Zied2019_IndianJournalofMicrobiology

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ORIGINAL RESEARCH ARTICLE
Study of Waste Products as Supplements in the Production
and Quality of Pleurotus ostreatus var. Florida
Diego Cunha Zied1 • Arturo Pardo-Giménez2 • George Azevedo de Oliveira3 •
Jaime Carrasco4 • Maria Luiza Zeraik3
Received: 15 February 2019 / Accepted: 12 April 2019 / Published online: 30 April 2019
� Association of Microbiologists of India 2019
Abstract Among the cultivated mushroom, the genus
Pleurotus is the second largest produced worldwide and the
most produced in Brazil. The application of agricultural
by-products (wastes) as substrate supplement is an effec-
tive approach to generate quality food while promoting a
circular economy in agriculture. The manuscript evaluates
the three key aspects of this practice: (1) the response of
different mushroom strains to supplementation, (2) the use
of agricultural by-products with different N content, and
(3) the efficacy of certain preliminary treatments applied to
the supplements. To this end, production and nutritional
quality of the mushroom were tested along the crop cycle.
Compared to the control substrate, the yield increased by
11, 26, 30 and 42% in the first flush and by 86 and 31% in
the second flush. Supplementation resulted in an increment
of the fiber and protein content of mushroom and a decline
of carbohydrate and lipid content of mushroom.
Keywords Edible mushrooms � Bioconversion �
Supplements composition � By-products � Yield
Introduction
Fivemain genera constitute approximately 85%of theworld’s
mushroom supply. Lentinula is the major genus, contributing
around 22% of the world’s cultivated mushrooms. With 5–6
cultivated species, Pleurotus is a close second, constituting
approximately 19% of the world’s mushroom output, whereas
Auricularia contributes around 17%. The other two main
genera, Agaricus and Flammulina, are responsible for 15 and
11% of the supply, respectively [1]. In Brazil, the species
Pleurotus ostreatus var. Florida is the most cultivated, with a
production of* 7475 tons, which represents around 48% of
the total mushroom produced in the country.
Different from the addition of materials rich in nitrogen
during the formulation of the substrate, at the end of
composting, the nitrogen content and C/N value may be
corrected by supplementation conducted either at spawning
(inoculation with mycelium) or at casing (the addition of a
layer of peat, clay and gypsum at the surface of the com-
post to induce fruiting) to enhance performance. Yields
generally increase by 5–20% and occasionally by more [2].
This technique became commercial in the beginning of
the year 2000, with North American and European com-
panies making available the supplements for the growers,
which had the advantage of being applied at spawning or at
casing, reducing the losses and wastage, not dispensing the
addition of supplements at the moment of the substrate
formulation. Research has shown that a large amount of
nutrients can be added, with yield increases that are almost
proportional to the amount added up to certain level, and
then decline significantly [3, 4].
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s12088-019-00805-1) contains sup-
plementary material, which is available to authorized users.
& Diego Cunha Zied
dczied@gmail.com
1 Faculdade de Ciências Agrárias e Tecnológicas (FCAT),
Universidade Estadual Paulista (UNESP), Câmpus de
Dracena, Rod. Cmte João Ribeiro de Barros, km 651, Bairro
das Antes, Dracena, SP 17900-000, Brazil
2 Centro de Investigación, Experimentación y Servicios del
Champiñón (CIES), C/Peñicas, s/n, Apdo. 63,
16220 Quintanar del Rey¸Cuenca, Spain
3 Departamento de Quı́mica, Universidade Estadual de
Londrina (UEL), Londrina, PR 86051-990, Brazil
4 Department of Plant Sciences, University of Oxford, S Parks
Rd, Oxford OX1 3RB, UK
123
Indian J Microbiol (July–Sept 2019) 59(3):328–335
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The increase in temperature that occurs immediately
after supplementing due to the high metabolic activity of
the fungus and the presence of other competing microor-
ganisms in compost can be problematic when using sup-
plementation [2, 3, 5, 6]. Certain practices have been
proposed to avoid this problem, such as the application of
different formaldehyde concentrations to the supplement,
the efficiency of which has subsequently been confirmed
[2, 5, 7, 8]. However, due to the toxicity of formaldehyde,
other types of treatments resulting in protein tanning need
to be investigated specifically for the organic production of
mushrooms.
Although the initial research with supplementation was
performed with Agaricus bisporus [3, 9, 10] considerable
research is also currently available regarding the applica-
tion of supplements to P. ostreatus, obtaining a substantial
increase in yield [11, 12]. In addition to commercial sup-
plements available for the producers in the market, the use
of low-cost agricultural by-products available at the pro-
ductive regions is a promising approach [5]. Different
researches suggest that substrates supplemented with cot-
ton seed cake provides the best response concerning yield
of Pleurotus spp. [13]. Naraian et al. [12] verified that
cotton seed cake was the best substrate supplement while
soybean meal was the second best additive for cultivation
of Pleurotus ostreatus var. Florida. Recently Pardo-
Giménez et al. [14] proposed the use of pistachio meal
waste, a material with a low N content, applied in the dose
of 15 g kg-1 compost, which provided a yield increase up
to 34.4%, in the cultivation of P. ostreatus.
We know that the nutritional behavior differs between A.
bisporus and P. ostreatus, as does the ability to degrade
lignin; thus, the appropriate method of substrate preparation
and the N content of the substrate should be determined for
each species, necessitating further research. Therefore, the
present paper evaluates the following 3 factors to improve
the supplementation techniques used in P. ostreatus culti-
vation, (1) the response of different strains to supplemen-
tation, (2) the use of different agricultural by-products with
different N sources, and (3) the efficacy of different treat-
ments applied to the supplements. For these purposes, we
analyzed both mushroom production parameters (the yield,
biological efficiency, number and size of mushroom and
size of clusters) and their quality/nutritional parameters (the
protein, ash, fiber, carbohydrate, and fat contents).
Materials and Methods
Spawn
Strains POS 16/01, 16/02 and 16/03 of Pleurotus ostreatus
var. Florida were used and collected from commercial
growers in the cities of Mogi-das-Cruzes, Presidente Pru-
dente and Cabreúva in Sao Paulo State (Brazil). The cri-
terion used for strain selection was the quantity and scale of
mushrooms produced by the grower/company (superior to
3 tons of fresh mushrooms harvested per month). Currently
these strains represent approximately 80% of total pro-
duction of P. ostreatus in Brazil. The procedures adopted
by Zied et al. [15, 16] were followed for spawn production.
The strains are deposited in the public culture collection of
Sao Paulo State University, Câmpus de Dracena, which
provides open access to interested researchers.
Substrate
The substrate was prepared according to the standards
adopted for the commercial production of P. ostreatus in
Brazil: a total of 16 days of substrate preparation (semi-
compost system), with 10 days in Phase I and 6 days in
Phase II. During the Phase I process, Brachiaria dicty-
oneura and sugarcane bagasse bagasse (bulk materials)
were moistened for 4 days; a pile was assembled on the 5th
day; and on the 6th day, the concentrated materials (riceand wheat bran, calcitic limestone and gypsum) were
added, and the pile was turned. The pile was subsequently
turned 3 more times (once a day) before the substrate was
transferred to a pasteurization chamber on the 10th day
(Phase II). The substrate was pasteurized at 72–65 �C for
12 h and subsequently conditioned at 55–48 �C for 4 days.
In Supplementary Section, Table 1 presents the chemical
characteristics of the substrate after the Phase II process.
The moisture, organic matter, C and N content and pH
were evaluated following the methodology presented by
Zied et al. [17]. The methodology used to evaluate ash,
fiber, fat and carbohydrate content are described in the
Proximate analysis section.
Supplementation at Spawning
Six different supplements were used: 2 of them were
commercial supplements used in several countries (Spawn
Mate II SE� and Pro Mycel Gold�, Amycel and Spawn
Mate company, Watsonville, US), recommended for the
production of P. ostreatus and A. bisporus, respectively.
The other supplements were made with the following
typical agricultural by-products from Brazil: (1) a mixture
of soybean, cotton and corn bran (33.3% each); (2) waste
from the acerola juice agro-industry; (3) waste from the
peanut agro-industry (80% peanut hulls ? 20% grain); (4);
and a combination of supplements i, ii and iii (33.3% each).
Altogether, 6 supplements and a control that consisted of
substrate without supplements were used. In Supplemen-
tary Section, Table 1 presents the chemical characteristics
of the supplements. The respective application rates for the
Indian J Microbiol (July–Sept 2019) 59(3):328–335 329
123
supplement and the spawn were 10 and 20 g kg-1 of wet
substrate. The supplement was applied together with the
spawn at the end of the Phase II process.
Supplement Treatment
The supplements based on the Brazilian agricultural by-
products were treated with either formaldehyde or heating,
as follows: (1) 360 ml of formaldehyde solution (45 ml of
formalin with a concentration of 35–40% formaldehyde
mixed with 315 ml of water) was applied to 3 kg of sup-
plement, resulting in a formaldehyde concentration of
6000 ppm [8], or (2) the supplement was subjected to dry
heat at 68 �C for 24 h. These treatments were performed
7 days before spawning and were applied to supplements
that had all been dried to a 4–6% moisture content and then
crushed before sifting with a\ 0.5-mm sieve.
Growing Cycle
The spawn run was completed on day 17, and the harvest
phase lasted 43 days; thus, the growing cycle totaled
60 days. The temperature and relative humidity were
26 ± 2 �C and 70 ± 5%, respectively, during the spawn
run and 26 ± 5 �C and 80 ± 10%, respectively, during the
harvest phase. The cultivation was conducted in a green-
house used specifically for P. ostreatus growth, with the
control of temperature, relative humidity and aeration. Four
flushes were harvested over the cultivation cycle. The
mushrooms were collected twice a day during each flush
and identified, weighed and counted for the analysis of the
production parameters.
Parameters Evaluated
The following production parameters were evaluated: (1)
the yield calculated as 100 times the f.w. of mushrooms
divided by the f.w. of compost, expressed as a percentage
(1st, 2nd, 3rd, and 4th flush and total yield); (2) biological
efficiency calculated as 100 times the f.w. of mushrooms
divided by the d.w. of compost, expressed as a percentage;
(3) the number of mushrooms harvested; (4) the weight per
mushroom, expressed in g (total fresh weight harvested
during the cycle divided by the number of mushrooms);
and (5) the weight per cluster, expressed in g (total fresh
weight harvested during the cycle divided by the number of
clusters), as previously described by Pardo-Giménez et al.
[14] and Zied et al. [17].
Proximate Analysis
To evaluate the quality/nutritional parameters, the har-
vested mushrooms of each strain and supplement treatment
were mixed to consider only the supplement compositions.
Subsequently, the content of ash, fiber, fat, protein and
carbohydrate were evaluated, according to the methods of
the Association of Official Analytical Chemists [18]. To
determine the ash content, the samples were burned at
550 �C for at least 6 h until a constant weight was reached.
The value for nitrogen, which is a precursor to the protein
content, was determined by the Kjeldahl method [19],
involving mushroom digestion, distillation and titration.
The protein content was calculated by multiplying the total
nitrogen by a conversion factor of 4.38 [20]. The crude fat
content was determined by a Soxhlet extraction method,
using the solvent n-hexane. The crude fiber value was
determined by a digestion method, submitting the sample
to two consecutive digestions with sulfuric acid and
sodium hydroxide solutions [21]. The total carbohydrate
content was calculated by subtracting the sum of the crude
protein, total fat, ash and fiber contents from the total
weight of the mushrooms.
Statistical Analyses
The experiment was performed considering the three
studied factors in a randomized complete block design (3
strains, 7 supplement compositions and 2 supplement
treatments), with 6 replicates per treatment. The factors
that presented significant differences were separated for a
better evaluation. ANOVA was used to analyze the data,
followed by the least significant difference (LSD) test at
p\ 0.05.
Results
The strain and supplement composition factors presented a
significant difference when evaluated the factors alone. The
POS 16/01 strain responded directly to supplementation,
and supplement using Brazilian by-product (mixture of
corn, soybean and cotton bran) produced a response equal
to that produced by the internationally distributed com-
mercial supplement (Spawn Mate II SE). By contrast,
neither the formaldehyde nor heat treatment showed dif-
ference (Table 1).
To better understand the results for the factors that
presented significance, the production parameters were
evaluated by separating the strain and supplement com-
position factors. In addition, the yield values were sepa-
rated by flush to verify a delayed-release nutrient effect of
the supplements treated with formaldehyde.
In the 1st flush (Table 2), the greatest yield increases
compared with the control occurred for the POS 16/01
strain with the supplement iv/heating treatment (an
increase of 11%), for the POS 16/02 strain with the
330 Indian J Microbiol (July–Sept 2019) 59(3):328–335
123
supplement i/heating and supplement i/aldehyde treatments
(respective increases of 26 and 30%), and finally for the
POS 16/03 strain with the supplement ii/heating treatment
(an increase of 42%). In the 2nd flush (Table 2 in Sup-
plementary Section), the greatest increases compared to the
control occurred for the POS 16/01 strain with Spawn Mate
II SE (an increase of 23%) and for the POS 16/02 strain
with the supplement iv/aldehyde treatment (an increase of
31%). In the 3rd flush (Table 2—Supplementary Section),
the greatest increase compared with the control occurred
for the POS 16/03 strain with Pro Mycel Gold, producing
an increase of 2%. In the 4th and final flush (Table 2—
Supplementary Section), the greatest significant increase
compared to the control occurred for the POS 16/01 strain
with the supplement iii/aldehyde treatment (increase of
50%); for the POS 16/03 strain, the control showed the
greatest yield.
Evaluating the total yield, supplement iv/heating pro-
vided an increase of 16% compared with the control in the
POS 16/01 strain, whereas supplement i/heating, supple-
ment iii/heating and Pro Mycel Gold provided respective
increases of 8, 2 and 3% in POS 16/03 (Table 2).
Table 1 Total yield analyzed
by the factor alone (strain,
supplement composition and
supplement treatment), without
the unfolding, with simple
factorial design
Factor Strain (Factor 1) Supplementcomposition (Factor 2) Supplement treatment (Factor 3)
Yield, % POS 16/01 30.51a Control (no supplement) 28.77ab Aldehyde 26.98
POS 16/02 27.35b Supplement i 29.11a Heating 29.07
POS 16/03 26.42b Supplement ii 27.69ab
Supplement iii 26.46b
Supplement iv 28.91ab
Spawn Mate II SE 29.43a
Pro Mycel Gold 27.42b
Mean 28.09 28.25 28.02
Supplement i is a mixture of corn, soybean and cotton bran (33.3% each). Supplement ii is waste from the
acerola juice agro-industry. Supplement iii is waste from the peanut agro-industry (80% hulls ? 20%
grains). Supplement iv is a combination of supplements i, ii and iii (33.3% each). Spawn Mate II SE and
Pro Mycel Gold are commercial supplements produced by the company Amycel. Values followed by
different lowercase letters within a column are significantly different at p\ 0.05
Table 2 Results obtained for yield and biological efficiency assessed in the cultivation of Pleurotus ostreatus var. Florida by the supplement/
treatment combination, flush and strain
Supplement/treatment 1st flush yield, % Total yield, % Biological efficiency, %
POS
16/01
POS
16/02
POS
16/03
POS
16/01
POS
16/02
POS
16/03
POS
16/01
POS
16/02
POS 16/03
Control (no
supplemented)
14.18AB 11.90AB 8.65AB 30.19ABC 28.82 27.30AB 137.25ABC 131.01 124.10AB
Supplement i/heating 13.39AB 14.98A 11.18AB 31.51ABC 29.49 29.44A 143.27ABC 134.04 133.82A
Supplement ii/heating 11.79AB 13.38AB 12.34A 31.09ABC 28.48 27.10AB 141.35ABC 129.45 123.21AB
Supplement iii/heating 11.26AB 8.83B 10.50AB 31.05ABC 25.38 27.92A 141.17ABC 115.37 126.91A
Supplement iv/heating 15.75A 10.07AB 10.11AB 35.16a A 25.47b 26.72b AB 159.84a A 115.77b 121.48b AB
Supplement i/aldehyde 12.29ab AB 15.49a A 8.39b AB 29.73ABC 29.17 24.91AB 137.21ABC 132.62 113.23AB
Supplement ii/aldehyde 10.33AB 10.05AB 5.59B 27.55BC 27.21 24.72AB 125.26BC 123.70 112.38AB
Supplement iii/aldehyde 8.36B 12.19AB 6.82AB 26.27ab C 27.95a 20.20b B 119.43ab C 127.05a 91.84b B
Supplement iv/aldehyde 12.18AB 8.67B 10.86AB 32.45ABC 26.50 27.16AB 147.50ABC 120.50 123.48AB
Spawn mate II SE 10.39AB 9.77AB 7.94AB 34.23AB 26.96 27.10AB 155.61AB 122.54 122.88AB
Pro Mycel Gold 8.68B 11.85AB 7.93AB 28.92ABC 25.40 28.04A 131.49ABC 115.46 127.49A
Mean 10.79 28.18 128.11
Supplement i is a mixture of corn, soybean and cotton bran (33.3% each). Supplement ii is waste from the acerola juice agro-industry.
Supplement iii is waste from the peanut agro-industry (80% hulls ? 20% grain). Supplement iv is a combination of supplements i, ii and iii
(33.3% each). Spawn Mate II SE and Pro Mycel Gold are commercial supplements produced by the company Amycel. Values followed by
different lower case letters within a line and upper case letters within a column are significantly different at p\ 0.05
Indian J Microbiol (July–Sept 2019) 59(3):328–335 331
123
Therefore, no supplement proved to be significantly supe-
rior in all three strains used regarding the total yield;
however, one combination—the supplement i/heating—
showed higher total yield than that of the control in all the
strains, with values of 31.51, 29.49 and 29.44% compared
to respective control values of 30.19, 28.82 and 27.30% for
the POS 01/16, 02/16 and 03/16 strains, respectively. POS
16/02 was the only strain that did not respond to supple-
mentation (total yield), neither positively nor negatively.
The significance values obtained of the supplements and
the strains for biological efficiency were equal to the total
yield, showing the same performance.
The large number of mushrooms harvested as well as the
large cluster size can explain the high total yield obtained
by supplement iv/heating in the POS 16/01 strain; and the
large cluster size can explained the high total yield
obtained by supplement i/heating for POS 16/03 (Table 3).
The POS 16/01 strain showed important differential mor-
phological characteristics in the substrate control: a small
size of individual mushrooms and large cluster size,
important mushroom market characters (small size and
little fibrosity) appropriate for the food served in Japanese
restaurants.
The proximate analyses of P. ostreatus mushrooms
cultivated with different supplements are shown in Table 4.
The ash content ranged from 56.91 to 61.47 g kg-1 of dry
weight among the mushrooms cultivated on the different
supplements. The carbohydrate content found in P.
ostreatus mushrooms produced with supplemented
substrate (from 494.47 to 637.41 g kg-1) was lower than
that in mushrooms produced with unsupplemented sub-
strate (661.12 g kg-1), except for supplement ii. One of the
most significant results was the higher fiber contents of the
supplemented mushrooms (reaching values of
266.76 g kg-1) compared to the unsupplemented substrate
(107.21 g kg-1). Significant variation in lipid content was
also recorded, with lower values in the supplemented
mushrooms, except for those cultivated with supplement
iii, due to the high levels of lipid rich-peanuts found in this
supplement (there are approximately 50 g of total fat in
100 g of peanut, USDA National Nutrient data base).
The use of supplements in mushroom cultivation
increased the amount of protein per mushroom to a level
approximately 10% higher than that presented by the
control (unsupplemented substrate). The commercial sup-
plement Pro Mycel Gold produced a protein content of
174.91 g kg-1, which was the highest observed and could
be explained by the nitrogen-rich nature (7.7%) of this
supplement (Table 1—Supplementary Section).
Discussion
It is essential to create new technology to better understand
the processes involved in mushroom cultivation based on
the interaction of fungal development, substrate supple-
mentation and any treatment applied to the supplement.
Table 3 Results obtained for the production parameters assessed in the cultivation of Pleurotus ostreatus var. Florida by the supplement/
treatment combination, flush and strain
Supplements/treatments Number of mushrooms, u Weight per mushroom, g Weight per cluster, g
POS
16/01
POS
16/02
POS
16/03
POS
16/01
POS 16/02 POS
16/03
POS 16/01 POS
16/02
POS
16/03
Control (no
supplemented)
263AB 206 217AB 1.96b 2.40a ABC 2.13ab AB 37.46ABCD 35.50AB 33.06AB
Supplement i/heating 259AB 209 250AB 2.13 2.42AB 2.00ABC 34.90CD 40.57AB 43.63A
Supplement ii/heating 279a AB 239ab 194b B 1.91b 2.07ab ABCDE 2.43aA 51.83a ABC 37.29ab AB 32.93bAB
Supplement iii/heating 263AB 226 238AB 2.06 1.98CDE 2.06ABC 36.40BCD 37.03AB 33.63AB
Supplement iv/heating 312a A 229b 247ab AB 1.90 1.91E 1.89BC 54.12a A 34.52b AB 34.01bAB
Supplement i/aldehyde 278AB 214 246AB 1.83b 2.34a ABCD 1.76b BC 52.37AB 35.75AB 37.41AB
Supplement ii/aldehyde 231B 251 229AB 2.03 1.88E 1.87BC 33.74D 45.41A 29.64AB
Supplement iii/aldehyde 228B 239 197B 2.03 2.02BCDE 1.68C 35.45ab BCD 45.27a A 22.71b B
Supplement iv/aldehyde 263a AB 231 217AB 2.21 1.95DE 2.15AB 38.64ABCD 38.08AB 40.87A
Spawn Mate II SE 284a AB 192b 271a A 2.06ab 2.47a A 1.75b BC 46.14ABCD 30.32AB 40.46A
Pro Mycel Gold 251AB 216 262AB 1.99 2.04BCDE 1.83BC 37.11ABCD 27.41B 36.90AB
Mean 240.39 2.03 37.90
Supplement i is a mixture of corn, soybean and cotton bran (33.3% each). Supplement ii is waste from the acerola juice agro-industry.
Supplement iii is waste from the peanut agro-industry (80% hulls ? 20% grain). Supplement iv is a combination of supplements i, ii and iii
(33.3% each). Spawn Mate II SE and Pro Mycel Gold are commercial supplements produced by the company Amycel. Values followed by
different lower case letters within a line and upper case letters within a column are significantly different at p\ 0.05
332 Indian J Microbiol (July–Sept 2019) 59(3):328–335
123
First, the use of a strain that responds to the application
of supplements rich in nutrients, especially N, is crucial.
Similar results were reported by other authors [22, 23]. In
our research, the N content of the supplements ranged
between 1.7 and 7.7%, which consequentlyprovided a C/N
ratio ranging from 6/1 to 28/1 (Table 1—Supplementary
Section), which may not be a primordial condition for the
metabolic development of P. ostreatus mushrooms such as
verified for the POS 16/02 strain (Table 3). This strain did
not show a significant increase in total yield despite pre-
senting a positive response in the first and second flushes. If
the research objective in this case was to evaluate the
precocity (only in the first and second flushes) and a short
cycle (a 35-day crop cycle) of production, the POS 16/02
strain could have important characteristics. In nature,
Pleurotus spp. mostly grow on dead plant parts, which are
generally poor in nutrients and vitamins [12]. The substrate
used as a control had a C/N ratio of 64/1. Fungi of Pleu-
rotus spp. do not need a substrate with chemical/microbial
selectivity, since they can grow in nutritional media with a
C/N ratio comprised in a wide range of values, at least
between 30 and 300/1 [9], without thermophilic beneficial
communities.
The methods of substrate preparation for P. ostreatus
production are highly diversified [4, 9, 11, 24–26]. With
commercial production, cultivation using bags is the most
recommended method, due to the high substrate volume to
be produced and the use of pasteurization tunnels having a
capacity exceeding 30 tons of substrate. This method of
substrate production can be accomplished adopting a rapid
production system (4 or 5 days of Phase I and 2 days of
Phase II) or a semicompost system (7–11 days of Phase I
and 5–7 days of Phase II). The present research used a
semicompost system, adopted in Brazil, which favors the
generation and succession of mesophilic to thermophilic
microorganisms and uses labile carbon sources, helping to
degrade some types of compost in the substrate. This
method of substrate production may accelerate the release
to P. ostreatus of certain nutrients that could have been
provided by the application of supplement; perhaps for this
reason, a yield response to supplement use was not so
evident in the present research but was confirmed, mainly
in the first and second flush for 3 different strains (media
values of 10.79%, Table 2 and 8.46%, Table 2—Supple-
mentary Section).
Second, the quality, availability and price of the sup-
plement to be used in commercial cultivation are extremely
important. Supplement i can be prepared anywhere in
Brazil and in other agricultural countries around the world,
since it used as base noble materials and relatively
expensive, such as soybean bran (a high-N source) [2, 5],
cotton bran (providing several amino acids) [12, 27] and
corn bran (a nutritional reserve source) [28]. Supplements
ii and iii, both low-N sources (Table 1—Supplementary
Section), were prepared with agricultural by-products
(waste) found in some regions of Brazil and available
without cost (no commercial price). Finally, supplement iv
was simply a mix of supplements i, ii and iii. The highest
yield responses were observed with supplement i and the
commercial Spawn Mate II SE supplement (Table 1), even
the strain POS 16/03 that presented the highest total yield
in the supplement Pro Mycel Gold, which demonstrates its
response to the application of N-rich supplements
(Table 2).
The addition of supplement iv/heating in the substrate
inoculated with the POS 16/01 strain provided a yield
increase of 16% compared to the control, and the addition
of Spawn Mate II SE in the substrate inoculated with the
POS 16/01 strain provided a 13% increase. The best results
were obtained with the POS 16/01 strain and the
Table 4 Nutritional composition of Pleurotus ostreatus var. Florida cultivated with different supplements
Material Ashes, g kg-1 Carbohydrates, g kg-1 Fibers, g kg-1 Lipids, g kg-1 Proteins, g kg-1
Substrate 61.09a 661.12a 107.21c 35.46a 135.12c
Supplement i 56.91b 637.41ab 151.70bc 16.94b 137.05c
Supplement ii 48.03d 662.33a 148.56bc 17.64b 123.45d
Supplement iii 54.63bc 494.47c 266.76a 33.82a 150.32b
Supplement iv 61.47a 583.23b 190.41b 16.06b 148.84b
Spawn mate II SE 51.66cd 613.58ab 179.59b 18.42b 136.76c
Pro Mycel Gold 57.97ab 599.09ab 149.82cb 18.21b 174.91a
Mean 55.97 607.32 170.58 22.36 143.78
Supplement i is a mixture of corn, soybean and cotton bran (33.3% each). Supplement ii is waste from the acerola juice agro-industry.
Supplement iii is waste from the peanut agro-industry (80% hulls ? 20% grain). Supplement iv is a combination of supplements i, ii and iii
(33.3% each). Spawn Mate II SE and Pro Mycel Gold are commercial supplements produced by the company Amycel. Values followed by
different lowercase letters within a column are significantly different at p\ 0.05
Indian J Microbiol (July–Sept 2019) 59(3):328–335 333
123
supplement iv with a C/N ratio of 18/1. Zied et al. [2],
reviewing the application of soy-based products as sup-
plements, mentioned that a yield increase of approximately
5–20% or more can be verified. Regarding costs and profits
of compost supplementation with commercial additives in
A. bisporus cultivation, with an estimation that the cost of
supplementation is covered by a 1.5 kg m-2 increase in
yield [2], and approximately 30 kg of mushrooms are
harvested per square meter in modern facilities, a mush-
room yield increase of 5% would be needed to cover the
cost of supplementation. In the present study, the following
supplements and strains provided a more than 5% increase
in yield compared with the control: supplement iv/heating
(combining supplements i, ii and iii), supplement iv/alde-
hyde and Spawn Mate II SE with the POS 16/01 strain and
supplement i/heating with POS 16/03.
Third, in the present situation studied, formaldehyde
was found to be useful for sanitary treatment and for
delaying the release of nutrients. As shown in the results
section, the yield increases compared to that of the control
substrate were 11, 26, 30 and 42% in the first flush and 23
and 31% in the second flush. In the third flush, the only
strain that responded significantly grew best on the control
substrate, which was not supplemented, and that supple-
mented with Pro Mycel Gold (treated with formaldehyde).
In the fourth and final flush, two strains presented a sig-
nificant effect of supplementation, with the highest yields
shown by POS 16/01 treated with formaldehyde (supple-
ment iii) and POS 16/03 grown on the control substrate.
Therefore, evidence for high yields were verified in the 1st
and 2nd flushes (6 reports, with 3 for supplements treated
using heating and 3 for supplements treated using
formaldehyde) and in the last two flushes (4 reports, with 2
for the control substrate and 2 for the substrate with
formaldehyde-treated supplement). Note that the Spawn
Mate II SE supplement is also treated with formaldehyde.
One of the purposes of the present study was to collect 4
flushes to describe fungal metabolism in relation to yield.
Therefore, the heating treatment of supplement provided a
precocity yield (first flush) and formaldehyde treatment
provided higher yield in the 2nd, 3rd and 4th flushes.
The 5% formaldehyde dose used was extremely effec-
tive in avoiding the presence in the substrate of contami-
nant molds. Similarly, the heating treatment was also
extremely effective. Both treatments have positive and
negative points; the main benefit of heating is the exclusion
of human and environmental toxins, whereas the chemical
treatment is much cheaper and thus more economically
competitive. A restricted study of energy expenditure
should be performed to determine the most appropriate
supplement treatment from a cost–benefit standpoint. Many
people and companies are seeking to replace chemically
based practices with cleaner ones.
The results for the quality/nutritional parameters mea-
sured in harvested P. ostreatus mushrooms show that
supplementation produced mushrooms with lower carbo-
hydrate and greater fiber and proteincontents, except when
using supplement ii. The cultivation of mushrooms with
supplement ii also resulted in a low total yield (Table 2).
However, the use of supplements increased not only the
yield but also important mushroom nutritional parameters,
such as protein and fiber, which offer several benefits to
human health, exemplified by the study of Ng et al. [29].
These authors showed that the dietary incorporation of
fiber-rich oyster mushroom (Pleurotus sajor-caju) powder
improves the postprandial glycemic response. Thus, the use
of supplements increases both the yield and quality of
mushrooms.
A recent study showed as companies can potentially the
quality of fungal foods providing the consumer with
selected products, such as marketing only the mushroom
part of pilei that has protein values of 339.6 g kg-1 while
the whole mushroom has 290.3 g kg-1 [30]. The present
manuscript also demonstrates that supplementation of
substrate can provides a selected product, with approxi-
mately 30%, more protein than convectional mushroom
harvested in unsupplemented substrate (a situation verified
for the Pro Mycel Gold supplement).
Conclusion
The practice of substrate supplementation using by-product
is viable, mainly in terms of increase precocity and quality
of mushroom, decreasing environmental damage. The
choose of correct strain with the appropriated supplement
(C/N ratio between 11 and 18/1) are fundamental condi-
tions for the practice success. Supplement treatment could
be done in by-products, and formaldehyde and heating may
be used.
Acknowledgements We would like to thank the Fundação de
Amparo a Pesquisa do Estado de São Paulo for the financial support
Granted (FAPESP 15/15306-3 and 15/24810-7).
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	Study of Waste Products as Supplements in the Production and Quality of Pleurotus ostreatus var. Florida
	Abstract
	Introduction
	Materials and Methods
	Spawn
	Substrate
	Supplementation at Spawning
	Supplement Treatment
	Growing Cycle
	Parameters Evaluated
	Proximate Analysis
	Statistical Analyses
	Results
	Discussion
	Conclusion
	Acknowledgements
	References