Vinegar Extract Produced Using Ripe Fruits of Wild Genotype of Citrus depressa Hayata in Okinawa

Ten grams of shikuwasa obtained from three fruits was pulverized in 40 mL of spirit vinegar (Kraft Heinz, USA) using a mill mixer (IFM-800, Iwatani, Japan) as one of three repetition samples. The mixture was blended four times for 10 s each. Consequently, the samples mixed with vinegar spirit were then centrifuged at 1190 g for 20 min and were finally filtered using No. 2 qualitative filter paper (Advantec, Japan) (Hirose et al. 2017a).

Reference samples were prepared from ascorbic acid (reduction type, Nacalai Tesque, Japan) dissolved in spirit vinegar to concentrations of 30, 40, 50, 60, 70, 80, 90, and 100 mg·L⁻¹. A volume of 5 μL of these standard solutions was injected onto a Wakosil-5C18 HPLC column (4.6 × 250 mm, Wako Pure Chemical, Japan) at a flow rate of 1.0 mL·min⁻¹. The solvent was 5 mmol·L⁻¹ phosphate buffer solution. Samples were measured at the wavelength of 246 nm using an SPD-20A ultraviolet (UV) detector (Shimadzu, Japan). The ascorbic acid concentration in the samples was measured using an RQ flex reflectometer (Kanto Chemical, Japan) (Takebe & Yoneyama 1995) and was corrected using a regression formula for the relation between the HPLC and RQ flex results.

The polyphenols concentration in shikuwasa vinegar extract was determined using the Folin–Ciocalteu colorimetric method (Furuta et al. 1998). The shikuwasa vinegar samples (50 uL) were mixed with 50 μL of the Folin–Ciocalteu reagent (Wako Pure Chemical, Japan) and 50 μL of 5% Na₂CO₃ solution, and the absorbance was measured at 750 nm after 20 min of mixing at room temperature. The polyphenols contents are expressed as equivalents of mg per L gallic acid.

Ten milliliter of vinegar extract was mixed with 100 mL of ultrapure water produced by RFU424BB (Advantec), which was titrated with 0.5 mol·L⁻¹ NaOH until pH 8.2 ± 0.3 using a pH meter by acid-base titration (Hashimoto et al. 2008).

Oxygen radical absorbance capacity (ORAC) assay values were evaluated as described in a previous publication (Mikami et al. 2009). In short, 110.7 nmol·L⁻¹ fluorescein sodium salt (Sigma-Aldrich, Japan) solution and 31.7 mmol·L⁻¹ AAPH 2,2-azobis (2-amidinopropane) solution were prepared in 75 mmol·L⁻¹ phosphate buffer solution (pH 7.4). After dilution with the assay buffer, the vinegar extract was subjected to the ORAC assay. Diluted vinegar extract and fluorescein solution were applied to a microplate. After a 10-min incubation at 37 °C, fluorescence (excitation 485 nm; emission 528 nm) was recorded for the first time in a Multi-Detection Microplate Reader SH-9000 (Corona, Japan) equipped with a temperature-controlled incubation chamber.

The microplate was taken out of the SH-9000, and AAPH solution was added to the microplate, after which the plate was placed in the SH-9000 again. Fluorescence (excitation 485 nm; emission 528 nm) was then recorded every 2 min over 1.5 h using the SF6 software. Three duplicates of the measurements were expressed as μmol of Trolox equivalents (TE) per 100 mL of vinegar extract (μmol of TE per 100 mL).

Polymethoxyflavone (PMF) extraction was performed as previously described (Ichinokiyama et al. 2012). For PMF extraction from shikuwasa fruits, 100 mg of freeze-dried powder sample with 1 mL of methanol:DMSO (1:1) were subjected to ultra-sonic wave for 10 min (M1800-J, Yamato science) and centrifuged at 2000 g for 2 min. This was performed three times and extract solutions were obtained by diluting to a volume of up to 5 mL. For PMF extraction from vinegar extract, a volume of 1 mL of vinegar extract mixed with 1 mL of ethanol was subjected to an ultrasonic wave for 30 min (M1800-J, Yamato Scientific, Japan) and the insoluble constituent was removed by centrifugation at 2000 g for 2 min. Sample solutions were prepared for HPLC after the above extract solution had been filtered (0.20 μm hydrophilic PTFE from Advantec). Sinensetin (Wako Pure Chemical), nobiletin, and tangeretin (Sigma-Aldrich, Japan) were dissolved in ethanol to a concentration of 0.1 mg·mL⁻¹ respectively as standards. A quantitative analysis of each PMF was performed as previously described (Hirose et al. 2017a). A volume of 5 μL of the sample solutions was injected onto a Union UK-C18 HPLC column (3 × 100 mm, Imtakt, Japan) at a flow rate of 0.4 mL·min⁻¹ at 40 °C. The solvent used was acetonitrile/water/trifluorosetic acid (50/50/0.05). Analyses were monitored at a wavelength of 340 nm using an LC-20A UV detector (Shimadzu). PMF levels were calculated as the sum of sinensetin, nobiletin, and tangeretin.

Limonoid extraction was performed as previously described (Hirose et al. 2017a). For limonoid extraction from shikuwasa fruits, 100 mg of freeze-dried powder sample was mixed with 2 mL of acetic acid and 5 mL of ethyl acetate then vortexed for 1 min. For limonoid extraction from vinegar extracts, 0.4 mL of vinegar extract was mixed with 1 mL of ethyl acetate then vortexed for 1 min. Ethyl acetate phase was collected after centrifugation at 2000 g for 2 min. These steps were repeated three times and the supernatant was completely evaporated under reduced pressure. The extract solution was obtained by reconstituting with 0.4 mL of acetonitrile.

Sample solutions for HPLC were prepared after the above extract solution had been filtered (0.20 μm, hydrophilic PTFE from Advantec). Limonin (Wako Pure Chemical) was dissolved in acetonitrile to a concentration of 0.1 mg·mL⁻¹ respective to their standards. Quantitative analysis of limonin was performed as previously described (Hirose et al. 2017a). A volume of 2 μL of the sample solutions were injected onto Cadenza CD-C18 HPLC column (3 × 100 mm, Imtakt) using a flow rate of 0.4 mL·min⁻¹ at 40 °C. The solvent was acetonitrile/water/formic acid (40/60/0.1). Analyses were monitored at a wavelength of 210 nm using an LC-20A UV detector (Shimadzu).

Flavor components were evaluated as previously described (Hirose et al. 2017b). Using 1.0 mL vinegar extract, the insoluble constituent was removed by centrifugation at 2000 g for 2 min. Next, 1 mL sample was placed in a vial, and 50 μL 0.1% n-hexanol (special grade, Wako Pure Chemical) was added as an internal standard. Monotrap monolithic silica adsorbent (MonoTrap RGPS TD from Gl Sciences, Tokyo, Japan) was used to trap the odor component out of contact. Flavor components were trapped by keeping the vial heated to 50 °C for 1 h. Flavor components in the adsorbent were injected by split ratio of 1:10 onto a InertCap Pure WAX ProG 2M column (0.25 mm × 60 m, df = 0.25 μm, Gl Sciences) at a flow rate of 1.0 mL·min⁻¹ helium at 40 °C (5 min) − (6 °C per min) − 250 °C in the GCMS-QP2010 Ultra (Shimadzu) system using thermal desorption from an OPTIC-4 multimode inlet unit (Gl Sciences, Netherlands).

As MS conditions, ion source and interface temperature were 200 °C and 250 °C and ionization method was EI at a voltage of 70 eV. Based on the data obtained from GCMS, the NIST11 Mass Spectrum Library (National Institute of Standards and Technology, USA) and FFNSC 2 Flavor and Fragrance Natural and Synthetic Compounds GCMS Library (Chromaleont, Italy) were used to identify the odor components. Results were reported as mean values of peak area ratio to an internal standard. Monoterpenes represent the sum of α-pinene, β-myrcene, and limonene. Monoterpene alcohols represent the sum of linalool, terpinen-4-ol, and α-terpineol.

Vinegar extract was diluted 10 times with distilled water as a sample for measurement. The CIELAB color space of the diluted vinegar samples was measured using a CM-2600d spectrophotometer (Konica Minolta, Japan) using a D65 illuminant with 10° observer angle. Calibration was conducted according to the manufacturer protocol. Data were expressed in terms of L*, a*, and b* (Stintzing et al. 2003).

The vinegar extracts were diluted 10 times with distilled water and caster sugar was added at the concentration of 4% (w/w) for taste tests. Six men and women, ages 20–50, evaluated the shikuwasa aroma, bitterness, green smell, and general flavor on a scale of 1 to 5 (Hanagasaki 2021). For the aroma, 5 represented the strongest shikuwasa aroma and 1 represented the weakest shikuwasa 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 general flavor, 5 represented “like” and 1 represented “dislike”.

The Tukey–Kramer test was used for statistical analysis and was run in Excel statistics 2012 (Social Survey Research Information).

The Ishikunibu fruits tended to be smaller than the ‘Kugani’ fruits (Table 1). The fruit color of both genotypes was mainly green when harvested in December, then turned orange as the fruits ripened. In February, both genotypes were ripe, and their color was orange (Fig. 1).

Figure 1.

PMF and limonin in Ishikunibu fruits significantly decreased from December to February. As for the ‘Kugani’ fruits, PMFs, sinensetin, and tangeretin were significantly lower in February, but limonin was significantly higher in February (Table 2). According to Hanagasaki (2021), PMFs of ‘Kugani’ in December and February was approximately half of the value in August. Every PMF was higher in the ‘Kugani’ than the Ishikunibu fruits in December and February. Further, limonin in the Ishikunibu was lower than that in the ‘Kugani’ fruit in February and vice versa in December (Table 2).

The vinegar volume showed no significant difference across genotypes and harvesting time (Table 3). The titratable acidity and ascorbic acid contents in vinegar extract did not differ between genotypes and harvesting times, except that the titratable acidity of extract with ‘Kugani’ harvested was lower than that with Ishikunibu in February (Table 3).

Polyphenols contents in vinegar extract did not differ between genotypes and harvesting times. ORAC assay can be used to measure antioxidant capacity (Mikami et al. 2009). Here, ORAC values did not show significant differences across genotypes and harvesting time (Table 3). The vinegar extracts had ORAC values ranging from 4.9 to 7.1 μmol TE·mL⁻¹. The PMF ORAC values are not high, as they only contain a methoxy group and lack a hydroxy group. For instance, the ORAC value of nobiletin was about 1.45 mmol TE·g⁻¹. A nobiletin content of 7.3 mg·100 mL⁻¹ in the sample from Ishikunibu harvested in February was equivalent to only 0.11 μmol TE·mL⁻¹ of the total ORAC value of 5.7 μmol TE·mL⁻¹. Therefore, other components, such as polyphenols, contributed to the ORAC activity to a greater extent. The vinegar extracts showed a total polyphenol content ranging from 158.1 to 191.7 μg·mL⁻¹ (Table 3). The polyphenols content and the ORAC value in extracts with Ishikunibu were higher than those with ‘Kugani’ harvested in December and February, yet there were no significant differences. Every PMF in vinegar extracts with ‘Kugani’ was significantly higher than those with Ishikunibu harvested in both December and February (Table 3).

The limonin level in vinegar extracts with Ishikunibu was lower than that in ‘Kugani’ samples in both terms of harvesting, although differences were not significant because of the variance in the values (Table 3). Limonin levels were scattered because they depend on whether the sample includes more seeds. However, the bitterness of the vinegar extract made with Ishikunibu was rated “higher” than the extracts with ‘Kugani’ harvested in February, i.e., the vinegar extract with Ishikunibu tasted less bitter (Table 3). The monoterpene contents showed no significant differences among all categories (Table 3). However, monoterpene alcohols in the vinegar extracts made with Ishikunibu harvested in December and February were significantly higher than those with ‘Kugani’ (Table 3). Monoterpene alcohols are known to be important contributors to the citrus aroma as well as the sweet floral and herbal resinous aromas (Choi et al. 2002). The shikuwasa aroma and green-smell ratings of the vinegar extract made with Ishikunibu harvested in February were higher than those with ‘Kugani’, yet the difference was insignificant (Table 3).

Interestingly, the vinegar extract made with Ishikunibu looked different and was more transparent and more brilliant when compared with that made with ‘Kugani’ (Fig. 2). The clarity of the vinegar extract improved because albedo or other parts of the Ishikunibu harvested in February might not contain substantial pectin, cellulose, and hemicellulose, which are nearly insoluble fibers (Rafiq et al. 2018). Ishikunibu harvested in February was fresher and had less fruit puffing owing to its late-ripening feature. Since the Ishikunibu harvested in February had a deep orange color (Fig. 1), the vinegar extract with the fruits harvested in February exhibited a deep and striking color (Fig. 2). Ishikunibu is known to contain a considerable amount of β-cryptoxanthin; the β-cryptoxanthin content in Ishikunibu was the highest with respect to the seven genotypes native to Okinawa when averaged, based on the harvests obtained in 2013 and 2014 (Sugawara et al. 2017). Additionally, Ishikunibu had more β-cryptoxanthin and β-carotene contents than ‘Kugani’ and ‘Nakamoto Seedless’ (Hanagasaki 2021) harvested in February 2018 (Mitsube & Sugawara 2021). Cryptoxanthin is a red crystalline solid with a metallic luster in its pure form. Further, the a* value generally represents the degree of red, which was significantly higher in the vinegar extract with Ishikunibu harvested in February than in that made with ‘Kugani’ (Table 3). Therefore, the Ishikunibu fruit, which was deep orange (Fig. 1), most likely contributed to the bright color of the vinegar extract because of its β-cryptoxanthin content (Sumiasih et al. 2018).

Figure 2.

β-cryptoxanthin performs various functions and has been reported to prevent the development of metabolic syndrome (Sugiura et al. 2015a), type-2 diabetes (Sugiura et al. 2015b), and the early pathogenesis of non-alcoholic liver disease (Sugiura et al. 2016). Vinegar is effective for treating fatigue and promoting health (Miyagi et al. 2013), and the vinegar extracts in this study contained different amounts of nutritional components. Therefore, using wild genotypes of shikuwasa, such as Ishikunibu, can yield beneficial vinegar extracts with increased sourness.