Scientia Horticulturae 247 (2019) 101–106

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Scientia Horticulturae
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Effect of maturity and after-ripening on the formation of gel in the syrup T

made from Japanese apricot ‘Suiko’ fruits
⁎
Yasuhisa Tsuchidaa, , Sayo Onishib, Nobuki Gatob, Yoshiaki Nakaa, Takaaki Oea, Noriaki Jomuraa
a
Japanese Apricot Laboratory, Wakayama Fruit Tree Experiment Station, Minabe, Wakayama, 645-0021, Japan
b
Food Science Research Laboratory, Nakano BC Co. Ltd., Kainan, 642-0034, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: To process clear syrup from ‘Suiko’ Japanese apricots, the effect of fruit changes on the amount of gel with pectin
After-ripening characteristics formed upon natural ripening or after-ripening was investigated. Little gel was formed in the
Gel syrup extracted from early-mature fruits subjected to after-ripening for 0 to 4 days, and from moderately mature
Pectin fruits subjected to after-ripening for 0 to 2 days; conversely, a large amount of gel was formed in the syrup
Prunus mume Siebold & zucc.
extracted from fully mature drop fruits. Uronic acid was found at a higher concentration and had a higher degree
Syrup
of esterification in the gel than in the liquid phase. Moreover, the gel contained larger amounts of high-mole-
cular-weight polysaccharides than the liquid phase. In raw fruits, an increase in the duration of natural ripening
and after-ripening reduced high-molecular-weight water-soluble pectin content, and increased the low-mole-
cular-weight polymer. This suggests that low-molecular-weight pectin would actively convert into syrup in ri-
pened fruits, while high-methoxyl pectin would recombine under conditions of low pH and high soluble solid
content, thus resulting in gel formation. We conclude that early-matured fruits after a 4-day after-ripening
treatment were best suited for processing because they showed the least gel formation in the clarified syrup,
while preserving aroma.

1. Introduction
In Japan, most Japanese apricot (Prunus mume Siebold et Zucc.)
fruits are processed to prepare pickle, liquor, juice, and confectionery
because the raw flesh is too sour to eat due to its high content of citric
acid. In Wakayama prefecture, the yield of Japanese apricot fruits is the
largest in the country (MAFF statistical report, 2016). One of the main
cultivars in Wakayama prefecture is ‘Nanko’, which occupies 85% of
the apricot producing area; fruits of this cultivar are mainly processed
to pickled ume (Umeboshi in Japanese) consumed as part of the rice
meal. However, the decline of the rice-based diet has reduced con-
sumption of pickled ume, causing a severe fall of the market price of
‘Nanko’ apricots. A solution to this dilemma requires introducing other
apricot cultivars that show attractive characteristics and that can be
processed to obtain other products for the market.
Japanese apricot ‘Suiko’ is a cultivar developed at the Institute of
Fruit Tree and Tea Science, NARO in 2009 (Yaegaki et al., 2014).
‘Suiko’ apricots emit a pear-like fragrance that is enhanced during
maturation and may be further induced by after-ripening. ‘Suiko’ fruits
are not suitable for processing into pickled ume because of the frequent
occurrence of gumming; instead, it is well suited for beverage
production. The syrup made with sugars extracted from ‘Suiko’ fruits by
osmotic pressure shows fruity flavour. However, a gel substance re-
garded as an impurity in manufacture often develops in the syrup.
Pectins are long-chain polysaccharides that cause coagulation and
haze formation in the extracted juice, making it highly viscous, thus
rendering subsequent clarification processing by filtration rather diffi-
cult (Rai and De, 2009), which in turn results in important yield losses.
Based on these reports and our knowledge of the syrup making process,
we hypothesised that the gel that forms in ‘Suiko’ syrup should consist
of water-soluble pectin (WSP) polysaccharides.
Pectin is thought to consist mainly of uronic acid (UA) units joined
in chains by α-(1–4) glycosidic linkages (Mukhiddinov et al., 2000). UA
residues on the pectin backbone are esterified to varying degrees,
usually with methanol. The ratio of methyl esterified UA groups to total
UA groups is termed the degree of methyl esterification (DE) (Brejnholt,
2010; Srivastava and Malviya, 2011). Pectins are divided into high-
methoxyl (HM) and low-methoxyl (LM) pectins (Sriamornsak, 2003;
Baker et al., 2005). HM pectin undergoes gel formation in the presence
of high sugar (sucrose) content under acid conditions (Baker et al.,
2005). Not only DE, molecular weight may also affect the gelling
properties of pectin (Rakitikul et al., 2016).

⁎
Corresponding author.
E-mail address: tsuchida_y0001@pref.wakayama.lg.jp (Y. Tsuchida).

https://doi.org/10.1016/j.scienta.2018.12.005
Received 30 July 2018; Received in revised form 1 December 2018; Accepted 6 December 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Y. Tsuchida et al.
Contents and molecular weight of pectin in Japanese apricot fruits
changes with growth stage (Otoguro and Kaneko, 2004; Tsuchida et al.,
2014). In addition, storage duration changes the amount and molecular
weight of pectin polysaccharides in many fruits (McCollum et al., 1989;
Siddiqui et al., 1996; Sakurai and Nevins, 1997; Terasaki et al., 2001;
Tsuchida et al., 2004). These reports advocate the need for elucidating
the effects of fruit ripening conditions on the characteristics of the gel
forming in the syrup.
In this study, we investigated the relationship between gel char-
acteristics in the syrup (amount, pectin volume, molecular weight, and
DE) and characteristics of ‘Suiko’ fruits (pectin volume and molecular
weight), as they change during the progress of natural maturation and
upon when the fruits are subjected to after-ripening.
2. Materials and methods
2.1. Fruit sampling and after-ripening
Nineteen-year-old Japanese apricot ‘Suiko’ trees planted in the open
at the Japanese Apricot Laboratory in Minabe, Wakayama prefecture
(33°82′ N, 135°35′ E) were used for fruit sampling. Fruits were collected
to represent fruit maturity stages: early-mature (June 10), moderately
mature (June 13) and drop at full maturity (June 17). Early-mature
fruits were obtained when 30% surface of the fruit skin showed lustre
caused by falling trichomes, based on visual assessment, demonstrated
by Oe et al. (2012). Drop fruits were obtained just after dropping.
Moderately mature fruits were obtained at harvest midterm, between
early-mature and drop fruit stage.
After harvest, fruits (approximately 1 kg) were subsequently ripened
by incubation at 20 °C. Ripening treatment was applied for 0, 2, 4, or 6
days to early-mature fruits; moderately mature fruits were ripened for
0, 2, or 4 days, while drop fruits were ripened for 0 or 2 days. Four
fruits were sampled from each group to determine fruit firmness,
polysaccharide content and molecular weight analysis in the flesh,
while the rest of the sampled fruits were stored at −30 °C until syrup
extraction. Fruit firmness was measured by rheometry (COMPACK-100;
Sun Scientific, Tokyo, Japan) as the index of ripeness, because this
parameter decreases with maturation (Tsuchida et al., 2014). Firmness
measured by rheometry was defined as the maximum load by moving a
fruit on the stage to the columnar probe (5 mm in diameter) upward at
a speed of 1 mm·s−1 into the fruit to a depth of 1 mm.
2.2. Extraction and contents measurement, and molecular weight analysis
of water-soluble pectin polysaccharides (WSPs) from raw fruits
WSPs were extracted according to the method described by Sakurai
and Nevins (1997). Briefly, 7-g samples of flesh (mesocarp) from four
fruits were chopped with a knife. Next, samples were boiled for 15 min
in 80% EtOH, and then grinded with a glass homogeniser (model HOM;
As one, Osaka, Japan). Homogenates were centrifuged for 10 min at
30,000× g (CR 21E Centrifuge; Hitachi Koki, Ibaraki, Japan); the su-
pernatants containing monosaccharides or disaccharides were removed.
Precipitates were dispersed in Na-acetate buffer (pH 6.0) and incubated
for 2 h at 80 °C with α-amylase (Bacillus amyloliquefaciens α-amylase;
Sigma Aldrich, St. Louis, MO, USA). The mixtures were centrifuged
(10 min at 30,000 × g) and supernatants were discarded to remove
starch. Precipitates were dispersed in 10 mL of distilled water, heated
for 10 min at 100 °C and centrifuged for at 30,000 × g for 10 min; this
treatment was repeated three times. The combined supernatants were
designated as the WSP fraction. WSP fractions were subjected to the m-
hydroxydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973) to
determine UA content. Three millilitres of sulfuric acid/sodium tetra-
borate solution was added to 0.5 mL of diluted WSP fractions in the
tubes refrigerated in crushed ice. The mixture was shaken with a tube
mixer then the tubes heated in a water bath at 100 °C for 10 min. After
cooling in a water-ice bath, 50 μL of the m-hydroxydiphenyl reagent
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Scientia Horticulturae 247 (2019) 101–106
(0.15% solution of m-hydroxy-diphenyl in 0.5% NaOH) was added. The
tubes were shaken, and absorbance measurements made at 520 nm
using a spectrophotometer (V-550; JASCO, Tokyo, Japan). Data were
analysed for statistical significance by Tukey’s multiple-range test.
Molecular weight analysis of polysaccharides in the WSP fraction
was conducted by the following method. WSPs were injected into an
HPLC (model LC-10AD; Shimadzu, Kyoto, Japan) equipped with a gel-
filtration column (TSK-Gel G5000 PW, 7.5 mm × 30 cm; Tosoh,
Yamaguchi, Japan). The samples were eluted with 50 mM Na-phos-
phate buffer (pH 7.2) at a flow rate of 0.5 mL·min−1. Fractions were
collected at 0.5-min intervals and then UA contents at each retention
time (Rt) were estimated by the method described above. Standard
dextrans of known molecular weights were run through the column for
molecular calibration.
2.3. Making syrup from fruits
Three sets of approximately 300 g of frozen fruits from each after-
ripening treatment were placed in a 4-L glass jar and filled with
granulated sugar. Osmotic pressure extracted fluid from fruits, which
melt the granulated sugar. Syrup making was completed after 7 d with
fully melting of the granulated sugar. Syrup was double-boiled for
sterilisation until its temperature reached 85 °C.
2.4. Gel amount and polysaccharides analysis of the syrup
Syrup (10 mL) was centrifuged for 10 min at 30,000 × g and di-
vided into the precipitated gel and the liquid phase. The weight of the
gel was measured; then, gel and liquid phase were sealed in a dialysis
membrane (MWCO: 3.5 kD; Spectrum Laboratories, Inc., Rancho
Dominguez, USA) and dialysed for 3 d against distilled water to elim-
inate monosaccharides or disaccharides. The content of UA in the dia-
lysates was estimated by the method mentioned above. Data were
analysed for statistical significance by Tukey’s multiple-range test.
Subsequently, lyophilizates of the gel and the liquid phase were dis-
solved with 50 mM Na-phosphate buffer (pH 7.2) and introduced into
an HPLC system and molecular weight distribution was analysed by the
method described above.
2.5. Determination of the DE in the syrup
Methyl ester content of pectin in the gel and liquid phase was
analysed by the method described by Klavons and Bennett (1986).
Dialysates prepared as described above were added with 1.0 M KOH to
be hydrolysed at 20 °C. After 30 min the hydrolysates were titrated with
diluted phosphoric acid to pH 7.5, the optimum pH of alcohol oxidase.
Alcohol oxidase from Pichia pastoris (Sigma Chemical, St. Louis, USA)
was added to the hydrolysates and standard methanol solution diluted
with potassium phosphate buffer in a range from 0 to 0.002% at a rate
of 0.0005% and incubated at 25 °C for 15 min. Next, 0.02 M 2, 4-pen-
tanedione eluted in 2.0 M ammonium acetate-0.05 M acetic acid com-
pounded solution as colouring reagent was added and the mixture in-
cubated at 60 °C for 15 min. Absorbance at 412 nm was measured to
determine methanol concentration in the pectin samples. DE was cal-
culated as the percentage of molar number of methanol relative to that
of UA. Data were arcsine transformed and analysed for significant dif-
ferences between gel and liquid phase by Student´s t-test.
3. Results
3.1. Changes in fruit firmness and content and molecular weight analysis of
pectin polysaccharide fraction from flesh
Fruit firmness of ‘Suiko’ apricots steadily decreased as maturity
progressed with ripening date (Fig. 1).
UA content in the WSP from the fruits showed a declining trend
Y. Tsuchida et al.
Fig. 1. Changes in fruit firmness of Japanese apricots ‘Suiko’. Data are means
( ± SE) of four measurements. Vertical bars indicate standard errors.
Fig. 2. Uronic acid (UA) content in water-soluble pectin (WSP) extracted from
Japanese apricot ‘Suiko’ fruits. Data are the mean ( ± SE) of four measure-
ments. Different letters indicate significant differences at the 5% level by
Tukey’s multiple-range test.
with ripening (Fig. 2). An increase in the number of after-ripening days
also reduced UA content in all but not drop fruits.
High-molecular-weight UA polymer content in WSP was largest in
early-mature fruits (Fig. 3). Maturity reduced the content of the high
molecular-weight UA polymer (retention time [Rt] 12–19 min) and
increased that of the low molecular-weight polymer (Rt, 24–29 min).
After-ripening accelerated the increase in low-molecular-weight UA
polymer, especially in drop fruits.
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Scientia Horticulturae 247 (2019) 101–106
Fig. 4. Gel formation amount in the syrup made from Japanese apricot ‘Suiko’
fruits. Data are means ( ± SE) of three measurements. Gel amount was de-
termined by weighing precipitate obtained from centrifugation of syrup.
Different letters indicate significant differences at the 5% level as per Tukey’s
multiple-range test.
3.2. Gel amount and pectin content in the gel
Gel formation in the syrup steadily increased with after-ripening
treatment progress; this increment was accelerated as fruit maturity
advanced (Fig. 4). Early- and moderately mature fruits were ripened by
incubation for 0–4 d and 0–2 d at 20 °C, respectively, and produced gel
in such minute amounts that they could hardly be detected visually; in
contrast, drop fruits produced large amounts of gel, regardless of after-
ripening treatment.
UA content in the gel (Fig. 5) followed the same trend as gel for-
mation. Thus, UA contents in the early-mature fruits subjected to after-
ripening for 0–4 d and in the moderately mature fruits subjected to
after-ripening for 0–2 d, were significantly low; however, it increased
dramatically thereafter. On the other hand, drop fruits accumulated a
large amount of UA in the gel in 0-d after-ripening treated fruits, which
reached the highest level in 2-d after-ripening-treated fruits.
3.3. Molecular weight distribution of pectin in the syrup
Molecular weight distribution profiles of gel and liquid phase from
syrup are shown in Fig. 6. Total peak area represents the amount of UA
extracted from 1-g samples. The amount of high-molecular-weight UA
polymer (Rt, 12–19 min) in the gel was significantly larger than that in
the liquid phase. The high-molecular-weight UA polymer in the gel
decreased with an increase in after-ripening days in early-mature fruits.
Fig. 3. Molecular distribution profile of WSP
from flesh of Japanese apricot ‘Suiko’ fruits.
The fraction was subjected to HPLC with a gel-
filtration column. Left, middle, and right pa-
nels show molecular distribution of Early-ma-
ture, Moderately mature, and Drop fruit, re-
spectively. UA was estimated by the m-
hydroxydiphenyl assay. Total peak area re-
presents the amount of UA extracted from 1 g
FW of tissue. The column was calibrated with
dextrans of known molecular mass (413, 148,
65.5, 39, 9.9 kDa), as shown in the upper hor-
izontal axis.
Y. Tsuchida et al.
Fig. 5. UA content in the gel obtained by centrifugation of syrup extracted from
Japanese apricot ‘Suiko’ fruits. Data are means ( ± SE) of three measurements.
Different letters indicate significant differences at the 5% level as per Tukey’s
multiple-range test.
In moderately mature and drop fruits, the amount of high-molecular-
weight polymer was lower than that in early-mature fruits, and they
remained constant regardless of ripening treatment. UA content in the
liquid phase slightly increased with progress of maturity, while after-
ripening accelerated the increase in UA content in drop fruits.
3.4. DE of pectin in the syrup
DE showed a tendency to be higher in the gel than in the liquid
phase, especially in moderately mature and drop fruits (Fig. 7). DE
ranged from 31.3% to 94.1%, from 42.7% to 69.7%, and from 48.6% to
49.3% in the gel of early-mature, moderately mature, and drop fruits,
respectively; whereas that in the liquid phase ranged from 23.6 to
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Scientia Horticulturae 247 (2019) 101–106
Fig. 7. Degree of esterification (DE) of the UA in the gel and liquid phase of the
syrup extracted from Japanese apricot ‘Suiko’ fruits. ** indicates data of arcsine
transformation were significant different by t-test (n = 3).
76.5%, from 20.7 to 43.5%, and from 22.4 to 33.3%, respectively.
4. Discussion
In raw fruits, firmness decreased with maturation and after-ripening
date (Fig. 1), concomitant with a decrease in high-molecular-weight UA
(Fig. 3). Similar results were shown in a previous report on Japanese
apricot fruits during progress of ripening stage (Tsuchida et al., 2014);
in addition, increase in the low-molecular-weight UA was detected
(Fig. 3). These results suggest that the degradation of polysaccharides
was accompanied by maturation, and that this occurred after the ri-
pening process. Decreased molecular size in pectin increases solubility
(De Veau et al., 1993; Vicente et al., 2007), suggesting that increased
low-molecular-weight pectin in fully ripe fruit easily converts to syrup.
Pectin content in the gel (Fig. 5) correlated with the amount of gel
Fig. 6. Molecular distribution profile of UA in
the gel (left column) and liquid phase (right
column) from syrup made from Japanese
apricot ‘Suiko’. An aliquot of the lyophilised
powder of gel and liquid phase was subjected
to HPLC with a gel-filtration column. Total
peak area represents the amount of UA ex-
tracted from 1 g of gel and liquid phase. The
column was calibrated with dextrans of known
molecular mass (413, 148, 65.5, 39, 9.9 kDa),
as shown right above the upper panels.
Y. Tsuchida et al.
formed in the syrup (Fig. 4). This result suggests that pectin is the major
component of the gel. HM and LM pectin are typically classified DE at
levels higher than 43% and lower than 43%, respectively (Danno,
2011). In the syrup made from moderately mature and drop fruits, DE
in the gel and liquid phase were nearly over 43% and below 43%, re-
spectively (Fig. 7). This result suggests that pectin in the gel should be
HM pectin and that in the liquid phase should be LM pectin at those two
ripening stages. One of the factors that control viscosity of pectin is DE
(Sriamornsak, 2003). Gelation of HM pectin requires low pH and low
water activity, pH must be between 2.5 and 3.8 and the soluble solid
content should be between 55% and 85% (Brejnholt, 2010). As pH is
lowered, ionisation of the carboxylate groups in HM pectin is sup-
pressed, causing a reduction in hydration of the carboxylic acid groups
(Watase and Nishinari, 1993; Srivastava and Malviya, 2011). Under low
ionisation, polysaccharide molecules no longer repel each other over
their entire length and can associate and form a gel (Srivastava and
Malviya, 2011). In addition, sugars stabilise hydrophobic interactions
between the methyl ester groups (Oakenfull and Scott, 1984; Brosio
et al., 1993; Rao et al., 1993). The syrup made from ‘Suiko’ fruits was
about pH 3, due to the abundant citric acid in the pulp, and soluble
solid content was around 55% (data not shown). From these results, HM
pectin from moderately mature and drop fruits in the syrup aggressively
formed the gel under the preferred prevalent conditions.
On the other hand, in the syrup made from early-mature fruits, DE
of the pectin in the liquid phase with after-ripening treatment for 0 to 4
d ranged from 49.5% to 76.5% (Fig. 7), thus being HM category, while
DE of the pectin in the gel made from fruits of day 6 was 31.3%, i.e.
category LM. These contradictory results would be determined by
density and molecular weight of pectin. The viscosity of pectin is re-
ported to increase by raising its concentration and the presence of high
molecular-weight polysaccharides (Brejnholt, 2010). In the liquid
phase, UA concentration was lower than at other ripening stages
(Fig. 6). This result suggests that diffusion of pectin polysaccharides
prevents aggregation. The gel from 6-d after-ripening treatment con-
tained a relatively large amount of high molecular-weight UA (Fig. 6).
This result suggests that even low DE pectin can form gel.
Gel formation occurred to a low extent in the syrup made from
early-mature fruits and from moderately mature fruits that were after-
ripened for 0 to 4 and 0 to 2 days, respectively (Fig. 4). In a previous
study (Katagiri et al., 2017), we observed that syrup made from early-
mature fruits after-ripened for 4 to 6 d produced abundant aroma
components. These results support the conclusion that early-mature
fruits subjected to after-ripening by incubation for 4 d at 20 °C are best
suited syrup production.
‘Suiko’ fruits produce a gel that represents an inconvenience during
syrup making. On the other hand, this cultivar has abundant pectin. To
take advantage of this characteristic, ‘Suiko’ fruits would be adequate
raw material of pectin-rich foods, such as jam. In the future, further
research should aim to expand the industrial applications of this cul-
tivar.
5. Conclusions
The amount of gel formed in the syrup made from Japanese apricots
‘Suiko’ increased with maturity and with the number of days of after-
ripening treatment of raw fruits. This phenomenon was likely caused by
accelerated solubility of water-soluble pectin in the fruits accompanied
by the degradation of polysaccharides. Solubilised high-methoxyl
pectin will recombine under conditions of low pH and high soluble solid
content in the syrup, particularly in fully matured drop fruits, resulting
in the formation of a large amount of gel.
Funding
This research was supported by grants from the Project of the NARO
Bio-oriented Technology Research Advancement Institution (the special
105
Scientia Horticulturae 247 (2019) 101–106
scheme project on regional developing strategy).
Declaration of interests
None.
Acknowledgements
We wish to express our gratitude to Professor Yoshihiko Ozaki in the
Department of Science and Technology on Food Safety of the Faculty of
Biology-Oriented Science and Technology at Kinki University, for his
helpful advice in measuring DE.
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In Japanese with English abstract.