INTRODUCTIONShikuwasa (Citrus depressa L.) is a common citrusfruit in Okinawa, the southernmost prefecture of Japan.Shikuwasa is very sour and also called Hirami lemon orthin-skinned flat lemon. In Okinawan dialect, shikuwasameans “to eat vinegar”, however it does not containvinegar, and the name refers to its strong sour taste. Thisnative Okinawan citrus fruit became popular all overJapan and is used to garnish dishes as well as to makejuices and jams.As it grows, shikuwasa changes color from darkgreen to yellow or orange. Unripe (green) fruit isextremely sour and is used to make condiments. Ripe(yellow) shikuwasa has better taste from December tothe end of January. Shikuwasa harvested from Augustto September is usually used as an acidifying agent invinegar substitution. Fruit harvested from Septemberto December is used in juice production and fromDecember to January can be eaten in the raw form.Shikuwasa is mainly cultivated in the northernpart of Okinawa. In 2017, 3398 t of shikuwasa wereproduced, with the fruiting area of shikuwasa being363 ha. Shikuwasa contains high levels of nutritionalcomponents, 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 otherfunctional components from shikuwasa can contribute tohealthy food products.Among known varieties of shikuwasa in Okinawa,Ogimi Kugani (hereinafter Kugani) is the most popular.Kugani is a commercially available superiorlandrace which is widely used in the food industry, fresh or processed [5]. Nakamoto Seedless (hereinafterSeedless) is a seedless variety of shikuwasa that isbecoming popular, due to its characteristics, which aresimilar to those of Kugani [6].Citrus tree cultivation involves thinning youngfruits to improve fruit size and quality. Thinning isperformed from June to July because shikuwasa is nottypically used during this period. Thinned fruits aswell as normally harvested fruits unsuitable for sale, forexample bruised fruits, should be effectively utilized toobtain processed food products such as vinegar extracts.To produce a vinegar extract with low bitterness,Hirose et al. used only the peels of residual substancesfrom Kugani, and Hanagasaki et al. used residualsubstances from Tankan (Citrus tankan Hayata) [7, 8].Using this technology, Ponzu sauce (Japanese citrusvinegar soy sauce) production is already beingcommercialized in Okinawa. In addition, the wholeSeedless fruit harvested from June to August is useful toproduce vinegar extract because it has no seeds, containslow levels of limonin, and has low bitterness [9]. In spiteof the fact that vinegar extract from the whole Kuganifruit is expected to be bitter because it typically contains14 seeds per fruit, the potential use of the whole unripeKugani should be explored to avoid wastes [6].The aim of the present study was to develop vinegarextract from the whole unripe Kugani fruit and comparethe characteristics of this extract with those of Seedlessvinegar extracts to improve the profitability of the fruit.STUDY OBJECTS AND METHODSShikuwasa (Nakamoto Seedless and Ogimi Kuganivarieties) has been cultivated in Okinawa AgriculturalResearch Center (26º37’ N, 127º59’ E; 40 masl) since2005. The Seedless and Kugani fruit under studywere harvested in June 8, July 6, and August 3 (2018),respectively.The objectives of the study were vinegar extractsmade from Seedless (20% fruit) and Kugani (5, 10, and20%). The fruit weight was defined by averaging theweights of 70~450 fruits, equal to approximately 1 kg.The fruit diameter was determined by averaging thediameters of 20 fruits. Moisture content was determinedby calculating the weight difference before and afterfreeze-drying.Production of vinegar extract. As for shikuwasaof Kugani variety harvested in different months, thefruit was broken down using a Bamix M200 mixer(Bamix, Switzerland). Afterwards, a 20% samplewas prepared by stirring 10 g of the fruit and 40 mLof spirit vinegar (Kraft heinz, USA). Stirring wasperformed with a 4 cm stirrer bar at 2.0×g for 10, 30, 60,and 90 min.Similarly, we prepared 20% Seedless vinegarsamples. To compare vinegar extracts from Kugani andSeedless harvested in July and August, the sampleswere stirred for 60 min. The mixed samples werecentrifuged at 1190×g for 20 min and filtered usingNo. 2 qualitative filter paper (Advantec Co. Ltd., Japan).To compare vinegar extracts from shikuwasaharvested in June, we used 20% Seedless as well as 5and 10% Kugani vinegar samples. The Kugani sampleswere obtained by mixing 2.5 or 5 g of Kugani fruit with47.5 and 45 mL of vinegar and stirring for 60 min.Freeze-dried powder process. To analyzepolymethoxyflavone 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 (IwataniCorp, Japan) using a 1.4 mm mesh.Titratable acidity determination. A volume of10 mL of the vinegar extracts was mixed with 100 mLof pure water and titrated with 0.5 mol/L NaOH untilpH 8.2 ± 0.3 using a pH meter. We applied acid-basetitration [10].Polymethoxyflavone analysis. Polymethoxyflavoneextraction was performed as described by Ichinokiyamaet al. [11]. To extract polymethoxyflavones fromshikuwasa, 100 mg of freeze-dried powder sampleswith 1 mL of methanol:DMSO (1:1) were subjectedto ultrasonic wave for 10 min (M1800-J, Japan) andcentrifuged at 2000×g for 2 min. We performed it threetimes. Extract solutions were obtained by diluting to avolume of up to 5 mL.To obtain polymethoxyflavones from vinegarextracts, 1 mL of the vinegar extracts with 1 mL ofethanol were subjected to ultrasonic wave for 30 min.The insoluble component was removed by centrifugationat 2000×g for 2 min. The solutions were prepared forHPLC, namely filtered using a 0.20 μm hydrophilicPTFE (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 of0.1 mg/mL and used as standards. Quantitativeanalysis of each polymethoxyflavone wasperformed as described by Hirose et al. [7].The sample solutions (5 μL) were injected ontoa Union UK-C18 HPLC column (3×100 mm,0.4 mL/min flow rate, Japan) at 40ºC. The solventwas acetonitrile/water/trifluorasetic acid (50/50/0.05).Analyses were performed at 340 nm using anLC-20A UV detector (Shimadzu corp., Japan). Amountsof polymethoxyflavones were calculated as the sum ofsinensetin, nobiletin, and tangeretin.Limonoid analysis. To extract limonoid fromshikuwasa, 100 mg of the freeze-dried sample wasmixed with 2 mL of acetic acid and 5 mL of ethylacetate and then vortexed for 1 min. To obtain limonoidfrom vinegar extracts, 0.4 mL of the vinegar extractswas mixed with 1 mL of ethyl acetate and then vortexedfor 1 min. Both ethyl acetate phases were collectedafter centrifugation at 2000×g for 2 min. These stepswere repeated three times, and the supernatant wascompletely evaporated under reduced pressure.Extract solutions were obtained by reconstitutingthe powdered samples with 2 and 0.4 mL of acetonitrilefor shikuwasa and vinegar extract, respectively. The312Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316bFigure 1 Nakamoto Seedless (a) and Ogimi Kugani (b)harvested from June to August (from left to right)Table 1 Characteristics of Nakamoto Seedlessand Ogimi Kugani harvested in different monthsHarvestingdateVariety Fruitweight,gFruitdiameter,cmMoisturecontent,%June 8, 2018 NakamotoSeedless2.27 1.67 75.9OgimiKugani3.51 1.92 80.4July 6, 2018 NakamotoSeedless5.48 2.18 82.0OgimiKugani8.81 2.58 82.0August 3, 2018 NakamotoSeedless10.07 2.81 83.6OgimiKugani15.31 3.24 82.9aTable 2 Chemical components of Nakamoto Seedless and Ogimi Kugani harvested in different monthsComponent Nakamoto Seedless Ogimi KuganiJune July August June July AugustPolymethoxyflavones, 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 ± 0.04cSinensetin 1.50 ± 0.12a 1.04 ± 0.01b* 0.90 ± 0.02b* 1.21 ± 0.05a 0.90 ± 0.01b 0.56 ± 0.01cNobiletin 11.01 ± 0.89a 7.31 ± 0.04b 6.22 ± 0.08b* 11.03 ± 0.42a 7.94 ± 0.05b* 4.81 ± 0.03cTangeretin 6.66 ± 0.47a 3.98 ± 0.02b* 3.69 ± 0.05b* 5.65 ± 0.20a 3.60 ± 0.03b 2.50 ± 0.02cLimonin, 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 monthPolymethoxyflavones represent the sum of sinensetin, nobiletin, and tangeretinn.d. – not detectedsolutions were prepared for HPLC by filtering. Limoninand nomilin (Wako Pure Chemical Corp., Japan) wereeach dissolved in acetonitrile to a concentration of0.1 mg/mL and used as standards.Quantitative analysis of limonin and nomilin wasperformed as described by Hirose et al. [7]. A volume of2 μL of the sample solutions was injected into CadenzaCD-C18 HPLC column (0.4 mL/min, 40ºC). The solventwas acetonitrile/water/formic acid (40/60/0.1). Theanalyses were carried out at a wavelength of 210 nmusing an LC-20A UV detector (Shimadzu corp., Japan).Sensory evaluation. Vinegar extracts were diluted10 times with distilled water, and 4% (w/w) castersugar was added. Six men and six women aged 20–50evaluated aroma, bitterness, green smell, and overallacceptability of the samples using a 5-point scale.For the shikuwasa aroma, 5 represented the strongestshikuwasa aroma and 1 represented the weakestaroma or the strongest vinegar aroma. For bitterness,5 represented the lowest bitterness and 1 represented thestrongest bitterness. For green smell, 5 represented thelowest green smell and 1 represented the strongest greensmell. For overall acceptability, 5 represented “like” and1 represented “dislike”.RESULTS AND DISCUSSIONFigure 1 demonstrates shikuwasa (NakamotoSeedless and Ogimi Kugani varieties) harvested fromJune to August. The weight, diameter, and moisture ofboth fruit varieties were increasing during the time(Table 1). The characteristics of Seedless were lowerthan those of Kugani.Changes in chemical components of Seedless andKugani with time are shown in Table 2, changes inchemical components of both extracts with time areshown in Tables 3 and 4. In both varieties, the contentof polymethoxyflavones was the highest in Juneand decreased with time (P < 0.01 for Seedless andP < 0.001 for Kugani). Comparing polymethoxyflavonecontent in both varieties, there was no significant313Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316difference in June, but in August it was significantlyhigher in Nakamoto Seedless compared withKugani (Table 2). Additionally, polymethoxyflavonesin the vinegar extracts from both varieties significantlydecreased with time (Seedless: P < 0.001, Kugani:P < 0.001) (Tables 3, 4). However, the recovery ratio ofpolymethoxyflavones from material increased with time.Hence, earlier harvested fruit contain higher amountsof polymethoxyflavones, which possess bioactiveproperties.Limonin content did not show a decreasing trend inSeedless, but decreased in Kugani with time (P = 0.217for Seedless and P < 0.001 for Kugani). Limonin levelTable 3 Characteristics of vinegar extracts from Nakamoto Seedless and Ogimi Kugani harvested in JuneParameter Nakamoto Seedless Ogimi Kugani20% 20% 10% 5%Vinegar volume, mL 31.5 ± 0.4c 31.9 ± 0.2c 38.4 ± 0b 42.2 ± 0.2aTitratable acidity, % 4.70 ± 0.10c 4.82 ± 0b 4.87 ± 0.10b 4.99 ± 0.10aPolymethoxyflavones, 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)dSinensetin 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)dNobiletin 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)dTangeretin 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)dLimonin, 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)bShikuwasa aroma 2.75 ± 0.21 3.17 ± 0.20 2.83 ± 0.26 2.58 ± 0.25Green smell 4.08 ± 0.33 3.75 ± 0.24 3.75 ± 0.36 4.25 ± 0.34Bitterness 3.67 ± 0.30a 2.42 ± 0.33b 3.42 ± 0.28ab 4.25 ± 0.27aOverall accaptability 3.83 ± 0.20a 2.83 ± 0.24b 3.50 ± 0.19ab 3.67 ± 0.14aData 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 tangeretinValues in parentheses indicate the recovery ratio of fruitTable 4 Characteristics of vinegar extracts from Nakamoto Seedless and Ogimi Kugani harvested in July and AugustParameter Nakamoto Seedless Ogimi Kugani Nakamoto Seedless Ogimi KuganiJuly August20%Vinegar volume, mL 29.8 ± 1.1b 36.4 ± 1.1a 21.8 ± 0.6b 27.5 ± 0.4aTitratable acidity, % 5.04 ± 0.10a 4.66 ± 0b 5.01 ± 0.10a 4.78 ± 0bPolymethoxyflavones, 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)bSinensetin 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)bNobiletin 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)bTangeretin 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)bLimonin, 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)aShikuwasa aroma 2.54 ± 0.18 3.00 ± 0.27 2.46 ± 0.33 2.85 ± 0.35Green smell 4.08 ± 0.34 3.69 ± 0.30 4.31 ± 0.27 4.65 ± 0.18Bitterness 3.92 ± 0.23a 3.15 ± 0.27b 4.31 ± 0.28 4.31 ± 0.22Overall acceptability 3.85 ± 0.15 3.54 ± 0.14 3.62 ± 0.28 3.54 ± 0.22Data 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 tangeretinValues in parentheses indicate the recovery ratio of fruitwas significantly higher in the Kugani than in Seedlessvariety. Nomilin was not detected in both varieties. Ingeneral, the Seedless vinegar extract contained lowerconcentrations of limonin than the extract from Kugani.Changes in polymethoxyflavone and limoninlevels during the stirring process are demonstrated inFigs. 2 and 3.The amount of polymethoxyflavones in vinegarextracts from Kugani harvested from June to Augustsignificantly increased (P < 0.001 in June, P < 0.01in July, and P < 0.001 in August) during the stirringprocess (Fig. 2). Limonin levels in the extractssignificantly decreased in July but increased in August314Hanagasaki 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 inAugust) during the process (Fig. 2).Limonin levels in the vinegar extract from Kuganiharvested in August were not changed during thestirring time, whereas those in the extract from thefruit harvested in June and July significantly decreased(Fig. 3).The stirring time of 10–60 min decreased but that of60–90 min slightly increased the limonin concentrationin the vinegar extracts from Kugani harvested in Juneand July. Thus, 60 min was selected as the optimalstirring time, and this time we used to compare thelimonin level in Kugani extracts with that in Seedless.We also analyzed 5 and 10% vinegar extractsfrom Kugani harvested in June. The obtained vinegarvolumes and titratable acidity significantly increasedwith the decreasing fruit concentration. As forpolymethoxyflavones and limonin, their levels dependeddirectly on the fruit amount (Table 3).Comparison of the vinegar extracts from Seedlessand Kugani are shown in Tables 3 and 4. There wasno significant difference in vinegar volume in 20%extracts from Seedless and Kugani harvested in June.However, the 5 and 10% Kugani extracts demonstratedsignificantly higher vinegar volume than the Seedlessextracts in all the months.The titratable acidity of the 5% Kugani vinegarextract was the highest in June, whereas that of theSeedless extract was the lowest (Table 3). In Julyand August, the Seedless extract showed highertitratable acidity compared to Kugani (Table 4). Totalpolymethoxyflavone levels in the vinegar extractfrom Seedless in June were the highest, while the5% Kugani extract contained the lowest amount ofpolymethoxyflavones.Furthermore, there was a similar tendency for eachpolymethoxyflavone, namely sinensetin, nobiletin, andtangeretin. In July, there was no significant differencein each polymethoxyflavone between the Seedlessand Kugani vinegar extracts. In August, the Seedlessvinegar extract demonstrated higher level of thesepolymethoxyflavones than the Kugani extract.Sensory evaluation showed no significant differencein aroma and green smell among all the samples. The20% extract from Seedless fruit had significantly higherbitterness than the 20% Kugani sample in July, but therewas no significant difference in August (Table 4). As forJune, there was no significant difference between the20% Seedless extract and the 5 and 10% Kugani samples(Table 3).Overall acceptability of the Seedless vinegarextracts was significantly higher than that of the 20%Kugani samples, but there was no significant differencebetween the 5 and 10% Kugani extracts in June. Overallacceptability of the Seedless and Kugani vinegarextracts in July and August showed no significantdifferences.As mentioned above, limonin levels in the Kuganivinegar extract decreased during the stirring process inJune and July but did not change in August (Fig. 3). Thisimplies that limonin was protected from volatilizing orother enzymatic reactions during the stirring process.This can also explain the increasing level of somecomponents in Kugani harvested from July to August. Inaddition, it may also be associated with the fact that thevinegar extract from Kugani harvested in August wasnot bitter despite a high content of limonin, which is abitter compound present in the vinegar extract.Hirose et al. reported that vinegar extract from wastepeel of shikuwasa harvested in October and obtained bythe direct pulverizing method tasted bitter and containedhigh levels of limonin [7]. Dea et al. found thatincreasing levels of sucrose or citric acid decreased theperception of bitterness induced by limonin in orangejuice [12].In our study, sucrose or citric acid contained inKugani fruit juice could have masked the bitter taste oflimonin in the vinegar extract from Kugani harvestedin August. Therefore, the whole Kugani fruit harvestedafter August can be used as a raw material for vinegarextracts production by the stirring method. Not all thevinegar extracts made from Seedless fruit tasted bitterFigure 2 Levels of polymethoxyflavones in vinegar extractsfrom Ogimi Kugani depending on harvest monthand stirring timeFigure 3 Levels of limonin in Ogimi Kugani vinegar extractsdepending on harvest month and stirring timeabcabaab b ba a bc10152025300 20 80 100Polimethaxyflavone contentmg/mL40 60Stirring time, minJune July Augustaabbaab cbcaaaa012340 20 80 100Limonin content, mg/mL40 60Stirring time, minJune July Augusta a b100 20 80 100Polimethaxyflavone 40 60Stirring time, minJune July Augustaabbaab cbcaaaa012340 20 80 100Limonin content, mg/mL40 60Stirring time, minJune July AugustPolymethoxyflavone content,315Hanagasaki T. Foods and Raw Materials, 2021, vol. 9, no. 2, pp. 310–316as expected. However, the 20% Kugani extract wasbitter, especially in June. Therefore, we developed amethod to reduce bitterness of Kugani harvested in June.There are other methods to diminish bitterness.Hirose et al. reported that limonin was not detected invinegar extract from only Kugani peel [7]. In otherwords, segment membranes and seeds contain limoninbut removing them is laborious and time-consuming.In the present study, the 5 and 10% Kugani extracts didnot taste bitter and there was no significant difference inbitterness among them and the Seedless vinegar extractin June. Moreover, there were no significant differencesin shikuwasa aroma and green smell. Thus, the flavor ofthe Kugani (5 and 10%) and Seedless vinegar extractswas considered to be the same.Incidentally, some locals in Okinawa prefer the bittertaste of shikuwasa products, such as juice and jam.Demand for bitter-tasting vinegar extract can be met byusing 20% Kugani fruit harvested in June. Seedless ishardly cultivated commercially in Okinawa. To producevinegar extracts with lower bitterness, it is possibleto use only Kugani, as showed in this study. It is alsodesirable to effectively use thinned or bruised Seedlessand Kugani, which are not suitable for sale.CONCLUSIONWe produced vinegar extracts from the whole unripefruit of Ogimi Kugani and Nakamoto Seedless harvestedfrom June to August. The stirring method was appliedto obtain the extracts. The 20% extract from Kuganiharvested in June and July was bitter compared tothe Seedless extract. However, the 5 and 10% Kuganisamples did not differ in bitterness from the 20%Seedless extract in JuneIn addition, the 20% extracts from Kugani andSeedless fruit harvested in August showed similarbitterness, in spite of the fact that limonin levels in theKugani extract were higher than those in the Seedlesssample. Both vinegar extracts from Kugani and Seedlesscontained polymethoxyflavones, which decreased fromJune to August. The flavor of both vinegar extracts wassimilar.The extraction technique applied in this study iseasy to use and requires simple equipment, whichminimize hygienic problems. Producing vinegar extractsfrom shikuwasa would allow creating a broad range ofproducts such as Ponzu soy sauce, salad dressings, andfruit vinegar drinks. Moreover, vinegar extracts fromshikuwasa can be applied to any type of citrus fruitworldwide.CONFLICT OF INTERESTThe author declares that there is no conflict ofinterest.ACKNOWLEDGMENTSI thank Mr. N. Hirose, Senior supervisor of OkinawaIndustrial Technology Center for his valuable idea andadvice. I also thank Mr. F. Mitsube, Researcher, forproviding shikuwasa fruits. I appreciate the technicalsupports by Ms. K. Kyan and Mr. T. Kadekawa.