Itoman, Япония
Introduction. Nakamoto Seedless, a variety of shikuwasa (Citrus depressa L.) in Okinawa, can be used to produce vinegar extracts because it has no seeds causing bitter taste. However, Nakamono Seedless is hardly cultivated commercially in Okinawa. This research was aimed to develop vinegar extract from Ogimi Kugani, another major variety of shikuwasa, and compare its characteristics with those of extracts from Nakamoto Seedless. Study objects and methods. The study featured vinegar extracts from the whole shikuwasa of Nakamoto Seedless (20% fruit) and Ogimi Kunagi (5, 10, and 20% of fruit) varieties. The fruit was harvested in June, July, and August. We tested the samples for limonin and polymethoxyflavones content and sensory attributes, especially bitterness. Results and discussion. Vinegar extracts with 20% of Ogimi Kugani harvested in June and July tasted bitter compared to those from Nakamoto Seedless harvested in August, but extracts from Ogimi Kugani harvested in August were not bitter. In addition, 5 and 10% vinegar extracts from Ogimi Kugani harvested in June had lower bitterness. The vinegar extracts from both shikuwasa varieties contained polymethoxyflavones ‒ bioactive compounds ‒ and similar flavor. Conclusion. The whole shiluwasa fruit can be used to produce vinegar drinks, Ponzu soy sauce, salad dressings, etc.
Okinawan citrus, Ogimi Kugani, Nakamoto Seedless, vinegar extract, polymethoxyflavones, sinensetin, nobiletin, tangeretin
INTRODUCTION
Shikuwasa (Citrus depressa L.) is a common citrus
fruit in Okinawa, the southernmost prefecture of Japan.
Shikuwasa is very sour and also called Hirami lemon or
thin-skinned flat lemon. In Okinawan dialect, shikuwasa
means “to eat vinegar”, however it does not contain
vinegar, and the name refers to its strong sour taste. This
native Okinawan citrus fruit became popular all over
Japan and is used to garnish dishes as well as to make
juices and jams.
As it grows, shikuwasa changes color from dark
green to yellow or orange. Unripe (green) fruit is
extremely sour and is used to make condiments. Ripe
(yellow) shikuwasa has better taste from December to
the end of January. Shikuwasa harvested from August
to September is usually used as an acidifying agent in
vinegar substitution. Fruit harvested from September
to December is used in juice production and from
December to January can be eaten in the raw form.
Shikuwasa is mainly cultivated in the northern
part of Okinawa. In 2017, 3398 t of shikuwasa were
produced, with the fruiting area of shikuwasa being
363 ha. Shikuwasa contains high levels of nutritional
components, including polymethoxyflavones [1],
which are well known for their biological activities.
Polymethoxyflavons demonstrate anti-tumor, antiinflammation,
and anti-hyperglycemic properties [1–4].
Therefore, extracting polymethoxyflavones and other
functional components from shikuwasa can contribute to
healthy food products.
Among known varieties of shikuwasa in Okinawa,
Ogimi Kugani (hereinafter Kugani) is the most popular.
Kugani is a commercially available superior
landrace which is widely used in the food industry, fresh or processed [5]. Nakamoto Seedless (hereinafter
Seedless) is a seedless variety of shikuwasa that is
becoming popular, due to its characteristics, which are
similar to those of Kugani [6].
Citrus tree cultivation involves thinning young
fruits to improve fruit size and quality. Thinning is
performed from June to July because shikuwasa is not
typically used during this period. Thinned fruits as
well as normally harvested fruits unsuitable for sale, for
example bruised fruits, should be effectively utilized to
obtain processed food products such as vinegar extracts.
To produce a vinegar extract with low bitterness,
Hirose et al. used only the peels of residual substances
from Kugani, and Hanagasaki et al. used residual
substances from Tankan (Citrus tankan Hayata) [7, 8].
Using this technology, Ponzu sauce (Japanese citrus
vinegar soy sauce) production is already being
commercialized in Okinawa. In addition, the whole
Seedless fruit harvested from June to August is useful to
produce vinegar extract because it has no seeds, contains
low levels of limonin, and has low bitterness [9]. In spite
of the fact that vinegar extract from the whole Kugani
fruit is expected to be bitter because it typically contains
14 seeds per fruit, the potential use of the whole unripe
Kugani should be explored to avoid wastes [6].
The aim of the present study was to develop vinegar
extract from the whole unripe Kugani fruit and compare
the characteristics of this extract with those of Seedless
vinegar extracts to improve the profitability of the fruit.
STUDY OBJECTS AND METHODS
Shikuwasa (Nakamoto Seedless and Ogimi Kugani
varieties) has been cultivated in Okinawa Agricultural
Research Center (26º37’ N, 127º59’ E; 40 masl) since
2005. The Seedless and Kugani fruit under study
were harvested in June 8, July 6, and August 3 (2018),
respectively.
The objectives of the study were vinegar extracts
made from Seedless (20% fruit) and Kugani (5, 10, and
20%). The fruit weight was defined by averaging the
weights of 70~450 fruits, equal to approximately 1 kg.
The fruit diameter was determined by averaging the
diameters of 20 fruits. Moisture content was determined
by calculating the weight difference before and after
freeze-drying.
Production of vinegar extract. As for shikuwasa
of Kugani variety harvested in different months, the
fruit was broken down using a Bamix M200 mixer
(Bamix, Switzerland). Afterwards, a 20% sample
was prepared by stirring 10 g of the fruit and 40 mL
of spirit vinegar (Kraft heinz, USA). Stirring was
performed with a 4 cm stirrer bar at 2.0×g for 10, 30, 60,
and 90 min.
Similarly, we prepared 20% Seedless vinegar
samples. To compare vinegar extracts from Kugani and
Seedless harvested in July and August, the samples
were stirred for 60 min. The mixed samples were
centrifuged at 1190×g for 20 min and filtered using
No. 2 qualitative filter paper (Advantec Co. Ltd., Japan).
To compare vinegar extracts from shikuwasa
harvested in June, we used 20% Seedless as well as 5
and 10% Kugani vinegar samples. The Kugani samples
were obtained by mixing 2.5 or 5 g of Kugani fruit with
47.5 and 45 mL of vinegar and stirring for 60 min.
Freeze-dried powder process. To analyze
polymethoxyflavone and limonin levels in shikuwasa,
the samples were freeze-dried using an FD-550 freezedrier
(Tokyo rikakikai Co. Ltd., Japan). After drying,
the samples were crushed in an IFM-800 mill (Iwatani
Corp, Japan) using a 1.4 mm mesh.
Titratable acidity determination. A volume of
10 mL of the vinegar extracts was mixed with 100 mL
of pure water and titrated with 0.5 mol/L NaOH until
pH 8.2 ± 0.3 using a pH meter. We applied acid-base
titration [10].
Polymethoxyflavone analysis. Polymethoxyflavone
extraction was performed as described by Ichinokiyama
et al. [11]. To extract polymethoxyflavones from
shikuwasa, 100 mg of freeze-dried powder samples
with 1 mL of methanol:DMSO (1:1) were subjected
to ultrasonic wave for 10 min (M1800-J, Japan) and
centrifuged at 2000×g for 2 min. We performed it three
times. Extract solutions were obtained by diluting to a
volume of up to 5 mL.
To obtain polymethoxyflavones from vinegar
extracts, 1 mL of the vinegar extracts with 1 mL of
ethanol were subjected to ultrasonic wave for 30 min.
The insoluble component was removed by centrifugation
at 2000×g for 2 min. The solutions were prepared for
HPLC, namely filtered using a 0.20 μm hydrophilic
PTFE (Advantec Co. Ltd.).
Sinensetin (Wako Pure Chemical Corp., Japan),
as well as nobiletin, and tangeretin (Sigma-Aldrich,
Japan) were dissolved in ethanol to a concentration of
0.1 mg/mL and used as standards. Quantitative
analysis of each polymethoxyflavone was
performed as described by Hirose et al. [7].
The sample solutions (5 μL) were injected onto
a Union UK-C18 HPLC column (3×100 mm,
0.4 mL/min flow rate, Japan) at 40ºC. The solvent
was acetonitrile/water/trifluorasetic acid (50/50/0.05).
Analyses were performed at 340 nm using an
LC-20A UV detector (Shimadzu corp., Japan). Amounts
of polymethoxyflavones were calculated as the sum of
sinensetin, nobiletin, and tangeretin.
Limonoid analysis. To extract limonoid from
shikuwasa, 100 mg of the freeze-dried sample was
mixed with 2 mL of acetic acid and 5 mL of ethyl
acetate and then vortexed for 1 min. To obtain limonoid
from vinegar extracts, 0.4 mL of the vinegar extracts
was mixed with 1 mL of ethyl acetate and then vortexed
for 1 min. Both ethyl acetate phases were collected
after centrifugation at 2000×g for 2 min. These steps
were repeated three times, and the supernatant was
completely evaporated under reduced pressure.
Extract solutions were obtained by reconstituting
the powdered samples with 2 and 0.4 mL of acetonitrile
for shikuwasa and vinegar extract, respectively. The
312
Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316
b
Figure 1 Nakamoto Seedless (a) and Ogimi Kugani (b)
harvested from June to August (from left to right)
Table 1 Characteristics of Nakamoto Seedless
and Ogimi Kugani harvested in different months
Harvesting
date
Variety Fruit
weight,
g
Fruit
diameter,
cm
Moisture
content,
%
June 8, 2018 Nakamoto
Seedless
2.27 1.67 75.9
Ogimi
Kugani
3.51 1.92 80.4
July 6, 2018 Nakamoto
Seedless
5.48 2.18 82.0
Ogimi
Kugani
8.81 2.58 82.0
August 3, 2018 Nakamoto
Seedless
10.07 2.81 83.6
Ogimi
Kugani
15.31 3.24 82.9
a
Table 2 Chemical components of Nakamoto Seedless and Ogimi Kugani harvested in different months
Component Nakamoto Seedless Ogimi Kugani
June July August June July August
Polymethoxyflavones, mg/g d.w. 19.18 ± 1.46a 12.33 ± 0.06b 10.81 ± 0.14b* 17.89 ± 0.66a 12.44 ± 0.08b 7.87 ± 0.04c
Sinensetin 1.50 ± 0.12a 1.04 ± 0.01b* 0.90 ± 0.02b* 1.21 ± 0.05a 0.90 ± 0.01b 0.56 ± 0.01c
Nobiletin 11.01 ± 0.89a 7.31 ± 0.04b 6.22 ± 0.08b* 11.03 ± 0.42a 7.94 ± 0.05b* 4.81 ± 0.03c
Tangeretin 6.66 ± 0.47a 3.98 ± 0.02b* 3.69 ± 0.05b* 5.65 ± 0.20a 3.60 ± 0.03b 2.50 ± 0.02c
Limonin, mg/g d.w. 0.29 ± 0.04 0.18 ± 0.04 0.28 ± 0.03 1.55 ± 0.09a* 0.94 ± 0.03b* 0.68 ± 0.05c*
Nomilin, mg/g d.w. n.d. n.d. n.d. n.d. n.d. n.d.
Data represent mean ± SE (n = 3)
Different letters among the same variety indicate significant differences by Tukey–Kramer’s test (P < 0.05)
Asterisks indicate high significance by Tukey–Kramer’s test (P < 0.05), compared with the other variety in the same month
Polymethoxyflavones represent the sum of sinensetin, nobiletin, and tangeretin
n.d. – not detected
solutions were prepared for HPLC by filtering. Limonin
and nomilin (Wako Pure Chemical Corp., Japan) were
each dissolved in acetonitrile to a concentration of
0.1 mg/mL and used as standards.
Quantitative analysis of limonin and nomilin was
performed as described by Hirose et al. [7]. A volume of
2 μL of the sample solutions was injected into Cadenza
CD-C18 HPLC column (0.4 mL/min, 40ºC). The solvent
was acetonitrile/water/formic acid (40/60/0.1). The
analyses were carried out at a wavelength of 210 nm
using an LC-20A UV detector (Shimadzu corp., Japan).
Sensory evaluation. Vinegar extracts were diluted
10 times with distilled water, and 4% (w/w) caster
sugar was added. Six men and six women aged 20–50
evaluated aroma, bitterness, green smell, and overall
acceptability of the samples using a 5-point scale.
For the shikuwasa aroma, 5 represented the strongest
shikuwasa aroma and 1 represented the weakest
aroma or the strongest vinegar aroma. For bitterness,
5 represented the lowest bitterness and 1 represented the
strongest bitterness. For green smell, 5 represented the
lowest green smell and 1 represented the strongest green
smell. For overall acceptability, 5 represented “like” and
1 represented “dislike”.
RESULTS AND DISCUSSION
Figure 1 demonstrates shikuwasa (Nakamoto
Seedless and Ogimi Kugani varieties) harvested from
June to August. The weight, diameter, and moisture of
both fruit varieties were increasing during the time
(Table 1). The characteristics of Seedless were lower
than those of Kugani.
Changes in chemical components of Seedless and
Kugani with time are shown in Table 2, changes in
chemical components of both extracts with time are
shown in Tables 3 and 4. In both varieties, the content
of polymethoxyflavones was the highest in June
and decreased with time (P < 0.01 for Seedless and
P < 0.001 for Kugani). Comparing polymethoxyflavone
content in both varieties, there was no significant
313
Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316
difference in June, but in August it was significantly
higher in Nakamoto Seedless compared with
Kugani (Table 2). Additionally, polymethoxyflavones
in the vinegar extracts from both varieties significantly
decreased with time (Seedless: P < 0.001, Kugani:
P < 0.001) (Tables 3, 4). However, the recovery ratio of
polymethoxyflavones from material increased with time.
Hence, earlier harvested fruit contain higher amounts
of polymethoxyflavones, which possess bioactive
properties.
Limonin content did not show a decreasing trend in
Seedless, but decreased in Kugani with time (P = 0.217
for Seedless and P < 0.001 for Kugani). Limonin level
Table 3 Characteristics of vinegar extracts from Nakamoto Seedless and Ogimi Kugani harvested in June
Parameter Nakamoto Seedless Ogimi Kugani
20% 20% 10% 5%
Vinegar volume, mL 31.5 ± 0.4c 31.9 ± 0.2c 38.4 ± 0b 42.2 ± 0.2a
Titratable acidity, % 4.70 ± 0.10c 4.82 ± 0b 4.87 ± 0.10b 4.99 ± 0.10a
Polymethoxyflavones, mg/100 mL 33.48 ± 0.13 (25.6)a 27.14 ± 0.30 (26.6)b 13.01 ± 0.19 (12.7)c 7.76 ± 0.02 (7.6)d
Sinensetin 2.89 ± 0.04 (38.2)a 2.20 ± 0.03 (44.0)b 1.10 ± 0.02 (22.1)c 0.63 ± 0.01 (12.7)d
Nobiletin 22.80 ± 0.10 (40.9)a 19.15 ± 0.21 (42.1)b 8.63 ± 0.13 (19.0)c 5.03 ± 0.01 (11.1)d
Tangeretin 7.78 ± 0.02 (23.1)a 5.80 ± 0.07 (24.9)b 3.27 ± 0.05 (14.0)c 2.09 ± 0.01 (9.0)d
Limonin, mg/100 mL 0.79 ± 0.04 (45.7)c 2.73 ± 0.09 (42.9)a 2.55 ± 0.02 (40.1)a 1.31 ± 0.05 (20.1)b
Shikuwasa aroma 2.75 ± 0.21 3.17 ± 0.20 2.83 ± 0.26 2.58 ± 0.25
Green smell 4.08 ± 0.33 3.75 ± 0.24 3.75 ± 0.36 4.25 ± 0.34
Bitterness 3.67 ± 0.30a 2.42 ± 0.33b 3.42 ± 0.28ab 4.25 ± 0.27a
Overall accaptability 3.83 ± 0.20a 2.83 ± 0.24b 3.50 ± 0.19ab 3.67 ± 0.14a
Data represent mean ± SE, n = 3 (except sensory evaluation)
Different letters among each sample indicate significant differences by Tukey–Kramer’s test (P < 0.05)
Polymethoxyflavones represent the sum of sinensetin, nobiletin, and tangeretin
Values in parentheses indicate the recovery ratio of fruit
Table 4 Characteristics of vinegar extracts from Nakamoto Seedless and Ogimi Kugani harvested in July and August
Parameter Nakamoto Seedless Ogimi Kugani Nakamoto Seedless Ogimi Kugani
July August
20%
Vinegar volume, mL 29.8 ± 1.1b 36.4 ± 1.1a 21.8 ± 0.6b 27.5 ± 0.4a
Titratable acidity, % 5.04 ± 0.10a 4.66 ± 0b 5.01 ± 0.10a 4.78 ± 0b
Polymethoxyflavones, mg/100 mL 19.60 ± 0.51 (31.7) 21.42 ± 1.22 (35.7) 17.95 ± 0.06 (48.9)a 15.47 ± 0.12 (55.7)b
Sinensetin 1.71 ± 0.05 (43.9) 1.74 ± 0.10 (55.1) 1.46 ± 0.01 (47.8)a 1.04 ± 0.01 (52.7)b
Nobiletin 13.32 ± 0.34 (48.7) 15.11 ± 0.87 (54.3) 12.12 ± 0.04 (57.5)a 10.32 ± 0.08 (60.8)b
Tangeretin 4.56 ± 0.11 (30.7) 4.57 ± 0.26 (36.2) 4.37 ± 0.02 (34.9)a 4.10 ± 0.04 (46.5)b
Limonin, mg/100 mL 0.58 ± 0.01 (70.9)b 1.26 ± 0.15 (27.3)a 0.92 ± 0.03 (78.8)b 2.85 ± 0.02 (98.2)a
Shikuwasa aroma 2.54 ± 0.18 3.00 ± 0.27 2.46 ± 0.33 2.85 ± 0.35
Green smell 4.08 ± 0.34 3.69 ± 0.30 4.31 ± 0.27 4.65 ± 0.18
Bitterness 3.92 ± 0.23a 3.15 ± 0.27b 4.31 ± 0.28 4.31 ± 0.22
Overall acceptability 3.85 ± 0.15 3.54 ± 0.14 3.62 ± 0.28 3.54 ± 0.22
Data represent mean ± SE, n = 3 (except sensory evaluation)
Different letters among each sample from the same harvest date indicate significant differences by Tukey–Kramer’s test (P < 0.05)
Polymethoxyflavones represent the sum of sinensetin, nobiletin, and tangeretin
Values in parentheses indicate the recovery ratio of fruit
was significantly higher in the Kugani than in Seedless
variety. Nomilin was not detected in both varieties. In
general, the Seedless vinegar extract contained lower
concentrations of limonin than the extract from Kugani.
Changes in polymethoxyflavone and limonin
levels during the stirring process are demonstrated in
Figs. 2 and 3.
The amount of polymethoxyflavones in vinegar
extracts from Kugani harvested from June to August
significantly increased (P < 0.001 in June, P < 0.01
in July, and P < 0.001 in August) during the stirring
process (Fig. 2). Limonin levels in the extracts
significantly decreased in July but increased in August
314
Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316
(P < 0.001 in June, P < 0.001 in July, and P < 0.05 in
August) during the process (Fig. 2).
Limonin levels in the vinegar extract from Kugani
harvested in August were not changed during the
stirring time, whereas those in the extract from the
fruit harvested in June and July significantly decreased
(Fig. 3).
The stirring time of 10–60 min decreased but that of
60–90 min slightly increased the limonin concentration
in the vinegar extracts from Kugani harvested in June
and July. Thus, 60 min was selected as the optimal
stirring time, and this time we used to compare the
limonin level in Kugani extracts with that in Seedless.
We also analyzed 5 and 10% vinegar extracts
from Kugani harvested in June. The obtained vinegar
volumes and titratable acidity significantly increased
with the decreasing fruit concentration. As for
polymethoxyflavones and limonin, their levels depended
directly on the fruit amount (Table 3).
Comparison of the vinegar extracts from Seedless
and Kugani are shown in Tables 3 and 4. There was
no significant difference in vinegar volume in 20%
extracts from Seedless and Kugani harvested in June.
However, the 5 and 10% Kugani extracts demonstrated
significantly higher vinegar volume than the Seedless
extracts in all the months.
The titratable acidity of the 5% Kugani vinegar
extract was the highest in June, whereas that of the
Seedless extract was the lowest (Table 3). In July
and August, the Seedless extract showed higher
titratable acidity compared to Kugani (Table 4). Total
polymethoxyflavone levels in the vinegar extract
from Seedless in June were the highest, while the
5% Kugani extract contained the lowest amount of
polymethoxyflavones.
Furthermore, there was a similar tendency for each
polymethoxyflavone, namely sinensetin, nobiletin, and
tangeretin. In July, there was no significant difference
in each polymethoxyflavone between the Seedless
and Kugani vinegar extracts. In August, the Seedless
vinegar extract demonstrated higher level of these
polymethoxyflavones than the Kugani extract.
Sensory evaluation showed no significant difference
in aroma and green smell among all the samples. The
20% extract from Seedless fruit had significantly higher
bitterness than the 20% Kugani sample in July, but there
was no significant difference in August (Table 4). As for
June, there was no significant difference between the
20% Seedless extract and the 5 and 10% Kugani samples
(Table 3).
Overall acceptability of the Seedless vinegar
extracts was significantly higher than that of the 20%
Kugani samples, but there was no significant difference
between the 5 and 10% Kugani extracts in June. Overall
acceptability of the Seedless and Kugani vinegar
extracts in July and August showed no significant
differences.
As mentioned above, limonin levels in the Kugani
vinegar extract decreased during the stirring process in
June and July but did not change in August (Fig. 3). This
implies that limonin was protected from volatilizing or
other enzymatic reactions during the stirring process.
This can also explain the increasing level of some
components in Kugani harvested from July to August. In
addition, it may also be associated with the fact that the
vinegar extract from Kugani harvested in August was
not bitter despite a high content of limonin, which is a
bitter compound present in the vinegar extract.
Hirose et al. reported that vinegar extract from waste
peel of shikuwasa harvested in October and obtained by
the direct pulverizing method tasted bitter and contained
high levels of limonin [7]. Dea et al. found that
increasing levels of sucrose or citric acid decreased the
perception of bitterness induced by limonin in orange
juice [12].
In our study, sucrose or citric acid contained in
Kugani fruit juice could have masked the bitter taste of
limonin in the vinegar extract from Kugani harvested
in August. Therefore, the whole Kugani fruit harvested
after August can be used as a raw material for vinegar
extracts production by the stirring method. Not all the
vinegar extracts made from Seedless fruit tasted bitter
Figure 2 Levels of polymethoxyflavones in vinegar extracts
from Ogimi Kugani depending on harvest month
and stirring time
Figure 3 Levels of limonin in Ogimi Kugani vinegar extracts
depending on harvest month and stirring time
a
b
c
ab
a
ab b b
a a b
c
10
15
20
25
30
0 20 80 100
Polimethaxyflavone content
mg/mL
40 60
Stirring time, min
June July August
a
a
b
b
a
ab c
bc
a
a
a
a
0
1
2
3
4
0 20 80 100
Limonin content, mg/mL
40 60
Stirring time, min
June July August
a a b
10
0 20 80 100
Polimethaxyflavone 40 60
Stirring time, min
June July August
a
a
b
b
a
ab c
bc
a
a
a
a
0
1
2
3
4
0 20 80 100
Limonin content, mg/mL
40 60
Stirring time, min
June July August
Polymethoxyflavone content,
315
Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316
as expected. However, the 20% Kugani extract was
bitter, especially in June. Therefore, we developed a
method to reduce bitterness of Kugani harvested in June.
There are other methods to diminish bitterness.
Hirose et al. reported that limonin was not detected in
vinegar extract from only Kugani peel [7]. In other
words, segment membranes and seeds contain limonin
but removing them is laborious and time-consuming.
In the present study, the 5 and 10% Kugani extracts did
not taste bitter and there was no significant difference in
bitterness among them and the Seedless vinegar extract
in June. Moreover, there were no significant differences
in shikuwasa aroma and green smell. Thus, the flavor of
the Kugani (5 and 10%) and Seedless vinegar extracts
was considered to be the same.
Incidentally, some locals in Okinawa prefer the bitter
taste of shikuwasa products, such as juice and jam.
Demand for bitter-tasting vinegar extract can be met by
using 20% Kugani fruit harvested in June. Seedless is
hardly cultivated commercially in Okinawa. To produce
vinegar extracts with lower bitterness, it is possible
to use only Kugani, as showed in this study. It is also
desirable to effectively use thinned or bruised Seedless
and Kugani, which are not suitable for sale.
CONCLUSION
We produced vinegar extracts from the whole unripe
fruit of Ogimi Kugani and Nakamoto Seedless harvested
from June to August. The stirring method was applied
to obtain the extracts. The 20% extract from Kugani
harvested in June and July was bitter compared to
the Seedless extract. However, the 5 and 10% Kugani
samples did not differ in bitterness from the 20%
Seedless extract in June
In addition, the 20% extracts from Kugani and
Seedless fruit harvested in August showed similar
bitterness, in spite of the fact that limonin levels in the
Kugani extract were higher than those in the Seedless
sample. Both vinegar extracts from Kugani and Seedless
contained polymethoxyflavones, which decreased from
June to August. The flavor of both vinegar extracts was
similar.
The extraction technique applied in this study is
easy to use and requires simple equipment, which
minimize hygienic problems. Producing vinegar extracts
from shikuwasa would allow creating a broad range of
products such as Ponzu soy sauce, salad dressings, and
fruit vinegar drinks. Moreover, vinegar extracts from
shikuwasa can be applied to any type of citrus fruit
worldwide.
CONFLICT OF INTEREST
The author declares that there is no conflict of
interest.
ACKNOWLEDGMENTS
I thank Mr. N. Hirose, Senior supervisor of Okinawa
Industrial Technology Center for his valuable idea and
advice. I also thank Mr. F. Mitsube, Researcher, for
providing shikuwasa fruits. I appreciate the technical
supports by Ms. K. Kyan and Mr. T. Kadekawa.
1. Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M. Antiproliferative activity of flavonoids on several cancer cell lines. Bioscience, Biotechnology and Biochemistry. 1999;63(5):896-899. https://doi.org/10.1271/bbb.63.896.
2. Hirata T, Fujii M, Akita K, Yanaka N, Ogawa K, Kuroyanagi M, et al. Identification and physiological evaluation of the components from Citrus fruits as potential drugs for anti-corpulence and anticancer. Bioorganic and Medicinal Chemistry. 2009;17(1):25-28. https://doi.org/10.1016/j.bmc.2008.11.039.
3. Choi SY, Ko HC, Ko SY, Hwang JH, Park JG, Kang SH, et al. Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biological and Pharmaceutical Bulletin. 2007;30(4):772-778. https://doi.org/10.1248/bpb.30.772.
4. Kunimasa K, Kuranuki S, Matuura N, Iwasaki N, Ikeda M, Ito A, et al. Identification of nobiletin, a polymethoxyflavonoid, as an enhancer of adiponectin secretion. Bioorganic and Medicinal Chemistry Letters. 2009;19(7):2062-2064. https://doi.org/10.1016/j.bmcl.2009.02.002.
5. Ishikawa R, Badenoch N, Miyagi K, Medoruma K, Osada T, Onishi M. Multi-lineages of Shiikuwasha (Citrus depressa Hayata) evaluated by using whole chloroplast genome sequences and its bio-diversity in Okinawa, Japan. Breeding Science. 2016;66(4):490-498. https://doi.org/10.1270/jsbbs.15151.
6. Medoruma K, Higa A, Kinjo H, Zukeyama H, Awaguni Y, Miyagi T, et al. Characteristics of seedless Citrus depressa, Nakamoto seedless. Bulletin of the Okinawa Prefectural Agricultural Research Center. 2011:5-10.
7. Hirose N, Maeda G, Onda S, Shoda M, Miyagi K, Wada K, et al. Development of vinegar extract from the waste peels of Shiikuwasha. Nippon Shokuhin Kagaku Kogaku Kaishi. 2017;64(2):81-89. https://doi.org/10.3136/nskkk.64.81.
8. Hanagasaki T, Hirose N, Maeda G, Onda S, Wada K. Vinegar extract of fruit waste from juice production using Tankan (Citrus tankan Hayata) native to Okinawa, Japan. Food Science and Technology Research. 2019;25(5):667-676. https://doi.org/10.3136/fstr.25.667.
9. Hirose N, Maeda G, Miyagi K, Wada K, Ohta H. Characteristics of vinegar extract from immature seedless Shiikuwasha (Citrus depressa Hayata, Nakamoto Seedless). Food Preservation Science. 2019;45:215-221. (In Jap.).
10. Hashimoto Y, Chuda Y, Suzuki T, Yasui A. Method validation for determination of total acid in vinegar based on potentiometric titration by interlaboratory study. Bunseki kagaku. 2008;57(6):453-459. https://doi.org/10.2116/bunsekikagaku.57.453.
11. Ichinokiyama H, Maegawa T, Goto M. Flavonoid contents of whole fruit and various tissues of a new acid citrus, ‘Niihime’. Horticultural research (Japan). 2012;11(3):387-391. https://doi.org/10.2503/hrj.11.387.
12. Dea S, Plotto A, Manthey JA, Raithore S, Irey M, Baldwin EA. Interactions and thresholds of limonin and nomilin in bitterness perception in orange juice and other matrices. Journal of Sensory Studies. 2013;28(4):311-323. https://doi.org/10.1111/joss.12046.
