Solid-state fermentation of apple pomace using Phanerocheate ...

Solid-state fermentation of apple pomace using Phanerocheate chrysosporium
– Liberation and extraction of phenolic antioxidants
C.M. Ajila a , S.K. Brar a,⇑ , M. Verma b , R.D. Tyagi a , J.R. Valéro a
a INRS-ETE, Université du Québec, 490, Rue de la Couronne, Québec, Canada G1K 9A9
b Institut de recherche et de développement en agroenvironnement inc. (IRDA), 2700 rue Einstein, Québec, Canada G1P 3W8
article info
Article history:
Received 24 August 2010
Received in revised form 25 October 2010
Accepted 19 November 2010
Available online 26 November 2010
Keywords:
Solid-state fermentation
Phanerocheate chrysosporium
Apple pomace
Phenolic compounds
Antioxidants
1. Introduction
abstract
There is an increasing global trend towards the efficient utilisation
of natural resources. The direct disposal of agro industrial
by-products as a waste in the environment represents a major
cause for environmental pollution and also an important loss of
biomass which could be used for the production of different
metabolites with added commercial value (Vendruscolo, Pitol,
Koch, & Ninow, 2007). Sustainable food production and valueaddition
of wastes is the most important issue in the agro and food
processing industry. Apple and apple products are one of the major
fruit and fruit products consumed all over the world. Several million
tonnes of apple pomace are generated during the processing
of apple products, such as apple juice, jelly, cider, among others
(Bhushan, Kalia, Sharma, Singh, & Ahuja, 2008), which contributes
about 20–35% of the total fruit production. Apple pomace has been
used as a source for several applications, such as pectin recovery
(Schieber et al., 2003), enzyme production (Favela-Torres, Volke,
& Viniegra, 2006), animal feed (Sehm, Lindermayer, Dummer,
Treutter, & Pfaffl, 2007), organic acids production (Shojaosadati &
⇑ Corresponding author. Address: Institut national de la recherche scientifique
Centre Eau, Terre & Environnement/Centre for Water, Earth and Environment 490
de la Couronne, Québec (Qc), Canada G1K 9A9. Tel.: +1 418 654 3116; fax: +1 418
654 2600.
E-mail address: satinder.brar@ete.inrs.ca (S.K. Brar).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.11.129
Food Chemistry 126 (2011) 1071–1080
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Apple pomace is a by-product from the apple processing industry and can be used for the production of
value-added phenolic compounds. A study was carried out to understand the changes and liberation of
phenolic compounds and improvement in antioxidant activity during solid-state fermentation of apple
pomace using Phanerocheate chrysosporium. The solid-state fermentation of apple pomace using P. chrysosporium
mobilised the polyphenolic compounds and improved the nutraceutical properties. The polyphenol
content in acetone extract increased and the results were statistically significant (P < 0.05) from
4.6 to 16.12 mg GAE/g dry weight during solid-state fermentation. The effect of various solvents, temperature,
time and detergents were also investigated for the extraction of polyphenolics by ultrasonication
and microwave-assisted extraction methods. The polyphenol content of the extracts was found to be in
the range of 5.78–16.12 mg GAE/g DW of samples, depending on the solvent, extraction time and temperature.
Antioxidant activities of polyphenol extracts were tested using the 2,2-diphenyl-1-picryhydrazyl
(DPPH) radical methods, where the IC50 ranged from 12.24 to 23.42 lg DW sample, depending on the
extraction conditions and the antioxidant activities correlated well with the polyphenol concentrations.
Ó 2010 Elsevier Ltd. All rights reserved.
Babaeipour, 2002), ethanol production (Paganini, Nogueira, Silva,
& Wosiacki, 2005), aroma compounds (Medeiros, Pandey, Vandenberghe,
Pastore, & Soccol, 2006), natural antioxidants (Foo & Lu,
1999), among others.
Diets rich in fruits and vegetables are gaining increased importance
due to their significant role in reducing the risk of certain
types of cancer, cardiovascular diseases and other chronic diseases
(Joshipura et al., 2001; McCann et al., 2007; Suárez et al., 2010).
Fruits and vegetables contain many antioxidant compounds
including phenolic compounds, carotenoids, anthocyanins and tocopherols
(Naczk & Shahidi, 2006). Apple is an important source of
bioavailable polyphenols, such as flavonols, monomeric and oligomeric
flavonols, dihydrochalcones, anthocyanidins, as well as others
(Escarpa & Gonzalez, 1998). The most abundant polyphenols
present in apples are chlorogenic acid, phloretin glucosides and
quercetin glucosides (Wijngaard, R€ossle, & Brunton, 2009). Other
polyphenolic compounds, such as catechins and procyanidins, have
also been identified, but are present in relatively small amounts
(Foo & Lu, 1999). The polyphenolic compound contents vary
greatly among different varieties and various parts of apples; apple
peels contain a higher concentration of phenolic compounds than
the flesh (Vrhosek, Rigo, Tonan, & Mattivi, 2004). Owing to the
increasing interest in new natural sources of antioxidant products,
apple pomace has been investigated as a potential source of
bioactive polyphenols during recent years (Cetkovic et al., 2008),
which have many applications in food, pharmaceutical and

1072 C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080
cosmetic industry by virtue of its antioxidant and antimicrobial
activities.
While the potential of apple pomace as a source of polyphenols
seems clear, there is less information on potential strategies for the
recovery of these compounds. Most of the polyphenolic compounds
are present in a bound form with carbohydrates, such as
glycosides in nature. This bound nature of polyphenolics as glycosides
reduces their ability to function as good antioxidants (Vattem
& Shetty, 2003). The lowered antioxidant activity has a direct effect
on the health functionality when these compounds are ingested
into the body via food or nutraceuticals, and may have to depend
on the probiotic status of the digestive system (Vattem & Shetty,
2003). Therefore, the release of free phenolics can improve the
health functionality of these phytochemicals. Many ligninolytic
and carbohydrate metabolising enzymes are produced by fungi
during fermentation of lignocellulosic wastes. These enzymes can
hydrolyse the phenolic glycosides and can release the free
aglycones, potentially having high antioxidant activity, making
them very useful for applications in food and beverage industries
(Vattem & Shetty, 2003).
In the present investigation, the ability of white rot fungi,
Phanerocheate chrysosporium to release phenolic antioxidants from
apple pomace by solid-state fermentation has been studied. The
extraction of polyphenolics from apple pomace and fermented apple
pomace was carried out by ultrasonic assisted extraction and
microwave-assisted extraction methods. The optimisation of solvents,
extraction time, extraction temperature, power and effect
of non-ionic surfactants for the extraction of polyphenolics has also
been investigated.
2. Materials and methods
Apple pomace samples from the apple processing industry, Lassonde
Inc., Rougemont, Montreal, Canada was collected and used
as the solid substrate for the solid-state fermentation. All chemicals
required for the experiments have been purchased from Fisher
Scientific (Fisher Scientific Company, Ontario, Canada), VWR chemicals
(VWR international, Quebec, Canada) and Sigma Chemicals
(Sigma–Aldrich Canada Ltd., Ontario, Canada) and were of analytical
grade.
2.1. Solid-state fermentation
Medium for fermentation: Apple pomace was used as natural substrate
for the solid-state fermentation. The apple pomace solids
were stored at 20 °C for its conservation prior to use. For the fermentation,
apple pomace was treated with inducers, such as copper
sulphate (2 mM), veratryl alcohol (2 mM) and Tween-80 (0.1%), before
the pH was adjusted to 4.5 and it was sterilised in an autoclave
for 30 min at 121 ± 1 °C. The moisture content in the apple pomace
was 72% w/v. P. Chrysosporium was propagated and stored at
20 °C, according to the procedure developed by Gassara, Brar,
Tyagi, Verma, and Surampalli (2010). The concentration of spore
suspension used in the experiments was 2.5 10 6 spores/g of solid.
Solid-state fermentation: The fermentation was carried out in
flasks. The media in flasks were autoclaved at 121 ± 1 °C for
30 min. The inoculation was performed using the spore suspension.
The fermentation was carried out in a controlled environment
with temperature at 37 ± 1 °C for 14 days.
2.2. Ultrasonic assisted extraction of polyphenolic compounds
Apple pomace and fermented apple pomace were accurately
weighed to 1 g and 20 ml of solvents was added. The samples were
placed in an ultrasonication bath (Elma Hans Schmidhauer GmbH
& Co. KG, Germany). The optimisation of solvents for the extraction
of phenolics by ultrasonication was carried out using different solvents,
such as water; 60% ethanol; 70% ethanol, 80% ethanol; 60%
acetone; 70% acetone and 80% acetone; 60% methanol; 70% methanol
and 80% methanol. The ultrasonic extraction was performed
for 30 min at 40 ± 1 °C. The optimisation of time for the extraction
of phenolics was carried out using different solvents, such as water,
80% acetone and 80% alcohol. Duplicates of each sample were
extracted at different time intervals of 20, 30 and 40 min at 40 °C.
The optimisation of temperature for the extraction of phenolics
was performed using water. Duplicates of each sample were extracted
at different temperatures of 30, 40, 50, 60, 70 and 80 °C
for 30 min. The effect of Tween-20 as a surfactant for the extraction
of polyphenolics was carried out using different concentrations of
Tween-20, such as 0.1%, 1%, 2% and 5% in v/v with water.
For each of the optimisation experiments, duplicates of each
sample were extracted at the same time. Each sample mixture
was centrifuged at 9268g for 20 min to obtain the supernatant.
The supernatant was used for determination of total phenolics
content.
2.3. Microwave-assisted extraction of polyphenolic compounds
Apple pomace and fermented apple pomace were accurately
weighed to 1 g and 20 ml of solvent was added. Each mixture
was taken in a sealed 100 ml green chem Teflon reactor vessel
and extracted for 10 min at 60 °C with a pressure of 692 kpa and
power 400 W, using a sophisticated microwave extractor (Mars,
CEM Corporation, Northcarolina, USA). The microwave extractor
allowed highly accurate control of pressure, power and temperature.
After 10 min of extraction, vessels were allowed to cool prior
to their removal from the extractor. Duplicates of each sample
were extracted and each sample mixture was centrifuged at
9268g for 20 min to obtain the supernatant. The optimisation of
solvents, extraction time, temperature and effect of detergents
for the extraction of phenolics was performed using the same protocol
as for ultrasonication.
2.4. Estimation of total phenolic content
The content of total polyphenolics in the phenolic extract of apple
pomace and fermented apple pomace was determined by the method
of Swain and Hillis (1959). The supernatant obtained after centrifugation
of ultrasonic and microwave-assisted extraction was used
for the determination of total phenolics content. The absorbance
was recorded at 725 nm. Gallic acid was used as a standard. The content
of total polyphenolics in the extract was expressed as gallic acid
equivalents (GAE) in g/dry weight of the samples.
2.5. Free radical scavenging activity of polyphenolic compounds
The effect of polyphenol extracts on DPPH radical was determined
according to the method by Brand-Williams, Cuvelier, and
Berset (1995). A 100 lM solution of DPPH in methanol was prepared
and polyphenol extract (200 ll) was mixed with 1 ml of
DPPH solution. The mixture was shaken vigorously and left in
the dark at room temperature for 20 min. The absorbance of the
resulting solution was measured at 517 nm. The control contained
all the reagents except the polyphenol extract. The capacity to
scavenge DPPH radical was calculated by following Equation:
Scavenging activityð%Þ ¼½1 ðAs=A0ÞŠ 100 ð1Þ
where A0 is the absorbance at 517 nm of the control and As is the
absorbance in the presence of polyphenol extract. The results
were plotted as the percentage of scavenging activity against
concentration of the sample. The half-inhibition concentration

(IC50) was defined as the amount of sample in dry weight required
for 50% of free radical scavenging activity. The IC 50 value was calculated
from the plots as the antioxidant concentration required for
providing 50% free radical scavenging activity.
2.6. Statistical analyses
All the experiments were conducted in triplicates and data presented
are an average of triplicates along with the standard deviation.
Statistical analysis was done for the parameters, such as
extraction methods, solvents, time and effect of detergents, to find
out the optimum conditions for polyphenolic extraction and antioxidant
activities. The database was subjected to an analysis of
variance (ANOVA) and multiple range tests among data were carried
out using the Statistical Analysis System Software (STAT-
GRAPHICS Centurion, XV trial version 15.1.02 year 2006,
StatPoint, Inc. USA). The results which have P < 0.05 were considered
as significant. The method used to discriminate among the
means is Fisher’s least significant difference (LSD) procedure. Using
this method, there is a 5.0% risk of calling each pair of means significantly
different when the actual difference equals 0.
3. Results and discussion
The polyphenol content in apple pomace and fermented apple
pomace was determined. It was found that the polyphenol content
increased from 15.53 to 17.2 mg GAE/g dry weight (DW) of samples
depending on the solvent used, extraction time and temperature.
Antioxidant activities of polyphenol extracts were tested
using the 2,2-diphenyl-1-picryhydrazyl (DPPH) radical methods
and the IC50 ranged from 12.24 to 23.42 lg DW sample, depending
on the extraction conditions and the antioxidant activities, correlated
well with the polyphenol concentrations. Extraction of polyphenols
by vortexing the sample with water, 80% acetone and 80%
ethanol was done as control experiment for the extraction techniques,
such as ultrasonication and microwave digestion. The polyphenol
content in water, 80% acetone and 80% ethanol was 1.24,
5.6 and 4.2 mg GAE/g dry weight sample. The polyphenol extraction
by vortexing was very low compared to other extraction
methods. Thus, further experiments on the optimisation of parameters
were conducted mainly using ultrasonication and microwave-assisted
extraction methods. The effect of solvents,
temperature, time and detergents during the extraction of polyphenolics
by ultrasonic and microwave-assisted extraction methods
was studied for the development of better extraction and
recovery method for polyphenolics.
The ultrasonic extraction is a simple and rapid method for the
extraction and fractionation of plant materials. The ultrasonication
was principally ascribed to mechanical and chemical effects of
acoustic wave, which break the biological cell and facilitate the
release of cell contents into the extraction medium (Toma,
Vinatoru, & Mason, 2001). Further, the cell fragmentation dramatically
increases the contact areas and accelerates the mass transfer
rate of target extracts into the extraction solvent. Microwaveassisted
extraction is a faster extraction process, where microwave
energy is efficiently delivered to materials through molecular
interaction with the electromagnetic field and offers a rapid transfer
of energy to the extraction solvent and raw plant materials
(Zhou & Liu, 2006).
3.1. Optimisation of solvents for the extraction of polyphenolics using
ultrasonic and microwave extraction
The average total polyphenol contents of apple pomace and fermented
apple pomace extracts tested for different solvents such as
C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080 1073
water, Ethanol 60%, Ethanol 70%, Ethanol 80%, Acetone 60%, Acetone
70%, Acetone 80%, Methanol 60%, Methanol 70% and Methanol 80%
by ultrasonication and microwave extraction are presented in
Fig. 1A and B. Acetone (80%) extracted a significantly higher amount
of phenolic compounds (P < 0.05) compared to other solvents, such
as acetone, methanol, ethanol and water. The extractability of polyphenols
by the different solvents followed used the order: acetone
80% > acetone 70% > acetone 60% > ethanol 80% > ethanol 70% >
ethanol 60% > methanol 80% > methanol 70% > methanol 60% >
water, as shown in the Fig. 1A and B. The average total phenol content
of acetone 80% extract (16.12 mg GAE/g DW) was at least three
times higher than that of aqueous extracts (5.78 mg GAE/g DW) and
was statistically significant (P < 0.05).
Water, methanol and ethanol were found to be inefficient for
the extraction of polyphenolics compared to acetone by both ultrasonic
and microwave extraction methods. The phenolic compounds
in the extracts are more often associated with other
biomolecules, such as protein, carbohydrates, lipids, terpenes,
chlorophyll and other organic compounds. Hence, there should
be a better solvent for extracting the polyphenolic compounds. It
has been already reported that ethanol/water or acetone/water extracted
better when compared to either ethanol or acetone alone
(Yilmaz & Toledo, 2006) during the extraction of grape seed polyphenolics.
The aqueous extraction of polyphenolics leaves a large
amount of residual polyphenols which can be extracted only by
an appropriate combination of solvents. The polyphenol content
in aqueous extraction was very low compared to other solvents
in both the extraction methods. The main drawback of the aqueous
extraction is the low yield in antioxidants, with low polarity or
lipo-soluble antioxidants. Solubility of polyphenols depends
mainly on the hydroxyl groups, the molecular size and the length
of hydrocarbon. Zhou and Yu (2004) reported that among the solvents
tested, 50% acetone extracts contained a higher level of total
phenolics from wheat whereas, ethanol was the least effective solvent,
which is also in agreement with our results. In the study carried
out by Yu, Ahmedna, and Goktepe (2005), 80% ethanol and 80%
methanol were found to be more efficient than water for extracting
total phenolics from peanut skin, which is also in concordance with
the results from this study.
The free radical scavenging activity of polyphenol extracts from
apple pomace and fermented apple pomace with different solvents
by ultrasonic assisted and microwave-assisted extraction was measured
as IC 50 value and the results are shown in Table 1. Solvents
used for polyphenol extraction also had significant effects on DPPH
scavenging capacity determination for polyphenol extracts from
apple pomace and fermented apple pomace (Table 1). DPPH method
is a widely used method for antioxidant activity studies. The
method is based on the reduction of alcoholic DPPH solutions at
517 nm, in the presence of a hydrogen donating antioxidant (Koleva,
Van Beek, Linssen, De Groot, & Evstatieva, 2002) and polyphenols
have been reported to be potent hydrogen donors to the DPPH
radical because of their ideal structural chemistry (Rice-Evans, Miller,
& Paganga, 1997). The polyphenol extracts with 80% acetone as a
solvent, in both apple pomace and fermented apple pomace by
ultrasonication and microwave extraction methods, showed significantly
higher DPPH radical scavenging activity (P < 0.05) than
those with other solvents and this trend was similar to that observed
for polyphenol content. The IC 50 values obtained from the
polyphenol extract by ultrasonic assisted extraction varied significantly
(P < 0.05) from 24.43 to 52.96 lg DW and 14.27 to
50.18 lg DW for apple pomace and fermented apple pomace,
respectively, as shown in the Table 1. Acetone extract (80%) of both
apple pomace and fermented apple pomace exhibited higher inhibitory
activity against DPPH radical in comparison with their corresponding
solvent extracts. This can be attributed to a higher
concentration of polyphenolics present in the extract. In both the

1074 C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080
Fig. 1. Optimisation of solvent for extraction of polyphenolics: (A) by ultrasonication and; (B) by microwave extraction. Values are expressed as means of three
replicates ± standard deviations; means indicated with different letters differ significantly (P < 0.05).
Table 1
Effect of solvents on free radical scavenging activity of apple pomace and fermented apple pomace polyphenol extract by ultrasonic and microwave-assisted extraction.
Solvents Ultrasonic assisted extraction Microwave-assisted extraction
Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg) Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg)
Water 52.96 ± 2.50 j
50.18 ± 2.00 j
42.24 ± 2.20 i
40.16 ± 2.40 i
Ethanol 60% 37.09 ± 1.80 h
27.68 ± 2.00 ef
32.46 ± 3.40 g
25.46 ± 3.20 e
Ethanol 70% 28.83 ± 1.80 f
26.96 ± 2.20 ef
25.24 ± 2.80 e
22.42 ± 3.20 d
Ethanol 80% 26.04 ± 4.20 ef
25.74 ± 2.40 e
22.38 ± 4.20 d
20.14 ± 2.70 c
Methanol 60% 30.99 ± 3.40 g
27.68 ± 1.40 ef
24.46 ± 4.40 e
22.14 ± 3.80 d
Methanol 70% 25.55 ± 2.60 e
26.21 ± 2.60 ef
22.52 ± 4.60 d
20.56 ± 2.20 c
Methanol 80% 23.43 ± 2.20 d
20.59 ± 2.10 c
20.80 ± 2.10 c
18.26 ± 2.40 b
Acetone 60% 26.04 ± 1.40 ef
25.74 ± 2.80 e
24.42 ± 4.20 e
22.38 ± 4.30 d
Acetone 70% 25.02 ± 4.20 e
17.19 ± 1.60 b
20.64 ± 2.60 c
14.25 ± 2.80 a
Acetone 80% 24.43 ± 1.80 e
14.27 ± 2.50 a
20.12 ± 2.40 c
12.24 ± 5.20 a
IC50 values were calculated from the dose responses curves. All data are the mean ± SD of three replicates. Mean followed by different letters in the same column differ
significantly (P < 0.05) between the parameters such as solvent, extraction method and fermentation. Bold cells represent optimal values.
apple pomace and fermented apple pomace, the order of radical
scavenging activity was : 80% acetone > 80% methanol > 70% acetone
> 70% methanol > 60% acetone > 80% ethanol > 70% ethanol
> 60% methanol > 60% ethanol > water.
The IC 50 values obtained from the polyphenol extract by microwave-assisted
extraction varied from 20.12 to 42.24 lg DW and
12.24 to 40.16 lg DW for apple pomace and fermented apple pomace,
respectively. Acetone extract (80%) of both apple pomace and
fermented apple pomace exhibited higher and significant inhibi-
tory activity (P < 0.05) against DPPH radical as in the case of ultrasonic
assisted extraction.
In both the apple pomace and fermented apple pomace, the order
of radical scavenging activity was: 80% acetone > 70% acetone
> 80% methanol > 80% ethanol > 70% methanol > 60%
acetone > 60% methanol > 70% ethanol > 60% ethanol > water. The
polyphenolic extracts by microwave-assisted extraction
(12.24 lg DW) showed higher free radical scavenging activity than
the extract by ultrasonic assisted extraction (14.27 lg DW). The

higher values of free radical activity may be due to the higher phenolic
contents extracted by microwave extraction, compared to
ultrasonication.
The UV spectra of the polyphenolic extracts with different solvents
have been analysed. Fig. 2 shows UV spectra of the polyphenolic
extracts with different solvents. The result shows the good
agreement between experimental polyphenol content data and
the UV spectral analysis of polyphenol extracts between 200 and
600 nm. It should be clearly understood that the UV spectra verified
the extraction of polyphenolic extract of apple pomace and apple
pomace based on spectral differences among the polyphenolic
compounds extracted by different solvents.
3.2. Optimisation of time for the extraction of polyphenolics by
ultrasonic and microwave extraction
The polyphenol content and free radical scavenging activity of
polyphenol extracts with ultrasonication and microwave-assisted
extraction at different time intervals is shown in Fig. 3A and B
and Table 2. In the first stage of extraction, the total phenol content
and free radical scavenging activity especially increased with
extraction time in both ultrasonic assisted extraction and microwave-assisted
extraction. In ultrasonic assisted extraction, the
phenolic extraction was maximum at 30 min of extraction. The
phenolic content in apple pomace increased from 1.41 to
2.81 mg GAE/g DW, 4.68 to 5.72 mg GAE/g DW and 8.13 to
12.28 mg GAE/g DW in water, 80% ethanol and 80% methanol,
respectively. In the case of fermented apple pomace, total phenol
content increased significantly (P < 0.05) from 6.74 to
9.76 mg GAE/g DW, 11.34 to 13.46 mg GAE/g DW and 18.97 to
19.90 mg GAE/g DW in water, 80% ethanol and 80%, methanol,
respectively. After 30 min of extraction, the phenolic content
decreased.
C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080 1075
Microwave assisted phenolic extraction was carried out at different
time intervals, such as 5, 10 and 15 min and the maximum
extraction was obtained in 10 min of extraction. The phenolic content
in apple pomace increased from 9.52 to 10.82 mg GAE/g DW,
10.69 to 15.72 mg GAE/g DW and 14.53 to 18.28 mg GAE/g DW in
water, 80% ethanol and 80% methanol, respectively at 10 min
of extraction. In the case of fermented apple pomace, total
phenol content increased significantly (P < 0.05) from 10.26 to
14.24 mg GAE/g DW, 11.28 to 17.24 mg GAE/g DW and 14.46 to
20.12 mg GAE/g DW in water, 80% ethanol and 80% methanol,
respectively, as shown in the Fig. 3A and B.
The extraction time impacted DPPH scavenging capacity determination
for polyphenol extracts from apple pomace and fermented
apple pomace. The free radical scavenging activity of
polyphenol extracts from apple pomace and fermented apple pomace
with different extraction times by ultrasonic assisted and
microwave-assisted extraction was measured as IC50 value and
the results are shown in Table 1. The IC50 values obtained from
the polyphenol extract by ultrasonic assisted extraction varied
from 24.43 to 28.24 lg DW and 14.27 to 18.24 lg DW for apple
pomace and fermented apple pomace, respectively. The IC 50 values
obtained from the polyphenol extract by microwave-assisted
extraction varied from 20.12 to 24.32 lg DW and 12.24 to
16.38 lg DW for apple pomace and fermented apple pomace,
respectively. The free radical scavenging activity increased with
enlarged extraction time and later it decreased in both ultrasonication
and microwave-assisted extraction. At 30 min extraction, apple
pomace and fermented apple pomace exhibited higher and
more significant inhibitory activity against DPPH radical in comparison
with 20 and 40 min for ultrasonic assisted extraction with
other solvent extracts. The antioxidant activity was significantly
different for the corresponding extraction time (P < 0.05). In fact,
in the case of microwave-assisted extraction, 10 min extraction
Fig. 2. UV–Visible spectra of polyphenolic extracts using different solvents (80% acetone, 80% ethanol, 80% methanol and water).

1076 C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080
Fig. 3. Optimisation of extraction time for polyphenolics: (A) by ultrasonication and; (B) by microwave extraction. Values are expressed as means of three
replicates ± standard deviations; means indicated with different letters differ significantly (P < 0.05).
Table 2
Effect of extraction time on free radical scavenging activity of apple pomace and fermented apple pomace polyphenol extract of 80% acetone by ultrasonic and microwaveassisted
extraction.
Time (min) Ultrasonic assisted extraction Microwave-assisted extraction
Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg) Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg)
5 – – 24.32 ± 2.80 f
16.38 ± 2.90 c
10 – – 20.12 ± 2.40 e
12.24 ± 5.20 a
15 – – 22.14 ± 2.60 e
15.67 ± 4.30 b
20 28.24 ± 2.60 h
18.24 ± 5.20 d
–
30 24.43 ± 1.80 f
14.27 ± 2.50 b
–
40 26.21 ± 2.60 g
16.34 ± 4.20 c
–
IC50 values were calculated from the dose responses curves. All data are the mean ± SD of three replicates. Mean followed by different letters in the same column differ
significantly (P < 0.05) between the parameters such as extraction methods, temperature and fermentation. Bold cells represent optimal values.
showed higher antioxidant activity compared to 5 and 15 min
extraction. This can be attributed to higher concentration of polyphenolics
present in the extract.
Extraction time affected the total phenolic content and antioxidant
activities. The decrease in polyphenolic content and antioxidant
activity after particular extraction time may be due to the
chemical and enzymatic degradation, which in turn affects the polyphenol
content and its antioxidant activity (Larrauri, Ruperez, &
Saura-Calixto, 1997). It has been suggested that polyphenols can
react with other plant components and decompose to form other
less active phenolic contents (Guillot, Malnoë, & Stadler, 1996). It
was also found that a longer extraction time increases the chance
of oxidation of phenolics, unless reducing agents are added to
the solvent system, which reduces the antioxidant activity (Naczk
& Shahidi, 2006).
3.3. Optimisation of temperature for the extraction of polyphenolics in
ultrasonic and microwave extraction
Extraction temperature is one of the important factors affecting
the extraction efficiency of polyphenols. The yield of polyphenol
extraction can be obtained at higher temperature. High temperature

solvent will promote distribution of polysaccharides in the cell wall
so that more solvent can be contacted with polyphenols (Sun, Chu,
Wu, & Liu, 2002).
Fig. 4A and B show the effect of temperature for the ultrasonic
and microwave assisted polyphenol extraction, for different
extraction temperatures ranging from 30 to 80 °C. Fig. 4A shows
that in the ultrasonic assisted extraction, with the increase of temperature
from 30–40 °C, the polyphenol concentration increased
significantly (P < 0.05) from 7.1 mg GAE/g DW to 9.2 mg GAE/
g DW, then reached the peak, and finally dropped from 50 to
80 °C. In the case of microwave-assisted extraction, the polyphenol
content increased significantly (P < 0.05) with temperature from
40 to 60 °C and slowly dropped after 60 °C as shown in Fig. 4B.
The extraction temperature also led to important effects on
DPPH scavenging capacity determination for polyphenol extracts
from apple pomace and fermented apple pomace. The free radical
scavenging activity of polyphenol extracts from apple pomace and
fermented apple pomace, with different extraction temperatures
ranging from 30 to 80 °C, by ultrasonic assisted and microwave-assisted
extraction, was measured as IC50 value and the results are
shown in Table 3. The IC 50 values obtained from the aqueous polyphenol
extract by ultrasonic assisted extraction varied from 52.96
to 58.11 lg DW and 50.22 to 56.26 lg DW for apple pomace and
fermented apple pomace, respectively. The IC50 values obtained
C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080 1077
from the polyphenol extract by microwave-assisted extraction varied
from 42.24 to 51.48 lg DW and 40.16 to 48.45 lgDW
(P < 0.05) for apple pomace and fermented apple pomace, respectively.
The free radical scavenging activity increased with increase
in extraction temperature and it was a maximum at optimum
extraction temperature and later it decreased in both ultrasonication
and microwave-assisted extraction. At 40 °C, both apple pomace
and fermented apple pomace exhibited higher and more
significant inhibitory activity (P < 0.05) against DPPH by ultrasonic
assisted extraction. Meanwhile, in the case of microwave-assisted
extraction, extraction at 60 °C showed higher antioxidant activity
compared to other temperatures.
The phenomenon of increase in polyphenol content with increase
in temperature could be due to the fact that with the increase
in temperature, solvent viscosity declined and the
movement of molecules gets accelerated, which in turn led to
the dissolution of polyphenols with the increase of diffusion coefficient
and stability. However, much higher temperature will cause
the loss of solvent, resulting in a lower yield and also promoting
the oxidation of polyphenols, which in turn affect the free radical
scavenging activity of the extract. Increased extraction of polyphenolic
groups with increasing temperature can be attributed to rapid
mass transfer of the solutes (Schwartzberg, Torres, & Zaman,
1982) and increased solubility, especially of polymeric fractions,
Fig. 4. Optimisation of temperature for extraction of polyphenolics: (A) by ultrasonication and; (B) by microwave extraction. Values are expressed as means of three
replicates ± standard deviations; means indicated with different letters differ significantly (P < 0.05).

1078 C.M. Ajila et al. / Food Chemistry 126 (2011) 1071–1080
Table 3
Effect of temperature on free radical scavenging activity of apple pomace and fermented apple pomace polyphenol aqueous extract by ultrasonic and microwave-assisted
extraction.
Solvents Ultrasonic assisted extraction Microwave-assisted extraction
Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg) Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg)
30 54.26 ± 4.20 f
53.28 ± 2.20 f
51.28 ± 3.20 e
48.26 ± 3.80 d
40 52.96 ± 2.50 ef
51.22 ± 1.80 e
50.24 ± 1.60 e
46.24 ± 2.40 c
50 53.28 ± 3.60 f
50.18 ± 2.00 e
46.28 ± 2.80 c
42.14 ± 2.80 b
60 54.46 ± 3.90 f
52.24 ± 1.70 ef
42.24 ± 2.20 b
40.16 ± 2.40 a
70 56.24 ± 1.20 g
54.48 ± 1.40 f
45.64 ± 3.20 c
44.92 ± 2.70 c
80 58.11 ± 2.30 h
56.26 ± 2.40 g
51.28 ± 4.20 e
48.45 ± 3.30 d
IC 50 values were calculated from the dose responses curves. All data are the mean ± SD of three replicates. Mean followed by different letters in the same column differ
significantly (P < 0.05) between extraction methods, time and fermentation. Bold cells represent optimal values.
Fig. 5. Effect of Tween-20 on extraction of polyphenolics: (A) by ultrasonication and; (B) by microwave extraction. Values are expressed as means of three
replicates ± standard deviations; means indicated with different letters differ significantly (P < 0.05).
at higher temperatures. Therefore, the optimal extraction temperature
was considered to be 40 °C for ultrasonic assisted extraction
and 60 °C for microwave-assisted extraction.
3.4. Effect of Tween-20 on extraction of polyphenolics using ultrasonic
and microwave extraction
The effect of surfactants on polyphenolic extraction was studied
using Tween-20 with different concentrations ranging from 0.1% to
5% v/v in aqueous extract by ultrasonic and microwave-assisted
extraction methods and the results are given in Fig. 5A and B.
Tween-20 is a polysorbate surfactant, which is both relatively sta-
ble and non-toxic, and is used as a detergent and emulsifier in
many domestic, scientific and pharmacological applications. 1%
v/v of Tween-20 increased the polyphenol extraction in both ultrasonic
assisted and microwave-assisted extraction. The effect of
these surfactants on free radical scavenging activity of aqueous
polyphenol extracts from apple pomace and fermented apple pomace
with different concentrations ranging from 0.1 to 5% v/v by
ultrasonic assisted and microwave-assisted extraction was measured
as IC50 value and the results are shown in Table 4. The IC50
values obtained from the aqueous polyphenol extract by ultrasonic
assisted extraction varied from 46.14 to 52.96 lg DW and 45.24 to
50.18 lg DW for apple pomace and fermented apple pomace,

Table 4
Effect of Tween-20 on free radical scavenging activity of apple pomace and fermented apple pomace polyphenol extract by ultrasonic and microwave-assisted extraction.
Tween 20 (%) Ultrasonic assisted extraction Microwave-assisted extraction
Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg) Apple pomace (IC50 in lg) Fermented apple pomace (IC50 in lg)
0 52.96 ± 2.50 g
50.18 ± 2.00 f
42.24 ± 2.20 c
40.16 ± 2.40 bc
0.1 48.26 ± 3.80 e
47.71 ± 1.20 e
41.62 ± 3.20 bc
38.22 ± 2.20 b
1 46.14 ± 2.20 d
45.24 ± 4.10 d
39.64 ± 2.80 b
35.42 ± 3.60 a
2 48.28 ± 3.20 e
46.45 ± 2.80 d
42.33 ± 3.60 c
38.44 ± 4.20 b
5 51.26 ± 5.20 f
48.22 ± 4.10 e
45.26 ± 3.20 d
42.28 ± 2.80 c
respectively. The IC50 values obtained from the polyphenol extract
by microwave-assisted extraction varied from 39.64 to 45.26 lg
DW and 35.42 to 42.28 lg DW for apple pomace and fermented
apple pomace, respectively. Aqueous polyphenol extract of apple
pomace and fermented apple pomace with 1% v/v Tween-20
exhibited statistically significant higher inhibitory activity
(P < 0.05) against DPPH by both microwave and ultrasonic assisted
extraction.
Tween-20 might have enhanced the extraction of polyphenols
due to two factors: (a) lowering surface tension and hence enhancing
the release of polyphenols especially entrapped in the complex
solid matrix; and (b) facilitating increased reaction rate due to the
increased contact between surfactant, polyphenols and other components
in the liquid phase. In fact, Tween has been commonly
used as a surfactant and extractant in many applications, including
proteins, cells, as well as others (Umesaki, Kawai, & Mutai, 1977)
4. Conclusion
The present study showed that the enrichment of apple pomace
with phenolic antioxidants can be carried out by solid-state fermentation
using white rot fungus, Phanerocheate chrysosporium.
The phenolic content was higher in fermented apple pomace than
the apple pomace and the antioxidant activity was correlated with
the increase in polyphenolic content. The extraction of polyphenol
content varied depending on the type of solvent, temperature of
extraction, extraction time and the method used. Solid-state fermentation
thus, not only resulted in value addition to the apple
pomace, but also improved the antioxidant activity.
Acknowledgements
The authors are sincerely thankful to the Natural Sciences and
Engineering Research Council of Canada (Discovery Grant
355254, Canada Research Chair), FQRNT (ENC 125216) and MAPAQ
(No. 809051) for financial support. The views or opinions
expressed in this article are those of the authors.
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