Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/multidisciplinary-digital-publishing-institute/comprehensive-evaluation-of-the-postharvest-antioxidant-capacity-of-d9bYArGcxp
- Publisher
- Multidisciplinary Digital Publishing Institute
- Copyright
- © 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
- ISSN
- 2076-3921
- DOI
- 10.3390/antiox8050136
- Publisher site
- See Article on Publisher Site
Abstract
antioxidants Article Comprehensive Evaluation of the Postharvest Antioxidant Capacity of Majiayou Pomelo Harvested at Di erent Maturities Based on PCA 1 1 1 , 1 , 2 , Zhengpeng Nie , Chunpeng Wan , Chuying Chen * and Jinyin Chen * Collaborative Innovation Center of Post-harvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province, Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits & Vegetables, Jiangxi Agricultural University, Nanchang 330045, China; nzp2018@126.com (Z.N.); chunpengwan@jxau.edu.cn (C.W.) Pingxiang University, Pingxiang Jiangxi 337055, China * Correspondence: cy.chen@jxau.edu.cn (C.C.); jinyinchen@126.com (J.C.); Tel.: +86-791-8381-3158 (C.C. & J.C.) Received: 10 April 2019; Accepted: 13 May 2019; Published: 17 May 2019 Abstract: Majiayou pomelo (Citrus grandis L. Osbeck, MP) is a famous local red pulp pomelo from the Jiangxi province in China that is rich in natural active substances. In order to investigate the postharvest antioxidant capacities of MP pulp and determine the optimal harvesting time, fruits that were harvested at three di erent maturities (185, 200, and 215 days after full bloom) were observed for 180 days of preservation at ambient temperature. An abundance of ascorbic acid and lycopene in the MP pulp was found during storage, and in Harvest I, these substances were significantly higher than in Harvest II and Harvest III fruit (p < 0.05). The activity of ascorbate peroxidase (APX), peroxidase (POD), and catalases (CAT) in Harvest I and Harvest II were far higher after 90 days. The radical scavenging ability of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion radical (O ), and hydroxyl radical (OH) in Harvest I and Harvest II were higher. There was a significantly positive correlation (p < 0.01) between the antioxidant components (ascorbic acid, lycopene, carotenoids, total phenols, and total flavonoids), enzyme activity, and radical scavenging ability. The comprehensive scores determined by principal component analysis (PCA) in Harvest I and II were higher than those in Harvest III. Therefore, the optimal harvesting period of MP for each year is determined to be early November. The study provides a theoretical basis for the maintenance of the postharvest fruit value and the regulation of fruit functional components. Keywords: Majiayou pomelo (MP); postharvest; antioxidant capacity; harvesting periods; PCA 1. Introduction Majiayou pomelo (Citrus grandis L. Osbeck, MP) is a fruit variety that resulted from the breeding of the local pomelo population in Majia Natural village, Danan town, Guangfeng district of Jiangxi province, China, which originated in the Chenghua period of the Ming dynasty (A.D. 1465) and has been cultivated for centuries [1]. In addition, it is listed in the national geographic indication for the protection of products and has been awarded the title of “China’s famous fruit”, resulting in the creation of the top 50 fruit companies in China. It is also the first health food that has passed clinical verification for the treatment of diabetes in China [2]. The MP industry has developed rapidly. The planting area of MP in Shangrao city reached nearly 1.4 thousand hectares (ha) by the end of 2016. The net benefit reached 200 thousand Yuan per ha, which has boosted local economic development [2]. However, the industry now is facing with fruits quality deterioration for excessive pursuit of yield and neglect of management of the harvesting process. Antioxidants 2019, 8, 136; doi:10.3390/antiox8050136 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 136 2 of 15 Pomelo fruit is popular among consumers due to its large size, high nutritional value, unique fragrance, and storability [3]. Many researchers have carried out studies on the utilization of pomelo peel [4], but relatively few studies have focused on the bioactive substances in the fruit pulp, which contains phenols, pigments, limonoids, and polysaccharides, providing free radical scavenging, antioxidant, cardioprotective, and anticancer properties [5–7]. As the characteristic red pulp pomelo variety, MP has attractive color and taste, and its pulp contains high levels of ascorbic acid, lycopene, beta-carotene, and phenols [8]. The majority of studies on MP have only focused on cultivation management, and the changes of the fruit’s functional components and antioxidant activity during postharvest storage have not yet been explored. During storage, various reactive oxygen species (ROS) can also be cleared by ROS scavenging systems, such as endogenous antioxidant substances and enzyme systems [9]. Ascorbate peroxidase (APX), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) are present as ROS scavengers. SOD turns O into H O , POD and CAT eliminate H O , and APX removes the H O by 2 2 2 2 2 2 2 using ascorbic acid as an electron donor. These antioxidant enzymes can scavenge free radicals, reduce the level of membrane lipid peroxidation and the degree of membrane damage, as well as maintain the dynamic balance of the cellular systems [9]. Fruit storability is closely related to harvest time, and di erent harvest times have di erent e ects on the functional components and antioxidant activity of the fruit [10–12]. Fruit harvested too late usually experiences severe tissue aging, accelerated water loss [13], degradation of nutrient components and bioactive substances, and a decline in free radical scavenging capacity [14]. Thus, the determination of optimal harvesting periods will provide a theoretical basis for reducing the postharvest loss of fruit and guide fruit farmers toward producing and harvesting in a scientific way. Generally, the antioxidant performance of fruits can be reflected by di erent indicators, but it is dicult to comprehensively analyze the influence of these dispersed indicators. Principal component analysis (PCA) is an analytical method that condenses many variables into a few comprehensive variables, combined with Pearson correlation analysis, which explains the correlation between di erent variables. Both of these analytical techniques can comprehensively analyze the influence of harvesting maturity on postharvest antioxidant capacity [15]. This method has already been widely applied in the quality evaluation of grapes [16], peaches [17], kiwifruit [18], pomelos [19], and many other fruits. In this study, the antioxidant capacity of MP pulp, stored at ambient temperature, was comprehensively experimentally evaluated by the determination of the main antioxidant components, the enzyme activity of POD, CAT, APX, and SOD, and the scavenging ability via 2,2-diphenyl- 1-picrylhydrazyl (DPPH), OH, O and ferric reducing antioxidant power (FRAP) assays. Combined with PCA and Pearson’s correlation analysis, we determined the optimal harvesting period and explored the changes of the functional components during postharvest storage, which provides a basis for the improvement of MP as a health food and reinforces the economic benefits of the MP industry. 2. Materials and Methods 2.1. Plant Material MP fruits, from 10-year-old trees grafted on Fructus aurantii rootstock, were used. Plants were grown at the base of Majiashan mountain (28.56 N, 117.99 E; 60 m above sea level), at the MP orchard of Jiangxi Guangfeng Xiafeng Green Agriculture Development Co., Ltd., (Shangrao, Jiangxi, China) with a subtropical monsoon humid climate. The management of trees followed local standard agronomic measures, the spacing in the rows and between rows were 3 4 m, and the fruits were all covered with paper bags. 2.2. Experiment Design and Storage Conditions According to the local harvesting time of each year, we set three di erent harvesting maturities for 2017, which were harvested at 185 days after full bloom (October 30th; Harvest I), 200 days after Antioxidants 2019, 8, 136 3 of 15 full bloom (November 14th; Harvest II) and 215 days after full bloom (November 29th; Harvest III). For scientific picking, we excluded the marginal trees in the orchard and randomly selected 20 trees, with the same growth and the same slope, for further research. At the time of picking, fruits with the same size, color, and maturity, and which were disease-free. Fruit in the middle part of the tree were picked (except for bore fruits), and at least 15 fruits were picked from each tree. Then, they were packed in plastic woven bags and were transported back to our laboratory. After four days of pre-storage under natural conditions, the paper bags were removed, and fruits were washed under running water and dried naturally, without any treatment. After each fruit was bagged with a 0.03 mm polyethylene bag, 300 fruits were picked for long-term storage in each harvesting maturity. Next, all of the 900 fruits were stored for 180 days at ambient temperature (85–95%, relative humidity), and the fruit samples were sampled with eight fruits for each time at day 0, day 30, day 60, day 90, day 120, day 150, and day 180. After blending with liquid nitrogen frozen, the pomelo fresh samples were preserved in a 80 C ultra-low temperature refrigerator for further study, and the samples were prepared in duplicate. 2.3. Determination of Antioxidant Components 2.3.1. Ascorbic Acid Assay The content was acquired by a 2,6-dichlorophenol indophenol (DCIP) titration method [20]. About 4 g of sample powder, ground with liquid nitrogen, was mixed with 50 mL of 2% (m/v) oxalic acid solution. Then, 10 mL of the solution was transferred to a triangular flask (50 mL), and the DCIP solution that had been calibrated was immediately performed for sample solution titration. The terminal point was recorded, with a reddish appearance at 15 s fadeless. The ascorbic acid content was calculated by the consumed volume of the DCIP solution. 2.3.2. Lycopene and Carotenoids Assay The extraction method of lycopene referred to the method that was optimized by Xu et al. [21]. Of the pulp sample powder, 1.0 g, ground in liquid nitrogen, was pretreated with 95% ethanol for 30 min, centrifuged at 4000 r/min for precipitation, and petroleum ether (3.5 mL) was added to the precipitation. Then, it was incubated at 30 C in a thermostatic oscillator, with aluminium-foil paper to avoid light, and extracted for 3.8 h to dissolve it in organic solvent. After extraction, it was kept in the dark for 10 minutes, and the organic phase in the upper layer was the extraction solution. The measurement method of lycopene was described by Martínez-Valverde et al. [22]. The content of lycopene was expressed as g/g and was calculated by the molar extinction coecient at a wavelength of 472 nm. Carotenoids were measured according to the method described previously [23], with few modifications. About 0.5 g of the sample powder was added with the extraction agent (15 mL, hexane:acetone:anhydrous alcohol = 2:1:1, v/v). Then, was ultrasonically extracted for 30 min, keeping it out of the sun in the meantime. Afterwards, it was centrifuged at 4000 r/min for 10 min to obtain the supernatant. The precipitate was extracted twice with the extraction agent (15 mL) in the same way until it was colorless, combining the supernatant and allowing a constant volume to 45 mL. The absorbance value was determined at a wavelength of 450 nm, with a blank extraction solvent. The content of carotenoids was expressed as g/g and was calculated by the average extinction coecient at a wavelength of 450 nm of the standard -carotenoids. 2.3.3. The Total Phenolic and Total Flavonoid Contents Assay The total phenolic content was assayed following the Folin–Ciocalteu (FC) method [24], with few modifications. The extraction solution (0.5 mL) was mixed with distilled water (5 mL) and FC reagent (0.5 mL). The mixed solution was set for 5 min after blending, then 1 mL of sodium carbonate solution (10% mass fraction) was added to it, and the absorbance at a wavelength of 725 nm was measured, after incubation in light for 1 h. Gallic acid was used as a standard sample to make the standard curve, and the total phenolic content was expressed as mg/g. Antioxidants 2019, 8, 136 4 of 15 The aluminium nitrate colorimetric method [25] was applied to determine the total flavonoid content, with certain alterations. About 0.3 mL of sodium nitrite (5% mass fraction) was added to the extraction solution (2.4 mL). After blending and incubating it for 6 min, 0.3 mL of aluminium nitrate (10% mass fraction) was added. Then, it was incubated for another 6 min, 4 mL of sodium hydroxide (1 M) was added to it, and the absorbance at a wavelength of 510 nm was measured, after incubation in light for another 15 min. Rutin was used as a standard sample to make the standard curve, and the total flavonoid content was expressed as mg/g. 2.4. Enzyme Extraction and Activity Assays 2.4.1. SOD, POD, and CAT Activity Assays A total of 3.0 g of frozen pulp powder, grinded by liquid nitrogen, was added to the precooling 5.0 mL of phosphate bu er solution (PBS, 50 mM, pH 7.8) and quartz sand (0.2 g) for ice-bath grinding. Later, the homogenate was centrifuged at 16,000 r/min for 20 min at 4 C, and the supernatant stored at 4 C was used for the determination of the SOD activity, POD activity and CAT activity. The SOD activity was determined by the nitro-blue tetrazolium (NBT) method [26], with some modifications. The reaction mixture was made of 1.5 mL PBS (50 mM, pH 7.8), 0.3 mL ethylene diamine tetraacetic acid disodium (EDTA-Na , 10 mM), 0.3 mL methionine (130 mM), 0.3 mL NBT (750 M), 0.3 mL riboflavin (40 M), 0.1 mL of crude enzyme extract and 0.5 mL of distilled water, and the control group received distilled water instead. After adding riboflavin, the control group was immediately placed in the shade, with a cover made of a double layer of black cardboard. Then, the reaction mixture was placed under a 4000 lx fluorescent lamp for 15–20 min at 25–35 C for the chromogenic reaction. Finally, the absorbance at a wavelength of 560 nm was measured, and one unit of the enzyme activity was equal to the amount of enzyme required to inhibit 50% of the NBT 1 1 photoreduction reaction. The SOD activity was expressed as U g h . The POD activity was acquired by the guaiacol method [26]. The reaction solution included 3.0 mL of guaiacol (25 mM), 0.5 mL of crude enzyme extract and 200 L of hydrogen peroxide (0.5 M). After hydrogen peroxide was added, the solution was quickly mixed to initiate the reaction and start the timer. An absorbance value at a wavelength of 470 nm at 15 s was the initial value, recording the consequent increase of absorbance in 5 min, and distilled water was the reference. The POD activity 1 1 was expressed as U g min . The CAT activity was obtained by the ultraviolet colorimetry method [26]. The reaction system consisted of 2.9 mL of hydrogen peroxide (20 mM) and 0.1 mL of crude enzyme extract, and distilled water was used as a control. An absorbance value at a wavelength of 240 nm at 15 s was recorded as the initial value, which was recorded every 30 s, and more than six data points were continuously 1 1 measured. The CAT activity was expressed as U g min . 2.4.2. APX Activity Assay The APX activity adopted the ultraviolet colorimetry method described by Amako and colleagues [27]. A weighted amount of 0.5 g of frozen fruit pulp powder was homogenized with 5 mL PBS (0.1 M, pH 7.5), pre-cooled at 4 C, containing EDTA (0.1 mM), ascorbic acid (1 mM) and polyvinylpyrrolidone (2%, PVPP). The homogenate was centrifuged at 12,000 r/min for 30 min at 4 C, and the resulting supernatant was used for the APX activity determination. The reaction mixture was made of 2.6 mL PBS (50 mM, pH 7.5), pre-cooled at 4 C, containing EDTA (0.1 mM) and ascorbic acid (1 mM), crude enzyme extract (0.1 mL) and hydrogen peroxide (2 mM), and distilled water was used as a control. The determination method was consistent with the CAT enzyme. An absorbance value at 1 1 a wavelength of 290 nm was recorded, and the APX activity was expressed as U g min . Antioxidants 2019, 8, 136 5 of 15 2.5. Assessment of Antioxidant Capacity 2.5.1. Antioxidants Extraction For the extraction of the antioxidants, 0.5 g of the pulp sample powder, grinded by liquid nitrogen, was added with 10 mL methanol. After shaking it well, it was extracted ultrasonically at 50 C for 30 min and centrifuged at 5000 r/min for 15 min to obtain the supernatant. The precipitate was extracted twice, with a 10 mL extraction agent, in the same way, until it was colorless. Then, the supernatant was combined, allowed a constant volume with methanol, and preserved in 40 C for the determination of the antioxidant assay. 2.5.2. DPPH Radical Scavenging Assay The DPPH assay [28] was used to evaluate the total antioxidant activity. DPPH reserve solution (0.2 mM) was prepared with anhydrous ethanol. After shaking it well, the mixed liquor was incubated at ambient temperature under light conditions for 30 min. The absorbance values of A (2.0 mL DPPH reserve solution and 2.0 mL 70% ethanol), A (2.0 mL DPPH reserve solution and 2.0 mL extraction solution) and A (2.0 mL 70% ethanol and 2.0 mL extraction solution) at a wavelength of 517 nm were measured for the DPPH scavenging capacity, which was expressed as follows: scavenging rate (%) = [(A A + A )/A ] 100%. 0 1 2 0 2.5.3. Superoxide Radical Scavenging Assay The superoxide anoin radical scavenging capacity was measured by the pyrogallol autoxidation method, with few modifications [29]. About 4.5 mL of Tris-HCl bu er (50 mM, pH 8.2) was mixed with distilled water (3.3 mL) and incubated for 20 min at 25 C. Then, 0.9 mL of extraction solution and 0.3 mL of catechol solution (3 mM, prepared with 10 mM HCl by 25 C pre-heat) were quickly added to it. The absorbance of the reaction solution at a wavelength of 320 nm was recorded within 5 min after blending and named V (absorbance changes after every minute). Following the method, described above, the extracted solution was replaced by distilled water for the reaction, and V was obtained. The scavenging rate (%) = [1 V /V ] 100%. 1 0 2.5.4. Hydroxyl radicals scavenging assay The hydroxyl radicals scavenging assay is based on the reaction between salicylic acid and the Fenton process [30]. Of the extract solution, 1.0 mL was added with 1.0 mL of ferrous sulfate solution (9 mM, FeSO 7H O) and 1 mL hydrogen peroxide solution (10 mM), and it was incubated at 37 C in a 4 2 water bath for 10 min. Then, 1 mL of salicylic acid solution was added (9 mM), maintaining it at 37 C for 30 min in a water bath, and the absorbance of the reaction solution at a wavelength of 510 nm was measured, named A . Following the method, described above, the extracted solution was replaced by distilled water for the reaction, and A was obtained. The scavenging rate (%) = [1 A /A ] 100%. 0 1 0 2.5.5. Ferric-Reducing Antioxidant Power Assay The FRAP was assayed following a previously described method [31], where 2.5 mL of the extraction solution was mixed with 2.5 mL PBS (0.2 mM, pH 6.6) and 2.5 mL potassium ferricyanide solution (1% mass fraction), and it was incubated at 50 C for 20 min. After cooling rapidly, 2.5 mL of trichloroacetic acid solution (10% mass fraction) was added to it. Then, the mixture was centrifuged at 3000 r/min for 10 min to obtain the supernatant. The absorbance at a wavelength of 700 nm of 2.5 mL supernatant, 2.5 mL distilled water and 0.5 mL ferric trichloride solution (0.1% mass fraction) was measured, and this represents the ferric reducing ability. In addition, the higher the absorbance value, the stronger the reducing ability. Antioxidants 2019, 8, 136 6 of 15 2.6. Statistical Analysis Data statistics and drawing were performed by Excel 2013 software and Origin 8.0 software, Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 16 respectively. All experimental results were expressed as the mean value and standard error of more Data statistics and drawing were performed by Excel 2013 software and Origin 8.0 software, than three repetitions. IBM SPSS 20.0 software (IBM-SPSS, Armonk, NY, USA) was used for the analysis respectively. All experimental results were expressed as the mean value and standard error of more of variance (ANOVA), Pearson correlation analysis and PCA. The significant di erence level is set at than three repetitions. IBM SPSS 20.0 software (IBM-SPSS, Armonk, NY, USA) was used for the p < 0.05 and p < 0.01. analysis of variance (ANOVA), Pearson correlation analysis and PCA. The significant difference level is set at p < 0.05 and p < 0.01. 3. Results 3.1. 3. R Changes esults of the Antioxidant Components of MP Ascorbic acid is an essential nutrient substance of the citrus variety, which has tremendous e ects 3.1. Changes of the Antioxidant Components of MP on fruit quality, disease resistance and anti-aging ability. The ascorbic acid in pomelo fruits decreased Ascorbic acid is an essential nutrient substance of the citrus variety, which has tremendous continuously along with the storage time (Figure 1A). While the ascorbic acid of Harvest II and Harvest effects on fruit quality, disease resistance and anti-aging ability. The ascorbic acid in pomelo fruits III decreased rapidly after 90 days of storage, that of Harvest I only dropped by 22.04% during the decreased continuously along with the storage time (Figure 1A). While the ascorbic acid of Harvest entire storage and always maintained a high level, which was significantly higher than the other two II and Harvest III decreased rapidly after 90 days of storage, that of Harvest I only dropped by (p < 0.05). 22.04% during the entire storage and always maintained a high level, which was significantly Lycopene and carotenoids are functional natural pigments of red fresh pomelo. Similar to the higher than the other two (p < 0.05). changes in ascorbic acid, lycopene also declined as the storage period was prolonged (Figure 1B). While Lycopene and carotenoids are functional natural pigments of red fresh pomelo. Similar to the carotenoids and total phenols first increased and then decreased (Figure 1C,D), the total flavonoids changes in ascorbic acid, lycopene also declined as the storage period was prolonged (Figure 1B). (Figure 1E) content had a slight fluctuation in its contents. Comparing the harvesting periods, While carotenoids and total phenols first increased and then decreased (Figure 1C,D), the total the lycopene of Harvest I was always higher than that of Harvest II and Harvest III, and there existed a flavonoids (Figure 1E) content had a slight fluctuation in its contents. Comparing the harvesting significant di erence between Harvest I and Harvest III (p < 0.05). The carotenoids of Harvest I and periods, the lycopene of Harvest I was always higher than that of Harvest II and Harvest III, and Harvest II were significantly higher than those of Harvest III after 60 days of storage (p < 0.05), and there existed a significant difference between Harvest I and Harvest III (p < 0.05). The carotenoids of the total phenols of Harvest I were significantly higher than those of Harvest III. In addition to the Harvest I and Harvest II were significantly higher than those of Harvest III after 60 days of storage flavonoids, (p < 0.05), Harvest and the total phenols o I and HarvestfII Harv canest I were maintain significantly higher than those of Harvest these functional components at a high level III. In and addition to the flavonoids, Harvest I and Harvest II can maintain these functional components at a maintain a strong biological activity and better defense against fruit oxidative stress. high level and maintain a strong biological activity and better defense against fruit oxidative stress. Harvest I 50 A Harvest I Harvest II Harvest II a Harvest III a a Harvest III a a ab 36 a a c b c a b b a 30 b b c 20 18 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d Harvest I Harvest I a D Harvest II a Harvest II Harvest III Harvest III a a a a b a b 18 100 a ab b b b ab a b b c 15 b 80 c c b 0 306090 120 150 180 0 306090 120 150 180 Storage time / d Storage time / d Figure 1. Cont. Carotenoid content (μg/g) Ascorbic acid content (mg/100g) Total phenols content (mg/g) Lycopene content (μg/g) Antioxidants 2019, 8, 136 7 of 15 Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 16 Harvest I Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 16 2.8 Harvest II Harvest III 2.4 Harvest I a 2.8 Harvest II Harvest IIIa 2.0 b a a b 2.4 a a a a b b 1.6 a c 2.0 b a a b c a a a a b b 1.2 1.6 b 0 30 60 90 120 150 180 1.2 Storage time / d 0 30 60 90 120 150 180 Figure 1. Dynamic changes for the antioxidant substance of Majiayou pomelo (MP) during storage at Figure 1. Dynamic changes for the antioxidant substance of Majiayou pomelo (MP) during storage at Storage time / d ambient temperature. (A) Changes for the ascorbic acid content; (B) Changes for the lycopene ambient temperature. (A) Changes for the ascorbic acid content; (B) Changes for the lycopene content; Figure 1. Dynamic changes fo content; r the antiox (C) C ida hang nt su es for the bstance of caroteno Majiayou pomelo (M id content; (D P) ) Cha during storage nges for the at total phenols content; (E) (C) Changes for the carotenoid content; (D) Changes for the total phenols content; (E) Changes for the ambient temperature. (A) C Changes for the total f hanges for the ascorbi lavonoids content. Th c acid content; (B) e differe Changes for the lycopene nt letters for the same storage time indicates a total flavonoids content. The di erent letters for the same storage time indicates a significant di erence content; (C) Changes for the sig caroteno nificant diff id co erence ( ntent; ( p < D 0.05 ) Cha ). nges for the total phenols content; (E) (p < 0.05). Changes for the total flavonoids content. The different letters for the same storage time indicates a 3.2. Changes of Antioxidant Enzyme of MP significant difference (p < 0.05). 3.2. Changes of Antioxidant Enzyme of MP The ROS scavenging enzyme system consisted of SOD, POD, CAT and APX, and is capable of The ROS scavenging enzyme system consisted of SOD, POD, CAT and APX, and is capable of 3.2. Changes of Antioxidant Enzyme of MP reducing the accumulation of free radicals to alleviate ROS-mediated damage. The SOD activity rose reducing the accumulation of free radicals to alleviate ROS-mediated damage. The SOD activity The ROS scavenging enzyme system consisted of SOD, POD, CAT and APX, and is capable of slightly after falling a little, and there was little fluctuation (Figure 2A). Meanwhile, its activity in rose slightly after falling a little, and there was little fluctuation (Figure 2A). Meanwhile, its activity reducing the accumulation of f Harvest II w ree radical as signific s to allevant iatl e y h ROS igher than in -mediated d Harvest amage. Th III e SOD (p < 0. act0 iv5i)t, and y rose there was no significant in Harvest II was significantly higher than in Harvest III (p < 0.05), and there was no significant slightly after falling a little, and there was little fluctuation (Figure 2A). Meanwhile, its activity in difference between Harvest I and Harvest II. The POD activity (Figure 2B) had almost the same di erence between Harvest I and Harvest II. The POD activity (Figure 2B) had almost the same Harvest II was significantly higher than in Harvest III (p < 0.05), and there was no significant variation trend as the CAT activity (Figure 2C). The POD and CAT activity of Harvest III reached a variation trend as the CAT activity (Figure 2C). The POD and CAT activity of Harvest III reached a peak difference between Harvest I and Harvest II. The POD activity (Figure 2B) had almost the same peak at 60 days of storage, while Harvest I and Harvest II maximized at 90 days of storage. After 90 at 60 days of storage, while Harvest I and Harvest II maximized at 90 days of storage. After 90 days variation trend as the CAT activity (Figure 2C). The POD and CAT activity of Harvest III reached a days of storage, Harvest I and Harvest II were much higher than Harvest III, and there was a of storage, Harvest I and Harvest II were much higher than Harvest III, and there was a significant peak at 60 days of storage, sign while ificant difference bet Harvest I and Harv ween Harvest I and Harv est II maximized at 90 daest III ys of st( orag p < e. 0.05 Af). Furthermore, Ha ter 90 rvest I and di erence between Harvest I and Harvest III (p < 0.05). Furthermore, Harvest I and Harvest II could days of storage, Harvest I Ha and rvest II coul Harvest II were d defer the a much hi dvent of the enz gher than Harvest III, a yme activi nty peak i d there wa n POD s a and CAT, and both defer the advent of the enzyme activity peak in POD and CAT, and both maintained a relatively significant difference between Harvest I and Harvest III (p < 0.05). Furthermore, Harvest I and maintained a relatively high enzyme activity in the middle and later periods of the storage. APX is a high enzyme activity in the middle and later periods of the storage. APX is a key enzyme in the Harvest II could defer the advent of the enzyme activity peak in POD and CAT, and both key enzyme in the ascorbic acid–glutathione cycle, and its variation was due to ascorbic acid. ascorbic acid–glutathione cycle, and its variation was due to ascorbic acid. Obviously, APX presented a maintained a relatively high enzyme activity in the middle and later periods of the storage. APX is a Obviously, APX presented a declining trend as a whole, and the decline rate was faster after 90 days declining trend as a whole, and the decline rate was faster after 90 days of storage (Figure 2D). Besides, key enzyme in the ascorbic acid–glutathione cycle, and its variation was due to ascorbic acid. of storage (Figure 2D). Besides, the APX activity of Harvest I and Harvest II were significantly higher the APX activity of Harvest I and Harvest II were significantly higher than that of Harvest III (p < 0.05), Obviously, APX presented than that a declining of tr Harvest III end as a who (p < le, and 0.05), but the dec there was no line rate wa signific s faster after ant d 90 ifference days between Harvest I and but of ther storag e was e (F no igure significant 2D). Besidi des er , tence he AP between X activity Harvest of HarvIest and I and Harvest Harve II. st II w Thus, ere s Harvest ignificant I and ly h Harvest igher Harvest II. Thus, Harvest I and Harvest II could delay the decline of the APX activity to a certain than that of Harvest III (p < 0.05), but there was no significant difference between Harvest I and II could delay the decline of extent, promoti the APX activity ng the resista to a certain nce to extent, strepr ss omoting for pomelo the frr uit d esistance uring to storage stress . for Harvest II. Thus, Harvest I and Harvest II could delay the decline of the APX activity to a certain pomelo fruit during storage. Harvest I a Harvest I extent, promoting the resistance to stress for pomelo fruit during storage. Harvest II Harvest II 100 a 12 Harvest III 10 Harvest III Harvest I a Harvest I b a 80 a B a a Harvest II Harvest II 100 a a a a a a 8 b Harvest IIa I 10 60 a Harvest III ab ab b a 80 a ab ab a a b a a b b b ab b b a ab a a b 8 6 b b a a ab b ab 20 b c b ab b b ab 40 b b b ab b ab b 6 4 b 0 30 60 90 120 150 180 0 30 60 90 120 150 180 20 b c b b Storage time / d c Storage time / d 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d Figure 2. Cont. -1 -1 SOD activity (U g h ) Total flavonoids content (mg/g) -1 -1 SOD activity (U g h ) Total flavonoids content (mg/g) -1 -1 POD activity (U g min ) -1 -1 POD activity (U g min ) Antioxidants 2019, 8, 136 8 of 15 Antioxidants 2019, 8, x FOR PEER REVIEW 8 of 16 90 Harvest I D Harvest I Harvest II Harvest II a a 75 a a Harvest III a Harvest III a a a 400 a 60 b a b a b b a b a 200 a 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d Figure 2. Dynamic changes for the antioxidant enzymes activities of Majiayou pomelo (MP) during Figure 2. Dynamic changes for the antioxidant enzymes activities of Majiayou pomelo (MP) during storage at ambient temperature. (A) Changes for the superoxide dismutase (SOD) activity; (B) storage at ambient temperature. (A) Changes for the superoxide dismutase (SOD) activity; (B) Changes Changes for the peroxidase (POD) activity; (C) Changes for the catalase (CAT) activity; (D) Changes for the peroxidase (POD) activity; (C) Changes for the catalase (CAT) activity; (D) Changes for the for the ascorbate peroxidase (APX) activity. The different letters for the same storage time indicates a ascorbate peroxidase (APX) activity. The di erent letters for the same storage time indicates a significant significant difference (p < 0.05). di erence (p < 0.05). 3.3. Changes of the Antioxidant Capacity of MP 3.3. Changes of the Antioxidant Capacity of MP Pomelo fruit contains phenols and other antioxidants, and thus its antioxidant capacity is also Pomelo fruit contains phenols and other antioxidants, and thus its antioxidant capacity is also of great concern. In this experiment, the DPPH radical scavenging method, which is widely applied of great concern. In this experiment, the DPPH radical scavenging method, which is widely applied and highly sensitive, combined with the hydroxyl radical scavenging method, superoxide anions and highly sensitive, combined with the hydroxyl radical scavenging method, superoxide anions radical scavenging method and FRAP assay, was used to determine the pomelo pulp’s antioxidant radical scavenging method and FRAP assay, was used to determine the pomelo pulp’s antioxidant capacity. The scavenging rate of DPPH, •OH and O2 • decreased, following storage time, as shown capacity. The scavenging rate of DPPH, OH and O decreased, following storage time, as shown in Figure 3A–C. Moreover, there was no significant difference between Harvest I and Harvest II in in Figure 3A–C. Moreover, there was no significant di erence between Harvest I and Harvest II in terms of the scavenging capacity of DPPH and •OH, but the scavenging capacity was significantly terms of the scavenging capacity of DPPH and OH, but the scavenging capacity was significantly higher for these two harvesting times than for Harvest III (p < 0.01). At the same time, the higher for these two harvesting times than for Harvest III (p < 0.01). At the same time, the superoxide superoxide radical scavenging ability of Harvest III was at its lowest level among the three radical scavenging ability of Harvest III was at its lowest level among the three harvesting times, and harvesting times, and it was significantly lower than that of Harvest I and Harvest II (p < 0.05). As it was significantly lower than that of Harvest I and Harvest II (p < 0.05). As for FRAP, it showed a for FRAP, it showed a tendency of increasing first and then decreasing during storage (Figure 3D), tendency of increasing first and then decreasing during storage (Figure 3D), and the reducing ability in and the reducing ability in Harvest II was significantly higher than in Harvest I and Harvest III, Harvest II was significantly higher than in Harvest I and Harvest III, after 60 days of storage (p < 0.05), after 60 days of storage (p < 0.05), which indicated that Harvest II could effectively slow its descent, which indicated that Harvest II could e ectively slow its descent, compared with the others. To sum compared with the others. To sum up, Harvest I and Harvest II had a stronger antioxidant capacity up, Harvest I and Harvest II had a stronger antioxidant capacity than Harvest III. than Harvest III. 100 60 Harvest I Harvest I 3.4. Correlation a Analysis of Di erent Indexes of MP Harvest II Harvest II ab Harvest III Harvest III Pearson correlation analysis can reveal the degree of correlation between two parameters. 55 a a a ab Antioxidant components are consumed continuously under ambient temperatur ab e storage, a and the ab 85 b a a a ab antioxidant enzyme activity and free radical a scavenging ability, whose internal relationships were a a 80 a expressed in the form of correlation coecient, are weakened accordingly. Obviously, there were multiple sets of significant correlations among di erent indexes (p = 0.05 or p = 0.01) during the storage of MP (Figure 4). As for the antioxidant components, there were significant positive correlations 0 30 60 90 120 150 180 0 30 60 90 120 150 180 among ascorbic acid, lycopene, carotenoid and total phenol (p = 0.01). The correlation between Storage time / d Storage time / d ascorbic acid and lycopene (r = 0.930, p = 0.01), total phenol and carotenoids (r = 0.879, p = 0.01) was 0.60 Harvest I C Harvest I higher. At the same time, the correlation between ascorbic D acid and lycopene (r = 0.930, p = 0.01), a Harvest II Harvest II total phenol and carotenoids (r = 0.879, p = 0.01) was quite high. However, the correlation coecient Harvest III Harvest III 0.56 a a 20 a ab of the total flavonoids and other antioxidant components (ascorbic acid, lycopene, acarotenoids and b a b a total phenols) 15 was relativelyalow and was considered a low b correlation. This was the same for all a b ab 0.52 b b of the antioxidant components and SOD and FRAP. These results indicated that the total flavonoids 10 b b b were probably not the main contributor to the ab antioxidant capacity of the pomelo fruit during storage, 0.48 c c and there were di erences among the di erent antioxidant evaluation systems, like SOD and FRAP. In addition, there were significant or extremely significant correlations among antioxidant components, 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d antioxidant enzymes and free radical scavenging ability. The high corr Sto elations rage time /wer d e between two -1 -1 - DPPH scavenging capacity (%) CAT activity (U g min ) O ⋅ scavenging capacity (%) -1 -1 FRAP (A) APX activity (U g min ) ⋅ OH scavenging capacity (%) Antioxidants 2019, 8, x FOR PEER REVIEW 8 of 16 90 a Harvest I D Harvest I Harvest II Harvest II a a 75 a Harvest III a Harvest III a 400 a 60 b a b a b a a 200 a 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d Figure 2. Dynamic changes for the antioxidant enzymes activities of Majiayou pomelo (MP) during storage at ambient temperature. (A) Changes for the superoxide dismutase (SOD) activity; (B) Changes for the peroxidase (POD) activity; (C) Changes for the catalase (CAT) activity; (D) Changes for the ascorbate peroxidase (APX) activity. The different letters for the same storage time indicates a significant difference (p < 0.05). 3.3. Changes of the Antioxidant Capacity of MP Pomelo fruit contains phenols and other antioxidants, and thus its antioxidant capacity is also of great concern. In this experiment, the DPPH radical scavenging method, which is widely applied and highly sensitive, combined with the hydroxyl radical scavenging method, superoxide anions radical scavenging method and FRAP assay, was used to determine the pomelo pulp’s antioxidant capacity. The scavenging rate of DPPH, •OH and O2 • decreased, following storage time, as shown in Figure 3A–C. Moreover, there was no significant difference between Harvest I and Harvest II in terms of the scavenging capacity of DPPH and •OH, but the scavenging capacity was significantly Antioxidants 2019, 8, 136 9 of 15 higher for these two harvesting times than for Harvest III (p < 0.01). At the same time, the superoxide radical scavenging ability of Harvest III was at its lowest level among the three antioxidant components (ascorbic acid and lycopene) and APX activity and free radical scavenging harvesting times, and it was significantly lower than that of Harvest I and Harvest II (p < 0.05). As ability for FRA (DPPH, P, it showed a ten OH, O ), dency of the other incr two easing (carotenoids first and then decr and total phenol) easing d and urin enzyme g storage (Fig activity ure (POD 3D), and and the reducing CAT), whose corr ability in Harves elation coecients t II was signific were all mor antly higher th e than 0.8, andan in Harvest were of a significant I and Harvest correlation III, (after p = 0.01). 60 days of storage ( These results p indicated < 0.05), wh that ichthese indica antioxidant ted that Harve components st II coulddetermined effectively slow the antioxidant its descent, performance compared wiof th t pomelo he others pulp . To sum to a lar up ge, extent, Harves and t I and the H evaluation arvest II h ra esults d a stwer ronger e of ant a high ioxid reliability ant capa.city than Harvest III. 100 60 Harvest I Harvest I Harvest II Harvest II ab Harvest III Harvest III 90 a a a ab ab ab b a a a a ab 80 a 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d 0.60 Harvest I C Harvest I Harvest II Harvest II Harvest III Harvest III 0.56 a a ab 15 a a b ab 0.52 b 10 b b b b c ab 0.48 c c 0 30 60 90 120 150 180 0 30 60 90 120 150 180 Storage time / d Storage time / d Figure 3. Dynamic changes for the antioxidant capacity of Majiayou pomelo (MP) during storage at ambient temperature. (A) Changes for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging capacity; (B) Changes for the hydroxyl radical (OH) scavenging capacity; (C) Changes for the superoxide anions radical (O ) scavenging capacity; (D) Changes for the ferric reducing antioxidant power (FRAP). The di erent letters for the same storage time indicates a significant di erence (p < 0.05). 3.5. PCA on the Postharvest Antioxidant Capacity of MP According to the preliminary experimental results, we standardized 13 indexes of MP in the postharvest storage process for PCA by using SPSS 20.0 software. Three principal components were extracted, and the cumulative variance contribution rate reached 85.633% (Table 1), which could reflect most information from the original data. According to the load values of each principal component in Table 2, the variance contribution rate of principal component 1 (45.124%) is the highest, and ascorbic acid, lycopene, APX activity, and scavenging capacity of DPPH, OH and O all had a bigger loading on it. As for the variance contribution rate of principal component 2, it was 30.256%, and the carotenoids, total phenols, POD activity, CAT activity and FRAP all had a large loading on it. Similarly, the variance contribution rate of principal component 3 was 10.252%, and the total flavonoids and SOD activity had a large load on it. The above three aspects constitute the comprehensive postharvest antioxidant capacity of MP. -1 -1 DPPH scavenging capacity (%) CAT activity (U g min ) O ⋅ scavenging capacity (%) -1 -1 FRAP (A) APX activity (U g min ) ⋅ OH scavenging capacity (%) Antioxidants 2019, 8, 136 10 of 15 Ascorbic acid Ascorbic acid 1 Lycopene Lycopene 0.930 ** 1 Carotenoid Carotenoid 0.711 ** 0.664 ** 1 Total phenols Total phenols 0.751 ** 0.705 ** 0.879 ** 1 Total flavonoids Total flavonoids 0.039 0.178 0.359 0.486 * 1 SOD SOD 0.136 0.362 0.058 0.175 0.277 1 POD POD 0.640 ** 0.572 ** 0.860 ** 0.805 ** 0.300 −0.028 1 CAT CAT 0.819 ** 0.753 ** 0.883 ** 0.852 ** 0.163 0.083 0.881 ** 1 APX APX 0.864 ** 0.87 ** 0.523 * 0.637 ** −0.112 0.412 0.488 * 0.700 ** 1 DPPH scavenging capacity DPPH scavenging capacity 0.827 ** 0.804 ** 0.503 * 0.597 ** −0.100 0.357 0.445 * 0.633 ** 0.914 ** 1 •OH scavenging capacity •OH scavenging capacity − 0.856 ** 0.926 ** 0.467 * 0.541 * 0.066 0.507 * 0.363 0.588 ** 0.858 ** 0.764 ** 1 O2 • scavenging capacity O2 • scavenging capacity 0.885 ** 0.912 ** 0.560 * 0.635 ** 0 0.507 * 0.514 * 0.726 ** 0.930 ** 0.825 ** 0.939 1 FRAP FRAP 0.211 0.155 0.360 0.541 * 0.218 0.092 0.343 0.274 0.339 0.504 * 0.019 0.117 1 Figure 4. Correlation matrix based on Pearson’s correlation coefficient between the different antioxidant substances, antioxidant enzymes and antioxidant capacities. The Figure 4. Correlation matrix based on Pearson’s correlation coecient between the di erent antioxidant substances, antioxidant enzymes and antioxidant capacities. color intensity and numbers are proportional to the correlation coefficients. Positive correlations are shown in red, and negative correlations, in blue. *,** means a The color intensity and numbers are proportional to the correlation coecients. Positive correlations are shown in red, and negative correlations, in blue. *,** means a significance of p = 0.05 and p = 0.01, respectively. significance of p = 0.05 and p = 0.01, respectively. Antioxidants 2019, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 136 11 of 15 In order to further compare the di erences of the postharvest antioxidant capacity at each harvesting period, combining the standardized indexes (X –X ) and factor load value, we obtained 1 13 the scores of each principal component, which is shown in the following formula: F = 0.844X + 0.868X + 0.382X + 0.436X 0.234X + 0.448X + 0.317X 1 1 2 3 4 5 6 7 +0.583X + 0.932X + 0.855X + 0.931X + 0.947X + 0.054X 8 9 10 11 12 13 F = 0.458X + 0.380X + 0.858X + 0.856X + 0.562X 0.153X + 0.858X 2 1 2 3 4 5 6 7 +0.737X + 0.245X + 0.270X + 0.118X + 0.229X + 0.539X 8 9 10 11 12 13 F = 0.130X + 0.117X 0.032X + 0.160X + 0.809X + 0.699X 0.137X 0.155X 3 1 2 3 4 5 6 7 8 +0.028X + 0.026X + 0.203X + 0.123X + 0.179X 9 10 11 12 13 Then, the score of each principal component was multiplied by the contribution rate of variance, that is, the weight of each principal component, and we obtained the comprehensive model of the postharvest antioxidant capacity of MP: F = 0.45124F + 0.30256F + 0.10252F 1 2 3 Figure 5 shows the comprehensive scores of MP in the postharvest storage at di erent harvesting periods. Taking the composite score of 0 as the benchmark, a positive value indicates a strong antioxidant capacity, and a higher value indicates a stronger capacity, while a negative value indicates a poor capacity. Obviously, the composite score rose gently, after a slight decline after 30 days. After storage for 90 days, the fall of the comprehensive scores was accelerated, with the worst oxidation resistance. Harvest III was negative during the whole storage period, and Harvest I and Harvest II became negative after storage for 150 days and 120 days, respectively, which was 60 days longer than for Harvest III. Furthermore, the comprehensive scores of Harvest I and Harvest II were much higher than those of Harvest III during the whole storage period. However, the scores of Harvest I and Harvest II were relatively close, so the antioxidant capacity di erence is not significant. Thus, considering the comprehensive postharvest antioxidant capacity, Harvest I and Harvest II are significantly better than Harvest III. Table 1. Eigenvalue and accumulative contribution rate of the postharvest quality evaluation of Majiayou pomelo (MP). Variance Contribution The Cumulative Variance Principal Component Characteristic Value Rate (%) Contribution (%) 1 5.866 45.124 45.124 2 3.933 30.256 75.381 3 1.333 10.252 85.633 Table 2. Rotated component matrix of the principle component analysis. Principal Components Standardized Code Name Indexes 1 2 3 X Ascorbic acid 0.844 0.458 0.130 X Lycopene 0.868 0.380 0.117 X Carotenoid 0.382 0.858 0.032 X Total phenols 0.436 0.856 0.160 X Total flavonoids 0.234 0.562 0.699 X SOD 0.448 0.153 0.809 X POD 0.317 0.858 0.137 X CAT 0.583 0.737 0.155 X APX 0.932 0.245 0.028 X DPPH scavenging capacity 0.855 0.270 0.026 X OH scavenging capacity 0.931 0.118 0.203 X O scavenging capacity 0.947 0.229 0.123 12 2 X FRAP 0.054 0.539 0.179 Table 1. Eigenvalue and accumulative contribution rate of the postharvest quality evaluation of Majiayou pomelo (MP). Characteristic Variance Contribution The Cumulative Variance Principal Component Value Rate (%) Contribution (%) 1 5.866 45.124 45.124 2 3.933 30.256 75.381 3 1.333 10.252 85.633 Table 2. Rotated component matrix of the principle component analysis. Principal Components Standardized Code Name Indexes 1 2 3 X1 Ascorbic acid 0.844 0.458 −0.130 X2 Lycopene 0.868 0.380 0.117 X3 Carotenoid 0.382 0.858 −0.032 X4 Total phenols 0.436 0.856 0.160 X5 Total flavonoids −0.234 0.562 0.699 X6 SOD 0.448 −0.153 0.809 X7 POD 0.317 0.858 −0.137 X8 CAT 0.583 0.737 −0.155 X9 APX 0.932 0.245 0.028 X10 DPPH scavenging capacity 0.855 0.270 0.026 X11 •OH scavenging capacity 0.931 0.118 0.203 Antioxidants 2019, 8, 136 12 of 15 X12 O2 • scavenging capacity 0.947 0.229 0.123 X13 FRAP 0.054 0.539 0.179 Harvest I Harvest II Harvest III 0 30 60 90 120 150 180 -3 -6 -9 Storage time / d Figure 5. Comprehensive scores of the postharvest antioxidant capacity for Majiayou pomelo (MP) at Figure 5. Comprehensive scores of the postharvest antioxidant capacity for Majiayou pomelo (MP) at di erent harvesting periods during storage at ambient temperature. different harvesting periods during storage at ambient temperature. 4. Discussion 4. Discussion Pomelo fruits aging during storage is a relatively complex process that is accompanied by a series Pomelo fruits aging during storage is a relatively complex process that is accompanied by a of physiological and biochemical changes [32]. Faced with abiotic stresses that lead to fruit tissue series of physiological and biochemical changes [32]. Faced with abiotic stresses that lead to fruit disorders in its various functional components, including ascorbic acid, flavonoids, carotenoids and tissue disorders in its various functional components, including ascorbic acid, flavonoids, phenolic compounds, occurring at di erent degrees of synthesis or degradation [33]. What we observed carotenoids and phenolic compounds, occurring at different degrees of synthesis or degradation in the storage process of MP at ambient temperature was consistent with previous studies. While most [33]. What we observed in the storage process of MP at ambient temperature was consistent with of the antioxidants, like ascorbic acid and lycopene, were constantly and gradually degraded by previous studies. While most of the antioxidants, like ascorbic acid and lycopene, were constantly consumption as respiratory substrates during the aging process of pomelo fruits. The carotenoids and and gradually degraded by consumption as respiratory substrates during the aging process of total phenols were increased before storage for 60 days and then declined. Xu and Deng [34] found that pomelo fruits. The carotenoids and total phenols were increased before storage for 60 days and then carotenoids might continue to be synthesized in the process of postharvest storage, initially leading to declined. Xu and Deng [34] found that carotenoids might continue to be synthesized in the process an increase in its contents at the early stage of storage. Besides, the total phenolic content is influenced of postharvest storage, initially leading to an increase in its contents at the early stage of storage. by ascorbic acid, flavonoids and phenolic acids, and the phenolic substances can be released from the Besides, the total phenolic content is influenced by ascorbic acid, flavonoids and phenolic acids, and proteins and sugars mixtures [35]. It is speculated that the synthesized amount of total phenols in the early stage of storage is higher than the decomposition amount of it, resulting in an increased content. Antioxidants 2019, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/antioxidants As for the little fluctuation of the total flavonoids content, it was probably due to its low contents in the pomelo pulp [36]. Besides, most of the pigments and phenolic substances reached their peak at 90 days of storage, which was enriched with nutritious compounds that are beneficial for health. Thus, the storage time at ambient temperature should be controlled within 90 days. After that, the fruit pigment will gradually decompose, and the original flavor disappears. The free radical scavenging ability reflects the antioxidant capacity of fruits to a certain extent, and accordingly, the free radical accumulation causes tissue damage and organ aging, which is thought to be a significant tool for measuring the aging of fruits and vegetables [37]. Generally, the higher the free radical scavenging rate, the lower the degree of aging, and the better the fruit quality. The Pearson’s correlation analysis showed a decrease in the scavenging rate of DPPH, and OH and O tended to be synchronized with the consumption of ascorbic acid and lycopene. This indicated that these various substances had a great contribution to the antioxidant capacity of pomelo fruit under storage [38]. When the correlation coecients of each nutrient component with the scavenging power of OH and O were compared, lycopene and ascorbic acid showed a stronger correlation than the rest of the components, followed by carotenoids and total phenols. No significant correlation with flavonoids was seen, which demonstrates that lycopene and ascorbic acid are probably the main contributors to the antioxidant capacity of MP during storage. In the process of fruit aging, the accumulation of intracellular ROS would continue to increase and enhance the membrane lipid peroxidation, damage the organic structure and finally lead to fruit decay [39]. Fruits being harvested too early or late may a ect the levels of ROS scavenging enzymes (POD, SOD, CAT and APX), thus disrupting the cellular homeostasis of redox metabolism [40]. The results showed that the enzyme activity (POD, CAT and APX) of di erent harvesting periods accelerated the decrease after 90 days of storage. This indicated that the harvesting period had a Comprehensive scores Antioxidants 2019, 8, 136 13 of 15 great influence on the redox metabolism during the long-term storage of pomelo fruits. In addition, the enzyme activities of Harvest I and Harvest II were both higher in the later storage period than in Harvest III, and the former delayed the arrival of the enzyme activity peak. The late harvesting of MP may cause the disturbance of redox metabolism in the later storage period, which is not conducive to the storage of pomelo fruits. PCA is an authentic tool that simplifies the multiple evaluation indexes and has become one of the main methods for the comprehensive quality evaluation of fruits, vegetables and foods [41,42]. Herein, the PCA was applied to extract 4 principal components from 13 antioxidant indexes, which reflected 85.633% of the information of all indexes. After the main indexes were standardized, the scores of the three principal components were calculated. The product of each score and the variance contribution percentage of the corresponding characteristic value was accumulated to obtain the comprehensive evaluation of the antioxidant capacity for each harvesting period. Obviously, the scores decreased sharply after 90 days of storage, indicating that the optimal storage time of pomelo fruit is 90 days, after the appropriate harvest. When the storage time was too long, the fruit had a poor anti-oxidative and anti-aging ability. In addition, the comprehensive score of Harvest I was always the highest among the three, which was close to Harvest II, with a strong antioxidant performance. Therefore, the periods, i.e., 185 to 200 days after full bloom, should be considered as a suitable harvesting period for MP. 5. Conclusions In this research, Harvest I and Harvest II significantly reduced the loss of functional components, maintained higher antioxidant enzyme activities and kept a stronger ability to scavenge free radicals after 180 days of storing MP at ambient temperature, indicating that it is better to maintain the fruit’s commodity value after harvesting. Besides, the comprehensive scores of Harvest I and Harvest II during storage were far higher than the other, so the recommended harvesting time of MP for each year is determined to be early November, namely, 185 to 200 days after full bloom. This study will not only provide guidance for production practices, but also a baseline reference for future research on the change rules of e ective functional components in the postharvest process of MP. Author Contributions: Z.N. carried out the experiments, analyzed the data, prepared tables and figures and wrote the original draft; C.C. conceived and designed the experiments; J.C. obtained research funding, provided the physiology laboratory; C.W. contributed the experimental design and revised the manuscript draft, analyzed the data and supervised the project implementation. Funding: This research was funded by the Modern Agricultural Technology System of the Citrus Industry and Synergetic Innovation Center of Postharvest Key Technology and Quality Safety of Fruits & Vegetables of Jiangxi Province (NO. JXARS-07 and No. JXGS-05). Conflicts of Interest: The authors declare no conflict of interest. References 1. Cao, L.X. The Investigation of Pummelo Germplasms and the Origin Analysis of Majiayou in Guangfeng, Jiangxi Province. Master ’s Thesis, Huazhong Agricultural University, Wuhan, China, 2012. 2. Yu, J.T. Study on the Development of Famous and Special Agricultural Products in Jiangxi Province—Above Example of Raomajia Grapefruit. Master ’s Thesis, Nanchang University, Nanchang, China, 2018. 3. Liu, S.Z.; Jiang, Y.L.; Li, X.M.; Zhang, Z.Q.; Hu, W.R. Research progress in postharvest physiology and storage technology of pomelo fruit. Food Sci. 2010, 31, 394–399. [CrossRef] 4. Ding, X.; Guo, L.; Zhang, Y.; Fan, S.; Gu, M.; Lu, Y.; Jiang, D.; Li, Y.; Huang, C.; Zhou, Z. Extracts of pomelo peels prevent high-fat diet-induced metabolic disorders in c57bl/6 mice through activating the PPARalpha and GLUT4 pathway. PLoS ONE 2013, 8, e77915. [CrossRef] [PubMed] 5. Jeong, Y.; Lim, J.W.; Kim, H. Lycopene inhibits reactive oxygen species-mediated NF-B signaling and induces apoptosis in pancreatic cancer cells. Nutrients 2019, 11, 762. [CrossRef] 6. Lim, H.K.; Moon, J.Y.; Kim, H.; Cho, M.; Cho, S.K. Induction of apoptosis in U937 human leukaemia cells by the hexane fraction of an extract of immature Citrus grandis Osbeck fruits. Food Chem. 2009, 114, 1245–1250. [CrossRef] Antioxidants 2019, 8, 136 14 of 15 7. Mokbel, M.S.; Hashinaga, F. Evaluation of the antioxidant activity of extracts from buntan (Citrus grandis Osbeck) fruit tissues. Food Chem. 2006, 94, 529–534. [CrossRef] 8. Xu, J.; Deng, X.X. Red juice sac of citrus and its main pigments. J. Fruit Sci. 2002, 19, 307–313. [CrossRef] 9. Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7, 405–410. [CrossRef] 10. Chen, S.W.; Hsu, M.C.; Fang, H.H.; Tsai, S.H.; Liang, Y.S. E ect of harvest season, maturity and storage temperature on storability of carambola ‘Honglong’ fruit. Sci. Hortic. 2017, 220, 42–51. [CrossRef] 11. Ceccarelli, A.; Farneti, B.; Frisina, C.; Allen, D.; Donati, I.; Cellini, A.; Costa, G.; Spinelli, F.; Stefanelli, D. Harvest maturity stage and cold storage length influence on flavour development in peach fruit. Agronomy 2018, 9, 10. [CrossRef] 12. Elena, G.M.; Moreno, D.A.; Cristina, G.V. Genotype and harvest time influence the phytochemical quality of Fino lemon juice (Citrus limon (L.) Burm. F.) for industrial use. J. Agric. Food Chem. 2008, 56, 1669–1675. [CrossRef] 13. Huang, X.M.; Wang, H.C.; Yuan, W.Q.; Lu, J.M.; Yin, J.H. A study of rapid senescence of detached litchi: roles of water loss and calcium. Postharvest Biol. Tech. 2005, 36, 177–189. [CrossRef] 14. Valero, D.; Guillén, F.; Martínezromero, D.; Castillo, S.; Zapata, P.J.; Díazmula, H.M.; Serrano, M. The quality and antioxidant capacity during storage of sweet cherries are a ected by ripening stage at harvest. Acta Hortic. 2010, 880, 57–64. [CrossRef] 15. Moore, B. Principal component analysis in linear systems: Controllability, observability, and model reduction. IEEE Trans. Autom. Control 2003, 26, 17–32. [CrossRef] 16. Liu, H.F.; Wu, B.H.; Fan, P.G.; Li, S.H.; Li, L.S. Sugar and acid concentrations in 98 grape cultivars analyzed by principal component analysis. J. Sci. Food Agric. 2006, 86, 1526–1536. [CrossRef] 17. Wu, B.; Quilot, B.; Kervella, J.; Génard, M.; Li, S. Analysis of genotypic variation of sugar and acid contents in peaches and nectarines through the Principle Component Analysis. Euphytica 2003, 132, 375–384. [CrossRef] 18. Soufleros, E.H.; Pissa, I.; Petridis, D.; Lygerakis, M.; Mermelas, K.; Boukouvalas, G.; Tsimitakis, E. Instrumental analysis of volatile and other compounds of Greek kiwi wine; sensory evaluation and optimisation of its composition. Food Chem. 2001, 75, 487–500. [CrossRef] 19. Cheong, M.W.; Shao, Q.L.; Zhou, W.; Curran, P.; Yub, B. Chemical composition and sensory profile of pomelo (Citrus grandis (L.) Osbeck) juice. Food Chem. 2012, 135, 2505–2513. [CrossRef] 20. Akah, N.P.; Onweluzo, J.C. Evaluation of water-soluble vitamins and optimum cooking time of fresh edible portions of elephant grass (Pennisetum purpureum L. Schumach) shoot. Nig. Food J. 2014, 32, 120–127. [CrossRef] 21. Xu, Y.; Wang, L.F.; Xu, X.Y.; Pan, S.Y. Optimization of lycopene extraction from red grapefruit by response surface methodology. Food Sci. 2010, 31, 255–259. [CrossRef] 22. Martínez-Valverde, I.; Periago, M.J.; Provan, G.; Chesson, A. Phenolic compounds, lycopene and antioxidant activity in commercial varieties of tomato (Lycopersium esculentum). J. Sci. Food Agric. 2002, 82, 323–330. [CrossRef] 23. Lee, H.S. Characterization of carotenoids in juice of red navel orange (Cara Cara). J. Agric. Food Chem. 2001, 49, 2563–2568. [CrossRef] [PubMed] 24. Goulas, V.; Manganaris, G.A. Exploring the phytochemical content and the antioxidant potential of Citrus fruits grown in Cyprus. Food Chem. 2012, 131, 39–47. [CrossRef] 25. Wang, Y.C.; Chuang, Y.C.; Hsu, H.W. The flavonoid, carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chem. 2008, 106, 277–284. [CrossRef] 26. Pasquariello, M.S.; Patre, D.D.; Mastrobuoni, F.; Zampella, L.; Scortichini, M.; Petriccione, M. Influence of postharvest chitosan treatment on enzymatic browning and antioxidant enzyme activity in sweet cherry fruit. Postharvest Biol. Tech. 2015, 109, 45–56. [CrossRef] 27. Amako, K.; Chen, G.X.; Asada, K. Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants. Plant Cell Physiol. 1994, 35, 497–504. [CrossRef] 28. Yamaguchi, T.; Takamura, H.; Matoba, T.; Terao, J. HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci. Biotech. Bioch. 1998, 62, 1201–1204. [CrossRef] 29. Li, X. Improved pyrogallol autoxidation method: a reliable and cheap superoxide-scavenging assay suitable for all antioxidants. J. Agric. Food Chem. 2012, 60, 6418. [CrossRef] Antioxidants 2019, 8, 136 15 of 15 30. Chang, C.Y.; Hsieh, Y.H.; Cheng, K.Y.; Hsieh, L.L.; Cheng, T.C.; Yao, K.S. E ect of pH on Fenton process using estimation of hydroxyl radical with salicylic acid as trapping reagent. Water Sci. Tech. 2008, 58, 873–879. [CrossRef] [PubMed] 31. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [CrossRef] [PubMed] 32. Ding, Y.; Chang, J.; Ma, Q.; Chen, L.; Liu, S.; Jin, S.; Han, J.; Xu, R.; Zhu, A.; Guo, J. Network analysis of postharvest senescence process in citrus fruits revealed by transcriptomic and metabolomic profiling. Plant Physiol. 2015, 168, 357. [CrossRef] 33. Silalahi, J. Anticancer and health protective properties of citrus fruit components. Asia Pac. J. Clin. Nutr. 2002, 11, 79–84. [CrossRef] 34. Xu, J.; Deng, X.X. Identification of main pigments in red flesh Navel Orange (Citrus sinensis L.) and evaluation of their concentration changes during fruit development and storage. Acta Hortic. Sin. 2002, 29, 203–208. [CrossRef] 35. Doshi, P.J.; Adsule, P.G. E ect of storage on physicochemical parameters, phenolic compounds and antioxidant activity in grapes. Acta Hortic. 2008, 785, 447–456. [CrossRef] 36. Yu, J.; Hou, S.; Wu, H.; Zhang, Z.; Lv, Z.; Zhou, Z. Phenolic compositions and antioxidant capacity of the fruit pulp of popular pomelo cultivars in chongqing. Food Sci. 2016, 37, 83–88. [CrossRef] 37. Seel, W.; Hendry, G.; Atherton, N.; Lee, J. Radical formation and accumulation in vivo, in desiccation tolerant and intolerant mosses. Free Rad. Res. Commun. 1991, 15, 133–141. [CrossRef] 38. Gardner, P.T.; White, T.A.C.; McPhail, D.B.; Duthie, G.G. The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices. Food Chem. 2000, 68, 471–474. [CrossRef] 39. Vera-Guzmána, A.M.; Aispuro-Hernándeza, E.; Vargas-Arispuroa, I.; Islas-Osunaa, M.A.; Martínez-Télleza, M.Á. Expression of antioxidant-related genes in flavedo of cold-stored grapefruit (Citrus paradisi Macfad cv. Rio Red) treated with pectic oligosaccharides. Sci. Hortic. 2019, 243, 274–280. [CrossRef] 40. Ahmadiafzadi, M.; Tahir, I.; Nybom, H. Impact of harvesting time and fruit firmness on the tolerance to fungal storage diseases in an apple germplasm collection. Postharvest Biol. Tech. 2013, 82, 51–58. [CrossRef] 41. Hossain, M.B.; Patras, A.; Barry-Ryan, C.; Martin-Diana, A.B.; Brunton, N.P. Application of principal component and hierarchical cluster analysis to classify di erent spices based on in vitro antioxidant activity and individual polyphenolic antioxidant compounds. J. Funct. Foods 2011, 3, 179–189. [CrossRef] 42. Patras, A.; Brunton, N.P.; Downey, G.; Rawson, A.; Warriner, K.; Gernigon, G. Application of principal component and hierarchical cluster analysis to classify fruits and vegetables commonly consumed in Ireland based on in vitro antioxidant activity. J. Food Compos. Anal. 2011, 24, 250–256. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Journal
Antioxidants – Multidisciplinary Digital Publishing Institute
Published: May 17, 2019