Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi
Phenolic compounds in Growth Substrate of
Ganoderma sessile and Ganoderma lingzhi
INTRODUCTION
Mushrooms and growth substrate as a
source of antioxidants
Mushroom cultivation is a huge industry,
with the global mushroom
projected to expand rapidly into 2030.
With the continued growth of the industry,
huge amounts of unprofitable mushroom
substrate waste are being
alongside, leading to discardment in
landfills, both a wastage of resources and
space (Ceurstemont, 2020).
Mushroom fruit bodies are well reported to
possess many compounds that serve as
natural antioxidants,
polysaccharides, tocopherols, phenolics,
carotenoids, ergosterol and ascorbic acid
found in fruit bodies, mycelium, and
culture (Sánchez, 2017), resulting in the
production many types of mushroom
supplements. However, it is noteworthy
that prior research has also established the
substantial presence of
compounds, such as polyphenols, in
mycelium (Chilanti et al., 2021). This
investigation aims to quantify
antioxidant content of
substrates, which are high in mycelium
content, in hopes of repurposing them for
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use as supplements and reducing waste
generation in the industry.
Two species of mushrooms, Ganoderma
lingzhi (also known as Ganoderma
market sichuanense) and Ganoderma sessile were
used to compare the levels of antioxidants
available in the fruit body and the
substrate, to determine the viability of
generated mushroom substrate as a potential source
of antioxidant supplement. G. sessile and
G. lingzhi are polypore fungi, native to
North America and East Asia respectively.
The Ganoderma species has been used in
traditional medicine for over 2000 years,
including particularly due to the presence of
bioactive compounds such as terpenoids,
steroids, phenols, nucleotides and their
derivatives (Wachtel-Galor et al., 2011).
Therefore, we chose to investigate these
species as their substrates are more likely
to have higher concentrations of these
bioactive compounds, hence exhibiting
bioactive more potential to be used as natural
antioxidant supplements.
the Application and uses of growth
mushroom substrate
As a natural byproduct of cellular
mechanisms, normal intracellular reactive
oxygen species (ROS) production has a
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi
beneficial role in synthesising cellular
structures. (Dröge, 2002). However, when
there is an overproduction and inefficient
removal of ROS by the antioxidant
defence system, oxidative stress occurs.
Antioxidant defence mechanisms are
essential in regulating ROS levels in cells,
donating an electron to a rampaging free
radical to reduce it, thus reducing its
capacity to damage (Lobo et al., 2010).
When endogenous antioxidant production
fails to efficiently remove ROS, exogenous
antioxidant supplements, such as those
found in mushroom substrate, may be
required to scavenge free radicals and
reduce oxidative stress. As such, an
investigation into natural antioxidant
supplements may help limit the levels of
ROS in the human body.
MATERIALS AND METHODS
Data Collection
G. sessile and G. lingzhi were grown from
mushroom grow kits (bewildersg.com)
with substrate derived from grain waste.
10 kits of each species were grown
simultaneously. Mushrooms were grown
under bright shade, misted 2-3 times a day,
with temperature kept around 24-28°C,
and relative humidity around 80-90%.
Fruit body was harvested and cut into
small pieces and freeze dried for at least
48h before being stored in a freezer.
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Proximal substrate and distal substrate
were harvested and stored in a freezer
without freeze-drying. Proximal substrate
is a mixture of surface mycelium and
substrate, while distal substrate is substrate
found at the core, containing fine particles
of mycelium (Figure 1). These
measurements were taken to determine the
effect of mycelia concentration on total
phenolic content (TPC), as proximal
substrate has a higher mycelia
concentration than distal substrate. TPC of
substrates were then taken to be the sum of
TPC of mycelia and TPC of non-
inoculated substrate. Non-inoculated
substrate was used as a negative control to
determine the changes in TPC that were
solely due to the inoculation of substrate.
Figure 1. Location of fruit body, proximal,
and distal substrate (mushrooms pictured
are not used in experimentation) (Islam,
2015)
Fruit body, surface mycelium, and core
substrate were ground in a blender. 0.1g of
sample was homogenised in
microcentrifuge tubes, with either 70%
 Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

ethanol or water, and left to rest for 15 75g/L solution of Na2CO3 was added.
minutes. Samples were then centrifuged, Absorbance values were measured using a
and the supernatant collected, before microplate reader to determine the TPC in
adding more 70% ethanol or water and each sample, measured in gallic acid
homogenising again. Three extractions equivalents (GAE). FC reagent, Na2CO3
were conducted in total (Figure 2). and gallic acid were analytical grade
reagents purchased from Sigma-Aldrich.
Folin-Ciocalteu Assay
The Folin-Ciocalteu (FC) assay was Statistical Analysis
conducted to determine the TPC in a OriginPro (Originlab Corporation, USA)
sample. Under alkaline conditions, was used for statistical analysis. The TPC
phenolic compounds in the sample results were analysed using repeated
dissociate to produce phenolate anions. measures 2-way ANOVA in which the
These anions then reduce the first factor is the type of sample, and the
phosphomolybdic-phosphotungstic acid second factor is the extraction solvent used
complexes in the FC reagent. As a result, to extract the phenolic compounds. The
blue molybdenum-tungsten complexes Bonferroni post-hoc test was conducted to
with maximum absorbance at 765 nm are determine which sample was statistically
formed (Everette et al., 2021). significantly different from each other. P-
0.2g/L solution of gallic acid was serially values of less than 0.05 indicate
diluted in DI water to prepare 0.1g/L, statistically significant differences.
0.05g/L, 0.025g/L, 0.0125g/L, and
0.00625g/L samples, along with 0g/L
sample prepared with DI water used for
standards. FC reagent was diluted 10x with
DI water. Using a 96-well transparent flat
well plate, 100µL of FC reagent was added
to 20µL of sample or standard, and
incubated for 3 minutes. Then, 80µL of

Figure 2. Preparation and extraction of samples

Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

RESULTS AND DISCUSSION mushrooms, where fruiting bodies

typically have higher concentrations of
G. sessile mushrooms were ready to
phenolic compounds, whereas mycelium
harvest after approximately 30 days.
found in substrates contains more of other
Mushrooms that were harvested had a
bioactives such as lovastatin (Berger et al.,
reddish-brown interior and a white exterior
2022).
fan-shaped cap with brown spores. Each
The results also indicate that the extraction
harvest yielded an average of 100g of fruit
solvent does not affect the TPC, with p >
bodies.
0.05 (Table 1). This was unexpected, as
G. lingzhi mushrooms were ready to
common phenolic compounds present in
harvest after approximately 45 days.
mushrooms include gallic acid,
Harvested mushrooms had a flat, reddish-
protocatechuic acid, and cinnamic acid
brown, kidney-shaped cap, with brown
(Chu et al., 2023), which are more soluble
spores. Each harvest yielded on average
in ethanol than in water. One possible
40g of fruit bodies. G. lingzhi mushrooms
explanation would be the presence of other
took longer than expected to be ready to
ethanol-soluble compounds such as
harvest. Potential reasons for this will be
lovastatin (Berger et al., 2022), also
discussed below.
prevalent in mushrooms (Kała et al.,
Results from the repeated measures 2-way
2020), which may decrease the phenolic
ANOVA indicated that the type of sample
compound extraction efficiency of ethanol,
strongly influenced the TPC with p < 0.05
mitigating the expected difference in
(Table 1), which was expected. This could
solubility between water and ethanol.
be explained by the tissue-specific
distribution of phenolic compounds in

Table 1. 2-way ANOVA results

Factor Sum of DF Mean F P-value
squares square
Type of sample 164.9 6 27.49 104.3 <0.001
Extraction 0.1450 1 0.1450 0.3377 0.5923
solvent
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi
*DF indicates degree of freedom and F is the F-statistic value
The Bonferroni post-hoc test was also
conducted to show significant differences
between each sample.
Results show that there is no significant
difference in the TPC between distal and
proximal substrates of both G. lingzhi and
G. sessile (Figure 3). This was not
predicted since proximal substrate has a
higher mycelium content, which should
indicate a higher TPC. There is also no
significant difference in the TPC between
the fruit and substrates of G. lingzhi
(Figure 3). TPC in fruit should have been
higher than TPC in substrates, much like
in G. sessile, thus this was also an
unexpected result. Both distal
proximal substrates of G. sessile, and the
distal substrate of G. lingzhi
significantly lower TPC than the non-
inoculated blank substrate. The proximal
substrate of G. lingzhi had no significant
difference in TPC with the non-inoculated
substrate (Figure 3). The inoculation of
substrate with fungi should have increased
the TPC, thus this result was also
unexpected.
One possible explanation is that some
fungi have been shown to be able to
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degrade phenolic compounds (Khalil et al.,
2021). This can cause TPC in all parts of
the fungi to be similarly low, leading to no
significant differences in TPC between
proximal and distal substrates. This would
also explain the lower TPC in inoculated
substrate, than in the non-inoculated
substrate. However, there is still the
possibility that the content of other
bioactive compounds derived from
mycelium have increased. A more detailed
investigation should be done on specific
bioactive compounds such as
ergothioneine to better elucidate the
antioxidant potential of mycelium in
mushroom substrates.
and
We also found that the fruits of G. sessile
had had a significantly higher TPC than its
substrates. This was the expected result, as
previous research had determined that
fungi in the genus Ganoderma had high
TPC (Kim et al., 2008). This could be due
to the antioxidant, antimicrobial, and anti-
inflammatory properties of phenolic
compounds (Abdelshafy et al., 2021), thus
they may be concentrated in the fruit for
protection against predation.
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

Figure 3. Mean total phenolic content (mg GAE/g) of different samples with Bonferroni post-
hoc test conducted. Means that do not share the same letter are significantly different (p <
0.05). Error bars are standard deviation. Orange and green bars indicate extraction with
water and ethanol. Abbreviations: GS = G. sessile; GL = G. lingzhi; Sub D = distal
substrate; Sub P = proximal substrate; Blank Sub = non-inoculated substrate used as
negative control.
Inconsistencies in the results could likely results, other electron-transfer based
be due to the limitations of the Folin- assays such as the FRAP assay and the
Ciocalteu assay conducted. The FC reagent DPPH radical scavenging assay could be
is not specific to phenolic compounds, and conducted in conjunction with the FC
can be reduced by other substances present assay, as they are more selective and can
in mushrooms such as reducing sugars give a better indication of the
and ascorbic acid (Ainsworth & Gillespie, concentration of potentially oxidizable
2007). The TPC quantified by the assay is polyphenols than the FC assay
hence likely to be higher than the actual (Danilewicz, 2015). There is also benefit
TPC present. To increase the accuracy of in using a hydrogen-transfer based assay
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi
such as ORAC (Munteanu & Apetrei,
2021), allowing for more accurate and
precise measurements of TPC.
Another potential source of error is the
growth conditions of the mushroom. The
mushrooms were not grown in an
environment where these factors were
strictly controlled, and the temperature and
humidity were not recorded. While both
species of mushrooms were grown
indoors, factors such as temperature,
humidity and air flow could not be
completely controlled, with the
temperature and humidity sensor recording
fluctuations of temperature between 24-
28oC and 70-90% relative humidity.
Higher temperatures caused the mushroom
substrate to dry out, while lower
temperatures caused the substrate to
become too wet, leading to the growth of
mould and other microorganisms. This
would influence mushroom development,
and hence the TPC recorded.
CONCLUSION
Despite the initial hypothesis that the TPC
of spent mushroom substrate would equate
the sum of TPC of non-inoculated
substrate and TPC of mycelium, our
findings showed that the TPC of non-
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inoculated substrate was significantly less
than TPC of spent mushroom substrate
(Figure 2). This points to the possible
degradation of phenolic compounds in G.
sessile and G. lingzhi, as documented in
other species of fungi (Khalil et al., 2021).
Additionally, the TPC present in samples
may have reduced, but there is still the
possibility that other bioactives such as
ergothioneine derived from mycelium have
increased which can be looked into in
future research.
The study can be extended to determine
the effects of other variables on TPC such
as temperature and humidity of the
environment in which the mushrooms are
grown, to better mimic the industrial
farming of mushrooms, making it more
applicable to industrial mushroom farmers
and informing them about the feasibility of
repurposing mushroom substrate waste.
Different species of mushrooms and
different types of substrates such as
sawdust and manure can also be included
to ensure that our conclusions remain true
in these cases. Analysis of antioxidant
ability should also be enhanced by
measuring the concentration of specific
antioxidative compounds present, such as
ergothioneine.
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

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APPENDIX

Table 2. Descriptive statistics of different samples

Sample Mean Std. Error 95.00% LCL 95.00% UCL

GS Sub D 0.87466 0.02258 0.81198 0.93734

GS Sub P 1.32129 0.15072 0.90283 1.73976

GS Fruit 5.85847 0.32006 4.96986 6.74709

GL Sub D 1.90359 0.05393 1.75386 2.05333

GL Sub P 2.40961 0.1542 1.98147 2.83774

GL Fruit 1.72108 0.06869 1.53036 1.9118

Blank 2.98839 0.10984 2.68344 3.29335

Table 3. Bonferroni test for different samples

Sample Index Mean Std. DF |t|value Prob>|t| Alpha Sig 95.00% 95.00%
Difference Error Flag LCL UCL

GS Sub D GS 0 -0.44664 0.24005 24 1.86057 1 0.05 0 -1.26185 0.36858

Sub P

GS Sub D GS 1 -4.98382 0.24005 24 20.76129 1.60839E-15 0.05 1 -5.79903 -4.1686

Fruit

GS Sub D GL 2 -1.02894 0.24005 24 4.28628 0.00536 0.05 1 -1.84415 -0.21372

Sub D

GS Sub D GL 3 -1.53495 0.24005 24 6.3942 2.73667E-5 0.05 1 -2.35017 -0.71973

Sub P
Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

GS Sub D GL 4 -0.84642 0.24005 24 3.52596 0.03628 0.05 1 -1.66164 -0.0312

Fruit

GS Sub D 5 -2.11374 0.24005 24 8.80528 1.16494E-7 0.05 1 -2.92895 -1.29852

Blank

GS Sub P GS 6 -4.53718 0.24005 24 18.90072 1.35118E-14 0.05 1 -5.3524 -3.72196

Fruit

GS Sub P GL 7 -0.5823 0.24005 24 2.42571 0.48648 0.05 0 -1.39752 0.23292

Sub D

GS Sub P GL 8 -1.08831 0.24005 24 4.53362 0.00286 0.05 1 -1.90353 -0.27309

Sub P

GS Sub P GL 9 -0.39978 0.24005 24 1.66539 1 0.05 0 -1.215 0.41544

Fruit

GS Sub P 10 -1.6671 0.24005 24 6.94471 7.36637E-6 0.05 1 -2.48232 -0.85188

Blank

GS Fruit GL 11 3.95488 0.24005 24 16.47501 2.9081E-13 0.05 1 3.13966 4.7701

Sub D

GS Fruit GL 12 3.44887 0.24005 24 14.3671 5.80674E-12 0.05 1 2.63365 4.26409

Sub P

GS Fruit GL 13 4.1374 0.24005 24 17.23533 1.0681E-13 0.05 1 3.32218 4.95262

Fruit

GS Fruit 14 2.87008 0.24005 24 11.95601 2.83195E-10 0.05 1 2.05486 3.6853

Blank

GL Sub D GL 15 -0.50601 0.24005 24 2.10791 0.95904 0.05 0 -1.32123 0.30921

Sub P

GL Sub D GL 16 0.18252 0.24005 24 0.76032 1 0.05 0 -0.6327 0.99773

Fruit

GL Sub D 17 -1.0848 0.24005 24 4.519 0.00296 0.05 1 -1.90002 -0.26958

Blank

GL Sub P GL 18 0.68853 0.24005 24 2.86823 0.17784 0.05 0 -0.12669 1.50375

Fruit

GL Sub P 19 -0.57879 0.24005 24 2.41109 0.50235 0.05 0 -1.39401 0.23643

Blank

GL Fruit 20 -1.26732 0.24005 24 5.27932 4.30937E-4 0.05 1 -2.08254 -0.4521

Blank

Table 4. Descriptive statistics of different extraction solvents

Sample Mean Std. Error 95.00% LCL 95.00% UCL

Water 2.48509 0.1104 2.17856 2.79163

Ethanol 2.39408 0.05667 2.23673 2.55142

Oliver Alexander Wang, Ganesan Gabalini, Tan Shu Han Breanna BI009
Phenolic compounds in Growth Substrate of G. sessile and G. lingzhi

Table 5. Bonferroni test for different extraction solvents

Sample Index Mean Std. Error DF |t|value Prob>|t| Alpha Sig Flag 95.00% 95.00%
Difference LCL UCL

Water 0 0.09102 0.12831 4 0.70932 0.51728 0.05 0 -0.26524 0.44727

Ethanol

Type of sample Extraction method Mean Standard Deviation

GS Sub D Water 0.90051 0.1171

GS Sub D EtOH 0.8755 0.06534
GS Sub P Water 1.29834 0.47093
GS Sub P EtOH 1.34425 0.30914
GS Fruit Water 6.59493 2.18158
GS Fruit EtOH 5.64286 1.3754
GL Sub D Water 2.48009 0.7169
GL Sub D EtOH 2.04575 0.38909
GL Sub P Water 3.27343 0.7635
GL Sub P EtOH 2.48887 0.57398
GL Fruit Water 2.04547 0.22494
GL Fruit EtOH 1.6945 0.22335
Blank Sub Water 2.50724 0.46268
Blank Sub EtOH 3.46955 0.26725

Table 6. Descriptive statistics of different samples with different extraction solvents (used in
Figure 3)