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Antioxidant Activity and Total Phenolic Content of Volvariella volvacea and

Schizophyllum commune Mycelia Cultured in Indigenous Liquid Media

Article · March 2016

DOI: 10.5943/mycosphere/7/2/4

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 Mycosphere 7 (2): 131–138 (2016) www.mycosphere.org ISSN 2077 7019
Article
Doi 10.5943/mycosphere/7/2/4
Copyright © Guizhou Academy of Agricultural Sciences

Antioxidant Activity and Total Phenolic Content of Volvariella

volvacea and Schizophyllum commune Mycelia Cultured in Indigenous
Liquid Media
Rich Milton R. Dulay1*, John Jeric A. Vicente2, Aileen G. Dela Cruz2, Jude
Michael Gagarin3, Whilma Fernando3, Sofronio P. Kalaw1, Renato G. Reyes1
1
Center for Tropical Mushroom Research and Development, Department of Biological Sciences, College of Arts and
Sciences, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines
2
Aliaga National School, Poblacion East II, Aliaga, Nueva Ecija, Philippines
3
Exequiel R. Lina School, Poblacion Norte, Licab, Nueva Ecija, Philippines

Dulay RMR, Vicente JJA, Dela Cruz AG, Gagarin JM, Fernando W, Kalaw SP, and Reyes RG
2016 – Antioxidant activity and total phenolic content of Volvariella volvacea and Schizophyllum
commune mycelia cultured in indigenous liquid media. Mycosphere 7(2), 131–138, Doi
10.5943/mycosphere/7/2/4

Abstract
In the current work, we evaluated the different indigenous liquid culture media for mycelial
production and antioxidant property based on radical scavenging activity and total phenolic content
of Volvariella volvacea and Schizophyllum commune. In V. volvacea culture, the maximum
mycelial biomass was significantly achieved in coconut water with 12.2 g, while S. commune
efficiently grew on rice bran broth that produced the highest yield of 12.5 g. Similarly, the highest
volume loss of spent was significantly recorded in these media for both mushrooms. Mycelia of V.
volvacea grown in coconut water and S. commune in rice bran broth showed the most potent radical
scavenging activity with 21.19% and 19.45%, respectively. The highest total phenolic content were
found in rice bran broth with 23.19 mg AAE/g sample for V. volvacea mycelia and in coconut
water with 25.52 mg AAE/g sample for S. commune mycelia. Therefore, mycelia these edible
mushrooms may have potential as natural antioxidants which are affected by the liquid media.

Key words – S. commune – V. volvacea – total phenolic – antioxidant – liquid media.

Introduction
Edible and medicinal mushrooms represent one of the world’s greatest untapped resources
of nutritious and healthy food. They are rich in protein, carbohydrates, minerals, vitamins,
unsaturated fatty acids, phenolic compounds, tocopherols, ascorbic acid and carotenoids (Reis et al.
2011). Mushrooms have shown a number of biologically active compounds with therapeutic
activities such as, modulation of the immune system, inhibition of tumor growth and inflammation,
have hypoglycaemic and antithrombotic activities, lower blood lipid concentrations, prevent high
blood pressure and atherosclerosis, and antimicrobial and other activities (Menaga & Ayyasamy
2012). In addition to this, mushroom fruiting bodies can produce possible resource of optimal
quantities of bioactive compounds that exhibit antioxidant properties (Preeti et al. 2012).

Submitted 2 February 2016, Accepted 20 March 2016, Published online 30 March 2016
Corresponding Author: Rich Milton R. Dulay – e-mail – [email protected] 131
 Antioxidants act as the major defense of the body against the damages caused by the free
radicals (Jose & Janardhanan 2000). Chennupati et al. (2012) reported that antioxidants address
several diseases like cancer, hepatopathic, nephropathic, retinopathic damage, atherosclerosis,
Alzheimer’s disease, adult respiratory distress syndrome and diabetes by protecting the cells from
oxidative stress; scavenging free radicals, chelating catalytic metals and halting lipid peroxidation
chain reactions. The different antioxidants that can derived from foods include phenolic compounds
(phenolic acids and flavonoids), carotenoids, tocopherol and ascorbic acids which are classified
phytochemicals (Barros et al. 2008). One of the leading techniques in evaluating the antioxidant
activity is through the use of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a stable free radical
(Chennupati et al. 2012).
Volvariella volvacea is one of the most important and mushrooms for culinary purposes. It
is commonly known as paddy straw mushroom or kabuteng saging by Filipinos. Fruiting body of
V. volvacea is rich in bioactive metabolites and nutritional qualities that contribute not only to its
unique and delightful umami taste and aroma but most importantly to its notable functional
activities such anti-coagulant, anti-inflammatory, and anti-hypertensive (Eguchi et al. 2015).
Schizophyllum commune, on the other hand, is one of the common gill-bearing bracket fungi of
world-wide distribution. It can be easily identified by the peculiar structure of its gills which cover
hymenium during unfavorable climatic conditions. This mushroom is of great importance in the
pharmaceutical and food industries since it produces metabolites which are essential in the
production of industrial products (Reyes et al 2009). Schizophyllan is a polysaccharide derived
from S. commune mycelia that exhibit considerable anticancer activity (Qui & Liu 2000).
Mushrooms have long been recognized for their unique umami flavour and important
biological activities such as antioxidant. Hence, the main objective of the present work was to
investigate the antioxidative properties of the two Philippine wild edible mushrooms, V. volvacea
and S. commune, based on their radical scavenging activity and total phenolic content.

Methods

Mushroom strains and inoculum

Cultures of V. volvacea and S. commune were obtained from the Center for Tropical
Mushroom Research and Development (CTMRD), Central Luzon State University, Science City of
Munoz, Nueva Ecija, Philippines. Mycelia were aseptically revived into sterilized potato dextrose
agar (PDA) plates and incubated at 30°C for 7 days. These cultures were served as inoculant
source.

Mycelial culture in liquid media

The different indigenous liquid media were used in the evaluation of mycelial production of
the two mushroom species. These include coconut water from mature coconut (Cocos nucifera),
rice bran decoction (50g of Oryza sativa/L of water), local yellow corn grit decoction (50g of Zea
mays/L of water) and potato sucrose broth (250g of Solanum tuberosum/L of water+10g of white
table sugar). Liquid media (100 ml) were dispensed into microwavable plastic container and
sterilized in an autoclave at 121°C, 15 psi for 30 minutes. There were five replicates per treatment.
After cooling, media were inoculated with mycelial discs (prepared using flame sterile 10-mm-
diameter cork borer) and incubated at 30°C in static condition for 10 days to allow fungal growth.
The mycelial mats were harvested and weighed (wet weight basis) and the volume loss of culture
spent was measured.

Extraction of active components

Culture spent and mycelia were homogenized using a food processor and then ethyl acetate
(10 ml) was added into each cultured broth to extract antioxidant compounds. The ethyl acetate
soluble portion was concentrated under reduced pressure and the concentrates were dissolved in
ethanol for antioxidant analysis.
132
Radical scavenging activity assay
The free radical scavenging activity of the samples was estimated using the stable
2,2’diphenyl-1-1picrylhydrazyl (DPPH) radical following the standard method of Shimada et al.
(1992) with minor modifications. A 100 µl of test sample in ethanol was added with 5 µl DPPH
solution (5 mg DPPH powder in 2 ml of ethanol) in 96-well microtitter plates. The mixture was
shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at
517 nm. The inhibition of DPPH free radicals was calculated.

Estimation of total phenolics

The total phenolic content was estimated using Folin-Ciocalteu method of Slinkard &
Singleton (1977) with modifications. Sample solution (50 μl) was mixed 500 μl of 10%
FolinCiocalteu reagent (Folin:Methanol, 1:1, v/v). After 2 min, 50 μl of 7.5% saturated was added
and kept in the dark for 1 h before absorbance was taken at 765 nm. A calibration curve was
obtained using various concentrations of ascorbic acid. The total phenolic content of the sample
was expressed as mg of ascorbic acid equivalents (AAEs) per gram of sample. Data were analyzed
using Analysis of Variance (ANOVA) in SAS Statistical Program.

Results and Discussion

Mycelial production in liquid media

In mushroom biomass production, culture medium and/or substrate is one of the most
important factors for efficient growth. In this present work, the mycelial growth responses of V.
volvacea and S. commune on the different liquid media were investigated and the results are shown
in Figure 1. It can be seen that in V. volvacea culture, the maximum mycelial biomass was
significantly achieved in coconut water having a mean of 12.2 g at 30°C for 12 days. However, rice
bran broth supported the most efficient mycelial growth of S. commune that produced the highest
yield of 12.5 g. These two indigenous liquid media were found statistically comparable with the
commercial potato broth (CPB). These results imply that coconut water and rice bran broth could
be an alternative media for mycelial biomass production of V. volvacea and S. commune,
respectively. On the other hand, corn grit broth registered the lowest yield of mycelia in both
evaluated species. Volume loss of the spent media was also determined after 12 days of incubation
and the results are presented in Figure 2. Coconut water (for V. volvacea) and rice bran broth (for S.
commune significantly recorded the highest volume loss of the spent whereas con grit broth had the
lowest volume loss in both mushrooms. Apparently, liquid media with highest yield of mycelia
registered the highest volume loss of the spent, there is, therefore, a positive correlation.
The superiority of coconut water in V. volvacea culture and rice bran broth in S. commune
culture could be attributed to a wide variety of their chemical components that support the efficient
mycelial growth. Coconut water is nutritious medium rich sugars, sugar alcohols, lipids, amino
acids, nitrogenous compounds, organic acids, enzymes, vitamins (B1, B2, B3, B5, B6, B7 and B9)
and minerals vital for physiological functions (Santoso et al. 1996, Yong et al. 2009). It is also
contains phytohormones like auxin, various cytokinins, gibberellins (GAs), ethylene, cytokinins,
and abscisic acid (ABA) that play crucial roles in regulating plant growth in a wide range of
developmental processes (Ma et al. 2008). Rice bran, on the other hand, contains micronutrients
such as oryzanol, tocopherols, tocotrienols, phytosterol and dietary fiber like beta-glucan, pectin
and gum (Nagendra et al. 2011). It is also contain vitamin B1 and thamine, which are essential for
mycelial growth (Silverio et al. 1981).
However, in our previous works, the maximum mycelial yield of Panaeolus cyanescens was
achieved in coconut water and rice bran broth (Bustillos et al. 2014) while potato broth produced
the most luxuriant mycelial growth of Panaeolus antillarium (Dulay et al. 2015a). Moreover, both
Lentinus tigrinus and Lentinus sajor-caju efficiently grew on rice bran broth which significantly
recorded the highest yield of mycelia and volume loss (Dulay et al. 2015b). These findings and the
results of the present study demonstrated that the efficient mycelial production of these

133
basidiomycetes depend on the liquid medium used in submerged cultivation. Submerged cultivation
is a standard technique of mycelial biomass production of mushrooms with high nutritional value
and functional bioactivity. So, the bioactivities, in particular, antioxidant property of submerged
culture of the two evaluated mushrooms was also carried out in this study.

Fig. 1 – Yield of mycelial biomass of V. volvacea and S. commune in different liquid media after
12 days of incubation. CPB; commercial potato broth.

Fig. 2 – Volume loss of culture spent of V. volvacea and S. commune after 12 days of incubation.
CPB; commercial potato broth.

Antioxidant activity
Antioxidant is any substance that inhibits the damaging effects of free radicals brought by
oxidation. Phenolic acids, flavonoids, carotenoids, tocopherol and ascorbic acids are the most
common antioxidants contained in foods (Barros et al. 2008). The appropriate method to obtain the
significant antioxidant compounds from medicinal mushrooms is through submerged cultivation
(Elisashvili 2012). The DPPH radical scavenging activity of the liquid culture of mycelia of the two
tested mushrooms was determined. Figure 3 shows the radical scavenging activities of V. volvacea
and S. commune grown in different indigenous liquid media. Apparently, in both mushrooms, all
liquid cultures possess radical scavenging activity against free radicals. Among the different
134
indigenous liquid culture of V. volvacea, coconut water significantly recorded the highest activity
of 21.19%. This was followed by rice bran and potato broth which respectively recorded 18.21%
and 12.95%. However, the lowest activity was noted in corn grit with 12.08%. In S. commune
culture, on the other hand, the highest radical scavenging activity was registered in rice bran broth
and coconut water respectively having means of 19.45% and 19.35%. Although these values are
found lower than the activity cathechin as control, this property observed in these liquid cultures of
both mushrooms suggests a promising potentials in the pharmaceutical industries since antioxidant
acts significant physiological functions in preventing free radical associated diseases.

Fig. 3 – Radical scavenging activities of V. volvacea and S. commune in different indigenous liquid
media. CPB; commercial potato broth.

Total phenolic content

One of the most important bioactive compounds with antioxidant activity is the total
phenolic. Phenolic compounds exhibit antioxidant activity that are known to prevent heart
ailments, fight cancer and even are anti-inflammatory agents (Doughari 2012). The total phenolic
contents of the studied mushrooms cultured in indigenous liquid media were also determined in the
present study and the results are presented in Figure 4. It can be seen that all media grown with V.
volvacea mycelia exhibit phenolic contents. Rice bran broth significantly recorded the highest
phenolic content of 23.19 mg AAE/g sample while the corn grit showed the lowest content of 19.18
mg AAE/g sample. On the other hand, S. commune grown in coconut water recorded the highest
phenolic content of 25.52 mg AAE/g sample, followed by rice bran broth with 25.18 mg AAE/g
sample. The lowest was registered in corn grit with a mean of 21.99 mg AAE/g sample. Based on
the data, the varying indigenous liquid media could affect the phenolic contents of V. volvacea and
S. commune. This finding proved the previous work that the level of observed antioxidant activity
depended on the bio-ecological differences of tested strains such as geographical origination, type
of wood substrate, mycelial growth rate, and morphology, as well as the experimental conditions
(Elisashvili 2012).
Several references are available on the positive correlation of total phenolic content in the
mushroom extracts and their antioxidant properties. For instance, out of 23 mushroom species,
Termitomyces heimii and Helvella crispa ranked as high-phenolics species and accordingly, by all
assays T. heimii and T. mummiformis were found to be very high antioxidant varieties (Puttaraju et
al. 2006). This observation is similar to that obtained in Ganoderma tsugae, Lentinus edodes,
Volvariella volvacea, Lentinus tigrinus and Lentinus sajor-caju (Bimla & Punita 2006, Cheung et
al. 2003, Dulay et al. 2015b). In addition, total phenolic is the major naturally occurring antioxidant
components found in Agrocybe cylindracea water extract, in the range of 23.74–30.16 mg/g, which
explains its high antioxidant properties.

135
Fig. 4 – Total phenolic contents of V. volvacea and S. commune in different indigenous liquid
media. CPB; commercial potato broth.

The antioxidant activity of mushroom may also vary in their fruiting body, mycelia and
spent media. The total antioxidant components of A. cylindracea were 30.46, 27.72, and 24.57
mg/g for fruiting bodies, mycelia, and filtrate, respectively (Tsai et al. 2006). The mycelia Pholiota
alnicola, Lepista personata, Trametes versicolor, Volvariella bombycina, Schizophyllum commune,
Suillus luteus, and Lentinus edodes showed more than 20% antioxidant activity (Badalyan 2003).
Evaluation of antioxidant properties of methanolic extracts from Grifola frondosa, Morchella
esculenta, and Termitomyces albuminosus mycelia showed high antioxidant (85.4%–94.7%) at 25
mg/ ml (Mau et al. 2004).
In the current study, therefore, we evaluated the different indigenous liquid media for the
mycelial production and antioxidant activity. It was found out that the different indigenous liquid
media influenced the mycelia growth, volume loss, radical scavenging activity and total phenolic
content of the two mushrooms. The total phenolic content in the mushrooms positively correlated
on their antioxidant properties. This information on the antioxidant potential of these edible
mushrooms would require further exploration for human health benefits.

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