The percent overrun was calculated using the following equation: Overrun %= weight of egg white− sugar solution g − weight of whipped foam g weight of whipped foam g ×100 Microstructur
Trang 1the Characteristics of Meringue
Siwaporn O’Charoen, Shigeru Hayakawa, Yoshiki Matsumoto, and Masahiro Ogawa
Abstract: Excessive intake of sugar-rich foods leads to metabolic syndrome d-Psicose (Psi) not commonly found in
nature, is noncalorie sweetener with a suppressive effect on the blood glucose level Thus, Psi has the potential to be
utilized as a sucrose (Suc) replacer in sugar-rich foods, including meringue-based confectionery (MBC) In this study,
we investigated the effect of Psi on the physical and chemical properties of meringue Meringue was made by whipping
egg white and Suc (at a weight ratio of 1:1) and baking at 93°C for 2 h Thirty percent of the total weight of Suc was
replaced with d-ketohexoses such as Psi, d-fructose, d-tagatose, and d-sorbose The meringues containing d-ketohexoses
had higher specific volume than the meringue not containing d-ketohexoses (Ct-meringue) Baking of meringue caused
differences between Psi and the other d-ketohexose meringues Meringue containing Psi (P30-meringue) had the highest
breaking stress (7.00 × 105
N/m2) and breaking strain (4.40%), resulting in the crunchiest texture In addition, P30-meringue also had the highest antioxidant activity (491.84μM TE/mg-meringue determined by ABTS method) and
was the brownest due to a Maillard reaction occurring during baking The replacement of Suc with Psi improved the
characteristics of baked meringue Thus, Psi was found to be useful in modifying the physical and chemical properties of
MBC
Keywords: d-psicose, egg white protein, meringue, sucrose, sugar replacement
Practical Application: d-Psicose, a noncalorie sweetener with a suppressive effect on the blood glucose level, can be
utilized as a sucrose replacer in sugar-rich foods such as meringue-based confectionery (MBC) d-Psicose creates a
crunchy texture and enhances the antioxidant activity of baked meringue Thus, d-psicose may be useful in modification
of MBC
Introduction
Meringue is a popular aerated confectionery made of egg white
(EW) and sucrose (Suc) Meringue is also an important base
ma-terial of many confectioneries such as souffl´es, macaroons, and
angel food cake in which it provides most of the structural
sup-port (Vega and Sanghvi 2012) EW provides a foamy structure,
while Suc provides sweetness and stabilizes the foamy structure
Since Suc has a high calorie count (4 kcal/g), the excessive
con-sumption of meringue and meringue-based confectionery (MBC)
results in a high calorie intake A high calorie intake can cause
obe-sity, which is a risk factor for type 2 diabetes and coronary heart
disease (Walker 1971; Roberts and Wright 2012; Song and others
2012) Elimination of Suc in meringue and MBC helps reduce the
calorie count, but it will simultaneously cause a loss of sweetness
and sponge texture of the MBC
Low- and noncalorie sweeteners have been used as substitutes
for Suc Recently, rare sugars, defined as “monosaccharides and
their derivatives not commonly found in nature” by the Intl
Soci-ety of Rare Sugars have been introduced as alternative sweeteners
(Levin 2002; Mu and others 2012) d-Psicose (Psi), the C-3 epimer
of d-fructose (Fru), is a rare sugar having a sweetness equivalent
to 70% of that of Suc but a much lower calorie count of
approx-imately 0.39 kcal/g Psi has a suppressive effect on blood glucose
MS 20140657 Submitted 4/18/2014, Accepted 9/30/2014 Authors are with
Dept of Applied Biological Science, Faculty of Agriculture, Kagawa Univ., 2393
Ikenobe, Miki, Kagawa, 761-0795, Japan Direct inquiries to author Hayakawa
(E-mail: hayakawa@ag.kagawa-u.ac.jp).
levels and an inhibitory effect on body fat accumulation (Mat-suo and others 2002; Chung and others 2012; Ochiai and others 2013) Psi has been approved as generally recognized as safe by the U.S Food and Drug Administration (Mu and others 2012)
An application for approval as food for specified health uses has been made to the Japanese Ministry of Health, Labour, and Wel-fare (Hishiike and others 2013) Psi is expensive because of the difficulty in mass production However, the price of Psi has been reduced by the utilization of recombinant enzymes in the produc-tion (Takeshita and others 2000; Izumori 2006) Because of the confirmed health benefits and the lowered price, Psi has a high potential for use as a Suc replacer in MBC
The characteristics of meringue are related to its microstructure which relies on a structure building process (foam formation) and
a structure disruption process (foam drainage) These processes are regulated by the foaming capacity of EW proteins (EWP) and the foam stability brought by the addition of sugar and other ingre-dients (Licciardello and others 2012) There is very limited in-formation of the effects of Psi on the characteristics of meringue
Therefore, we investigate the characteristics of meringue con-taining Psi The effects of Psi were compared with the other 3 d-ketohexoses, Fru, d-tagatose (Tag), and d-sorbose (Sor)
Materials and Methods
Materials and chemicals
Psi was obtained from Izumoring Co., Ltd (Kagawa, Japan)
Tag, Fru, and Sor were obtained from the Kagawa Rare Sugar Re-search Center 2,2’-Azinobis (3-ethlybenzothiazoline-6-sulfonic C
Trang 2acid) (ABTS) was purchased from Wako Pure Chemical Industries
Ltd (Osaka, Japan) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was
purchased from Nacalai Tesque Inc (Kyoto, Japan) Fresh chicken
egg and Suc were obtained from a local supermarket All other
chemicals were analytical grade
Preparation of meringue
Meringue was prepared as follows: EW (50 g) was whisked
to soft peaks by using a hand mixer (SHM-10H, Shinwa
Trad-ing Co., Ltd., Tottori, Japan) at a speed settTrad-ing of 3 for 2 min
Then, mixed sugars (0%, 10%, 20%, 30%, 40%, and 50% [w/w]
d-ketohexose/[Suc + d-ketohexose]) (50 g) were gradually added
while whipping for 13 min The foams were then placed into
circular-shaped silicone mold (dia 35 mm with 20 mm height) and
leveled off using a spatula Then, the foams were baked at 93°C
for 2 h in an oven (SCOB-4.5MP, Nichiwa Electric Corp., Osaka,
Japan) The control meringue (Ct-meringue) was the meringue
that did not contain d-ketohexoses for sucrose replacement
Determination of foaming capacity
EW–sugar solutions were prepared by carefully dissolving mixed
sugars (50 g) into EW (50 g) to avoid foam formation Then,
the foaming capacity was determined according to the method
described by Phillips and others (1990) EW–sugar solutions were
poured into a 30 mL measuring cup and weighed Then, the EW–
sugar solutions were whipped at a speed setting of 3 for 15 min
using the hand mixer The resultant foam samples were transferred
back into a 30 mL measuring cup Then, the foam was leveled off
using a spatula and weighed The percent overrun was calculated
using the following equation:
Overrun (%)=
(weight of egg white− sugar solution (g) − weight of whipped foam (g))
weight of whipped foam (g)
×100
Microstructural observation of meringue
Baked meringues were cut into small pieces (10 mm width× 10
mm length× 2 mm height) using a razor blade Then, the small
pieces of meringue were mounted on aluminum stubs (Nisshin
EM Corp., Tokyo, Japan) with electrically conducting carbon tape
(Nisshin EM Corp.) and dried using a vacuum freeze dryer The
dried pieces of meringues were coated with gold using a metal
coater (smart coater, JEOL Ltd., Tokyo, Japan) and observed using
a scanning electron microscope (JCM-6000, JEOL Ltd.) operated
at 15 kV The size of the observed meringue air bubbles was
measured manually on image of baked meringues
Determination of rheological properties
Breaking stress and strain of baked meringue were determined
by penetration test using a Rheoner RE-3305 creep meter
(Ya-maden Co., Ltd., Tokyo, Japan) equipped with a 3-mm-dia rod
plunger and a 2 kgf load cell The 20-mm height meringue was
measured at a penetration speed of 1 mm/s Stress and strain of
baked meringue were calculated using the following equation:
Stress (N/m2)= F (N)
A(mm2)× 10−6
A =π
a (mm)
2
2
Strain(%)=
H0(mm)− H(mm)
H0(mm)
× 100
where F is a compressive force applied to baked meringue, A is
the contact surface area before the plunger penetrates into baked
meringue, a is the diameter of the rod plunger, H0is the original
height of baked meringue, and H is the height of baked meringue
after applying compressive force Breaking stress and strain were obtained from the top of the 1st peak of stress against strain curve (see an example of stress–strain curve of baked meringue shown
in Figure 1)
Specific volume
Specific volume (SV) of baked meringue was measured by the rapeseed displacement method (Sahin and Sumnu 2006) First, the bulk density of rapeseeds was determined Rapeseeds were filled into a known-volume glass container through tapping and smoothing the surface Then the glass container containing
rape-seeds were weighed The densities of the rape-seeds (Ds) were calculated
from the weight of rapeseeds (Ws) and the volume of rapeseeds
placed in container (Vs) as follows:
Ds(g/cm3)= Ws(g)
Vs(cm3) Baked meringue and rapeseeds were then placed together in container and weighed The weight of only rapeseeds placed
together with baked meringue (Wsm) was calculated as follows:
Wsm(g)= Wt(g)− (Wm(g)+ Wc(g)),
where Wt is the total weight of baked meringue and rapeseeds
placed together in container, Wmis the weight of baked meringue,
and Wc is the weight of the container The volume of the
rape-seeds placed together with baked meringue (Vsm) was calculated
as follows:
Vsm(cm3)= Wsm(g)
Ds(g/cm3)
The volume of baked meringue (Vm) was calculated as follows:
Vm(cm3)=Vc(cm3)− Vsm(cm3)
SV was calculated as follows:
SV(cm3/g) = Vm(cm3)/Wm(g)
Determination of thermal denaturation
The thermal denaturation of EWP in the presence of mixed sugars was determined by differential scanning calorimetry (DSC; Micro DSC VII, Setaram Inc., Caluire, France) The samples were
Trang 3prepared by dissolving Suc (0.7 g) and d-ketohexoses (0.3 g) into
EW (1.0 g) The sample (0.4 g) and deionized water (0.4 g) were
placed in a sample pan and a reference pan, respectively The
pans were put into DSC vessels and heated at the heating rate of
0.5°C/min from 25 to 120 °C in an N2atmosphere The
calori-metric data were analyzed using thermal analysis software from
Setaram Inc
Determination of meringue color
The color of baked meringue was determined using a
colorime-ter (ND-300A, Nippon Denshoku Industries Co., Ltd., Tokyo,
Japan) The color was evaluated using the Hunter L, a, b color
scale This color scale is based on 1 channel for luminance or
lightness (L) and 2 color channels (a and b) The a-axis extends
from green (−a) to red (+a) and the b-axis from blue (−b) to
yellow (+b) Each piece of meringue was rotated 90° 4 times and
measured at each position
Determination of antioxidant activity
Antioxidant activity of baked meringue was determined by
measuring ABTS and DPPH radical scavenging activities of
ethanol extract from baked meringue Ethanol extract of baked
meringue was prepared according to the method described by
Sun and others (2008) Baked meringue (2.5 g) was suspended
in 99.5% ethanol (10 mL) and homogenized at a speed setting
of 7 for 1 min using a POLYTRON R PT 10–35 homogenizer
(Kinematica AG, Luzern, Switzerland) The slurry sample was
centrifuged at 10,000 × g and 4 °C for 20 min to remove
the insoluble materials The supernatant was collected and
di-luted by 16 times using 99.5% ethanol Then, the didi-luted baked
meringue extract was used as a sample for determining antioxidant
activities
The diluted baked meringue extract (0.3 mL) was mixed
with the ABTS working solution (2.7 mL) The absorbance
at 734 nm was recorded after incubation for 1 min The
rel-ative antioxidant activity was calculated using the following
equation:
ABTS radical scavenging activity (%)
=
Absorbanceblank− Absorbancesample
Absorbanceblank
× 100
The diluted baked meringue extract (0.5 mL) was also mixed with 0.125 mM DPPH in 99.5% ethanol (2.0 mL) After being left
in the dark for 30 min, the absorbance of the samples was measured
at 517 nm The relative antioxidant activity was calculated using the following equation:
DPPH radical scavenging activity (%)
=
Absorbanceblank− Absorbancesample
Absorbanceblank
× 100
The ABTS and DPPH radical scavenging activities of meringues were expressed as micro molar of Trolox equivalents (TE) per milligram of baked meringue (μM TE/mg meringue).
Statistical analysis
Analysis of variance (ANOVA) was performed using the SPSS 15.0 statistical analysis system (SPSS Inc., Chicago, Ill., U.S.A.),
and the Duncan multiple range test (P < 0.05) was used to
deter-mine the statistical significance
Results and Discussion
Preliminary preparation of Psi meringue
The study was conducted in 2 steps The 1st step was to deter-mine the Psi replacement ratio that yields meringue with desired properties The 2nd step was to investigate the effect of Psi on physical and chemical properties of meringue Meringues were initially prepared using Suc with a partial replacement with Psi (Psi replacement ratio, 0–50%) The meringues, where the Psi replacement ratio was 40% or more, showed undesirable qualities such as a wet surface and chewy texture The Psi replacement
Figure 1–An example of stress against strain curve of baked meringues.
Trang 4ratio had to be less than 30% to obtain a desirable dry surface
Therefore, meringues of which 30% Suc was replaced with Psi
and other d-ketohexoses were employed in this study
Physical properties of Psi meringue
Foamy structure is an important characteristic of meringue, so
the foaming capacity of EW was determined in the presence of
sugars (Figure 2) EWs having a replacement ratio of 30% had
a higher percent overrun than EW containing only Suc (Ct)
The meringues replaced with Psi (P30), Tag (T30), and Sor (S30)
showed significantly higher percent overruns than that with Fru
(F30) In general, lowering the interfacial tension of water phase
of water–air system results in increasing foaming capacity of EWP
(Foegeding and others 2006) Thus, Psi, Tag, and Sor may be
superior to Fru in lowering the interfacial tension of EWP foam
Even though the reason why d-ketohexoses increase foaming
capacity is not clear, we suppose that d-ketohexoses might bind
to EWP through hydrogen bonding, leading to a change in
sur-face hydrophobicity of EWP and the increase in foaming capacity
(Sun and others 2008) Hydrogen bonding network of hydroxyl
(OH) groups of saccharide and protein was affected by binding
orientation and binding energy (Toone 1994) The orientation of
OH groups of each ketohexose are different (Fukada and others
2010) The difference in orientation of OH groups contributes
to the difference in hydrogen bonding orientation and binding
distance between D-ketohexoses and EWP Thus, the position of
OH groups of these sugars possibly has influence on the interaction
between EWP and sugar
Physical properties of baked meringue containing Psi
Meringue is subjected to heat treatment, namely baking, to set
the foam structure by converting the foam from a liquid to a
solid state Baking induces water evaporation from meringue and
EWP denaturation, leading to the formation of rigid structure of
baked meringue Additionally, baking of meringue causes a gas
expansion of EW foam, resulting in an increase in the volume of
meringue body Figure 3 shows the SV of baked meringues
pre-pared using the mixed sugars (30% (w/w) ketohexose/[Suc +
d-ketoheoxse]) P30-, T30-, and S30-meringues had higher SV than
F30-meringue and Ct-meringue These results show that Psi, Tag,
a b
0 50 100 150 200 250 300 350 400 450 500
Ct F30 P30 T30 S30
Figure 2–Percent overrun of EW-sugar solution containing 30% D
-ketohexoses Data are presented as mean± SD (n = 6) and different
superscript letters (a to c) show the significant difference (P < 0.05).
and Sor have a higher expansion ratio of a gas in the foam network
of meringue than Fru and Suc
Baked meringue was freeze-dried before observed by a scan-ning electron microscope (SEM) in order to completely eliminate moisture content of baked meringue Freezing or freeze-drying might have only little impact on the structure of meringue be-cause of 2 reasons The 1st reason is the absolutely low moisture content of baked meringue (2.5–4%) Water in structure of baked meringue could not form large ice crystal that destroys the baked meringue structure during freezing In addition, the sublimation
of only small amount of ice crystal could not cause shrinkage of baked meringue structure during freeze-drying The 2nd reason
is the hard and porous structure of baked meringue In general, hard and porous structure in foods is stable and retards shrinkage during freezing or freeze-drying
The microstructure of baked meringue was observed by SEM and the air bubble size of the baked meringues was estimated from SEM images (Figure 4) Baked Ct-meringue had thin and rough meringue matrix and the largest-sized air bubbles (500μm) In
contrast, baked d-ketohexose meringues (F30, P30, T30, and S30) had thick and smooth meringue matrix and small-sized air bubbles (250, 125, 125 to 250, and 200 to 250μm, respectively) However,
we still think that there is no impact of freezing/freeze-drying on structure of baked meringue We have 2 reasons supported our opinion
The difference in the gas expansion and microstructure of baked meringues may relate to thermal denaturation of EWP Thus, ther-mal denaturation temperatures of EWP were determined by DSC (Table 1) EW showed 2 main endothermic peaks with peak
tem-perature (T d) at 64.51 and 78.00°C, corresponding to the denat-uration of ovotransferrin and ovalbumin, respectively (Rossi and
Schiraldi 1992) The T d values were increased by the addition of sugars by 9.8 to 11.2°C and 11.9 to 13.0 °C for ovotransferrin and ovalbumin, respectively The results imply that each sugar increased the heat stability of ovotransferrin and ovalbumin The stabilizing effect of sugar for the 2 proteins was approximately 1°C smaller
in P30- and T30-EWs than in Ct-, F30-, and S30-EWs, suggest-ing that P30- and T30-mersuggest-ingues, compared to Ct-mersuggest-ingue, induced protein denaturation at ca 1°C lower temperature in the baking process
0 1 2 3 4 5 6 7 8 9 10
Ct F30 P30 T30 S30
3 /g)
Figure 3–SV of meringue prepared using sugar containing 30% D -ketohexoses Data are presented as mean± SD (n = 9) and different
small superscript letters (a to b) show the significant difference (P < 0.05).
Trang 5The results of denaturation temperature indicate that EWP in
Ct-meringue were denatured at the highest temperature This
suggests that it was the most difficult to form rigid structure of
Ct-meringue Thus, the foam structure of Ct-meringue may have
collapsed because of disproportionation due to the increase in
Laplace pressure and overexpansion of gas during baking This
foam collapse results in the low SV, the large-sized air bubble,
and thin and rough meringue matrix of Ct-meringue It was
eas-ier to form rigid structure of F30- and S30-meringues than
Ct-meringue, but more difficult than T30- and P30-meringues This
solid structure formation results in smoother and thicker meringue
matrix compared to that of Ct-meringue The results of
denat-uration temperature also suggest that F30- and S30-meringues
should have higher SV than Ct-meringue However, only S30-meringue, not F30-S30-meringue, had higher SV than Ct-meringue
This may be due to the difference in air content (overrun) of F30-and S30-meringues Since the air content of S30-meringue was higher than that of F30-meringue (Figure 2), gas expansion rate
of S30-meringue is higher than that of F30-meringue, probably resulting in the higher SV of S30-meringue On the other hand, T30- and P30-meringues formed the solid structure the most eas-ily Thus, T30- and P30-meringues had small-sized air bubbles and thick and smooth meringue matrix In addition, air bubbles
of T30- and P30-meringues were smaller than that of S30- and F30-meringues T30- and P30-meringues had high SV because they had more air content than Ct- and F30-meringues
Figure 4–SEM observation of baked meringue prepared using sugar containing 30% D -ketohexoses Images were observed
at the resolution 27 (left) and 150 (right).
Trang 6Table 1–Thermal denaturation temperatures of EW proteins in the presence of different sugars determined by DSC.
Data are presented as mean± SD (n = 3) and different small superscript letters (a to e) show the significant difference (P < 0.05).
Table 2–Breaking stress and strain of baked meringue prepared
using sugar containing 30% D-ketohexoses.
Sample Breaking stress (10 5 N/m 2 ) Breaking strain (%)
Data are presented as mean± SD (n = 24) and different small superscript letters (a to d)
show the significant difference (P < 0.05).
Breaking stress and strain of baked d-ketohexose meringues are
shown in Table 2 The breaking stress and strain indicate
hard-ness and retard the deformation of baked meringue, respectively
The baked d-ketohexose meringues (F30, P30, T30, and S30) had
30% to 72% higher breaking stress than the baked Ct-meringue
Particularly, the breaking stress of P30-meringue was
outstand-ingly high, showing that the addition of Psi hardened the baked
meringue The high breaking stress of d-ketohexose meringues is
closely related to the thick meringue matrix seen by scanning
elec-tron microscopy (SEM) observation (Figure 4) The size of the air
bubbles might also affect the hardness of the baked meringue The
results of the breaking stress suggest that the formation of
small-sized air bubbles creates resulting hard meringue A structure of
baked meringue with large-size air bubble could fracture more
easily than that with small-size air bubble when a compressive
force is applied to meringue
For breaking strain, P30-meringue also showed the highest
value P30-meringue showed a higher breaking strain than Ct-,
F30-, T30-, and S30-meringues, suggesting that Psi, compared to
the other sugars including Suc, makes meringue harder to
crum-ble The overall results from the breaking test clearly shows that the
30% replacement of Suc with Psi in meringue causes a crunchy
texture It is of great interest that baking of meringue widens the
difference in the physical properties between Psi and the other
d-ketohexoses
Chemical properties of baked meringue containing Psi
Maillard reaction (MR) is a spontaneous reaction between
amino group of protein and carbonyl group of reducing sugar MR
begins with a condensation reaction of primary amino groups of
protein with carbonyl group of sugar to form Schiff base products
that are rearranged to Amadori/Heyn products Amadori/Heyn
products are degraded and then formed brown, water
insolu-ble compounds known as melanoidins (Liu and others 2012)
Melanoidins have the antioxidant activity due to their
reduc-tone groups having reducing activity and metal chelating ability
(Namiki 1988) Suc, a nonresucing sugar, can cause MR after
hydrolysis of disaccharide (Hodge and Osman 1976) Thus, MR
with Suc is difficult to occur compared to reducing sugar such as d-ketohexoses
It was considered that baked meringue would exhibit high antioxidant activities because of browning substances such as melanoidins generated by MR in baking process Baked meringues containing 30% d-ketohexoses, especially Psi, had higher antioxi-dant activities than Ct-meringue as detected by ABTS (Figure 5a) and DPPH (Figure 5b) radical scavenging methods Of all the meringue prepared, P30-meringue had the highest radical scav-enging activities This is possibly due to that d-ketohexoses cause
MR easily compared to Suc
Baking of meringue also generated color change (whitish to
brownish) L-, a-, and b-values of meringue are shown in Table 3 Ct-meringue had the highest L-value whereas P30-meringue had
the lowest one This indicates that P30-meringue is darkest The
a b
e d c
0 100 200 300 400 500 600
Ct F30 P30 T30 S30
A
a b
c
0 100 200 300 400 500
Ct F30 P30 T30 S30
B
Figure 5–Antioxidant activities of baked meringue prepared using sugar containing 30% D -ketohexoses determined by ABTS (a) and DPPH (b) methods Data are presented as mean± SD (n = 3) and different small
superscript letters (a to d) show the significant difference (P < 0.05).
Trang 7Table 3– −Color value of baked meringue prepared using sugar
containing 30% D-ketohexoses.
Data are presented as mean± SD (n = 16) and different small superscript letters (a to c)
show the significant difference (P < 0.05).
a-value of all meringues was positive, which indicates that all
meringues are tinged with red P30-meringue had the highest
a-value while Ct-meringue had the lowest one The b-value of
all meringues was positive, which indicates that all meringues are
tinged with yellow P30-meringue had much higher b-value than
Ct-meringue Overall, the results of L-, a-, and b-values indicate
that meringues containing 30% d-ketohexoses were browner than
Ct-meringue; especially P30-meringue was the brownest This
strong brownish color of baked P30-meringue will be responsible
for the high antioxidant activity seen in P30-meringue It also
implies that MR proceeds fast with Psi compared to the other
d-ketohexoses
Conclusion
The replacement of Suc with d-ketohexoses caused increase
in foaming capacity and decrease in heat denaturation
tem-perature of EWP These results seem to induce difference in
microstructure and SV of baked meringues Baked meringue
containing d-ketohexoses had smaller size air bubble compared
to the counterparts not containing d-ketohexoses The different
microstructures (e.g., air bubble size) of baked meringues led the
different texture—the meringues having smaller size air bubble
have crunchier texture In addition, d-ketohexoses enhanced
the antioxidant activity of baked meringues Psi-meringue had
the crunchiest texture and highest antioxidant activity Thus,
Psi may be helpful for modifying functional properties of baked
meringue
Acknowledgments
This work was partially supported by the City Area Program, the
Ministry of Education, Culture, Sports, Science and Technology,
Japan The authors also thank Peter Lutes, associate professor of
Faculty of Agriculture, Kagawa Univ., for language editing
Author Contributions
S O’Charoen planned the experiments, collected test data, did the statistical analysis, and drafted the manuscript Y Matsumoto collected test data S Hayakawa designed the study and interpreted the results M Ogawa planned the experiments, interpreted the results, and drafted the manuscript
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