Food and Nutrition Sciences, 2013, 4, 87-94
http://dx.doi.org/10.4236/fns.2013.49A2012 Published Online September 2013 (http://www.scirp.org/journal/fns)
Polyphenol Oxidase Inactivation by Microwave Oven
and Its Effect on Phenolic Profile of Loquat
(Eriobotrya japonica) Fruit
Yanet Chávez-Reyes1, Lidia Dorantes-Alvarez1, Daniel Arrieta-Baez2, Obed Osorio-Esquivel3,
Alicia Ortiz-Moreno1*
1Departamento de Ingeniería Bioquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F.,
México; 2Espectrometría de Masas y RMN Centro de Nanociencias y Micro y NanoTecnología, México D.F., México; 3Instituto de
Salud Pública, Universidad de Chalcatongo, Chalcatongo de Hidalgo, Oaxaca, México.
Received June 15th, 2013; revised July 15th, 2013; accepted July 22nd, 2013
Copyright © 2013 Yanet Chávez-Reyes et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The objective of this research was investigated the effect of polyphenol oxidase microwave treatment on phenolic com-
position, antioxidant activity and microstructure of loquat fruit. Phenolic profile of methanolic extracts prepared from
fresh, and microwave-treated samples were analyzed. Antioxidant activity was also evaluated by 2,2’-azinobis (3-ethyl-
benzothiazoline-6-sulfonic acid) (ABTS•+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH+) methods. In addition, polyphe-
nol oxidase inactivation was carried out using a response surface methodology to establish the optimal conditions of
treatment. The phenolic content of fresh mesocarp was 311 ± 0.60 mg gallic acid equivalents (GAE)/100g dry weight
(DW) and that of microwave-treated mesocarp was 1230 ± 0.36 mg GAE/100g DW. Total phenolic content of water/
methanol extract significantly increases after microwave treatment rather than methanolic extract of fresh loquat. Five
glycoside phenolics were identified by HPLC-DAD-MS as 3-caffeoylquinic acid, 3-p-coumaroylquinic acid, 5-caffeoyl-
quinic acid and quercetin-3-O-sambubioside. Methanolic extract of microwave-treated mesocarp showed higher anti-
oxidant activity than that of fresh mesocarp. Thus, polyphenol oxidase inactivation by microwave energy preserved the
integrity of phenolic compounds as well as antioxidant activity in mesocarp extracts prepared from loquat fruit. It was
also noted that phenolics were more abundant in the microwaved samples than in the fresh samples.
Keywords: Loquat; Phenolic Compounds; Polyphenol Oxidase; Microwave; Eriobotrya japonica
1. Introduction
The loquat (Eriobotrya japonica Lindl) plant is grown in
subtropical areas of China, Japan, India, Israel and the
Mediterranean [1]. However, its utilization is limited due
to enzymatic browning that occurs immediately after
removing the peel [2]. Enzymatic browning is an unde-
sirable reaction because of its unattractive appearance
and the resulting loss of quality [3] such as sensorial and
nutritional modifications, softening, off-flavor develop-
ment and darkening. Thus, enzymatic browning greatly
depreciates the potential of loquat as a food product [4,5].
The extent of browning depends on phenol content and
polyphenol oxidase (PPO) activity [5]. Polyphenol oxi-
dase enzymes in loquat juices have been inhibited using
sulfhydryl compounds [6] and the effectiveness of anti-
browning agents may be due to the concentration of endo-
genous phenolic compounds presenting in each fruit [3].
Recent research has reported the advantages of using
microwaves for processing food products. Microwaves
can penetrate material and deposit energy, and therefore
heat can be generated throughout the volume of the ma-
terial. Microwave treatment can reduce processing time
and enhance product quality [7]. Microwave energy is a
useful alternative in the processing of fruits and vege-
tables because of its rapid heating rate and its non-ther-
mal effect on enzyme inactivation. Moreover, microwave
energy reduces the impact of elevated temperature and
improves retention of thermolabile compounds such as
polyphenols, vitamins, carotenoids and other secondary
metabolites [8,9]. The main objective of this research
*Corresponding author.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit
88
was to apply microwave energy to inactivate PPO enzy-
me activity in loquat fruit (Eriobotrya japonica) and
evaluate the effect of this treatment on the fruit phenolic
profile and antioxidant activity.
2. Materials and Methods
2.1. Plant Material
Fresh Sample (FS)
Loquat fruits were purchased at a local market in Me-
xico City and sanitized with Citrus® solution (20 mL/L,
v/v). Pulp fruit (mesocarp) was separated manually prior
to analysis and peel and seeds were discarded.
2.2. Microwave Sample (MS)
Design Expert ver. 7 (Stat-Ease, Inc. Minneapolis) soft-
ware was used to determine the optimal conditions for
PPO inactivation. Time and sample weight were indepen-
dent variables, while PPO activity, total phenolic content,
and antioxidant activity were response variables. Meso-
carp tissue (ranging from 190 - 260 g, Table 1) was placed
in a glass plate and treated at 478 Watts in a microwave
oven (MS-1742AT, LG Corporation, Mexico). Tempera-
ture after microwave-treated was measured using an in-
frared thermometer (RAYST6LXE, Raynger ST, Califor-
nia, USA). Microwave-treated samples were stored at 4˚C
until further analyses. The microwave energy intensity (E)
was calculated as follows using Equation (1) [10].



kJg ofsample1000EWsg 

(1)
“E” is the microwave energy intensity, “W” represents
the microwave oven power (watt), “s” is the treatment
time (s), “g” is the sample weight (g) and 1000 J is a
conversion factor.
2.3. Microstructural Analyses
Each sample of mesocarp (FS, MS) was fixed, washed,
and dehydrated as described [11]. Inclusion with Epon
812 resin and polymerization was carried out at 60˚C for
24 hours. Thin cuts were then made with an ultrami-
crotome (Ultracut, Leica UCT) and images were taken
with an electron transmission microscope scope (Joel-
JEM 1010 Japon) at 6000-fold magnification.
2.4. Polyphenol Oxidase Enzyme Assay
Ten grams of mesocarp (FS and MS samples) were ho-
mogenized in 20 mL of 0.2 M sodium phosphate buffer
pH 7.0, and 1 g/L polyvinylpyrrolidone. The resulting
mixture was centrifuged at 21,000 × g for 20 minutes at
4˚C, and the supernatant was collected as a crude extract.
PPO activity was determined in a reaction consisting of
2.9 mL of 50 mM catechol as substrate, 0.1 mL of 0.2 M
phosphate buffer pH 7.0, and 0.1 mL of the PPO crude
extract. Change in absorbance at 420 nm was recorded
every 5 s for 5 min using a spectrophotometer (Genesys
10uv scanning, Thermo Spectronic, Rochester, NY. USA).
One unit of PPO activity was defined as the change in
absorbance of 0.001/min/mL of enzyme [12].
2.5. Phenolic Compound Extraction
Two-gram samples (FS and MS) of mesocarp were
mixed with 15 mL of methanol: water (1:1, v/v) and
ultra-sonicated for 1 h (Ultrasonic cleaner, USA). The
Table 1. Effect of microwave treatment on PPO enzyme activity, total phenolic content and antioxidant activity.
Weight (g) Time (s) E (kJ/g) PPO activity unit/g FW TPC mg GAE/100g ABTS (μM Trolox)
Fresh sample 0 0 31.1 ± 0.5 311.1 ± 0.60 20.2 ± 0.21
225 82 0.17 6.4 ± 0.4 318.5 ± 0.58 25.8 ± 0.34
250 120 0.22 1.3 ± 0.2 348.5 ± 0.32 38.8 ± 0.46
200 120 0.28 0.5 ± 0.4 366.4 ± 0.90 48.3 ± 0.39
260 210 0.38 0 685.7 ± 0.88 69.4 ± 0.16
225* 210 0.44 0 1162.7 ± 0.20 79.8 ± 0.22
225* 210 0.44 0 1152.1 ± 0.36 82.1 ± 0.63
225* 210 0.44 0 1288.4 ± 0.67 81.4 ± 0.25
225* 210 0.44 0 1224.7 ± 0.84 82.2 ± 0.15
189 210 0.52 0 601.9 ± 0.12 70.6 ± 0.17
250 300 0.57 0 568.7 ± 0.33 65.7 ± 0.13
*Central point; FW: fresh weight; E: microwave energy intensity.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit 89
mixture was allowed to stand for 15 min at room tem-
perature and the sonication was repeated for an addi-
tional 1 h. The mixture was centrifuged (Beckman J2-H2,
USA) at 13,000 × g for 5 min. The supernatant was
collected and analyzed immediately.
2.6. Determination of Total Phenolic Content
(TPC)
TPC was determined according to the Folin-Ciocalteu
method described by Sun et al. A 100 μL aliquot of me-
thanolic extract was mixed with 750 μL of Folin-Cio-
calteu phenol reagent (diluted 1:10 with water) and was
allowed to stand for 5 min at room temperature; 0.75 mL
of 6% sodium bicarbonate was added to the mixture. The
mixture was then incubated for 90 min at room tempe-
rature in the dark. Reactions were measured at 750 nm
using a spectrophotometer (Genesys 10uv scanning, Ther-
mo Spectronic, Rochester, NY, USA). A calibration
curve using gallic acid at concentrations ranging from 0
to 0.25 mg/mL was prepared and reactions were tested
under similar conditions. Results are expressed as mg
GAE/g DW ± standard deviation (SD) for 3 replicates
[13].
2.7. HPLC-DAD-MS/MS Analysis of Phenolic
Compounds
Methanolic extracts were analyzed using a UHPLC-sys-
tem (Ultimate 3000, Dionex, Sunnyvale, CA, USA)
equipped with on-line degasser, binary pump, auto sam-
pler, column heater, diode array detector and a 100 μL
loop coupled to a MS detector (MicrOTOF-QII, Bruker
Daltonics, Biellerica, MA, USA). Chromatographic ana-
lysis was performed using a C18-reversed phase column
(Kinetex C-18, 50 × 2.1 mm i.d. Phenomenex, Torrance,
CA, USA), with a particle size of 2.6 μm. Mobile phase
A was 100% methanol and mobile phase B consisted of
5% formic acid in water. Separation of phenolics was
achieved using the following gradient: 0 min—5% B, 3
min—15% B, 13 min—25% B, 25 min— 30% B, 35
min—35% B, 39 min—45% B, 42 min—45% B, 44
min—50% B, 47 min—70% B, 50 min—70% B, 56
min—75% B, 61 min—80% B and a flow rate of 0.9
mL/min. The chromatograms were recorded at 280 nm
for 65 min and the sample injection volume was 20 μL.
Identification of phenolic compounds was performed
on a HPLC-DAD-ESI-MS/MS system (Bruker micrO-
TOF-Q II, Bruker Daltonics, Bremem, Germany) using
Electrospray Ionization (ESI) analysis. Samples were
dissolved in 100% methanol and were injected directly
into the spectrometer. Polymer-related peaks were identi-
fied in positive and negative ion mode. The capillary
potential was −4.5 kV, the dry gas temperature was
200˚C and the drying gas flow was 4 L/min. Total ion
chromatograms from m/z 500 to 3000 were obtained.
2.8. DPPH Radical Assay
The activity of loquat methanolic extracts on the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical was esti-
mated according to the procedure described by Ferreres
et al. (2009). A 100 µL aliquot of methanolic extract (FS
and MS samples) was mixed with 2.9 mL of a metha-
nolic solution of 150 µM DPPH. The reaction mixture
was incubated for 30 min in darkness at room tem-
perature. The absorbance of the resulting solution was
measured at 515 nm in a spectrophotometer (Genesys
10uv scanning, Thermo Spectronic, Rochester, NY, USA).
The control sample was composed of methanol instead of
methanolic extracts. The antioxidant activity of the tested
samples was expressed as % inhibition of DPPH ± SD,
following Equation (2):

Acontrol Asample
Antioxidant activity%100
Acontr ol


(2)
2.9. ABTS free Radical Scavenging Assay
The antioxidant capacity of loquat extracts was also eva-
luated by using 2,2´-azinobis (3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS) free radical scavenging following
the modified method. Potassium persulfate was added to
7 mmol/L of ABTS•+ and the resulting solution was in-
cubated for 16 h at room temperature in the dark. The
ABTS•+ solution was diluted with ethanol and adjusted to
an absorbance of 0.70 ± 0.02 at 734 nm before analysis.
A 10 µL aliquot of loquat extract was added to 990 μL of
the ABTS•+ cation solution and mixed thoroughly. After
mixing, the absorbance was measured at 734 nm every
minute for 7 min. Readings taken after 5 min of reaction
were used to calculate the inhibition (%) of ABTS. The
blank was made with ethanol in place of extracts. Ab-
sorbance values were corrected for the solvent as follows
[14] according to Equation (3):


 
t=5sa
t=0 sat=0 sot=5sot=0 so
t=0 sa
AA
ΔAsa AAAA

 (3)
In this case, “sa” refers to the sample and “so” refers
to the solvent. A standard calibration curve was construc-
ted by plotting absorbance values against varying con-
centrations of Trolox. Antioxidant capacities of the sam-
ples were calculated in Trolox equivalents and % ABTS
inhibition using this curve. All measurements were made
in triplicate and these experiments were repeated twice.
Data are reported as means ± SD.
2.10. Statistical Analysis
Statistical analyses were conducted using Sigma Stat ver-

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit
90
sion 3.5 (Jandel Corp., San Raphael, CA). Data was
analyzed by one-way ANOVA and differences among
the means were compared using the Holm-Sidak method
with a level of significance of P < 0.05.
3. Results and Discussion
3.1. Polyphenol Oxidase Inactivation by
Microwave Treatment
Results of microwave treatment indicated that PPO was
inactivated in most combinations of microwave power
and time (Table 1). However, conditions were selected
in consideration of the E value, the amount of energy
required to inactivate the enzyme, which is an important
parameter in scaling up the process. Therefore, an opti-
mal combination was considered to be the one where
enzyme activity was inactivated and the largest fraction
of phenolic compounds were extracted using the lowest
microwave energy intensity. Matsui et al. reported that
microwave treatment improves retention of thermolabile
nutrients and sensorial characteristics [8]. The PPO en-
zyme activity in loquat pulp was inactivated at 83˚C,
which is consistent with published findings that purified
PPO from loquat fruit (Mespilus germanica L., Rosacea)
is heat-denatured at approximately 80˚C [4]. Compared
to fresh mesocarp, significant reductions in PPO enzyme
activity (approximately 79%, 96%, and 98%) were ob-
served at E = 0.17 kJ/g, 0.22 kJ/g and 0.28 kJ/g respec-
tively. Our study indicated that the optimal combination
for PPO inactivation in loquat fruit was 478 W for 210 s,
resulting in an E value of 0.44 kJ/g. Under these condi-
tions, the largest fraction of phenolic compounds, aproxi-
mately 311.1 ± 0.60 mg GAE/100g were retained com-
pare to the other combinations. The E value required for
enzyme inactivation depends on several factors such as
the chemical composition, dielectric properties, pH, and
other physical properties of the tissue [15]. Similar E
values have been previously reported for different fruits
and vegetables such as avocado puree (700 W/23 s, E =
0.80 kJ/g), potato cubes (600 W/300 s, E = 0.85 kJ/g),
and mamey (937 W/165 s, E = 0.90 kJ/g) [15-17]. Mi-
crowaves are useful because they transfer energy through-
out the volume of the material, reducing processing time
and enhancing overall quality by inactivating PPO activ-
ity [18].
3.2. Microstructural Analysis
Microstructural analysis of fresh samples and micro-
wave-treated samples (at 478 W/210 s) are shown in
Figure 1. In fresh mesocarp sample (Figure 1(a)), the
cell walls and vacuoles showed a defined structure. After
microwave treatment, morphological alterations such as
thinner cell walls and compromised vacuoles were ob-
500 nm
wc
v
(a)
2 microns
wc
(b)
Figure 1. Transmission electron micrographs of loquat tis-
sue. (a) Fresh mesocarp (30 K), (b) Microwave-treated me-
socarp (6000×). CW: cell wall, V: vacuole. The magnifica-
tion used is indicated on each micrograph.
served (Figure 1(b)). These morphological changes may
favor the release of compounds from vacuoles and sig-
nificantly increase the amount of extractable phenolics in
microwaved tissue compared to fresh tissue. In agree-
ment with our results, other labs have reported that anti-
oxidant activity and phenolic extraction levels increased
after microwave treatment [19]. The intense heat gener-
ated from microwaves creates a high vapor pressure and
temperature inside plant tissue, resulting in the disruption
of plant cell wall polymers [20]. Consequently, cell wall
phenolics or bond phenolics can be released, thus causing
even more phenolics to be extracted [21]. However, more
studies are needed to understand how structural modifi-
cations caused by microwaves influence the extraction of
phenolic compounds.
3.3. Effect of Microwave Treatment on Total
Phenolic Content (TPC)
A central composite design was used to study the influ-
ence of microwave energy on TPC. Factors studied were
the weight of the samples which varied from 200 g to

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit
91
300 g and the processing time of 1 to 5 min. Our results
indicated that the yield of TPC in methanolic extracts
increased with treatment time. The predicted response for
total phenolic content in terms of coded factors levels, is
given in the equation TPC = −28,606.54 + 240.10
(weight) + 1452.58 (time) – 1.88 (weight × time) – 0.52
(weight2) – 121.32 (time2). The R2 value for the model
was 0.9434, and the lack of fit was not significant
(0.0654 and P > 0.05) suggesting that the model could be
used to predict the TPC.
Lindl.) and reported approximately 2-fold lower TPC
levels in mesocarp compared to our study. Furthermore,
another group reported lower (1.5-fold) phenolic concen-
trations in mesocarp tissue derived from the same loquat
variety used in our study [25]. The differences observed
in TPC may be due to several factors such as species,
variety, light, the extent of ripeness, and environmental
conditions [26]. Our study offers an excellent means to
inactivate PPO enzyme activity by employing microwave
energy. This method improves phenolic extraction and
preserves fruit sensory characteristics.
Increased microwave radiation resulted in an increase
in TPC content of loquat water/methanol extract. The
TPC, expressed as gallic acid equivalents, was 311.1 ±
0.60 mg/100g for fresh loquat pulp. After microwave
treatment at E = 0.44 kJ/g, TPC increased approximately
two-fold in water/methanol extract compared to extract
from fresh pulp. Our results are in agreement with the
findings of Hayat et al., (2010) who found that micro-
wave treatment improved the phenolic extraction in man-
darin pomace and peel. A possible explanation is that
microwave energy can potentiate the bioavailability of
free natural compounds by preventing the binding of
polyphenols to the plant matrix [22]. Alternatively, the
inactivation of the PPO enzyme during heat treatment
may inhibit polyphenol degradation [23]. However, we
observed that extended microwave treatment (300 s) in-
creased degradation of phenolic compounds (Table 1).
3.4. Effect of Microwave Treatment on Phenolic
Profile
To further explore the nature of treated loquat mesocarp,
phenolic compounds were identified it. The resulting
chromatograms indicated significant differences in the
phenolic profile of the fresh sample compared to micro-
waved sample (Figure 2).
Six major peaks were apparent by HPLC-DAD-MS/
MS (Figures 2(a)-(b)) and identified by their retention
times, spectral characteristics and ion molecular mass
(Table 2) in mesocarp. Total ion scanning was used in
combination with selected ion monitoring of the follow-
ing molecular ions [M + H]: (1) m/z 353, (2) m/z 337, (3)
m/z 353, (4) m/z 447, (5) m/z 595, and (6) m/z 431. The
peaks were identified as (1) 3-caffeoylquinic acid; (2) 3-
p-coumaroylquinic acid; (3) 5-caffeoylquinic acid and (5)
quercetin-3-O-sambubioside. 3-caffeoylquinic acid was
the dominant phenolic acid in fresh and microwave
treated-samples (Figure 2(b)) followed by 3-p-couma-
roylquinic acid, 5-caffeoylquinic acid and quercetin-3-O-
Phenolic compounds help protect plants against ultra-
violet light and participate in defenses against pathogenic
micro-organisms [24]. Similar TPC values were reported
by Koba et al. (2007) in ethanolic extracts derived from
Eriobotrya japonica. Recently, Ferreres et al. (2009)
analyzed a distinct loquat variety (Eriobotrya japonica
0 5 10 15 20 25 30 35 40 45 50 55 60 65
(a)
(b)
300
200
150
100
50
0
300
250
200
150
100
50
0
Intens
Intens
1
2
3
5
6
RT [min]
RT [min]
1
2
3
4
5
6
Figure 2. HPLC elution pr ofile s of glycoside phenolics (λ = 280 nm) from the mesocarp of loquat fruit. (a) Fresh mesocarp, (b)
Microwave-treated mesocarp, (1) 3-caffeoylquinic acid; (2) 3-p-coumaroylquinic acid; (3) 5-caffeoylquinic acid, (4) Not iden-
tified, (5) quercetin-3-O-sambubioside, (6) Not identified.

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit
92
Table 2. HPLC analysis of phenolic compounds in fresh and microwave-treated loquat mesocarp.
Mesocarp mg/g DW
Compoundc name Retention time (min) UV (nm) [M-H]− (m/z) Fresh Microwave
(1) 3-CQA 6.5 325 353 2.5 ± 0.3 10.5 ± 0.2
(2) 3-p-CoQA 8.5 311 337 1.9 ± 0.2 2.1 ± 0.2
(3) 5-CQA 9.6 325 353 1.0 ± 0.1 4.4 ± 0.3
(4) NI 10.5 -a,b 447 - -
(5) Q-3-Sbb 12.2 265 595 trd
0.3 ± 0.28
(6) NI 15.3 -a,b 431 - -
aCompounds masked by others. Their UV spectra were not properly resolved. bUV of 11 + 12: 257, 265 sh, 297 sh, 280 nm. cQ: quercetin, CQA: caffeoylquinic
acid, p-CoQA: p-coumaroylquinic acid, Sbb: sambubioside. dtr, Trace amounts, NI: not identified.
sambubioside. The 3-caffeoylquinic acid content increa-
sed significantly from 2.5 ± 0.3 mg/g DW to 10.5 ± 0.2
mg/g DW in fresh compared to microwave-treated
mesocarp, respectively. Thus, our results indicated that
microwave-treated improves the capacity to extract phe-
nolic compounds. Similar results have been previously
reported for pomace tissue from different citrus fruits, in
which an increase in phenolic compound release was
observed after microwave treatment [27-29]. The libera-
tion of phenolics is consistent with findings previously
reported by T. Uslu et al., who reported that during mi-
crowave treatment, differential heating rates between
different phases promotes liberation of phenolics. Selec-
tive heating is a fundamental phenomenon associated
with microwave heating [30] and it is possible that re-
lease of phenolics is coupled with either the selective
heating of a number of the individual phenolics in the
microwave field or physical forces acting between the
phenolic and the plant matrix [27]. The phenolic gly-
cosides identified in our study were the same as those
reported for fresh Eriobotrya japonica Lindl mesocarp
[31]. Other phenolic compounds have been identified in
mesocarp and pericarp tissues, including 3-caffeoyl-
quinic acid, 4-caffeoylquinic acid, 5-feruloylquinic acid,
hydroxybenzoic acid, chlorogenic acid and cyanidine
glucoside [1,32].
Remarkably, microwave-treated sample was not af-
fected by enzymatic browning, and phenolic levels were
more extractable than even those of fresh sample (Fig-
ures 2(a) and (b)). Finally, levels of 3-caffeoylquinic
acid, 3-p-coumaroylquinic acid and 5-caffeoylquinic acid
increased significantly in microwave-treated sample (Ta-
ble 2).
3.5. Effect of Microwave Treatment on
Antioxidant Activity
The antioxidant activity of loquat methanolic extracts
was determined by ABTS and DPPH radical scavenging
assays. Extract derived from microwave treated meso-
carp exhibited higher ABTS and DPPH radical scaveng-
ing than the extract from fresh mesocarp. ABTS inhibi-
tion for fresh mesocarp was 16.3 ± 0.1%, and 40.7 ±
0.3% for microwave-treated mesocarp. A linear correla-
tion (R2 = 0.70) of TPC and % ABTS inhibition was
found for mesocarp. Similar trends were observed using
the DPPH assay: Percent inhibition for fresh (9.6 ± 0.3%),
and microwave-treated (50.3 ± 0.2) mesocarp extracts.
There was also a linear correlation (R2 = 0.73) between
% DPPH inhibition and TPC and the DPPH inhibition
mesocarp samples. Antioxidant activity results may vary
considerably, depending on the specific assay used and
the type of radical generated from a given compound.
Results obtained by different methods are therefore not
always comparable [33]. However, similar trend have
been reported for mandarin pomace treatment with mi-
crowaves at 250 W for 15 min [27]. The authors con-
cluded that antioxidant activity increased after micro-
wave-treated due to the release of a free phenolic fraction.
Other studies indicated that microwave processing en-
hanced antioxidant activity in broccoli, spinach, green
beans and pepper [34]. Yamaguchi et al. reported that
radical scavenging activity increases after thermal proc-
essing due to the suppression of oxidative enzymes and
the release of potent antioxidant compounds. The authors
also concluded that the internal temperature of fruits and
vegetables is associated with inactivation of oxidative
enzymes and the destruction of cell walls, both of which
are responsible for radical-scavenging activity [35]. Our
study is consistent with these others in that the release of
phenolic compounds after microwave-treated resulted in
increased antioxidant activity in extracts from loquat
fruit.
4. Conclusions
Microwave treatment was proved to be a promising ave-

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit 93
nue to inactivate PPO in loquat fruit. In addition, micro-
wave treatment resulted in microstructural modifications
that facilitated the release of phenolic compounds from
the fruit matrix. The combination of PPO inactivation
with increased TPC increased antioxidant activity in me-
socarp.
In the future, this technology may be scaled up to
produce novel products derived from loquat fruit and
made available throughout the year. However, further
studies are needed to evaluate the effect of microwaves on
biocompounds such as carotenes, vitamins, and other
nutritional chemicals in loquat fruit.
5. Acknowledgements
This research was partly funded by Consejo Nacional de
Ciencia y Tecnología (CONACyT) scholarship 286143,
Secretaria de Investigación y Posgrado-IPN Proyect num-
ber 20090612 and 20100788, Comisión de Operación y
Fomento de Actividades Académicas del IPN (COFAA-
IPN).
REFERENCES
[1] C.-K. Ding, K. Chachin, Y. Ueda, Y. Imahori and C. Y.
Wang, “Metabolism of Phenolic Compounds during Lo-
quat Fruit Development,” Journal of Agricultural and
Food Chemistry, Vol. 49, No. 6, 2001, pp. 2883-2888.
http://dx.doi.org/10.1021/jf0101253
[2] C.-K. Ding, K. Chachin, Y. Ueda and Y. Imahori, “Puri-
fication and Properties of Polyphenol Oxidase from Lo-
quat Fruit,” Journal of Agricultural and Food Chemistry,
Vol. 46, No. 10, 1998, pp. 4144-4149.
http://dx.doi.org/10.1021/jf980093s
[3] C.-K. Ding, K. Chachin, Y. Ueda and C. Y. Wang, “Inhi-
bition of Loquat Enzymatic Browning by Sulfhydryl
Compounds,” Food Chemistry, Vol. 76, No. 2, 2002, pp.
213-218.
http://dx.doi.org/10.1016/S0308-8146(01)00270-9
[4] B. Dincer, A. Colak, N. Aydin, A. Kadioglu and S. Güner,
“Characterization of Polyphenoloxidase from Medlar Fruits
(Mespilus germanica L., Rosaceae),” Food Chemistry,
Vol. 77, No. 1, 2002, pp. 1-7.
http://dx.doi.org/10.1016/S0308-8146(01)00359-4
[5] A. Altunkaya and V. Gökmen, “Effect of Various Inhibi-
tors on Enzymatic Browning, Antioxidant Activity and
Total Phenol Content of Fresh Lettuce (Lactuca sativa),”
Food Chemistry, Vol. 107, No. 3, 2008, pp. 1173-1179.
http://dx.doi.org/10.1016/j.foodchem.2007.09.046
[6] S. Sellés-Marchart, J. Casado-Vela and R. Bru-Martínez,
“Isolation of a Latent Polyphenol Oxidase from Loquat
Fruit (Eriobotrya japonica Lindl.): Kinetic Characteriza-
tion and Comparison with the Active Form,” Archives of
Biochemistry and Biophysics, Vol. 446, No. 2, 2006, pp.
175-185. http://dx.doi.org/10.1016/j.abb.2005.12.004
[7] M. S. Venkatesh and G. S. V. Raghavan, “An Overview
of Microwave Processing and Dielectric Properties of
Agri-Food Materials,” Biosystems Engineering, Vol. 88,
No. 1, 2004, pp. 1-18.
http://dx.doi.org/10.1016/j.biosystemseng.2004.01.007
[8] K. N. Matsui, J. A. W. Gut, P. V. de Oliveira and C. C.
Tadini, “Inactivation Kinetics of Polyphenol Oxidase and
Peroxidase in Green Coconut Water by Microwave Proc-
essing,” Journal of Food Engineering, Vol. 88, No. 2,
2008, pp. 169-176.
http://dx.doi.org/10.1016/j.jfoodeng.2008.02.003
[9] K. Hayat, X. Zhang, U. Farooq, S. Abbas, S. Xia, C. Jia,
F. Zhong and J. Zhang, “Effect of Microwave Treatment
on Phenolic Content and Antioxidant Activity of Citrus
Mandarin Pomace,” Food Chemistry, Vol. 123, No. 2,
2010, pp. 423-429.
http://dx.doi.org/10.1016/j.foodchem.2010.04.060
[10] A. Ortiz-Moreno, L. Dorantes, J. Galindez and R. Guz-
mán, “Effect of Different Extraction Methods on Fatty
Acids, Volatile Compounds, and Physical and Chemical
Properties of Avocado (Persea americana Mill) Oil,”
Journal of Agricultural and Food Chemistry, Vol. 51, No.
8, 2003, pp. 2216-2221.
http://dx.doi.org/10.1021/jf0207934
[11] Dykstra Biological Electron Microscopy, “Theory, Tech-
niques and Troubleshooting,” Plenum Publishing Corp,
New York, 1992.
[12] M. Oktay, O. I. Kufrevioglu, I. Kocacaliskan and H. Saki-
roglu, “Polyphenol Oxidase from Amasya Apple,” Jour-
nal of Food Science, Vol. 60, No. 3, 1995, pp. 494-496.
http://dx.doi.org/10.1111/j.1365-2621.1995.tb09810.x
[13] T. Sun, Z. Xu, C.-T. Wu, M. Janes, W. Prinyawiwatkul
and H. K. No, “Antioxidant Activities of Different Col-
ored Sweet Bell Peppers (Capsicum annuum L.),” Journal
of Food Science, Vol. 72, No. 2, 2007, pp. s98-s102.
http://dx.doi.org/10.1111/j.1750-3841.2006.00245.x
[14] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M.
Yang and C. Rice-Evans, “Antioxidant Activity Applying
an Improved ABTS Radical Cation Decolourization As-
say,” Free Radical Biology & Medicine, Vol. 26, No.
9-10, 1999, pp. 1231-1237.
http://dx.doi.org/10.1016/S0891-5849(98)00315-3
[15] G. Palma-Orozco, J. G. Sampedro, A. Ortiz-Moreno and
H. Nájera, “In Situ Inactivation of Polyphenol Oxidase in
Mamey Fruit (Pouteria sapota) by Microwave Treat-
ment,” Journal of Food Science, Vol. 77, No. 4, 2012, pp.
c359-c365.
http://dx.doi.org/10.1111/j.1750-3841.2012.02632.x
[16] C. Severini, A. T. Baiano and T. Pili, “Microwave Blan-
ching of Cubed Potatoes,” Journal of Food Processing
and Preservation, Vol. 27, No. 6, 2003, pp. 475-491.
http://dx.doi.org/10.1111/j.1745-4549.2003.tb00531.x
[17] L. Dorantes-Álvarez, G. V. Barbosa-Cánovas and G. Gu-
tiérrez-López, “Inovations in Food Processing, Food Pre-
servation Technology Series. Blanching of Fruit and Ve-
getables Using Microwaves,” Technomic Publishing Com-
pany, 2000, pp. 149-161.
[18] M. E. Latorre, P. R. Bonelli, A. M. Rojas and L. N. Ger-
schenson, “Microwave Inactivation of Red Beet (Beta
vulgaris L. var. conditiva) Peroxidase and Polyphenoloxi-

Polyphenol Oxidase Inactivation by Microwave Oven and Its Effect
on Phenolic Profile of Loquat (Eriobotr ya jap oni ca) Fruit
94
dase and the Effect of Radiation on Vegetable Tissue
Quality,” Journal of Food Engineering, Vol. 109, No. 4,
2012, pp. 676-684.
http://dx.doi.org/10.1016/j.jfoodeng.2011.11.026
[19] T. Blessington, M. N. Nzaramba, D. Scheuring, A. Hale,
L. Reddivari and J. C. Miller Jr., “Cooking Methods and
Storage Treatments of Potato: Effects on Carotenoids,
Antioxidant Activity, and Phenolics,” American Journal
of Potato Research, Vol. 87, No. 6, 2010, pp. 479-491.
http://dx.doi.org/10.1007/s12230-010-9150-7
[20] I. Hamrouni-Sellami, F. Rahali, I. Rebey, S. Bourgou, F.
Limam and B. Marzouk, “Total Phenolics, Flavonoids,
and Antioxidant Activity of Sage (Salvia officinalis L.)
Plants as Affected by Different Drying Methods,” Food
and Bioprocess Technology, Vol. 6, No. 3, 2013, pp. 806-
817. http://dx.doi.org/10.1007/s11947-012-0877-7
[21] S. Inchuen, W. Narkrugsa and P. Pornchaloempong, “Ef-
fect of Drying Methods on Chemical Composition, Color
and Antioxidant Properties of Thai Red Curry Powder,”
Kasetsart Journal of Natural Science, Vol. 44, 2010, pp.
142-151.
[22] A. Gulati, R. Rawat, B. Singh and S. D. Ravindranath,
“Application of Microwave Energy in the Manufacture of
Enhanced-Quality Green Tea,” Journal of Agricultural
and Food Chemistry, Vol. 51, No. 16, 2003, pp. 4764-
4768. http://dx.doi.org/10.1021/jf026227q
[23] A. M. Chuah, Y.-C. Lee, T. Yamaguchi, H. Takamura,
L.-J. Yin and T. Matoba, “Effect of Cooking on the An-
tioxidant Properties of Coloured Peppers,” Food Chemis-
try, Vol. 111, No. 1, 2008, pp. 20-28.
http://dx.doi.org/10.1016/j.foodchem.2008.03.022
[24] K. D. Croft, “The Chemistry and Biological Effects of
Flavonoids and Phenolic Acidsa,” Annals of the New York
Academy of Sciences, Vol. 854, No. 1, 1998, pp. 435-442.
http://dx.doi.org/10.1111/j.1749-6632.1998.tb09922.x
[25] J.-Y. Lin and C.-Y. Tang, “Determination of Total Phe-
nolic and Flavonoid Contents in Selected Fruits and Ve-
getables, as Well as Their Stimulatory Effects on Mouse
Splenocyte Proliferation,” Food Chemistry, Vol. 101, No.
1, 2007, pp. 140-147.
http://dx.doi.org/10.1016/j.foodchem.2006.01.014
[26] G. G. Duthie, P. T. Gardner and J. A. Kyle, “Plant Poly-
phenols: Are They the New Magic Bullet?” Proceedings
of The Nutrition Society, Vol. 62, No. 3, 2003, pp. 599-
603. http://dx.doi.org/10.1079/PNS2003275
[27] K. Hayat, X. Zhang, H. Chen, S. Xia, C. Jia and F. Zhong,
“Liberation and Separation of Phenolic Compounds from
Citrus Mandarin Peels by Microwave Heating and Its Ef-
fect on Antioxidant Activity,” Separation and Purifica-
tion Technology, Vol. 73, No. 3, 2010, pp. 371-376.
http://dx.doi.org/10.1016/j.seppur.2010.04.026
[28] G. Xu, X. Ye, J. C. Chen and L. D. Liu, “Effect of Heat
Treatment on the Phenolic Compounds and Antioxidant
Capacity of Citrus Peel Extract,” Journal of Agricultural
and Food Chemistry, Vol. 55, No. 2, 2007, pp. 330-335.
http://dx.doi.org/10.1021/jf062517l
[29] S.-M. Jeong, S.-Y. Kim, D.-R. Kim, S.-C. Jo, K. C. Nam,
D. U. Anh and S.-C. Lee, “Effect of Heat Treatment on
the Antioxidant Activity of Extracts from Citrus Peels,”
Journal of Agricultural and Food Chemistry, Vol. 52, No.
11, 2004, pp. 3389-3393.
http://dx.doi.org/10.1021/jf049899k
[30] T. Uslu, Ü. Atalay and A. I. Arol, “Effect of Microwave
Heating on Magnetic Separation of Pyrite,” Colloids and
Surfaces A: Physicochemical and Engineering Aspects,
Vol. 225, No. 1-3, 2003, pp. 161-167.
http://dx.doi.org/10.1016/S0927-7757(03)00362-5
[31] F. Ferreres, D. Gomes, P. Valentão, R. Gonçalves, R. Pio,
E. A. Chagas, R. M. Seabra and P. B. Andrade, “Im-
proved Loquat (Eriobotrya japonica Lindl.) Cultivars:
Variation of Phenolics and Antioxidative Potential,” Food
Chemistry, Vol. 114, No. 3, 2009, pp. 1019-1027.
http://dx.doi.org/10.1016/j.foodchem.2008.10.065
[32] K. Koba, A. Matsuoka, K. Osada and Y.-S. Huang, “Ef-
fect of Loquat (Eriobotrya japonica) Extracts on LDL
Oxidation,” Food Chemistry, Vol. 104, No. 1, 2007, pp.
308-316.
http://dx.doi.org/10.1016/j.foodchem.2006.11.043
[33] K. Krishnaswamy, V. Orsat, Y. Gariépy and K. Than-
gavel, “Optimization of Microwave-Assisted Extraction
of Phenolic Antioxidants from Grape Seeds (Vitis vinif-
era),” Food and Bioprocess Technology, Vol. 6, No. 2,
2013, pp. 441-455.
http://dx.doi.org/10.1007/s11947-012-0800-2
[34] N. Turkmen, F. Sari and Y. S. Velioglu, “The Effect of
Cooking Methods on Total Phenolics and Antioxidant
Activity of Selected Green Vegetables,” Food Chemistry,
Vol. 93, No. 4, 2005, pp. 713-718.
http://dx.doi.org/10.1016/j.foodchem.2004.12.038
[35] T. Yamaguchi, M. Taeko, K. Rie, K. Hiroko, M. Fumiko,
T. Junji, T. Hitoshi and M. Teruyoshi, “Radical-Scaveng-
ing Activity of Vegetables and the Effect of Cooking on
Their Activity,” Food Science and Technology Research,
Vol. 7, No. 3, 2001, pp. 250-257.
http://dx.doi.org/10.3136/fstr.7.250