested (A4). Controls were obtained by filling
boxes with non-spawned compost and sampling at the
same time as above.
Cultivation of Pleurotus spp.
Spawn was prepared with moistened sorghum (55%
humidity), sealed in polypropylene bags and sterilized
at 121 ◦ C for 1 h. After cooling, bags were inoculated
with mycelia pre-cultivated on malt agar. Incubation
was carried out at 27 ◦ C, in darkness, until mycelia
completely covered the grains.
Coffee pulp was collected from a local coffee
processing plant, sun dried (<20% of moisture)
and stored at room temperature. Wheat straw was
broken into fragments varying from 1 to 3 cm in
length. To prepare the substrates, both coffee pulp
and wheat straw were hydrated for 12 h, and then
the excess moisture was drained (reaching 60 ± 5%
humidity). Two types of samples were prepared:
(1) in polypropylene bags with 2 kg of wet material
(500 g dry weight) for the evaluation of fruiting bodies
production and (2) in polypropylene bags with 200 g
of wet material for the determination of enzyme
activities and H2 O2 concentration. Each sample was
sterilized at 121 ◦ C for 1 h, cooled and homogeneously
mixed with spawn (50 g kg−1 of wet substrate) and
incubated at 27 ◦ C in darkness. Two days later small
perforations were made in the polypropylene bags to
allow gas exchange. The small samples were incubated
for 16 days, after which the plastic coverings were
removed and the samples were transferred to the
production room, in which they were maintained
throughout adequate air temperature (24–28 ◦ C),
air humidity (88–95%), light (10–12 h day−1 ) and
aeration (600–900 m3 h−1 ), for the initiation of
primordia. The mushroom production samples were
also incubated under the same conditions for the
initiation of the primordia for 16 days (or fewer if
they had early development of primordial); then, the
samples were transferred without plastic coverings to
the production room, where they were maintained
until the complete 36 days of cultivation. Strain
productivity, reported as biological efficiency (BE),
was calculated as fresh weight percent of fruiting
bodies with respect to dry weight of substrate.
During the experiment, the following stages
(P) were collected and analysed: 4, 8, 12, and 16 days
of incubation (P1, P2, P3 and P4, respectively); first
appearance of primordia (P5); beginning of first har-
vest (P6); and 1 week after finishing first harvest (P7).
For each strain and substrate, enzyme activities and
H2 O2 concentrations were measured using one block
per sampling day during the first 16 days of incubation
(samples P1 to P4). In addition, enzyme activities were
J Sci Food Agric 87:1337–1344 (2007)
DOI: 10.1002/jsfa

monitored during the primordial initiation stage (P5),
first flush (P6) and 1 week after the first flush (P7).
Assays for laccases and manganese
peroxidases in cultivation substrates
At the sampling time, the content of each sampled
box or bag was divided into three parts. One was
frozen at −80 ◦ C before being lyophilized and ground
to pass through a 1 mm sieve, another one was used
for measurements of moisture content by drying at
80 ◦ C for 48 h, and the last was immediately used for
extraction of enzymes. Crude enzyme extracts were
prepared by mixing 20 g (wet weight) of cultivation
substrate previously milled in a domestic grinder, with
50 mL of water in 500 mL flasks. The flasks were
rotated end-over-end at 50 rpm for 20 min at room
temperature. The crude enzyme extract was obtained
as the supernatant after centrifugation for 20 min at
12 000 × g, 4 ◦ C. They were stored at −20 ◦ C until
measurement of oxidase activities.
Laccase activity (EC 1.10.3.2) was assayed by
the oxidation of syringaldazine (Sigma, St Louis,
MO) into its quinone in 0.1 mmol L−1 phosphate,
pH 6.0. Changes in absorbance at 526 nm (ε =
65 000 mol L−1 cm−1 ) were measured for every 2 s for
2 min.15
Manganese dependent peroxidase activity (EC
1.11.1.13) was assayed by the oxidation of 3-
(dimethylamino)benzoic acid (DMAB) and 3-
methyl-2-benzothiazolinone hydrazone hydrochloride
(MBTH) (Flucka Chemica, St Louis, MO, USA)
in 50 mmol L−1 succinate/lactate, pH 5.0 in the
presence of MnSO4 (0.05 mmol L−1 ) and H2 O2
(0.1 mmol L−1 ). Changes in absorbance at 590 nm
(ε = 32 900 mol L−1 cm−1 ) were measured every 2 s
for 2 min.16 Absorbance changes obtained under the
same conditions but omitting MnSO4 and H2 O2 were
used to obtain manganese peroxidase activity by dif-
ference. In Mn2+ controls, MnSO4 but not H2 O2 was
added and the differences of absorbance with the mea-
surement where both MnSO4 and H2 O2 were omitted
were recorded. These controls were used to check
if H2 O2 was already present in compost extracts or
generated during the assay.
Assays for H2 O2 concentration in cultivation
substrates
The standardized method used was as follows. For
preparation of crude extracts, 150 mg of lyophilized
and ground cultivation substrate was mixed with
4 mL of 0.05 mol L−1 phosphate buffer pH 6.0 and
incubated at 25 ◦ C for 15 min. The crude extracts
were obtained as the supernatant after centrifugation
for 10 min at 12 000 × g, 4 ◦ C and used immediately
for H2 O2 assays. H2 O2 concentration was determined
immediately after inactivation of enzymes by heating
the extracts to 80 ◦ C for 40 min, as proposed by
Guillén and Evans17 for limiting the interference
of polyphenol oxidases in H2 O2 measurements.
Standard curves with H2 O2 in 0.05 mol L−1 phosphate
J Sci Food Agric 87:1337–1344 (2007)
DOI: 10.1002/jsfa
Hydrogen peroxide in mushroom substrates
buffer pH 6.0 or in compost extract showed the
heating treatment had no significant effect on H2 O2
concentration measured (results not shown). After
cooling, the extracts were mixed with 40 µL of
polyethylenimine at 1%, and then centrifuged at
1000 × g for 5 min to remove polymers18 which could
interfere with H2 O2 assays. The standard mixture
contained 0.25 mL of solution to be assayed, 0.25 mL
of a 20 U mL−1 solution of horseradish peroxidase
(Sigma), and 0.625 mL of chromophore solution
containing 0.6 mmol L−1 of MBTH and 7.5 mmol
L−1 of DMAB in 0.05 mol L−1 phosphate buffer pH
6.0. The mixture was incubated for 3 min at 30 ◦ C and
oxidation of the dyes was monitored at 590 nm.
Variations in parameters of the method were tested
on compost incubated for 15 days with A. bisporus
strain HU1. Extraction temperatures of 20, 25, 30 and
35 ◦ C were compared for obtaining the crude extracts,
with the other parameters unchanged. Incubation
temperatures from 30 to 100 ◦ C were used in the
heat treatment for 40 min from extractions at 25 ◦ C
and measurements were performed both in absence
(controls) and presence (assays) of peroxidase. H2 O2
concentration was the difference between assays and
controls. Heat treatment at 80 ◦ C was applied for
different times from 10 to 120 min from extractions at
25 ◦ C.
Statistics
One-way ANOVA and Fisher’s least significant
difference tests were performed to compare means
of H2 O2 concentration or enzyme activities. Linear
regression analysis by the least squares method was
used to determine correlations between polyphenol
oxidase activities and H2 O2 concentrations. Analyses
were performed with the Systat package (SPSS Inc.,
Chicago, IL, USA).
RESULTS
Improvement of H2 O2 concentration
measurement in A. bisporus compost
In the method proposed here the sample are prepared
in two successive phases: extraction from compost
and incubation of the extract, before H2 O2 assay in
the extract. The best temperature for extraction was
25 ◦ C with a yield of 395 nmol H2 O2 g−1 , the worst was
20 ◦ C (200 nmol H2 O2 g−1 ). Variations in incubation
temperature and time applied on the extracts affected
H2 O2 concentrations measured in compost colonized
by A. bisporus. In controls where no peroxidase was
added, significant values of absorbance were obtained
for incubation temperatures of 30 and 40 ◦ C. At 50 ◦ C
and above, a constant low absorbance was measured
in the controls. Only low quantities of H2 O2 were
observed at 30, 40, 60 and 70 ◦ C when peroxidase
was added. The higher values were at 50 ◦ C and 90 ◦ C
followed by 80 ◦ C (Table 1). The quantity of H2 O2
measured increased with the incubation time at 80 ◦ C
(Table 2). Consequently, the measurement of H2 O2
1339
J-M Savoie, D Salmones, G Mata

concentrations in composts sampled at different times 250

during cultivation of several strains of A. bisporus were
performed with extractions at 25 ◦ C for 15 min and 200

nmol min–1 g–1

incubations at 80 ◦ C for 40 min. 150

Enzyme activities in A. bisporus cultivation 100

substrates
50
There was no laccase and no manganese peroxidase
measured in non-inoculated compost. Laccase and 0
manganese peroxidase activities were all dramatically C45 HU1 Bs78C Bs78F Bs78Y Bs256 Bs261 Bs270

lower in A4 (at harvest) than in A3 (primordia), A Agaricus bisporus strains

but manganese peroxidase activity still was at a A2 A3 A4

significant level whereas laccase activity was negligible 70
in A4 (Fig. 1). Laccase activity in A2 (15 days after
60
spawning) was not significantly (P < 0.05) different

nmol min–1 g–1

50
for HU1, Bs261 and Bs270 having the higher activities.
The significant lower activities were for Bs78C and 40

Bs78F. With manganese peroxidase activity in A2, 30

there was the group HU1 and Bs256 with high 20
activities, the group C45, Bs270 and Bs78Y with 10
middle activities and Bs261, Bs78C and Bs78F with
0
lower activities. C45 HU1 Bs78C Bs78F Bs78Y Bs256 Bs261 Bs270
The values in Mn2+ controls were significant for B Agaricus bisporus strains
all the strains at A3. At this sampling time they A2 A3 A4
reached levels close to the manganese peroxidase
activity indicating that there was enough available 70

H2 O2 in samples for the expression of this activity. For 60

HU1, control Mn2+ values were close to manganese
nmol min–1 g–1

50
peroxidase activities at each sampling time. 40
The more productive strains were C45 and Bs270.
30
One strain, Bs261 produced mushrooms with high dry
20
matter content (Table 3). There was no significant
10

0
Table 1. Effect of incubation temperature on the quantity of H2 O2 C45 HU1 Bs78C Bs78F Bs78Y Bs256 Bs261 Bs270
measured in extracts from mushroom compost colonized by C Agaricus bisporus strains
A. bisporus HU1
A2 A3 A4
Temperature (◦ C)
Figure 1. Polyphenol oxidase activities in extracts from compost
4 30 40 50 60 70 80 90 100 colonized by eight A. bisporus strains at different times of the culture.
(A) Laccases; (B) manganese peroxidases; (C) controls with Mn2+ .
Controlsa 403 363 301 149 104 141 149 144 141 A2: 15 days after spawning; A3: time of promordia appearance; A4:
H 2 O2 b 128 75 112 347 115 117 256 360 218 first flush.

a Controls were measurements without addition of peroxidase

expressed as nmol H2 O2 g−1 .

correlation between the yield and enzyme activities
b H O concentrations in the extracts expressed nmol H O g−1 were
2 2 2 2
whatever the sampling time (data not shown).
differences between the measures with peroxidase and controls.
Changes in H2 O2 concentration in A. bisporus
Table 2. Effect of incubation time at 80 ◦ C on the quantity of H2 O2
compost
measured in extracts from mushroom compost colonized by A. No H2 O2 was found in the compost before the
bisporus HU1 inoculation of A. bisporus. H2 O2 concentration at
A1 was significantly lower in compost colonized by
Time (min) HU1, Bs78C and Bs78F than in compost colonized
by the other strains. There was a significant increase
10 20 30 40 55 75 120
between A1 (9 days after spawning) and A2 for all
Controlsa 55 59 80 84 95 111 113 the strains. Between A2 and A3, significant increases
H2 O2 concentrationb 229 251 277 310 302 338 333 where observed only for Bs78C, Bs256 and Bs261.
a
Controls were measurements without addition of peroxidase,
At A4 there were three groups of strains: Bs256 and
expressed as nmol H2 O2 g−1 . Bs261 with the higher H2 O2 concentration, HU1,
b H O concentrations in the extracts expressed nmol H O g−1 were Bs78C and Bs270 with the lower H2 O2 concentration.
2 2 2 2
differences between the measures with peroxidase and controls. C45, Bs78F and Bs78Y were between the other strains

1340 J Sci Food Agric 87:1337–1344 (2007)

DOI: 10.1002/jsfa
 Hydrogen peroxide in mushroom substrates

Table 3. Yields of A. bisporus mushrooms in the three boxes used for the samples A4

A. bisporus strain

C45 HU1 Bs78C Bs78F Bs78Y Bs256 Bs261 Bs270

Sampling time for A4 (days after 34 37 38 38 37 38 38 43

spawning)
Mushroom harvested (g per 100 g of 11.1 (1.38)a 6.5 (2.03) 5.2 (3.18) 5.2 (3.15) 5.8 (0.65) 7.3 (0.59) 6.3 (1.21) 11.8 (4.03)
compost)
% dry matter in the mushrooms 12.6 8.6 7.1 7.9 8.4 7.8 20.9 8.1
a Mean and standard deviation.

900 35
800 30
700 25

nmol min–1 g–1

600
20
nmol g–1

500
15
400
10
300
5
200
100 0
IE38 IE49 IE121 IE137 IE218 IE225
0 A
Pleurotus strains
C45 HU1 Bs78C Bs78F Bs78Y Bs256 Bs261 Bs270
P1 P2 P3 P4 P5 P6 P7
A. bisporus Strains
A1 A2 A3 A4 35
30
Figure 2. Concentrations of H2 O2 measured in extracts from
nmol min–1 g–1

compost colonized by eight A. bisporus strains at different times of 25

the culture. A1: 9 days after spawning; A2: 15 days after spawning; 20
A3: time of primordia appearance; A4: first flush.
15
10
Table 4. Correlation between polyphenol oxidase activities and H2 O2
5
concentrations measured in extracts from compost colonized by
eight strains of A. bisporus at different times of the culture 0
IE38 IE49 IE121 IE137 IE218 IE225

Sampling time B Pleurotus strains

P1 P2 P3 P4 P5 P6 P7
Polyphenol oxidase activities A2 A3 A4
Figure 3. Laccase activities measured in extracts from wheat straw
Laccase activity (A) and coffee pulp (B) colonized by six strains of Pleurotus spp. at
A2 0.35a 0.34 0.22 different times of the culture. P1 to P4: 4, 8, 12 and 16 days after
A3 0.52 0.42 0.14 spawning; P5: time of primordia appearance; P6: first flush; P7:
A4 −0.70 −0.37 0.05 1 week after first flush.
Manganese peroxidase activity
A2 0.52 0.21 0.01
A3 0.41 0.62 0.64 negative correlation between laccase activity at A4 and
A4 0.14 0.43 0.71 H2 O2 concentration at A2.
Control Mn2+
A2 0.00 y −0.43 Enzyme activities in Pleurotus spp. cultivation
A3 0.21 0.79 0.53
substrates
A4 0.02 0.22 y
No laccase or manganese peroxidase activities were
a
Pearson correlation coefficients. Values in bold type are not observed in non-inoculated straw and coffee pulp. In
significantly different from 1 with Bonferroni probabilities ≤0.1. general, higher laccase activity was obtained during
substrate colonization than during reproductive stages
(Fig. 2). There was no significant correlation with the (Fig. 3). Most strains cultivated in wheat straw showed
mushroom yields. Correlations between the enzyme maximal values of laccase activity in P2 and P3; the
activities and H2 O2 concentrations were determined strain IE137 (P. pulmonarius) had the highest activity.
(Table 4). The only significant ones were positive In the coffee pulp samples, laccase activity was higher
correlations between, manganese peroxidase activity than in wheat straw samples, with maximal values in
and H2 O2 concentration at A3, manganese peroxidase P3 and P4 and for the strain IE121 (P. djamor). On the
activity at A3 and H2 O2 concentration at A4, control other hand the strain IE218 (P. djamor) showed low
with Mn2+ and H2 O2 concentration at A3, and a laccase activity in both wheat straw and coffee pulp

J Sci Food Agric 87:1337–1344 (2007) 1341

DOI: 10.1002/jsfa
J-M Savoie, D Salmones, G Mata

4000 Table 5. Yields in six strains of Pleurotus cultivated on wheat straw

3500 and coffee pulp
3000
Primordial
nmol min–1 g–1

2500 initiation
2000 (days after Yield (g Dry matter
1500 Substrate Strain spawning 100 g−1 ) (%)
1000
Wheat straw IE-38 18–19 50 (15.8)a 9.0
500
IE-49 17–19 54 (11.8) 6.7
0 IE-121 11–13 40 (10.4) 8.4
IE38 IE49 IE121 IE137 IE218 IE225
IE-137 18–19 66 (24.7) 8.4
A Pleurotus strains
IE-218 11–14 30 (18.5) 5.2
P1 P2 P3 P4 P5 P6 P7
IE-225 16–32 77 (43.8) 7.3
4000 Coffee pulp IE-38 18–32 72 (28.1) 8.9
3500 IE-49 17–31 86 (40.9) 5.9
3000 IE-121 13–20 40 (4.7) 6.7
nmol min–1 g–1

2500
2000
1500
1000
500
0 fructification stage (P6); except for IE121 which
IE38 IE49 IE121 IE137 IE218
B Pleurotus strains
P1 P2 P3 P4 P5 P6
Figure 4. Manganese peroxidase activities measured in extracts
from wheat straw (A) and coffee pulp (B) colonized by six strains of
Pleurotus spp. at different times of the culture. P1 to P7 are the
sampling times described in Fig. 3.
substrates, and differently to other strains its maximal
laccase value was during fructification stage.
Production of manganese peroxidase enzyme was
similar to that observed for laccase showing high levels
during colonization stage (Fig. 4). In wheat straw
maximal levels of manganese peroxidase activity were
recorded between P2 and P4. Otherwise, in coffee
pulp samples, high levels of manganese peroxidase
were observed from P1 to P4 and in P7. Strains IE137
and IE138 showed highest manganese peroxidase
values in both substrates. A decrease in manganese
peroxidase activity was observed during primordia
formation (P5) and fructification (P6) and a new
increase was obtained during post harvest stage (P7)
on both substrates.
The more productive strains on wheat straw were
IE137 and IE225 and on coffee pulp the strains
IE49 and IE225. Despite the precocity of primordia
formation, the productivity of P. djamor strains (IE121
and IE218) was low. The strain IE38 produced
mushrooms with high dry matter content on both
substrates (Table 5).
Changes in H2 O2 concentration in Pleurotus spp.
cultivation substrates
As in compost with A. bisporus, no H2 O2 was
measurable in wheat straw and coffee pulp before
inoculation of Pleurotus strains. Wheat straw samples
showed gradual increases in H2 O2 concentrations
starting from P1 and reaching maximal values during
IE-137 22–32 50 (27.9) 8.4
IE-218 15–23 62 (31.4) 5.3
IE-225 23–31 80 (34.4) 5.8
a Mean and standard deviation.
IE225
had similar H2 O2 levels from P3 to P6. Highest
H2 O2 concentrations were observed with IE137 and
P7
IE225 (P. pulmonarius) (Fig. 5). On coffee pulp
H2 O2 concentrations increased from P1 until P4,
P5 or P6 depending on the strain. The highest
values in this substrate were obtained with IE38
and IE49 (P. ostreatus). Correlations between oxidase
activities and H2 O2 concentration were determined
(data not shown) and the only one significant
correlation obtained was a positive correlation between
manganese peroxidase activity at P4 and H2 O2 at P5
(r = 0.88).
DISCUSSION
Measurement of H2 O2 concentration with peroxidase
and MBTH + DMAB after heating at 80 ◦ C
has been used previously with a fungus producing
high laccase activities.17 This high temperature was
used to inactivate laccases interfering with H2 O2
concentration measurements. We observed in the
present study that at 50 ◦ C and above, A. bisporus
laccases in compost were inactivated and there was
no interference on the assay. However, the quantity of
H2 O2 measured after incubation of compost samples
was time and temperature dependent. At 50 ◦ C, high
concentrations were measured and these may be
due to enzymatic generation of H2 O2 . The enzymes
responsible for this production were inactivated at
60 or 70 ◦ C. When the incubation temperature
reached 80 ◦ C, non-enzymatic oxidation of compost
components might be responsible for the production
of H2 O2 . It is known that oxidation of hydroquinones
and organic acids by lignilolytic peroxidases or laccases
in presence of Mn2+ leads to H2 O2 production.19 – 21
For instance, a versatile peroxidase of Pleurotus eryngii
oxidizes hydroquinone to semiquinone radicals and
autoxidation generates O2 •− and then H2 O2 .22 This

1342 J Sci Food Agric 87:1337–1344 (2007)

DOI: 10.1002/jsfa
 Hydrogen peroxide in mushroom substrates

1600 peroxidase activity or control with Mn2+ at the stage of

1400 primordia formation were observed for A. bisporus and
1200 between H2 O2 concentration at the stage of primordia
1000
nmol g–1

and manganese peroxidase activity at the end of incu-

800 bation for Pleurotus spp. These correlations strengthen
600 the implication of manganese peroxidases produced by
400 white-rot fungi in their production of hydroquinones
200 and generation of active forms of oxygen.
0
IE38 IE49 IE121 IE137 IE218 IE225
Because of the high variability between the replicates
A Pleurotus strains it was difficult to obtain significant correlations with
P1 P2 P3 P4 P5
the yields. However, the two Pleurotus strains (IE 49
and IE 225) having the higher yields in both wheat
1600 straw and coffee pulp were also those with the higher
1400 H2 O2 concentrations at the stage of primordia. At
1200 this stage of development, generation of H2 O2 and
1000
nmol g–1

of other active forms of oxygen via biotic or abiotic

800
600
400
200
0
IE38 IE49 IE121 IE137 IE218 IE225
B Pleurotus strains
P1 P2 P3 P4 P5
Figure 5. Concentrations of H2 O2 measured in extracts from wheat
straw (A) and coffee pulp (B) colonized by Pleurotus at different times
of the culture. P1 to P5 are the sampling times described in Fig. 3.
second abiotic phase could be amplified by high
temperatures. H2 O2 measured in compost after heat
treatment was probably not only H2 O2 present
effectively at the sampling time but also H2 O2
generated, during heating, from active oxygen species
produced by the activity of the mycelium on some
phenolic compounds and oxalate present in composts.
Actually, what we measured as H2 O2 concentration in
composts was potential H2 O2 concentration, as it was
probably an estimation of both H2 O2 and hydroxyl
radicals (• OH). The presence of these active forms of
oxygen was the result of A. bisporus activity, because
no H2 O2 was detected in the absence of its mycelium.
A direct correlation between the potential H2 O2
concentration and the level of active mycelial biomass
of A. bisporus was observed in liquid cultures and
could be used for A. bisporus biomass estimation in
compost.12 During cultures in compost, the mycelial
growth of a cultivar of A. bisporus was estimated by the
decrease of xylanase, cellulase and β-glucosidase activ-
ities and the increase of laccase or protease activities.4
In the present study, H2 O2 concentration increased
during the vegetative growth for all the strains of A.
bisporus and Pleurotus spp. and reached maximal values
during the fruiting stages, which was in agreement with
the hypothesis of direct relationships between H2 O2
concentration and active biomass of A. bisporus or
Pleurotus spp. in their cultivation substrate. However,
the ratios of H2 O2 concentration to laccase activity
or H2 O2 concentration to biomass development were
apparently strain dependent. Interesting positive cor-
relations between H2 O2 concentration and manganese
mechanisms should be an important physiological trait
of A. bisporus or Pleurotus spp.
In Pleurotus spp. cultures, different abilities for
colonizing wheat straw or coffee pulp were observed.
No correlation was detected between oxidase activities
and mushroom production measured as weight of
harvested fructifications. In general, both values of
enzyme activity and mushroom production were
higher when the strains were cultivated on coffee
pulp. Whereas higher oxidases were measured in coffee
pulp, lower H2 O2 concentrations were recorded. As
previously observed for A. bisporus,12 both oxidase
activities and H2 O2 concentrations were affected by
the quality of the substrate. Coffee pulp is a very rich
and complex substrate containing many substances
that induced enzyme production and many nutrients
allowing mushroom production.8
H2 O2 measurements as proposed in this work
could be used to characterize colonization kinetics of
lignocellulosic substrates by various white-rot fungi,
but for comparison of strains or substrates this
measurement, as the enzymes activities are, is of lesser
value.
ACKNOWLEDGEMENTS
This work was supported by the cooperation program
ECOS/ANUIES, M00-A01. We are grateful to
Nathalie Minvielle for excellent technical assistance.
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