Physiochemical properties, dietary fibers, and functional characterization of three yuzu cultivars at five harvesting times

Abstract

This research focused on physiochemical and nutritional properties and functional characterization of three cultivars of yuzu—Native, Tadanishiki yuzu, and Namhae1—during different seasons. According to the cultivar and harvest time, yuzu cultivars were analyzed for free sugar, dietary fiber, hesperidin, naringin, and flavonoid content as well as antioxidant and antihypertensive activity. During November, Namhae1 exhibited the highest fruit weight, °Brix/acidity ratio, and total dietary fiber content. Tadanishiki contained the highest fructose and sucrose levels, pectin and cellulose contents, and soluble dietary fiber. Tadanishiki also had the highest hesperidin content in October, while the naringin content and antioxidant activity were the greatest in November. Antihypertensive activity was also the strongest for Tadanishiki, which was picked in October and November. These results indicated that Tadanishiki in October or November was the best for consumption or favorable processing because of its excellent product quality and high levels of nutritional and functional compounds.

Introduction

Yuzu (Citrus junos) originated in China but also grows wild in Japan and Korea. It resembles a very small grapefruit with an uneven skin but is rarely eaten as a raw fruit because of its tart flavor. Yuzu has been described to produce a pleasant citrus fragrance with a floral overtone and is widely used in Japanese and Korean cuisine. Furthermore, yuzu has been used in traditional Chinese medicine as an aromatic stomachic and sweating medicine. A hot yuzu bath improves blood circulation and prevent colds (Hirota et al., 2010). Yuzu is effective in preventing certain diseases because of its anti-inflammatory (Hirota et al., 2010), antioxidant (Yoo and Moon, 2016), and anticarcinogenic properties (Kim et al., 2013). Recently, ethanol extract of yuzu peel was reported to exert antidiabetic effects via AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-gamma (PPARγ) signaling, both in vitro and in vivo (Kim et al., 2013). However, the underlying mechanisms of the anti-obesity activity of yuzu have not been explored.

In addition to the fruit, yuzu peel is also used in the formulation of cosmetics and medications. The peel of yuzu (~30% of the fruit weight) is primarily used as a useful dietary fiber (DF) in the cosmetic, food, and fertilizer industries as it is highly biodegradable because of its high pectin, DF, saccharide, and protein content. Consequently, numerous studies have attempted to utilize this waste as a source of pectin, protease, carboxymethyl cellulose, and other high-value-added compounds. Additionally, soluble dietary fiber (SDF) could potentially be recovered in acceptable yields from this DF-rich byproduct.

Yuzu, as a peel-eatable fruit, is mainly made into sugar-pickled tea. Yuzu tea is produced with yuzu peel and juice after removing seeds (Hirota et al., 2010), which account for nearly 30%–35% of the yuzu fruit. Previously reported findings (Hirota et al., 2010; Yoo and Moon, 2016) showed that the peels of yuzu fruit contain higher amounts of nutritionally valuable and biologically active components compared to the pulp. In this study, each yuzu cultivar has unique characteristics—Tadanishiki contains no seeds, and Namhae1 has thick peels.

To date, most studies on yuzu have focused on the physiochemical or nutritional properties or the functional characterization of some yuzu cultivars, but a simultaneous study of all of these factors has not been conducted for these three cultivars (Native, Tadanishiki yuzu, and Namhae1) during different seasons. This research focused on analyzing and comparing the physiochemical properties and dietary fibers content among the Korean yuzu varieties. Furthermore, the yuzu varieties were compared for functional properties concerning phenolic compounds, antioxidant activity, and antihypertensive effects using HPLC analysis and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging assays. More detailed knowledge of the variability of the physiochemical and functional properties of yuzu cultivars at certain harvesting times will aid in the selection of yuzu varieties with more beneficial nutritional and functional properties and favorable processing characteristics.

Materials and methods

Reagents and materials

The yuzu fruits used in this study were grown at the Fruit Research Center of the Jeonnam Agricultural Research and Extension Services (Wando, Jeonnam Province, Korea, 34° 20′ 42.69"N, 126° 41′ 29.74"E). Three cultivars of yuzu—Native, Tadanishiki yuzu, and Namhae1—were harvested from July to November, 2018. After harvest, slices of yuzu were freeze-dried and ground into powder. A 1 g freeze-dried sample was extracted with 50 mL of 80% methanol for 3 h at 50 °C. DPPH, Folin-denies reagent, gallic acid, quercetin, and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hesperetin, naringenin, narirutin, and neohesperidin were purchased from ChromaDex (Irvine, CA, USA). ABTS was purchased from BIO BASIC, Inc. (Ontario, Canada). All other reagents were of analytical grade.

Physicochemical properties

Weight, hardness, color, pH, acidity, and soluble solid and moisture content were analyzed using fresh yuzu of three cultivars at harvest time. The fresh weight was obtained by weighing 20 randomly chosen yuzu fruits using an analytical balance. Hardness was measured using a hardness tester (XforceP, Zwick/Roell, Germany). Half cut fruit (flesh part downwards) on the plate is pressed by 8 mm diameter tip to detect its hardness. Color values of the yuzu surface were measured with a color-difference meter (CR-400 m KONICA. MINOLTA, Japan). The moisture content was measured by drying at 105 °C. For soluble solid content (°Brix), pH and total acidity measurement, 20 mL of distilled water was added to 2 g of the sample, and the supernatant was obtained by centrifugation at 5000×g, for 10 min (Combi 514R, Hanil Scientific Co., Ltd., Incheon, Korea). The soluble solid content (°Brix) was determined using a digital refractometer (Pocket refractometer PAL-1, ATAGO Co., Ltd., Tokyo, Japan), and pH was determined using a pH meter (Ion S220, METTLER TOLEDO, Columbus, OH, USA). For total acidity measurement, two drops of 0.1% phenolphthalein were added to 4 mL of supernatant, and the solution was titrated until red with a 0.1 N NaOH solution. The consumption capacity (mL) of the NaOH solution was determined and converted into citric acid.

DF composition

Insoluble dietary fiber (IDF) and soluble dietary fiber (SDF)

Samples were analyzed for SDF and IDF fractions according to AOAC Method 991.43. Samples were suspended in MES-TRIS buffer and sequentially digested with heat-stable α-amylase at 95–100 °C, protease at 60 °C, and amyloglucosidase at 60 °C. Digested enzymes were filtered through tared fritted glass crucibles. Crucibles containing IDF were rinsed with dilute alcohol and acetone and dried overnight in a 105 °C oven. Filtrates and washing solution were mixed with four volumes of 95% ethanol to precipitate materials that were soluble in the digests. After 1 h, the precipitates were filtered through fritted glass crucibles.

Acid detergent fiber (ADF), neutral detergent fiber (NDF), and lignin

Samples (0.5 g) were suspended in 200 mL of the acid detergent (AD) solution (20 g of acetyl trimethyl ammonium bromide in 1 N H₂SO₄), 200 mL of the neutral detergent (ND) solution (30 g of SDS, 18.61 g of EDTA, 6.81 g of sodium borate, 4.56 g of disodium hydrogen phosphate), and 10 mL of 2-ethoxy ethanol and digested sequentially with heat stability at 100 °C for 1 h. Crucibles containing DF were rinsed with dilute alcohol, acetone, and dried overnight in a 105 °C oven. To measure lignin, a 0.5 g sample was suspended in 15 mL of 72% H₂SO₄ and stirred for 2 h. The mixture was diluted to 3% H₂SO₄ and digested sequentially with stable heat at 100 °C for 3 h. Crucibles containing DF were rinsed with 150 mL of water and dried overnight in a 105 °C oven.

Pectin

The pectin content in yuzu was measured using a pectin identification kit (Megazyme). The pectin content was calculated from a calibration curve where low ester pectin extracted from citrus peel was used as the standard.

Ash and crude protein

The ash content of dried yuzu powder samples was analyzed following the official methods (AOAC 1998). Samples were ashed in a muffle furnace at 525 °C for 5 h. Crude protein was measured in Kjeldahl nitrogen 6.25.

Total phenolic and flavonoid contents

The 1 g sample of freeze-dried (5% w/v) was extracted with 20 mL of 80% ethanol for 30 min using a sonicator and filtered through a 0.45-μm membrane filter. Yuzu extract were applied for functional composition analysis by HPLC and antioxidant or ACE inhibition activity detection.

The total phenolic content in the yuzu was measured using the Folin-Denis method. Samples (30 μL) were diluted with 32.5 μL water, 12.5 μL of Folin-Denies reagent was added, and the reaction was allowed to progress in darkness for 6 min. Next, 12.5 μL of 7% sodium carbonate and 250 μL of DW were added to the mixed solution. After incubation for 60 min in darkness, absorbance was read at 760 nm using a microplate spectrophotometer (Synergy HTX, Biotek Epoch, Agilent, Santa Clara, CA, USA). The total phenolic content was calculated from a calibration curve where gallic acid was used as the standard. Two-hundred microliters of diethylene glycol and 20 μL of 2 N sodium hydroxide were added to a 20 μL sample, and the reaction proceeded at 37 °C for 30 min. Absorbance was read at 420 nm using a microplate spectrophotometer (Synergy HTX, Biotek Epoch, Agilent, Santa Clara, CA, USA). The total flavonoid content was calculated from a calibration curve where quercetin was used as the standard.

Free sugars by HPLC

The contents of free sugars in yuzu were determined using LC-20A HPLC (Shimadzu, Kyoto, Japan). The column used for analysis was SUPELCOGEL C-610H (7.8 × 300 mm; SUPELCO, Bellefonte, PA, USA), and the mobile phase used for 45 min was 0.1% phosphoric acid (in DW). The flow rate, column oven, and injection volume were set at 0.5 mL/min, 40 °C, and 10 μL, respectively, and detection was performed using RID-10A (Shimadzu, Kyoto, Japan).

Functional phenolics by HPLC–DAD

The content of phenolic compounds in yuzu was determined using an Agilent 1216 Infinity LC series system (Agilent Technologies, Palo Alto, CA, USA). The column used for analysis was the ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5-Micron; Agilent Technologies) with mobile phases of 0.1% formic acid (solvent A) in DW and methanol–acetonitrile (solvent B). A gradient system was applied: starting with A:80, B:20 at 5–10 min; A:60, B:40 at 10–15 min; A:50, B:50 at 15–20 min; A:30, B:70 at 20–25 min; and A:0, B:100 at 25–30 min. The flow rate was set at 0.5 mL/min, the column temperature was 35 °C, with a 10 μL injection, and absorbance was measured at 280 nm.

DPPH and ABTS radical-scavenging activity

Samples (50 μL) were diluted consistently, 250 μL of 1 mM DPPH was added, and samples were incubated at 25 °C for 10 min. Absorbance was measured at 517 nm using a microplate spectrophotometer (Synergy HTX, Biotek Epoch, Agilent, Santa Clara, CA, USA). Ascorbic acid was used as the reference compound. The radical-scavenging activity was expressed as a percentage of the inhibition of DPPH· radicals using the following equation:

$$\text{DPPH radical-scavenging activity}\left(\%\right)=1-\left(\text{sample absorbance}/\text{control absorbance}\right)\times 100.$$

The ABTS radical cation (ABTS+·) was produced using a reaction of 950 mL of 7 mM 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) solution with 50 mL of 2.5 mM potassium persulfate, which was then allowed to stand in the dark at 4 °C for 12–16 h before use. The ABTS+ solution was diluted with ethanol to obtain an absorbance of 0.75–1.05 at 735 nm. Samples (50 μL) were allowed to react with 250 μL of the ABTS+ solution for 30 min in darkness, after which time the absorbance was measured at 735 nm with a microplate spectrophotometer. The radical-scavenging activity was expressed as the percentage of ABTS radical inhibition using the following equation:

$$\text{ABTS radical-scavenging activity}\left(\%\right)=\left(1-\text{sample absorbance}/\text{control absorbance}\right)\times 100.$$

Angiotensin-converting enzyme (ACE) inhibitory activity

This method is based on the liberation of hippuric acid from hippuryl-L-histidyl-L-leucine (HHL) catalyzed by ACE. A 20 μL of 0.3% (w/v) HHL solution was mixed with a 25 μL sample, followed by pre-incubation at 37 °C. Immediately before use, 0.33 units/mL of ACE was prepared in cold water. Then, 5 μL of the ACE solution was immediately mixed and incubated at 37 °C for 15 min. The reaction was stopped by adding 250 μL of 1 M hydrochloric acid. Ethyl acetate (1 mL) was added vigorously for 60 s and centrifuged for 2 min at 3000×g. After centrifugation, 1 mL of the supernatant was evaporated in a boiling water bath for 15 min under a hood. The evaporated solution was dissolved in 2 mL of distilled water, and the absorbance was read at 288 nm using a microplate spectrophotometer. Distilled water was used as the control, and 1 N NaOH solution was used as the blank instead of the sample. The ACE inhibitory effects were compared with each sample and positive control, Captopril® (Borung Pharm. Co., Seoul, Korea).

$$\text{ACE inhibition rate}\left(\%\right)=\left[B-A/B-C\right]\times 100,$$

where A is the absorbance of HA generated in the presence of ACE-inhibitor, B the Abs of HA generated without ACE-inhibitor and C the Abs of HA generated without ACE (corresponding to HHL autolysis in the course of enzymatic assay).

Statistical analysis

All experiments were conducted with three replicates, and the results are expressed as the mean ± standard deviation (SD). The data were analyzed using one-way analysis of variance, and the pairwise means of the groups were compared with Duncan’s multiple-range test using the SPSS program (SPSS version 23.0 for windows, SPSS Inc., Chicago, IL, USA). Values of p < 0.05 were considered significant in all cases.

Results and discussion

Physicochemical properties

The total increased weight, change in color, and hardness of the three yuzu cultivars are shown in Fig. 1 and Table 1. The weight of the three yuzu cultivars increased in the order Namhae1 > Native > Tadanishiki, depending on the harvest time from July to November. Tadanishiki yuzu without seeds exhibited the lowest weights and Namhae1 yuzu with thickened peels had the highest fruit weight. Regarding the Hunter value of the yuzu peel, the Tadanishiki yuzu cultivar had the highest L (lightness) and b (yellow) values at 69.14 and 43.23, respectively, in November. The Native cultivar exhibited the highest a (redness) value of 6.43 in November. The hardness of yuzu was high in all immature fruits in July, after which it declined continuously. From the three yuzu cultivars, Namhae1 expressed the highest hardness (42 ± 4 N). All three yuzu cultivars were grown under similar climatic and cultural conditions with harvest time from July to November. It is commonly assumed in Japan that the best yuzu harvest time is November when the yuzu fruit matures, changes to full yellow, and attains the best characteristic flavor (Phi and Sawamura, 2008). The chlorophyll content in the yuzu peel was high during the earlier green stage and then declined during the yellowish-orange stage. After the reduction in chlorophyll content, the carotenoid content of yuzu tended to increase rapidly, as shown in Fig. 1; similar pattern in chlorophyll contents have previously been discussed by Kon and Shimba (1987).

Fig. 1.

Maturity stages of cultivated Native, Tadanishiki, and Namhae1 yuzu from July to November 2018

Table 1.

Changes in physicochemical characteristics of yuzu according to harvest time and cultivar

	Weight (g)	Hardness (N)	Hunter's color value			Acidity (%)	Soluble solids (oBrix)	Soluble solids/Acidity
			L	a	b
Tadanishiki yuzu
July	35 ± 4h	443 ± 7a	30.41 ± 2.49fg	−3.96 ± 0.88cd	4.64 ± 1.02g	1.87 ± 0.43ef	4.3 ± 0.7f	2.32
August	56 ± 6f	371 ± 57b	26.26 ± 0.90i	−5.99 ± 0.63e	7.42 ± 0.67f	1.88 ± 0.08ef	7.0 ± 1.0de	3.72
September	56 ± 9f	198 ± 50d	31.20 ± 2.44fg	−9.53 ± 1.90f	11.00 ± 2.23e	3.34 ± 0.38b	8.1 ± 0.1bcd	2.42
October	57 ± 6f	126 ± 15e	60.78 ± 2.47c	−13.06 ± 0.43g	34.12 ± 1.31c	3.66 ± 0.21b	9.7 ± 0.4ab	2.64
November	70 ± 6e	31 ± 4f	69.14 ± 1.18a	1.21 ± 1.93b	43.23 ± 0.95a	4.48 ± 0.34a	8.8 ± 0.4abc	1.97
Native
July	29 ± 4i	420 ± 46a	31.27 ± 1.38f	−3.01 ± 0.69c	3.70 ± 0.89g	1.96 ± 0.17def	4.8 ± 0.80f	2.43
August	45 ± 6g	350 ± 94b	26.86 ± 1.89hi	−6.27 ± 1.29e	7.67 ± 1.53f	2.61 ± 0.48cd	9.3 ± 0.6ab	3.58
September	95 ± 10c	200 ± 24d	28.76 ± 1.39gh	−8.47 ± 0.98f	9.77 ± 0.94e	2.98 ± 0.08bc	9.5 ± 0.2ab	3.18
October	100 ± 7c	96 ± 13e	57.49 ± 2.05d	−14.12 ± 1.58g	32.95 ± 0.71c	2.58 ± 0.27cde	9.9 ± 0.7a	3.83
November	132 ± 11b	22 ± 3f	66.08 ± 1.80b	6.43 ± 1.08a	42.21 ± 1.08ab	4.66 ± 0.44a	8.7 ± 0.8abc	1.87
Namhae1
July	35 ± 5hi	434 ± 30a	30.46 ± 1.02fg	−3.07 ± 1.08c	3.82 ± 1.01g	2.08 ± 0.38def	5.8 ± 2.3ef	2.80
August	52 ± 6f	397 ± 65ab	25.77 ± 1.78i	−5.16 ± 1.34de	6.80 ± 1.31f	1.80 ± 0.39f	5.0 ± 1.0f	2.77
September	79 ± 11d	250 ± 38c	30.57 ± 1.62fg	−8.85 ± 1.08f	10.51 ± 1.32e	2.98 ± 0.31bc	7.7 ± 0.1cd	2.60
October	132 ± 11b	246 ± 24c	53.31 ± 3.56e	−13.92 ± 0.95g	30.76 ± 2.24d	2.47 ± 0.60cdef	9.6 ± 0.3ab	3.89
November	141 ± 20a	42 ± 4f	66.05 ± 0.62b	2.15 ± 0.76b	41.55 ± 0.51b	4.48 ± 0.65a	10.1 ± 0.9a	2.25

Means with the same letter in each column are not significantly different by Duncan’s multiple-range test (p < 0.05). Values represent the mean ± SD (n = 3)

Table 1 shows that the Tadanishiki yuzu cultivar exhibited a reduction in the °Brix/acidity ratio from 2.32 to 1.97 during cultivation time from July to November. The Native cultivar exhibited a reduction from 2.43 to 1.87, and the Namhae1 cultivar showed a reduction from 2.80 to 2.25. The matured Namhae1 cultivar (November) had the highest °Brix/acidity ratio. It has been shown that the °Brix/acidity of citrus cultivars Yuzu, Kjool (Citrus unshiu Marcow), and dangyooja (Citrus grandis Osbeck), grown in Korea, are 1.98, 1.68, and 1.49, respectively (Yoo et al., 2009). There was an inverse relationship between the degree of °Brix/acidity and harvest time—as the harvest time increased from July to November, the results showed an increase in both acid and sugar contents in yuzu cultivars.

DF composition

DF contents tended to continuously decrease with harvest time from July to November (Table 2). In matured fruit, total DF of the three yuzu cultivars increased in order of Namhae1 (37%) > Native (36%) > Tadanishiki (29%). The overall percentage of IDF was higher (12.3%–23.5%) as compared to that of SDF (13.5%–16.7%). The IDF and SDF content of yuzu was the highest in Namhae1 and Tadanishiki, respectively. The ratio of SDF/IDF was the highest in the Tadanishiki yuzu cultivar in November (1.36 fold), followed by Native (0.72 fold) and Namhae1 (0.58 fold). However, the SDF/IDF ratio was crucial for both dietary and functional attributes. It is commonly assumed that fiber sources that are suitable for use as a food constituent should have an SDF/IDF ratio close to 1:2 (Figuerola et al., 2005). In previous study the SDF/IDF percentages of different fruit cultivars were 5.3:1 (Valencia orange), 5.9:1 (Marsh grapefruit), 5.5:1 (Eureka lemon), and 4.5:1 (Royal Gala apple; Figuerola et al., 2005). Therefore, the SDF/IDF percentages of the three yuzu cultivars in the present study were accepted as a reliable source of DF because it was 1:2, which coincided with the previous findings. The changes in the amounts of DF could be affected by maturity, variety, harvest time, and growth condition (Alasalvar et al., 2001). The Namhae1 cultivar represented a higher total dietary fiber % content (37%) compared to the other two cultivars in November. TDF% of different fruits including tangerine/mandarin oranges, watermelons, and bananas were 40%, 20%, and 50%, respectively (Hoe and Siong, 1999). Fruits are a significant source of DF with a balanced ratio of insoluble and soluble fractions. DF possesses health-based attributes linked to physiological and functional properties. Moreover, yuzu fruits possess antioxidant properties derived from bioactive substances that are associated with DF (Morales et al., 2020). Biologically active, non-nutrient compounds found in citrus fruits, including phytochemical antioxidants, SDF, and IDF, have supported the reduction of cancers and chronic diseases such as diabetes, hepatitis, arthritis, and coronary heart disease (Peris et al., 2019; Nile and Park, 2014). DF is not considered a nutrient, but it still contributes to the promotion of good health.

Table 2.

Changes in dietary fiber contents according to harvest time and cultivar

	Total dietary fiber (%)	Insoluble dietary fiber (%)	Soluble dietary fiber (%)	Lignin (mg/g)	Cellulose (mg/g)	Hemicellulose (mg/g)	Pectin (mg/g)
Tadanishiki yuzu
July	55.76 ± 1.90b	40.91 ± 2.27a	14.85 ± 4.17cde	64.50 ± 21.30e	76.25 ± 10.95a	78.75 ± 4.95def	254.13 ± 9.69a
August	48.93 ± 1.80c	23.22 ± 4.01c	25.71 ± 5.81a	81.50 ± 0.10d	37.00 ± 7.30cd	76.85 ± 7.25ef	173.81 ± 5.63c
September	40.37 ± 1.55efg	18.50 ± 4.20de	21.87 ± 5.75ab	112.80 ± 0.00ab	41.07 ± 8.35c	90.10 ± 2.40cd	132.90 ± 0.30g
October	36.30 ± 4.64h	15.73 ± 4.21ef	20.57 ± 0.43abc	55.53 ± 10.75e	28.83 ± 4.57de	53.93 ± 6.21g	176.93 ± 0.65c
November	29.01 ± 1.24i	12.29 ± 0.86f	16.72 ± 2.10bcde	57.90 ± 1.70e	10.75 ± 1.85f	46.40 ± 1.10g	140.69 ± 1.25fg
Native
July	49.68 ± 0.59a	30.79 ± 0.98b	17.34 ± 3.90e	90.60 ± 15.00cd	67.00 ± 1.30b	66.70 ± 9.40f	225.69 ± 5.00b
August	44.83 ± 0.37d	22.49 ± 3.53c	21.89 ± 1.57ab	56.90 ± 0.30e	59.85 ± 1.25b	81.70 ± 5.10de	176.63 ± 2.19c
September	42.97 ± 0.95de	18.07 ± 3.25de	24.90 ± 4.10a	100.60 ± 4.20bc	57.47 ± 3.65b	81.10 ± 2.20de	150.40 ± 0.30ef
October	35.51 ± 1.86h	16.12 ± 2.23ef	19.39 ± 4.09abcde	90.53 ± 11.17cd	23.27 ± 1.69e	97.00 ± 6.80bc	142.27 ± 17.15fg
November	36.09 ± 1.24h	20.99 ± 2.34cd	15.10 ± 1.10cde	125.80 ± 7.40a	4.35 ± 1.15f	117.40 ± 17.30a	139.13 ± 0.32fg
Namhae1
July	48.27 ± 0.54c	34.10 ± 0.01b	14.17 ± 0.53de	94.80 ± 0.00cd	40.80 ± 3.50c	86.85 ± 2.55cde	225.69 ± 2.50b
August	40.70 ± 1.26ef	17.33 ± 1.95de	23.37 ± 3.21a	81.40 ± 9.60d	58.90 ± 7.80b	67.45 ± 3.05f	165.06 ± 6.25cd
September	40.40 ± 1.20efg	20.20 ± 0.20cde	20.20 ± 1.40abcd	115.00 ± 0.00ab	27.03 ± 7.75e	106.87 ± 2.75ab	159.13 ± 0.95de
October	37.29 ± 3.03fgh	15.33 ± 0.81ef	21.96 ± 3.84ab	117.27 ± 7.76a	26.33 ± 4.31e	114.37 ± 6.65a	171.00 ± 14.70cd
November	37.01 ± 2.17gh	23.49 ± 1.65c	13.52 ± 0.52e	118.10 ± 0.50a	6.55 ± 1.55f	78.15 ± 1.65def	132.25 ± 3.44g

Means with the same letter in each column are not significantly different by Duncan’s multiple-range test (p < 0.05). Values represent the mean ± SD

As shown in Table 2, the content of insoluble fibers such as cellulose and hemicellulose was higher in the early stages of the yuzu cultivars; thereafter it continuously decreased. In matured fruit, Tadanishiki yuzu contained the highest pectin and cellulose contents of 140.7 mg/g and 10.8 mg/g, respectively, whereas the Native cultivar represented the highest lignin content (125.8 mg/g). All three yuzu cultivars had higher amounts of IDF than SDF. IDF and SDF accounted for 63% to 70% and 37% to 30% of the total DF content in the citrus cultivars, respectively (Yoo et al., 2009). Yuzu comprises natural soluble and insoluble fibers such as hemicellulose and pectin, which reduce cholesterol absorption in the gut (Hamdan et al., 2011). Pectin is an influential constituent of the plant cell wall. It is chemically a polysaccharide comprising a linear chain of associated galacturonic acid (Zou et al., 2016). Citrus peel has significantly high aromatic compounds and pectin. Moreover, the waste peel from juice extraction is utilized to produce essential oils and flavorings as well as for medicinal purposes (Minamisawa et al., 2014). Washing removed soluble components, and the recovered fiber powder had greater NDF contents than ADF. Namhae1 in October had the highest NDF content (258.0 mg/g; data not shown).

Free sugars by HPLC

The concentrations of free sugars (e.g., sucrose, fructose, and glucose) in the three yuzu cultivars primarily depend on the cultivation time from July to November. Figure 2(A) shows that Tadanishiki yuzu exhibited the highest fructose and sucrose levels; however, its glucose levels were lower than those of Native and Namhae1 cultivars in November. Sugar contents increased in the order of fructose > sucrose > glucose. In previous study on the Kiyomi tangor fruit of Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka), which largely consisted of accumulated sucrose-type sugar, the content of sucrose was two-fold higher than that of fructose and glucose during the succeeding stage of fruit ripening (Dong et al., 2019). These findings are similar to our research findings. During the ripening process, sugars and organic acids develop and tend to increase with the importation of sugar from the plant and from the mobilization of the starch reserves in the fruit itself, as reported by Selvaraj et al. (1989). Therefore, as time increases, ripeness increases, which leads to an increase in sugar content.

Fig. 2.

Changes in (A) free sugars and (B) flavonoid contents of yuzu by HPLC analysis according to harvest time and cultivar. Means with the same letter in each column are not significantly different by Duncan’s multiple-range test (p < 0.05)

Functional phenolic content by HPLC–DAD

Tadanishiki yuzu had the highest flavonoid content when harvested in October, as shown in Fig. 2(B). In matured fruit, functional phenolic compounds were higher in Tadanishiki (818 mg/100 g) than in Native (536 mg/100 g) or Namhae1 (696 mg/100 g). The content of hesperidin and naringin was high in immature yuzu fruits fresh-picked in July and subsequently declined steadily, as previously reported by Yoo and Moon (2016). The key phenolic components in citrus fruits are flavonoids, which are known to exhibit beneficial health-promoting effects (Yoo and Moon, 2016). In Fig. 2(B), flavone aglycones were identified and quantified among the three yuzu cultivars. In previous studies, the hesperidin contents in yuzu and lemon were 267 ± 4.8 mg (Yoo et al., 2009) and 237.96 ± 0.12 mg (Yoo and Moon, 2016) per 100 g dried weight, respectively. However, the hesperidin (273.0 ± 21.2 mg/100 g dried weight) and naringin (96.9 ± 3.0 mg/100 g dried weight) contents were the highest in the Tadanishiki yuzu cultivar in October and November, respectively.

Citrus fruits are known for their fragrance, partially because of flavonoids and limonoids (a type of terpene) found in the peel (Nogata, 2005; Zarina and Tan 2013). Like other citrus fruits, yuzu comprises several bio-functional compounds such as flavonoids, carotenoids, and ascorbic acid. Flavonoids, found in the free form and as glycosides, are a large group of low-molecular-weight polyphenolics and are one of the most significant classes of biologically active compounds. Moreover, hesperidin and naringin were identified as the main flavonoid compounds in yuzu (Fig. 2(B)), similar to the findings of previous studies (Assefa et al., 2017; Yoo et al., 2009). Hesperidin is a bioflavonoid, which is a copious and modest byproduct of citrus cultivation (Garg et al., 2001). Narirutin, hesperidin, naringin, and neohesperidin are the most abundant flavonoids in the edible part of many cultivars of citrus fruits (Nile and Park, 2014). Among the citrus flavonoids, the antioxidant activity of naringin, hesperidin, and naringenin have commonly been studied (Zou et al., 2016). Moreover, Wu et al. (2009) found a high association between the scavenging activity and the flavonoid component in citrus branches. It was reported by Garg et al. (2001) that hesperidin, a glycosidic form of hesperetin, is encountered comprehensively in the plant kingdom, specifically in citrus fruits such as grapefruits and oranges, which are also commonly used in traditional medicines that cure diseases and provide health attributes.

Total phenolic and flavonoid contents

The total phenolic and flavonoid contents of the three yuzu cultivars were determined and are listed in Table 3, among which Tadanishiki yuzu had a higher phenolic content (3.67 ± 0.04) in comparison with that of the two other cultivars (2.97 ± 0.05) in November. The total phenolic contents in various fruits such as apples (2.963 ± 0.06 mg/g), red grapes (2.01 ± 0.02 mg/g), strawberries (1.60 ± 0.01 mg/g), bananas (0.90 ± 0.03 mg/g), peaches (0.846 ± 0.00 mg/g), lemons (0.819 ± 0.03 mg/g), oranges (0.812 ± 0.01 mg/g), pears (0.706 ± 0.016 mg/g), and grapefruits (0.496 ± 0.02 mg/g) have been reported (Sun et al., 2002). In our study, the total phenolic content of yuzu cultivars was similar to that reported in previous studies (Ferreira et al., 2018; Shetty et al., 2016). Moreover, phenolic compounds and redox properties are liable for antioxidant activity, and flavonoids exhibit high biological activity and show antioxidant, anti-mutagenic, anti-inflammatory, and anti-allergic properties (Ferreira et al., 2018; Shetty et al., 2016). Because of its anti-inflammatory properties, yuzu prevents the production of cytokines and reactive oxygen species and decreases eosinophil migration (Hirota et al., 2010). In the previous literature, yuzu has been reported to exhibit 1.6 times greater antioxidant activity than other citrus cultivars such as kjool and dangyooja (Yoo et al., 2009). Moreover, there was a trend of high antioxidant activity with high total phenolic content. To obtain the maximum antioxidant properties, it might be desirable to harvest yuzu when they are fully ripe and mature.

Table 3.

Changes in total phenolic or flavonoid content, and antioxidant activity of yuzu according to harvest time and cultivar

	Total phenolics	Total flavonoids	DPPH		ABTS
	(%)	(mg/g)	(%)	Vit. C eq μg	(%)
Tadanishiki yuzu
July	4.88 ± 0.00a	26.33 ± 0.32a	62.96 ± 0.42a	69.09 ± 0.47a	96.80 ± 0.25a
August	4.02 ± 0.05d	16.29 ± 0.04d	49.13 ± 0.31c	53.83 ± 0.34c	73.93 ± 0.67c
September	3.05 ± 0.04h	10.13 ± 0.07g	22.92 ± 0.14j	24.90 ± 0.16i	59.81 ± 0.42e
October	3.22 ± 0.03g	9.71 ± 0.14g	24.28 ± 0.40i	26.40 ± 0.44h	52.91 ± 0.04hi
November	3.67 ± 0.04f	9.11 ± 0.51h	26.72 ± 0.77g	29.10 ± 0.85f	55.81 ± 0.10g
Native
July	4.70 ± 0.01b	24.16 ± 0.28b	61.90 ± 0.64b	67.92 ± 0.70b	93.93 ± 0.39b
August	3.66 ± 0.00f	15.45 ± 0.11e	44.68 ± 0.97e	48.92 ± 1.08d	68.61 ± 0.53d
September	2.54 ± 0.03j	9.81 ± 0.07g	22.23 ± 0.01jk	24.13 ± 0.02ij	57.08 ± 0.09f
October	2.96 ± 0.05i	7.54 ± 0.14i	25.54 ± 0.38h	27.79 ± 0.42g	55.99 ± 0.20fg
November	2.97 ± 0.05i	6.15 ± 0.07j	25.63 ± 0.40h	27.90 ± 0.44g	54.04 ± 0.45h
Namhae1
July	4.38 ± 0.03c	20.63 ± 0.60c	45.50 ± 0.77d	49.82 ± 0.85d	95.96 ± 0.03a
August	3.93 ± 0.02e	13.54 ± 0.05f	34.77 ± 0.40f	37.97 ± 0.44e	67.81 ± 1.19d
September	2.36 ± 0.04k	8.76 ± 0.14h	18.23 ± 0.15l	19.72 ± 0.16k	56.95 ± 0.46fg
October	2.55 ± 0.02j	9.15 ± 0.07h	22.02 ± 0.10k	23.91 ± 0.11j	51.82 ± 1.38i
November	2.97 ± 0.05i	7.62 ± 0.11i	24.28 ± 0.40i	26.40 ± 0.44h	46.23 ± 1.47j

Means with the same letter in each column are not significantly different by Ducan’s multiple-range test (p < 0.05). The values represent the mean ± SD (n = 3). 80% ethanol extract of yuzu (500 μg/mL) were applied

Antioxidant activity (DPPH and ABTS)

In Table 3, antioxidant activities that were measured using ABTS and DPPH assays were given a comparable ranking of antioxidant activity for the yuzu cultivars at each harvest time from July to November. The percentage of ABTS assay showed higher scavenging activity than DPPH assay and was in agreement with the literature (Thaipong et al., 2006). The Tadanishiki yuzu cultivar showed a high reproducibility of 55.81 ± 0.10% in November than DPPH in comparison to Native and Namhae1.

ACE inhibition

In Fig. 3, Tadanishiki yuzu had a 35% higher ACE inhibition percentage in November, whereas Native and Namhae1 showed 30% ACE inhibition. Tadanishiki yuzu showed a similar ACE inhibitory effect to 30 mg captopril, which was applied for high-pressure treatment. In previous studies, the ACE inhibition percentages of pears, nectarines, and melons were 17.22%, 16.93%, and 8.19%, respectively (Park et al., 2019); however, the ACE inhibition percentages in our study were comparatively higher than those reported in previous findings. Our findings suggest that these three yuzu cultivars could be supplemented with higher ACE inhibition percentages. Moreover, Das and De (2013) reported that ACE is a fundamental constituent in the control of blood pressure considering the renin-angiotensin system. It has become the main target in the control of hypertension. Commonly, fruit consumption can provide health attributes by decreasing the risk of chronic diseases such as metabolic syndrome diseases including type 2 diabetes and cardiovascular disease.

Fig. 3.

Inhibition rate of angiotensin-converting enzyme (ACE) in yuzu according to harvest time and cultivar. 80% ethanol extract of yuzu (500 μg/mL) were applied. Means with the same letter in each column are not significantly different by Duncan’s multiple-range test (p < 0.05)

In conclusion, the results revealed in this paper are the most comprehensive comparison of three different yuzu cultivars (Tadanishiki yuzu, Native, Nmahea1). Data collected during this research allowed us to screen the most promising yuzu cultivars in terms of high DF and physiochemical and functional properties. Among the three yuzu cultivars, the mature Namhae1 cultivar showed the best physiochemical properties, including the highest fruit weight, the °Brix/Acidity ratio, and total DF content. However, the Tadanishiki yuzu, with no seeds, represented remarkable nutritional and functional characteristics that mainly depended on the harvest time from July to November. Tadanishiki yuzu contained the highest amounts of pectin, free sugars, enriched hesperidin, and naringin, as well as the strongest antioxidant activity and ACE inhibition effect. The findings presented in this study provided a quantitative survey of the physiochemical and functional properties of yuzu cultivars. This research highlighted the properties of the Tadanishiki yuzu cultivar. In the future, this research will be useful in exploring more physiochemical properties of different yuzu cultivars.

Compliance with ethical standards

Conflict of interest

None of the authors of this study has any financial interest or conflict with industries or parties.