Research Article
Received: 23 December 2014
Revised: 5 April 2015
Accepted article published: 30 April 2015
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7242
Potential use of Lentinus squarrosulus
mushroom as fermenting agent and source of
natural antioxidant additive in livestock feed
Noorlidah Abdullah,* Ching-Ching Lau and Siti Marjiana Ismail
Abstract
BACKGROUND: Fermenting feed has gained a lot of popularity in recent years owing to its renowned benefits to the livestock
and feed quality. In the current study, Lentinus squarrosulus mushroom mycelium was tested for its potential as a fermenting
agent and source of natural antioxidant in the feed.
RESULTS: Phenolic content of methanolic and hot water extracts of the mycelium culture and its fermented maize ranged from
94.01 to 386.59 mg gallic acid equivalents g−1 extract while the DPPH radical scavenging, CUPRAC, reducing power (RPA) and
𝜷-carotene bleaching (BCB) antioxidant activity had EC50 values ranged from 15.30 to 120.3, 0.74 to 4.71, 1.86 to 13.5 and 0.01 to
0.21 mg mL−1 , respectively. At 1.0–20.0 mg mL−1 , the extracts retarded 21.02–55.35% of lipid deterioration. Pearson correlation
analysis revealed the phenolic content of the extracts has moderate correlation with DPPH (r = 0.589) and RPA (r = 0.580), also
a high correlation with BCB antioxidant activity (r = 0.872). In principal component analysis, DPPH, CUPRAC and RPA were seen
to be clustered tightly together while BCB antioxidant activity was grouped with the phenolic content.
CONCLUSION: Overall, L. squarrosulus mycelium functioned as antioxidants via several mechanisms, involving either electron
transfer or hydrogen transfer-based reactions suggesting it as a promising fermentation agent to enhance feed nutrition and
the fermented maize as a valuable feed resource.
© 2015 Society of Chemical Industry
Keywords: mushrooms; mycelium; submerged fermentation; solid substrate fermentation
INTRODUCTION
Rancidity is one of the major issues involving feed quality. Prolonged exposure to air, heat, sunlight and poor storage conditions
are among the factors promoting oxidative rancidity in the feed.
The drawbacks of long-term supply of deteriorated feed to animals
are documented in several studies, which include low growth performance, weak immune system, increase mortality rate and producing animal-based products with lower nutritional quality.1 – 3
Moreover, oxidised feeds are considered to be toxic, hence this has
provoked fears about the safety of food products derived from the
affected animals.
Antioxidant supplementation is found as a practical solution to
overcome this circumstance.4 Furthermore, antioxidant properties
in the feed are perceived to be deposited in the muscle and cellular membranes upon consumption leading to the production of
meat, milk and eggs with improved nutritional quality and oxidative stability.3 Besides, inclusion of antioxidants directly in the animal’s diet was found to provide better meat quality compared to
post-mortem application in processed meat.5 Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) are widely used
as antioxidant supplements but their potential carcinogenic effect
is a health concern.6 Fermentation is an efficient and natural way
that could augment antioxidative properties in the final product.7
Fermenting feed has gained a lot of popularity in recent years
owing to the benefits of which includes an improvement of
J Sci Food Agric (2015)
gut health8 and reduced pathogen infection in the animals,9 as
well as increased feed digestibility resulting in enhanced animal
feed intake and growth performance.10 Additionally, the performance of animals fed with fermented diets has characteristics
similar to that of animals inoculated with antibiotic growth promoters. This makes it a good alternative to the use of antibiotics, which have fallen under scrutiny due to the potential
risk of the emergence of drug-resistant bacteria over long-term
administration.10
Bacteria and yeast are among the most common inoculants
used in feed.7,10 Recently, mushroom mycelium has come into
the limelight as a potential fermenting agent from natural origin.
Examples of reported studies include fermentation of straw and
wheat by Trametes versicolor11 and Pleurotus ostreatus,12 respectively. Lentinus squarrosulus used in the current study is a wild
edible mushroom found in many parts of Asia, and contains high
nutrients and medicinal properties.13,14 Moreover, its fermented
product was found to produce a pleasant aroma which is liked by
animals.15
∗
Correspondence to: Noorlidah Abdullah, Mushroom Research Centre, Institute
of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia. E-mail: [email protected]
Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia
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© 2015 Society of Chemical Industry
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Many biochemical changes may occur during the fermentation
process which affects the bioactivity of the final product.16 The biological analysis of fermented feed in terms of microbial properties
and nutritional values has been widely discussed.12,17 The high levels of lactic acid bacteria with low pH and Enterobacteria counts
are deemed as the main factors responsible for the enhanced fermented feed quality.8 Nevertheless, antioxidant could also play a
role in protecting the feed against spoilage and improving the animal’s performance. However, to the best of our knowledge, there is
no report on the antioxidant potential of L. squarrosulus mycelium
and its fermented product tested based on several mechanisms of
action. Therefore, the objective of this work was to evaluate the
antioxidant efficacy of L. squarrosulus mycelium and the potential
of its fermented product (maize as a model) as dietary antioxidant
in livestock feed.
MATERIALS AND METHODS
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide,
trichloroacetic acid (TCA), ferric chloride, ferrous sulfate,
𝛽-carotene, linoleic acid, neocuproine, copper(II) chloride dihydrate, gallic acid and BHA were purchased from Sigma–Aldrich
(St. Louis, MO, USA). Folin–Ciocalteu phenol reagent, ammonium
acetate, sodium phosphate monobasic and sodium phosphate
dibasic were purchased from Merck (Darmstadt, Germany).
Thiobarbituric acid was obtained from AppliChem (Darmstadt,
Germany). All chemicals and reagents were of analytical grade.
Preparation of Lentinus squarrosulus inoculum
L. squarrosulus culture was authenticated by morphological and
molecular methods by experts in the Mushroom Research Centre,
University of Malaya, Malaysia. The mycelium was cultured on
GYMP agar consisting of glucose (1.5%), yeast extract (0.8%), malt
extract (0.8%) and peptone (0.8%) (w/v). The culture was deposited
in the Mushroom Research Centre culture collection, University of
Malaya, and assigned a culture code (KUM 50016).
Sample preparation of methanol and hot water extract from
Lentinus squarrosulus mycelium broth
L. squarrosulus mycelium broth (LSMB) was acquired by submerged fermentation. Ten Erlenmeyer flasks (250 mL) containing
50 mL of GYMP liquid media were autoclaved at 121 ∘ C for 15 min
followed by cooling to 25 ∘ C. Each flask was inoculated with
five 9-mm diameter mycelia plugs cut from the periphery of a
7-day-old colony, incubated at 25 ∘ C for 14 days, and the mycelial
broth lyophilised. Methanol extract (designated as LSMB-M) was
obtained by soaking the lyophilised sample in methanol for 2
days, the filtrates was concentrated by rotary evaporation and
stored at −20 ∘ C. Hot water extraction (designated as LSMB-HW)
was carried out by boiling the LSMB powder with distilled water
for 30 min, the filtrates collected was lyophilised and stored at
−20 ∘ C until analysis.
Sample preparation of methanol and hot water extract from
maize fermented with Lentinus squarrosulus mycelium
L. squarrosulus fermented maize (LSFM) was acquired by solid
substrate fermentation. Ten Erlenmeyer flasks (250 mL) containing maize–peptone–calcium carbonate at a ratio of 97:1:2 (w/w)
were autoclaved at 121 ∘ C for 15 min and cooled to room temperature. Each flask was inoculated with five 9-mm diameter mycelial
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N Abdullah, C-C Lau, S M Ismail
plugs cut from the periphery of a 7-day-old colony. After 14 days
incubation at 25 ∘ C, the grains and mycelia were crushed and
lyophilised. Hot water and methanol extraction was carried out following the method as described in the section above. The extracts
obtained were designated as ‘L. squarrosulus fermented maize
methanolic’ (LSFM-M) and ‘L. squarrosulus fermented maize hot
water’ (LSFM-HW), respectively.
DPPH radical scavenging assay
Scavenging effect of the test samples against DPPH radicals were
measured according to the method of Daker et al.18 Test solution
(0.1 mL) was added to 3.9 mL of a 0.06 mmol L−1 DPPH–methanol
solution, and the absorbance at 517 nm was recorded at 0, 1, 2
and every 15 min thereafter until the reaction reached a plateau
(against a methanol blank). BHA was used as the positive control.
The scavenging activity of DPPH was calculated as: % radical scavenging activity = [1 − (As /Ao )] × 100, where As is the absorbance of
the reaction mixture and Ao is the absorbance of 0.06 mmol L−1
methanolic DPPH. The EC50 (mg mL−1 ) value was the effective concentration at which 50% of the DPPH radicals were scavenged and
was obtained by interpolation from the linear regression analysis.
Cupric ion reducing antioxidant capacity (CUPRAC)
CUPRAC was determined according to the method of Apak et al.19
Test samples (1 mL) were added to a mixture containing 1 mL each
10 mmol L−1 copper(II), 7.5 mmol L−1 neocuproine and 1 mol L−1
ammonium acetate buffer (pH 7.0). The mixture was reacted at
room temperature for 30 min and absorbance at 450 nm recorded
against a reagent blank. BHA was used as the positive control.
The EC50 (mg mL−1 ) value was the effective concentration at which
the absorbance was 0.5 for cupric ion reducing capacity and was
obtained by interpolation from the linear regression analysis.
Reducing power activity (RPA)
The RPA of test samples was determined as described by
Abdullah et al.,20 where test samples (1 mL) were mixed with
2.5 mL 0.2 mol L−1 phosphate buffer (pH 6.6) and 2.5 mL potassium ferricyanide (1%), incubated at 50 ∘ C for 20 min. Then, 2.5 mL
of TCA (10%) was added and the mixture centrifuged at 1000 × g
for 10 min. Supernatant (2.5 mL) was mixed with 0.5 mL ferric
chloride (0.1%) and absorbance measured at 700 nm. BHA was
used as the positive control. The EC50 (mg mL−1 ) value was the
effective concentration at which the absorbance was 0.5 for
reducing power and was obtained by interpolation from the linear
regression analysis.
𝜷-Carotene bleaching (BCB) antioxidant assay
The procedure was carried out as described by Abdullah et al.20
𝛽-Carotene (2 mg) was dissolved in 10 mL chloroform and mixed
with 40 μL linoleic acid and 400 μL Tween 80. One millilitre was
pipetted into a 100 mL round-bottom flask, the chloroform evaporated under vacuum and 100 mL distilled water added with
vigorous shaking. Aliquots of this emulsion (4.8 mL) were transferred into tubes containing test samples (0.2 mL) and the zero
time absorbance measured immediately at 470 nm. The tubes
were incubated at 50 ∘ C for 2 h and another reading taken. Distilled water was used as the control and a blank (no 𝛽-carotene)
was prepared for background subtraction. Degradation rate (DR)
was calculated as: DR = ln(At=0 /At=120 ) × 1/120, where At=0 is the
absorbance of the reaction mixture at time 0 min and At=120 is the
© 2015 Society of Chemical Industry
J Sci Food Agric (2015)
L. squarrosulus as a fermenting agent and antioxidant additive in feed
Anti-lipid peroxidation (ALP) effect
The ability of test samples to inhibit lipid peroxidation was determined according to the method of Daker et al.18 The reaction mixture contained 1 mL of egg yolk emulsified with 0.1 mol L−1 phosphate buffer, pH 7.4 (final concentration of 25 g L−1 ) and 100 μL of
1 mmol L−1 Fe2+ . After the addition of test sample, the mixture was
incubated at 37 ∘ C for 1 h. Then, 0.5 mL of freshly prepared 15%
trichloroacetic acid and 1.0 mL 1% thiobarbituric acid were added
to the mixture. The reaction tubes were further incubated in a boiling water bath for 10 min, cooled to room temperature and centrifuged at 3500 × g for 10 min to remove precipitated proteins.
The formation of thiobarbituric acid reactive substances (TBARS)
in 100 μL of supernatant was measured at 532 nm against the
buffered egg yolk/Fe2+ (control). The % inhibition was calculated
by: % inhibition = (1 − As /Ao ) × 100, where As is the absorbance of
the sample and Ao is the absorbance of the control.
Estimation of total phenolic content (TPC)
The TPC of the test samples was estimated by the Folin–Ciocalteu
colorimetric assay as described by Abdullah et al.20 Briefly, 250 μL
sample was added to 250 μL 10% Folin–Ciocalteu phenolic
reagent and incubated at room temperature for 3 min. The mixture was incubated for another hour after the addition of 500 μL
10% sodium carbonate solution. The absorbance of the mixture was read at 750 nm and the phenol content determined
by comparing the absorbance values of samples to a gallic acid
standard curve. The concentration of total phenols was expressed
in mg gallic acid equivalent per gram of test sample (mg GAE g−1
extract).
Statistical analysis
Antioxidant analyses and estimation of TPC were carried out in
triplicate (n = 3) and the results reported as mean ± standard deviation. The Pearson correlation test was used to calculate the correlations between the five antioxidant assays and TPC of LSMB and
LSFM extracts. Differences of P < 0.05 were considered statistically
significant and P < 0.01 was taken to be statistically highly significant. Principal component analysis (PCA) was employed to study
clusters of the antioxidant assays and TPC. The data analysed by
PCA was displayed as bi-plot. All the data were analysed using SPSS
22.0 package (SPSS Inc., Chicago, IL, USA).
EC50 (mg mL–1)
LSMB-M
: 15.30 ± 0.97
LSMB-HW : 18.23 ± 0.66
: 45.78 ± 1.53
LSFM-M
LSFM-HW : 120.3 ± 2.11
BHA
: 0.11 ± 0.01
100
% radical scavenging activity
absorbance of the reaction mixture after 120 min. The antioxidant
activity was calculated using the following formula: antioxidant
activity = [(DRcontrol − DRsample )/DRcontrol ] × 100.
The EC50 (mg mL−1 ) value was the effective concentration at
which 50% of the 𝛽-carotene oxidation was inhibited.
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90
80
70
60
50
40
30
20
10
0
0
10
LSMB-M
20
30
40
Concentration (mg ml–1)
LSMB-HW
LSFM-M
50
LSFM-HW
Figure 1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability
of methanolic (M) and hot water extracts (HW) of L. squarrosulus mycelium
broth (LSMB) and L. squarrosulus fermented maize (LSFM). Data expressed
as mean ± standard deviation (n = 3). The EC50 (mg mL−1 ) value was the
effective concentration at which 50% of the DPPH radicals were scavenged.
Butylated hydroxyanisole (BHA) was used as a positive control.
DPPH radical scavenging assay
The DPPH radical scavenging assay measured the neutralising ability of extracts toward DPPH free radicals by transferring an electron
to DPPH where decolorisation of the solution was inversely correlated with the radical scavenging activity of the extracts. The result
obtained in Fig. 1 showed the methanolic extract has exhibited a
higher DPPH antiradical activity compared to its water extract. This
is in agreement with the finding that the DPPH reagent has a high
affinity toward lipophilic antioxidants as opposed to hydrophilic
ones.22
At 10–50 mg mL−1 , the scavenging abilities of LSMB-M and
LSMB-HW were 34.4–92.1% and 23.6–92.3%, respectively, with
EC50 values of 15.30 and 18.23 mg mL−1 , respectively. When the L.
squarrosulus was used as an inoculant in feedstuff, the fermented
maize produced has lower scavenging ability. At 10–50 mg mL−1 ,
LSFM-M and LSFM-HW scavenged the DPPH radical at 9.9–53.4%
and 1.0–19.3%, respectively, with EC50 values of 45.78 and
120.3 mg mL−1 , respectively. A study by Miyamoto and co-workers
showed oral administration of fermented feed increased DPPH
antiradical activity in the animal products, although the feed itself
had little of its activity.23 Our previous investigation revealed L.
squarrosulus fermented maize has 1.5 times better DPPH antiradical effect than its unfermented counterpart.24 Hence, the
true effect of administering L. squarrosulus fermented feed to the
production of animal products with antiradical activity warrants
further investigation.
RESULTS AND DISCUSSION
Evaluation of the antioxidant potentials in the extracts
Antioxidant compounds may act in vivo through different mechanisms which include termination of chain initiation by radical
scavenging, chelating of metal ions and elimination of peroxides.21
As such, a single antioxidant assay was inadequate to provide an
overview of the total antioxidant capacity of the samples. In the
present study, antioxidant activities of LSMB and LSFM (extracted
with methanol and hot water) were evaluated for DPPH radical scavenging activity, CUPRAC, RPA, BCB antioxidant and ALP
activity.
J Sci Food Agric (2015)
CUPRAC and RPA assays
The CUPRAC and RPA assay represented the capability of antioxidants to reduce cupric [Cu(II)] and ferric [Fe(III)] ions to cuprous
[Cu(I)] and ferrous [Fe(II)] ions, respectively. The amount of reduced
ions formed is monitored spectrophotometrically, where a higher
absorbance value indicated higher antioxidant activity.
Presently, CUPRAC assay is not commonly used among
researchers working on antioxidant studies, and little has been
published on the evaluation of cupric ion-reducing ability of
mushroom mycelia. However, this antioxidant assay claimed to
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Absrobance (450 nm)
A 1.4
1
0.8
0.6
0.4
0.2
0
0
0.5
LSMB-M
B 0.45
1
1.5
2
Concentration (mg mL–1)
LSMB-HW LSFM-M LSFM-HW
EC50 (mg mL–1)
0.4
Absorbance (700 nm)
than LSMB-HW while LSFM-M exhibited a two-fold higher RPA
than LSMB-M and LSFM-HW had a three-fold lower activity than
LSMB-HW. This may suggest that most of the organic soluble
antioxidants which are active cupric and ferric ion reductor in L.
squarrosulus mycelium remained active following the solid state
fermentation process as opposed to its water soluble counterpart.
There might be some structural modifications of the components
in maize due to enzymes liberated by L. squarrosulus mycelium
during fermentation that resulted in the variations in the reducing
capacity of the cupric and ferric ions. Numerous reports have
been published on the transformation of antioxidant properties and phytochemical composition in fermented solid based
products.16,31
Copper ions are chemically more reactive than iron ions, resulting in faster kinetics in redox reactions.19 The LSFM in the present
study was high in both CUPRAC and RPA, suggesting its high possibility to counteract the action of oxidants, hence has prolonged
shelf life. This criterion is important as feed tends to be exposed to
poor storage conditions that promote oxidation.
EC50 (mg mL–1)
LSMB-M : 0.85± 0.01
LSMB-HW : 0.74± 0.02
LSFM-M
: 1.07± 0.02
LSFM-HW : 4.71± 0.07
BHA
: 0.02± 0.00
1.2
LSMB-M
LSMB-HW
LSFM-M
LSFM-HW
BHA
0.35
0.3
0.25
N Abdullah, C-C Lau, S M Ismail
: 3.29± 0.08
: 4.45± 0.01
: 1.86± 0.01
: 13.5± 0.51
: 0.07± 0.01
0.2
0.15
0.1
0.05
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Concentration (mg mL–1)
LSMB-M
LSMB-HW
LSFM-M
LSFM-HW
Figure 2. (A) Cupric ion reducing antioxidant capacity and (B) reducing
power activity of methanolic (M) and hot water extracts (HW) of L. squarrosulus mycelium broth (LSMB) and L. squarrosulus fermented maize (LSFM).
Data expressed as mean ± standard deviation (n = 3). The EC50 (mg mL−1 )
value was the effective concentration at which the absorbance was 0.5.
Butylated hydroxyanisole (BHA) was used as a positive control.
offer many advantages over other electron-transfer based methods which include: the working pH of the method is close to
physiological (i.e. pH 7.0), it can be used with hydrophilic and
lipophilic antioxidants, the selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly found
in foodstuffs and the capacity to assay SH-bearing antioxidants.19
In the current study, the absorbance values recorded by LSMB-M
and LSMB-HW were 0.04–1.09 and 0.09–1.30, respectively, when
tested at 0.1–2.0 mg mL−1 while its simulation as animal feedstuff
(LSFM-M and LSFM-HW) had absorbance values of 0.07–0.91
and 0.03–0.20, respectively (Fig. 2A). This was comparable to the
values of hot water extracts from 14 culinary mushroom species
which had absorbances ranging from 0.137 to 1.058.20
As shown in Fig. 2B, the RPA of LSMB-M and LSMB-HW has
EC50 of 3.29 and 4.45, respectively. This was comparable with
EC50 values of methanolic (6.89 mg mL−1 ) and hot water extract
(3.97 mg mL−1 ) from Agrocybe cylindracea mycelium.25,26 A higher
RPA was recorded by Ganoderma tsugae and Armillaria mellea
mycelia with EC50 values lower than 1.00 mg mL−1 .27 – 29 LSFM-M
has a substantially high RPA where at 0.1–1.5 mg mL−1 ; it had
absorbances of 0.033–0.406 and an EC50 of 1.86 mg mL−1 . This
value is higher compared to yeast fermented soybean curd
and bacteria fermented peanut meal hydrolysate (both had
EC50 > 2.0 mg mL−1 ).7,30 Compared to LSFM-M, its water extract
counterpart has lower RPA with absorbances of 0.002–0.06.
Overall, LSFM-M exhibited only a slightly lower CUPRAC activity
than LSMB-M but LSFM-HW showed a four-fold lower activity
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BCB antioxidant assay
𝛽-Carotene is extremely susceptible to free-radical mediated oxidation. The BCB antioxidant method is based on the discoloration
of oxidised 𝛽-carotene due to the attack of linoleate free radicals
in the system. Samples without antioxidants had a rapid decrease
in absorbance, while in the presence of antioxidant, the colour was
retained for a longer time.21 The antioxidant activity was expressed
as % inhibition of discoloration relative to the control.
Referring to the result in Fig. 3, 𝛽-carotene in the system underwent rapid discoloration in the presence of low concentrations
of extracts, while at a higher concentration (∼0.10 mg mL−1 ),
the 𝛽-carotene colour was retained after the 2 h of incubation.
LSMB extracts showed high protective effect to hinder the
extent of 𝛽-carotene bleaching, where LSMB-M and LSMB-HW at
0.01 mg mL−1 showed 37.6% and 49.03% BCB antioxidant activity,
respectively, and at 0.5 mg mL−1 , the % inhibition increased to
91.61% and 89.85%, respectively. The extracts had EC50 values
of 0.03 and 0.01 mg mL−1 , respectively. Its fermented product
showed a slightly lower activity where the % inhibition of LSFM-M
and LSFM-HW increased from 17.31% to 60.95% and 19.67%
to 56.16%, respectively with EC50 values recorded at 0.21 and
0.19 mg mL−1 , respectively. Both LSMB and LSFM extracts in the
present study had a higher BCB antioxidant activity compared to
extracts of 24 mushroom species mycelia reported by Asatiani
and colleagues where most of the investigated species have
EC50 > 2.0 mg mL−1 .32
The presence of antioxidants involved in inhibiting the oxidation
of 𝛽-carotene can reduce the extent of 𝛽-carotene destruction in
feed ingredients, hence ensuring a sufficient supply of the compound to the animals. In addition to its role as a vitamin A precursor, 𝛽-carotene is important to the animal’s health by enhancing
their immunity and increases their reproductive efficiency.33 This
result is consistent with a report concerning the study on tomato.
It was found that the loss of BCB antioxidant and DPPH antiradical capacity in the fruit due to the skin and seed removal were
also accompanied by a significant decrease in their 𝛽-carotene
content.34
ALP effect
This method is based on the ability of the antioxidants to inhibit
ferrous-induced peroxidation of egg yolk phospholipids, with
© 2015 Society of Chemical Industry
J Sci Food Agric (2015)
100
% Antioxidant activity
90
80
70
60
50
EC50 (mg mL–1)
40
LSMB-M
LSMB-HW
LSFM-M
LSFM-HW
BHA
30
20
10
0
0
0.1
0.2
0.3
: 0.03 ± 0.02
: 0.01 ± 0.00
: 0.21 ± 0.01
: 0.19 ± 0.05
: 0.79 x 10–3± 0.01
0.4
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Total phenolic content (mg GAE g–1 extract)
L. squarrosulus as a fermenting agent and antioxidant additive in feed
450
TPC of BHA = 630.04 ± 21.78 mg GAE g–1 extract
400
350
300
250
200
150
100
50
0
0.5
LSMB
Methanol (M)
LSFM
Hot Water (HW)
Concentration (mg mL–1)
LSMB-M
LSMB-HW
LSFM-M
LSFM-HW
Figure 3. Inhibition (%) of 𝛽-carotene bleaching (BCB) activity of methanolic (M) and hot water extracts (HW) of L. squarrosulus mycelium broth
(LSMB) and L. squarrosulus fermented maize (LSFM). Data expressed as
mean ± standard deviation (n = 3). The EC50 (mg mL−1 ) value was the effective concentration at which 50% of BCB activity was inhibited. Butylated
hydroxyanisole (BHA) was used as a positive control.
60
% inhibition by 10 mg mL–1 BHA = 77.92 ± 2.02
% Inhibition
50
40
30
20
10
0
1.0
5.0
8.0
10.0
15.0
20.0
Concentration (mg mL–1)
LSMB-M
LSMB-HW
LSFM-M
LSFM-HW
Figure 4. Anti-lipid peroxidation abilities of methanolic (M) and hot water
extracts (HW) of L. squarrosulus mycelium broth (LSMB) and L. squarrosulus
fermented maize (LSFM). Data expressed as mean ± standard deviation
(n = 3). Butylated hydroxyanisole (BHA) was used as a positive control.
the production of malondialdehyde as the hallmark of the peroxidation process. At 1.0–20.0 mg mL−1 , LSMB-M and LSMB-HW
have retarded 21.02–43.86% and 27.19–48.46% of lipid deterioration, respectively (Fig. 4). The fermented maize has shown
better activity with 23.36–33.42% and 48.49–55.35% inhibited by
LSFM-M and LSFM-HW, respectively. Furthermore, L. squarrosulus
fermented maize was documented to have 1.6 times better ALP
ability than those unfermented counterpart.24 Apparently, the
biochemical changes involved during maize fermentation by
L. squarrosulus mycelium had increased the ALP activity in the
final product. In a study on maize fermented by Marasmiellus sp.,
the methanolic extract shows 50% inhibition at 6.00 mg mL−1 ,18
while hot water extracts of culinary mushroom fruiting bodies
inhibited peroxidation 33.33–57.18% at 10 mg mL−1 .20 These data
signified that the LSMB and LSFM have potent lipid peroxidation
suppression activity.
Lipid peroxidation has been recognised as a predominant cause
of quality deterioration in lipid-rich products. The potent ALP
ability of LSFM makes it a good choice of feedstuff. Not only does
it attenuate rancidity in feed and ensures a healthy feed supply,
J Sci Food Agric (2015)
Figure 5. Total phenolic content (TPC) of methanolic and hot water
extracts of L. squarrosulus mycelium broth (LSMB) and L. squarrosulus fermented maize (LSFM). TPC values are designated as mg gallic
acid equivalents in 1 g extract (mg GAE g−1 extract) and expressed as
mean ± standard deviation (n = 3). Butylated hydroxyanisole (BHA) was
used as a positive control.
but the deposition of its ALP compound in the muscle and cellular
membranes upon consumption can increase oxidative and colour
stability of animal products, hence extending the retail display life.
Estimation of TPC
The Folin–Ciocalteu method was used to estimate the TPC of
LSMB and LSFM extracts based on the reducing power of phenolic
hydroxyl groups.21 Referring to the result in Fig. 5, LSMB-M and
LSMB-HW had a TPC of 163.42 and 386.59 mg GAE g−1 extract,
respectively, while LSFM-M and LSFM-HW contained 122.63 and
94.01 mg GAE g−1 extract, respectively. These were substantially
higher than the amount reported for other mushrooms mycelia.
Hot water extracts of G. tsugae, A. cylindracea and A. mellea
mycelial cultures contain 41.03, 27.28 and 30.90 mg GAE g−1
extract, respectively, and methanolic extracts have 35.60, 15.55
and 27.10 mg GAE g−1 extract, respectively,25 – 29 while methanolic and hot water extracts of corncob fermented by G. sinensis
contained 24.0 and 35.3 mg GAE g−1 extract, respectively.35 Our
previous investigation has revealed L. squarrosulus fermentation
increases the TPC in maize by 1.3 times,24 suggesting that it has
enormous potential to enhance the nutritional and antioxidant
properties of the feed.
Inter-relationship of antioxidant activities and TPC in the
LSMB and LSFM extracts
Phenolic compounds have been frequently reported as bioactive
components associated with antioxidant properties. Regression
analysis was performed for the correlations among antioxidant
assays and TPC in the extracts from LSMB and LSFM (Table 1).
Apparently, the result of TPC in the current study has moderate correlation with DPPH (r = 0.589) and RPA (r = 0.580), also a
high correlation with BCB antioxidant activity (r = 0.872). This is
in agreement with previous studies which indicated that phenolic substances generally has a better correlation with DPPH
antiradical activity, ferric reducing antioxidant assay36 and BCB
antioxidant activity.37 On the other hand, there was no significant
correlation between TPC and their ability to reduce cupric ions. It
has been suggested that not all of the phenolic compounds are
active cupric ion reductor.38 Therefore, the type of phenolic compounds present in the extract is more influential to the CUPRAC
activity than their total content. It is noteworthy that there are
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Table 1. Correlation coefficients (r) between TPC and antioxidant
potential of extracts from Lentinus squarrosulus mycelial broth and L.
squarrosulus fermented maize
Parameter
TPC
ALP
BCB
RPA
CUPRAC
DPPH
CUPRAC
RPA
BCB
ALP
0.589*
0.526
0.580*
0.872**
0.147
0.067
0.246
0.253
0.306
–
0.654*
0.513
0.556
–
–
0.980**
0.997**
–
–
–
0.977**
–
–
–
–
** Correlation is significant at P < 0.01.
* Correlation is significant at P < 0.05.
DPPH, 2,2-Diphenyl-1-picrylhydrazyl radical scavenging; CUPRAC,
cupric ion reducing antioxidant capacity; RPA, reducing power activity;
BCB, 𝛽-carotene bleaching antioxidant activity; ALP, anti-lipid peroxidation antioxidant assay; TPC, total phenolic content.
significant (P < 0.01) correlations between DPPH-RPA (r = 0.980)
and DPPH-CUPRAC (r = 0.977) methods used to determine the
antioxidant capacity of the studied LSMB and LSFM samples.
Similar correlation coefficients (r = 0.8199–0.9999) between the
three antioxidant assays were found in the rapeseed samples.38
The results of the LSMB and LSFM extracts were subjected to PCA
using SPSS 22.0 package. Principal component analysis revealed
the presence of two components with eigenvalues exceeding 1,
which explained a total of 87.39% of the variance, with Component
1 contributing 65.59% and Component 2 contributing 21.78%
(Fig. 6). Oblimin rotation was performed to aid in the interpretation of these two components. The result obtained was consistent
N Abdullah, C-C Lau, S M Ismail
with the Pearson correlation test in Table 1. DPPH, CUPRAC and RPA
were seen being clustered tightly together while BCB antioxidant
activity was being grouped with TPC. Based on reaction mechanisms involved, antioxidant capacity assays can be divided into
two major groups, i.e. those involving electron transfer reactions
and others based on hydrogen transfer reactions.21 Hence, this
may explain the phenomenon of classification illustrated in Fig. 6
as DPPH, CUPRAC and RPA antioxidant assays are electron transfer
based method while BCB antioxidant activity and ALP are hydrogen atom transfer based method. TPC utilises both electron transfer and hydrogen atom transfer based reaction mechanism. The
close proximity of TPC with BCB antioxidant activity in the PCA
bi-plot suggests TPC from LSMB and LSFM may act predominantly
on hydrogen transfer based method.
Referring to the Pearson regression and PCA analysis, ALP assay
was seen to have low association with the other antioxidant capacity assays. However, they may provide synergistic effect on the
overall ALP activity of the feed. For instance, it has been well recognised that transition metal ion such as iron is an important catalyst
for the generation of free radicals that initiate lipid peroxidation
process. Hence, effective anti-radical agents and antioxidants with
reducing capability to reduce the oxidants may afford protection
against the generation of reactive oxygen species, thereby inhibiting peroxidation in the feed.
Several studies have documented a positive correlation between
culture conditions and the antioxidant properties of mushroom
mycelia,39,40 and a comprehensive overview of the production
of phenolic compounds using different solid substrate fermentation conditions is available.31 Geese fed with fermented feed
incubated at different time duration have shown variations on
Figure 6. Principal component analysis (PCA) loading plot of total phenolic content (TPC) and antioxidant potential of extracts from L. squarrosulus
mycelium broth and its fermented maize. Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging; CUPRAC, cupric ion reducing antioxidant
capacity; RPA, reducing power activity; BCB, 𝛽-carotene bleaching antioxidant activity; ALP, anti-lipid peroxidation antioxidant assay.
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© 2015 Society of Chemical Industry
J Sci Food Agric (2015)
L. squarrosulus as a fermenting agent and antioxidant additive in feed
the growth performance and oxidative stability of the organs.41
Therefore, the antioxidant properties of maize fermented by L.
squarrosulus mycelium may be further improved by optimising
culture conditions for carbon and nitrogen source, temperature,
moisture content, pH, aeration and growth time.
CONCLUSION
Although the positive control (BHA) had significantly higher activity than LSMB and LSFM extracts in all the antioxidant assays, it
can be used as a feed additive at milligram levels only due to its
carcinogenic potential in high doses, while mushroom mycelia is a
natural product that could be consumed at gram levels as a feed.
The total antioxidant capacity of feed may rely not only on the
effect of the most potent antioxidants, but rather on the combined
synergy action of all the antioxidants present in the diet. Besides,
there is a growing consensus among researchers that using a combination of antioxidants may be more effective than single compounds on a long-term basis. The ability of LSMB and LSFM extracts
in the current study to function as antioxidants via several mechanisms makes it a good source of antioxidant compounds. The
present work had proven LSMB as a valuable fermentation agent,
hence it can be commercialised as a starting culture for feed fermentation. Future work can be performed in vivo to verify the true
effect of LSFM in the biological system before applying them to
commercial animal feed and feed ingredient.
ACKNOWLEDGEMENTS
The authors are grateful to the Mushroom Research Centre, University of Malaya, for the facilities, and the High Impact Research MoE
Grant UM.C/625/1/HIR/MoE/SC/02 from the Ministry of Education
Malaysia.
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