Effects of fruit bagging on the physiochemical changes of grapefruit (Citrus paradisi)

Abstract

Fruit bagging is a commonly used cultivation measure to protect citrus fruit from insects and adverse environments. The present study aimed to comprehensively investigate the effects of bagging on the physiochemical characteristics of grapefruit. The grapefruit were bagged at approximately 110–120 d after anthesis with a one-layer kraft paper bag with black coating inside (SL), a double-layer kraft paper bag with one black paper as the inner layer (DL), and a three-layer kraft paper bag with two black papers as inner layers (TL), respectively. Ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) technique was used to identify a total of 19 flavonoids, 2 phenylpropanoids, 9 coumarins, and 5 limonoids. By using UPLC, 50 carotenoids were identified. Gas chromatography–mass spectrometry was used to identify 3 soluble sugars, 3 organic acids, and 11 amino acids. In the quantitated components in the peel (albedo and flavedo), the chlorophylls and the carotenoids components (such as luteoxanthin, violaxanthin, 9-cis-violaxanthin, xanthophyll, zeaxanthin and β-carotene) were significantly downregulated by bagging, causing the surface color of bagged fruit to turn yellow earlier but paler than that of the unbagged control, particularly in the three-layer kraft bag treatment. Unlike the peel, the color and the carotenoid content of the juice sacs were less affected. The physiochemical compounds other than pigments, including soluble sugars, organic acids, amino acids, flavonoids, coumarins and limonoids, were minimally affected by bagging treatments. Our results indicated that bagging at approximately 110–120 d after anthesis exerted influence mainly on peel color, but less on sugars, acids, amino acids, flavonoids, limonoids and coumarins of grapefruit.

grapefruit, fruit bagging, physiochemical characteristics, fruit quality

Introduction

Fruit bagging is a commonly used technology in fruit production. The farmers cover the fruit with bags to protect them from insect attack, damage from adverse environments such as excessively strong sunlight and wind, as well as the infection by pathogens. Various bags are used, including paper or plastic bags, transparent or not, depending on the fruit varieties and comprehensive consideration of cost and benefits (Hiratsuka et al., 2012; Magwaza et al., 2013; Lado et al., 2015, 2019; Promkaew et al., 2020). In practice, people found additional biological effects of fruit bagging, including effects on fruit quality, due to the changes in microenvironment around the fruit.

In citrus production, bagging is commonly used in some varieties, such as pomelo, orange and some citrus hybrids. Kraft paper bags with black coatings inside or lined with black paper inside are most widely used. A considerable number of studies have investigated the effect of fruit shading on citrus. Most of these studies report that bagging improves the commodity appearance of citrus fruit, with smoother peel surface and fewer defective spots (Magwaza et al., 2013, 2019); bagging also makes the rind color develop earlier (Sun et al., 2014; Lado et al., 2019; Promkaew et al., 2020; Wu et al., 2020). However, some negative effects of bagging have are been reported in some studies, such as decreasing the total soluble solids (TSS) content and soluble sugar content (Wang et al., 2005), and weakening the fragrance (Jiang et al., 2020). However, the results of different studies varied, which might be attributed to different bagging times. As reported by Wang et al. (2006), the effects of bagging on the reduction of soluble sugars and TSS are more significant in fruit bagged early. In addition, different bags and citrus varieties also lead to different results (Chun et al., 2008; Yang et al., 2019; Tang et al., 2020; Qiu et al., 2020).

Grapefruit is a popular fresh edible and processed citrus. It is characterized by a juicy taste and delicate fragrance. It is also rich in secondary metabolic compounds such as flavonoids, coumarins, and carotenoids, which have been reported to affect human health. The accumulation of some secondary metabolites even leads to bitter taste. In a previous study on red grapefruit, fruit shading using black plastic bags was reported to enhance the accumulation of total carotenoids and promote the development of rind color (Lado et al., 2015). The present study aimed to carry out a comprehensive study on the effects of bagging on grapefruit, through an extensive investigation on the dynamic changes in physiochemical characteristics.

Materials and Methods

Plant materials and treatments

Fruit bagging was conducted in the greenhouse of Yunzeying Modern Agricultural Demonstration Garden in Quzhou City, Zhejiang Province, China. Plants with similar tree vigor and under the same cultivation management were randomly selected. Fruit was bagged on 20 August 2020 (approximately 110–120 d after anthesis), with a one-layer kraft paper bag with a black coating inside (SL), a double-layer kraft paper bag with one black paper as the inner layer (DL), and a three-layer kraft paper bag with two black papers as inner layers (TL). For each bagging treatment, 20 trees were selected, with 15 fruit from different directions on each tree bagged. The unbagged fruit in the same tree were used as control. The fruit were harvested on 15 October (approximately 175–185 d after anthesis, S1), 30 October (approximately 190–200 d after anthesis, S2), 15 November (approximately 205–215 d after anthesis, S3), 30 November (approximately 220–230 d after anthesis, S4), and 15 December (approximately 235–245 d after anthesis, S5). At least 20 healthy fruits were collected for each treatment at each time point. The peel (flavedo and albedo) and juice sacs at the equator site were quickly frozen in liquid nitrogen and stored at –80 °C for later use.

Light transmission ratio of the bags

The light transmission ratio of the bags in the range 200–2500 nm was determined using a U-4100 UV/visible/near-infrared spectrometer (Hitachi, Tokyo, Japan), and that in the range 2500–8000 nm was determined by Thermo Nicolet iS10 Fourier transform infrared spectroscopy (Thermo Fisher Scientific, Waltham, MA, USA). Three repetitions were performed for each kind of bag, and the mean value was obtained.

Rind color and TSS analysis

The rind color of the fruit was evaluated according to a previously reported method (Cao et al., 2019). The L*, a*, b* values of the rind were measured by a Hunter Lab MiniScan XE Plus Colorimeter (Hunter Associates Laboratory, Inc., Reston, VA, USA) at four evenly distributed equatorial sites on the fruit surface. Twenty fruit repetitions were performed for each group. Citrus color index (CCI) was calculated as 1000×a*/(L*×b*).

TSS contents were detected by a refractometer PR101-a (Atago, Tokyo, Japan) with two measurements per fruit, and 20 fruit repetitions for each group.

Total chlorophylls and total carotenoids analysis

The total chlorophylls and carotenoids were analyzed according to the previously reported method (Fadeel, 1962). The peel and juice sac samples were lyophilized and ground into fine powder. Approximately 0.1 g of each sample was extracted with 4 mL cold 80% acetone for 30 min. The supernatants were obtained by centrifugation at 4000 r/min for 10 min. The extraction was repeated, and the supernatants of the two extractions were combined and added to 80% acetone to make up the final volume to 10 mL. The absorbance of the extracts was detected at 445, 644, and 662 nm, respectively, by UV/Vis spectrophotometer UV-2600 (Shimadzu, Kyoto, Japan). The concentration of chlorophyll a (C_a) was calculated as 9.78×A662–0.99×A644, the concentration of chlorophyll b (C_b) was calculated as 21.4×A644–4.65×A662, and the concentration of total carotenoids was calculated as 4.69×A445–(C_a+C_b)×0.268. The content was represented as μg/g dry weight of the sample.

Identification and quantitation of phenolics, coumarins and limonoids

The lyophilized samples were ground into fine powder. The ground samples (100 mg peel, 300 mg juice sacs) were extracted with 2 mL methanol for 30 min with the assistance of an ultrasonic water bath. The extraction was carried out three times, and the supernatants of the three extractions were combined and filtered through 0.22-μm syringe filters. The AB Sciex Triple TOF 5600+ system (AB Sciex, Framingham, MA, USA) coupled with an electrospray ionization source (ESI) system was used for the identification of compounds. Compounds were separated by HSS T3 analytical column (ACQUITY UPLC, 2.1 mm×150 mm, 1.8 μm; Waters Corp., Milford, MA, USA). A volume of 2 μL sample was injected under a column temperature of 25 °C. The mobile phase consisted of H₂O (A) and acetonitrile (B) at a flow rate of 0.3 mL/min. The gradient program was as follows: 0–8 min, 2%–20% B; 8–15 min, 20%–28% B; 15–17 min, 28%–50% B; 17–22.5 min, 50%–98% B. The compounds were detected at 280 nm. The parameters for the mass analysis were as follows: source voltage of 5500 V (positive mode) and 4500 V (negative mode), curtain gas of 0.24 MPa, ion source temperature of 600 °C (positive mode) and 500 °C (negative mode), declustering potential of 100 V and collision energy of 10 V. Compounds were quantitated by the UPLC-DAD system (Waters Corp.) coupled with the HSS T3 analytical column (ACQUITY UPLC, 2.1 mm×150 mm, 1.8 μm; Waters Corp.). The mobile phase and gradient program were the same as those used in the compound identification. Authentic standards of 4ʹ-β-d-glucosyl naringenin 7-O-rutinoside, eriocitrin, neoeriocitrin, scolymoside, narirutin, naringin, hesperidin, neohesperidin, poncirin, epoxyaurapten, isosinensetin, meranzin, limonin, nobiletin, nomilin, tangeritin, and auraptene (Yuanye Bio-Technology Co., Ltd., Shanghai, China) were used to assist compound identification.

Carotenoids composition determination

The carotenoid composition was determined according to a documented method with minor modifications (Xu et al., 2006). Ground, lyophilized samples of 100 mg were extracted with a mixture solution of 350 μL methanol, 700 μL chloroform and 350 μL water. The organic phase was collected by centrifugation at 10 000 r/min for 10 min. The residues were extracted repeatedly for an additional two times, and the organic phases from the three extractions were combined. The solvent in the extract was removed in a vacuum centrifugal concentrator at 30 °C. The residue was reacted with 350 μL of 6% KOH methanol solution in the dark at 60 °C for 30 min. The reacted mixture was added with 700 μL of chloroform and 350 μL of water and mixed well. Then, the mixture was centrifuged at 10 000 r/min for 5 min. The chloroform phase was collected, and then washed with 700 μL of water repeatedly until the water layer became neutral. The chloroform phase was concentrated under vacuum (30 °C) in a rotary evaporator and the samples were dissolved in 100 μL chromatographic ethyl acetate and filtered through 0.22-μm syringe filters. High-performance liquid chromatography (HPLC) analysis was carried out in a Waters Alliance system (2695 pump, 2996 diode array detector; Waters Corp.) coupled with a C30 column (250 mm×4.6 mm i.d., 5 μm), with a column temperature of 25 °C and an injection volume of 20 μL. The mobile phases consisted of methanol (A), 80% methanol containing 0.2% ammonium acetate (B), and tert-butyl methyl ether (C) at a flow rate of 1 mL/min. The gradient program was as follows: 0–6 min, 95% A and 5% B, 7–12 min, a linear gradient to 80% A, 5% B, and 15% C, 13–18 min, 80% A, 5% B, and 15% C, 19–30 min, gradient changed to 30% A, 5% B, and 65% C, 30–35 min, 30% A, 5% B, and 65% C, 35–37 min, gradient changed to 95% A and 5% B, 38–45 min, 95% A and 5% B. The compounds were detected under the scanning wavelength of 200–600 nm. The phytofluene was quantitated at 340 nm, and the other compounds were detected at 450 nm. Authentic standards of α-carotene (Anjiekai Biological Medicine Technology Co., Ltd., Shanghai, China), β-carotene, zeaxanthin (Solarbio Life Sciences, Beijing, China), β-cryptoxanthin and xanthophyll (Yuanye Bio-Technology Co., Ltd.) were used for the compounds’ determination.

Determination of soluble sugars, organic acids, and amino acids

The soluble sugars, organic acids and amino acids content were evaluated using the gas chromatography–mass spectrometer (GC-MS) method as previously described by Cao et al. (2020). The ground, lyophilized juice sac of 50 mg was mixed with 1.4 mL methanol and extracted by vertexing at 70 °C for 15 min. The supernatant was collected by centrifugation at 10 000 r/min for 10 min. Then 1.5 mL of ddH₂O and 750 μL of trichloromethane were added and fully mixed. After centrifugation at 4000 r/min for 10 min, 100 μL of the supernatant was obtained for the derivatization. Each sample was added to 10 μL of ribitol at 0.2 mg/mL, and then dried in a vacuum centrifugal concentrator at 30 °C for 3 h. The residues were derivatized with 60 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) by vertexing for 90 min at 37 °C. Then 40 μL NSTFA (99:1) (Sigma-Aldrich, St. Louis, MO, USA) was added and the mixture was vortexed at 37 °C to obtain the derivatives. The GC-MS analysis system consisted of a GC system (Agilent 7890GC, Agilent Technologies, Inc., Santa Clara, CA, USA), a fused silica capillary column (60 m×0.25 mm, 0.25 μm, HB-5MS stationary phase) and a mass spectrometer detector (Agilent 5975 MSD). The program for analysis was run at an initial column temperature at 100 °C for 1 min, then ramped to 184 °C at a rate of 3 °C/min, followed by a second ramp to 190 °C at a rate of 0.5 °C/min, and a third ramp to 250 °C at a rate of 10 °C/min, held for 1 min, then finally ramped to 280 °C at a rate of 5 °C/min, held for 3 min. The MS conditions were as follows: the ion source temperature was 230 °C, the ionization mode was electron bombardment ionization, the electron energy was 70 eV, the temperature of the fourth stage rod was 150 °C, and the temperature of the transmission line was 250 °C. The content of soluble sugars, organic acids and amino acids were calculated according to the standard curves of authorized compounds.

Data analysis

All experiments were performed with at least three repetitions. One-way analysis of variance (ANOVA) with Tukey’s method was applied to determine significant differences of means via SPSS 23.0 (IBM Corp., Armonk, NY, USA).

Results

Effects of bagging on fruit appearance and general quality index

UV-B (280–315 nm), UV-A (315–400 nm) and blue light (400–500 nm) were almost completely shut out by all three types of bags. The three-layer bag almost completely shut out the ambient lights of different wavelengths. For the two-layer bag, a very small proportion of the light (1500–2500 nm, <0.2%) can be transmitted through the bag. For the single-layer bag, a certain proportion of light in the range of >500 nm can be transmitted through the bag, and the transmission rate increased with the wavelength of the light in the range of 500–2500 nm (Fig. 1A–1B).

Figure 1.

Effects of bagging on the appearance and general quality indexes of grapefruit. (A) Light transmission ratio of different bags at 200–2500 nm; (B) light transmission ratio of different bags at 2500–7500 nm; (C, D) appearance of the peel and pulp of fruit; (E) chlorophyll a; (F) chlorophyll b; (G) total carotenoids; (H) citrus color index (CCI) of the fruit; (I) total soluble solids (TSS); S1, 15 October (approximately 175–185 d after anthesis); S2, 30 October (approximately 190–200 d after anthesis); S3, 15 November (approximately 205–215 d after anthesis); S4, 30 November (approximately 220–230 d after anthesis); S5, 15 December (approximately 235–245 d after anthesis); CK, unbagged control; SL, single-layer bag; DL, double-layer bag; TL, three-layer bag; DW, dry weight.

The color in the peel and the juice sac developed with the maturity of the fruit. Bagging accelerated the fading of the green color in the peel, with the effects intensified with the increase of bag layers. The three-layer bagged fruit completely turned yellow on 15 October, approximately 45 d ahead of the control (Fig. 1C), but the color of the juice sac showed no significant difference between the treatments and control (Fig. 1D).

The chlorophyll contents of the peel, especially chlorophyll a, were significantly reduced in the bagged treatments during S1 to S3 stages. However, the decrease of chlorophylls (especially chlorophyll b) in the late mature stage was inhibited by bagging, and even elevated at S5 (Fig. 1E–1F). With the development of the maturity process, the unbagged fruit turned yellow, and the difference of chlorophyll contents narrowed. After 30 December, the bagged and unbagged fruit became similar in color appearance and pigment contents (Fig. 1E–1H). The total carotenoid content was reduced in the bagged treatments in all the development stages (Fig. 1G).

TSS content fluctuated at the five time points, indicating that the inner taste quality of the fruit had been completely formed and became stable after 15 October. Bagging treatments had no significant effect on the TSS content (Fig. 1I).

Identification of metabolites

By chemical derivatization and GC-MS analysis, a total of 11 amino acids, 3 organic acids, and 3 sugars were identified in the juice sac of the grapefruit. Citric acid was the chief organic acid, followed by malic acid and succinic acid. Glucose and fructose were the chief sugars, followed by sucrose (Table S1, Fig. S1).

A total of 34 compounds were identified in the peel, and 22 compounds were identified in the juice sac, combining ion fragments of UPLC-HRMS, characteristic absorbance wavelength, retention time of authentic standards and the reference works of previous studies (Table 1, Fig. S2). The identified compounds in the peel included 1 phenylpropanoid, 9 coumarins, 11 flavanones, 7 flavones, 1 flavonols and 5 limonoids, and those in the juice sac included 2 phenylpropanoids, 8 coumarins, 11 flavanones and 1 flavonol. The limonoids, diosmetin-7-O-(6ʹʹ-O-acetyl)-neohesperidoside, 5,7,3ʹ,4ʹ-tetramethoxy flavone, and osthol existed in trace amounts in the peel, as well as apiosylskimmin. 1-O-trans-feruloyl-d-glucopyranoside, scolymoside, diosmetin-7-O-(6ʹʹ-O-acetyl)-neohesperidoside, naringin-6ʹ-rhamnoside, poncirin, epoxyaurapten, osthol, and meranzin existed in a trace amount in the juice sac. These compounds were difficult to be accurately quantitated and thus were not considered in the following analysis (Table S2).

Table 1.

Identification of flavonoids, limonoids and coumarins in the peel and juice sac of the grapefruit

Peak No.	TR (min)	Identification result	Molecular formula	Parent ion	λmax (nm)	Detected MW	MS2 ions	Class	Authorized	Distribution
1	5.2761	Apiosylskimmin (Luo et al., 2022)	C20H24O12	[M+H]+	235, 320, 389	457.1341	427, 391, 325, 187, 177, 163, 145	Coumarins		P, JS
2	5.8950	Umbelliferone 7-O-α-l-rhamnopyranosyl (1->2) β-d-glucopyranoside	C21H26O12	[M+H]+	240, 318, 389	471.1497	373, 218, 163	Coumarins		P, JS
3	6.0554	Scopoletin 7-O-α-l-rhamnopyranosyl-(1ʹ‘→6’)-β-d-glucopyranoside	C22H28O13	[M+H]+	242, 330, 380	501.1603	355, 193, 133	Coumarins		P, JS
4	6.2678	1-O-trans-Feruloyl-d-glucopyranoside (Liu et al., 2021)	C16H20O9	[M-H]-	230, 282, 330	355.1035	295, 193, 178, 160, 149, 134, 132	Phenylpropanoids		JS
5	6.5543	Trans-p-Sinapoyl β-d-glucopyranoside(Hilary et al., 2020)	C17H22O10	[M-H]-	242, 330	385.114	385, 247, 223, 205, 190, 175, 164	Phenylpropanoids		P, JS
6	6.7223	Citrusin A(Zhang et al., 2022)	C26H34O12	[M+FA-H]-	223, 330	583.2032	583, 537, 375, 327, 312, 195, 165, 150	Coumarins		P, JS
7	7.3024	4ʹ-β-d-glucosyl naringenin 7-O-rutinoside	C33H42O19	[M+FA-H]-	214, 285, 330	787.2302	741, 479, 433, 271, 151, 119	Flavanones	Std	P, JS
8	7.7840	Naringin 4ʹ-glucoside (Guo et al., 2021)	C33H42O19	[M+FA-H]-	214, 285, 330	787.2302	741, 579, 459, 271	Flavanones		P, JS
9	8.6810	Eriocitrin	C27H32O15	[M-H]-	282	595.1668	595, 459, 287, 175, 151, 135	Flavanones	Std	P, JS
10	9.1670	Neoeriocitrin	C27H32O15	[M-H]-	282	595.1668	595, 459, 339, 287, 235, 191, 151, 135	Flavanones	Std	P, JS
11	9.4193	Scolymoside	C27H30O15	[M-H]-	207, 254, 349	593.1512	593, 285, 284	Flavonols	Std	P, JS
12	9.6775	Naringin-6ʹʹ-rhamnoside (Guo et al., 2021)	C33H42O18	[M-H]-		725.2298	725, 605, 271, 151	Flavanones		P, JS
13	10.0042	Narirutin	C27H32O14	[M-H]-	282, 328	579.1719	579, 271, 151	Flavanones	Std	P, JS
14	10.4894	Naringin	C27H32O14	[M-H]-	282, 328	579.1719	579, 459, 339, 313, 271, 235, 177, 151, 119	Flavanones	Std	P, JS
15	11.0300	Hesperidin	C28H34O15	[M-H]-	282	609.1825	609, 301	Flavanones	Std	P, JS
16	11.5631	Neohesperidin	C28H34O15	[M-H]-	282	609.1825	609, 489, 343, 301, 286, 242	Flavanones	Std	P, JS
17	12.5634	Diosmetin-7-O-(6ʹʹ-O-acetyl)-neohesperidoside (Guo et al., 2021)	C30H34O17	[M-H]-	204, 256, 323	665.1723	621, 501, 271, 151	Flavanones		P, JS
18	12.809	Nomilin 17-β-d-glucopyranoside(Bennett et al., 1991)	C34H46O15	[M-H]-	/	693.2764	693, 565	Limonoids		P
19	13.1651	Nomilinic acid 17-β-d-glucopyranoside(Bennett et al., 1991)	C34H48O16	[M-H]-	/	711.287	711, 651, 607	Limonoids		P
20	14.2030	Deacetylnomilin 17-β-d-glucopyranoside(Bennett et al., 1991)	C32H44O14	[M-H2O-H]-	/	633.2553	633, 427, 383	Limonoids		P
21	15.6487	Poncirin	C28H34O14	[M-H]-	226, 282, 328	593.1876	595, 594, 593, 287, 286, 285	Flavanones	Std	P, JS
22	17.6775	Epoxyauraptene (Guo et al., 2021)	C19H22O4	[M+H]+	202, 323	315.1591	297, 175, 163, 153, 135	Coumarins		P, JS
23	17.8178	Isosinensetin	C20H20O7	[M+H]+	204, 249, 270, 339	373.1282	374, 358, 344, 316, 279, 247, 232, 217, 202, 189, 161, 153	Flavones	Std	P
24	18.0550	Meranzin (Guo et al., 2021)	C15H16O4	[M+H]+	202, 256, 320	261.1121	247, 189, 131, 103	Coumarins	Std	P, JS
25	18.1739	5,7,3ʹ,4ʹ-Tetramethoxy flavone (Guo et al., 2021)	C19H18O6	[M+H]+	216, 268, 304, 328	343.3152	343, 328, 313, 285, 181, 153	Flavones		P
26	18.4050	Limonin	C26H30O8	[M+H]+	/	471.2013	362, 300, 256, 146, 132	Limonoids	Std	P
27	18.5330	7-Methoxy-coumarin-8-yl-acetaldehyde	C12H10O4	[M+H]+	207, 259, 330	219.0652	219, 161, 146, 133, 72	Coumarins		P
28	18.6914	Nobiletin	C21H22O8	[M+H]+	209, 247, 270, 332	403.1387	403, 388, 373, 358, 327, 183	Flavones	Std	P
29	18.7661	Nomilin	C28H34O9	[M+H]+	/	515.2276	515, 503, 488, 461, 431, 412, 373, 286, 219, 163	Limonoids	Std	P
30	19.0120	3,3ʹ,4ʹ,5,6,7,8-Heptamethoxyflavone (Guo et al., 2021)	C22H24O9	[M+H]+	207, 254, 339	433.1493	433, 418, 417, 403, 385, 345	Flavones		P
31	19.1503	Tangeritin	C20H20O7	[M+H]+	270, 323	373.1282	373, 358, 343, 297, 211, 183	Flavones	Std	P
32	19.6576	5-Hydroxy-6,7,8,3ʹ,4ʹ-pentamethoxyflavone	C20H20O8	[M+H]+	202, 254, 282, 339	389.1231	389, 374, 373, 359, 341, 197, 169	Flavones	Std	P
33	19.8515	8-Hydroxy-5,6,7,3ʹ,4ʹ,5ʹ- hexamethoxy-flavone (5HPMF) (Guo et al., 2021)	C21H22O9	[M+H]+	204, 256, 285, 330	419.1337	419, 404, 403, 389, 371, 361, 328, 165	Flavones		P
34	20.1980	Osthol (Guo et al., 2021)	C15H16O3	[M+H]+	226, 273, 301, 330	245.1172	189, 131, 103	Coumarins		P, JS
35	21.4963	Auraptene	C19H22O3	[M+H]+	202, 320	299.1642	163, 107	Coumarins	Std	P, JS

TR, retention time; P, peel; JS, juice sac; Std, standard compounds; ‘/’ indicates that the information was not available.

A total of 45 and 50 carotenoids compounds were detected and quantitated in the peel and the juice sac of grapefruit, respectively, and 14 of them were identified by combining the characteristic absorbance wavelength, retention time of authentic standards or the reference works of previous studies (Table 2, Fig. S3).

Table 2.

Identification of carotenoids in the peel and juice sac of the grapefruit

Peak No.	Compounds	TR (min)	Characteristic wavelength (nm)	Distribution
1	Unidentified 1	3.857	319	JS
2	Unidentified 2	4.141	321	JS
3	Unidentified 3	4.818	353, 380, 392	JS
4	Unidentified 4	5.087	334, 438	P, JS
5	Unidentified 5	5.407	392, 412, 449	JS
6	Unidentified 6	5.882	406, 438, 450	JS
7	Unidentified 7	6.161	438, 554	P, JS
8	Unidentified 8	6.848	420, 449	P, JS
9	Unidentified 9	7.404	450, 517	P, JS
10	Unidentified 10	8.147	424, 450	P, JS
11	Unidentified 11	8.733	402, 421, 440	P, JS
12	Unidentified 12	9.33	410, 434, 461	P, JS
13	Unidentified 13	9.556	405, 427, 461	P, JS
14	Unidentified 14	10.143	412, 434, 459	P, JS
15	Violaxanthin	11.241	415, 438, 468	P, JS
16	Unidentified 15	11.999	410, 435, 463	P, JS
17	Unidentified 16	12.479	410, 435, 458	P, JS
18	Luteoxanthin	13.011	398, 421, 447	P, JS
19	Unidentified 17	13.751	405, 425, 445	P, JS
20	Auroxanthin	14.397	379, 401, 424	P, JS
21	β-Citraurin	14.701	406, 429, 459	P, JS
22	9-Cis-violaxanthin	15.197	411, 434, 463	P, JS
23	Unidentified 18	15.648	395, 416, 443	P, JS
24	Unidentified 19	16.099	395, 416, 443	P, JS
25	Unidentified 20	16.805	408, 424, 450	P, JS
26	Xanthophyll	17.308	422, 445, 472	P, JS
27	Unidentified 21	17.735	406, 430, 450	P, JS
28	Unidentified 22	18.474	393, 420, 440	P, JS
29	Unidentified 23	18.999	383, 426, 450	P, JS
30	Zeaxanthin	19.714	430, 450, 477	P, JS
31	Unidentified 24	20.165	417, 434, 464	P, JS
32	Unidentified 25	21.227	418, 440, 467	P, JS
33	Unidentified 26	22.197	415, 440, 443	P, JS
34	Phytofluene	22.439	330, 346, 364	P, JS
35	Phytofluene	23.097	330, 347, 364	P, JS
36	Unidentified 27	24.216	430, 438, 468	P, JS
37	Unidentified 28	24.473	420, 450, 469	P, JS
38	Phytofluene	25.195	331, 345, 364	P, JS
39	Unidentified 29	25.724	424, 450, 472	P, JS
40	Unidentified 30	25.777	423, 450, 467	P, JS
41	β-Cryptoxanthin	26.097	425, 450, 478	P, JS
42	Phytofluene	26.66	331, 347, 364	P, JS
43	Unidentified 31	27.154	424, 447, 468	P, JS
44	Unidentified 32	27.772	424, 447, 468	P, JS
45	α-Carotene	28.664	423, 444, 473	P, JS
46	Unidentified 33	29.1	440, 450, 471	P, JS
47	Unidentified 34	29.502	413, 450, 473	P, JS
48	β-Carotene	29.896	426, 451, 478	P, JS
49	Unidentified 35	30.577	426, 444, 469	P, JS
50	Unidentified 36	31.11	411, 435, 462	P, JS

TR, retention time; P, peel; JS, juice sac.

Identification of differential compounds

Volcano plots were applied to demonstrate a general understanding of the effects of bagging on the metabolic characteristics of grapefruit, using a total of 72 compounds that can be accurately quantitated in the peel (Fig. 2) and 83 compounds that can be accurately quantitated in the juice sac (Fig. 3).

Figure 2.

Volcano plots of the effects of bagging on the metabolites in the peel. S1, 15 October (approximately 175–185 d after anthesis); S2, 30 October (approximately 190–200 d after anthesis); S3, 15 November (approximately 205–215 d after anthesis); S4, 30 November (approximately 220–230 d after anthesis); S5, 15 December (approximately 235–245 d after anthesis); CK, unbagged control; SL, single-layer bag; DL, double-layers bag; TL, three-layer bag.

Figure 3.

Volcano plots of the effects of bagging on the metabolites in the juice sac. S1, 15 October (approximately 175–185 d after anthesis); S2, 30 October (approximately 190–200 d after anthesis); S3, 15 November (approximately 205–215 d after anthesis); S4, 30 November (approximately 220–230 d after anthesis); S5, 15 December (approximately 235–245 d after anthesis); CK, unbagged control; SL, single-layer bag; DL, double-layer bag; TL, three-layer bag.

In the peel, most of the significantly changed compounds were downregulated. With delaying harvest time, the number of significantly differential compounds decreased in the single-layer bag, indicating the narrowing of the difference between the single-layer bag treatment and the control. The changes in differential compounds between the bagging treatments and the control were greater in S1–S3 than in S4–S5, and increased with the number of layers of the bags (Fig. 2).

In the juice sac, there were fewer differential compounds than in the peel. Some upregulated compounds were found, including several carotenoids, flavanones, coumarins, soluble sugars, organic acids and amino acids. No significant rule can be found for the changes in compounds among different treatments and harvest times (Fig. 3).

Dynamic changes in differential compounds

By pairwise comparison among different samples, a total of 58 differential compounds were found in the peel. The dynamic changes in these compounds are shown by heatmap (Fig. 4A). These compounds were classified into three clusters according to their patterns of change. In one pattern, the compounds increase with maturity. A total of 14 differential compounds in the peel changed in such a pattern, all of which were carotenoids, including 9-cis-violaxanthin and luteoxanthin, which were the chief carotenoids in the peel of ripe grapefruit. In the other pattern, the compounds showed a trend of decrease with maturity. A total of 8 carotenoids, 1 coumarin and 1 flavonoid in the peel changed in such a pattern, including β-citraurin, β-carotene, α-carotene and phytofluene, which were the chief carotenoids in the peel of green ripe (S1) grapefruit. Auraptene and 5-hydroxy-6,7,8,3ʹ,4ʹ-pentamethoxyflavone (5HPMF) were also changed in this pattern. In another pattern, the compounds remained stable or fluctuated irregularly. A total of 34 differential compounds in the peel changed in such a pattern, including 16 carotenoids, 13 flavonoids, 4 coumarins and 1 phenylpropanoid.

Figure 4.

Dynamic changes of differential compounds in the peel. (A) Heatmap of differential compounds; (B) Venn diagram of differential compounds; (C) Dynamic changes of carotenoids composition; CK1–CK5, unbagged controls harvested at S1 to S5 stages; SL1–SL5, single-layer bag treatments harvested at S1 to S5 stages; DL1–DL5, double-layer bag treatments harvested at S1 to S5 stages; TL1–TL5, three-layer bag treatments harvested at S1 to S5 stages; C1–C9, unidentified carotenoids 4, 7–14; C10, violaxanthin; C11–C12, unidentified carotenoids 15–16; C13, luteoxanthin; C14, unidentified carotenoids 17; C15, auroxanthin; C16, β-citraurin; C17, 9-cis-violaxanthin; C18–C20, unidentified carotenoids 18–20; C21, xanthophyll; C22– C24, unidentified carotenoids 21–23; C25, zeaxanthin; C26–C28, unidentified carotenoids 24–26; C29–C30, phytofluene; C31–C32, unidentified carotenoids 27–28; C33, phytofluene; C34–C35, unidentified carotenoids 29–30; C36, β-cryptoxanthin; C37, phytofluene; C38–C39, unidentified carotenoids 31–2; C40, α-carotene; C41–C42, unidentified carotenoids 33–34; C43, β-carotene; C44–C45, unidentified carotenoids 35–36; DW, dry weight.

Bagging downregulated most of the carotenoids, and the degree of reduction increased with the bag layers. As shown in the Venn diagram, the peel from all the developmental stages shared 4, 11 and 8 differential compounds in the single-layer, double-layer and three-layer bagging treatments, respectively, all of which were downregulated compared to the unbagged control. All of these commonly changed compounds were carotenoids (Fig. 4B; Fig. 4C). This indicated that the accumulation of most of the carotenoids in the peel was induced by light, and the decrease of light transmission ratio led to the decrease of carotenoid contents in the peel. In S5, the total carotenoid contents increased to a certain degree both in the unbagged control and the bagged treatments (Fig. 4C), indicating that there were inducers other than light for carotenoid biosynthesis in the mature fruit.

A total of 72 differential compounds were found in the juice sac (Fig. 5A). The contents of most carotenoids in the juice sac increased with maturity. Apiosylskimmin and umbelliferone 7-O-α-l-rhamnopyranosyl (1->2) β-d-glucopyranoside decreased, while citrusin A increased with maturity. The flavonoids fluctuated except for naringin-4ʹ-glucoside and eriocitrin, which decreased with maturity. The soluble sucrose contents increased first and peaked in S3 or S4, while the organic acids fluctuated slightly in the five stages. The juice sac from all developmental stages shared 1, 0, and 0 differential compounds in the SL, DL, and TL, respectively, compared to the unbagged control (Fig. 5B). The carotenoid contents and compositions showed similar change patterns in the bagged treatments and unbagged control (Fig. 5C).

Figure 5.

Dynamic changes of differential compounds in the juice sac. (A) Heatmap of differential compounds; (B) Venn diagram of differential compounds; (C) Dynamic changes in carotenoids composition. CK1–CK5, unbagged controls harvested at S1 to S5 stages; SL1–SL5, single-layer bag treatment harvested at S1 to S5 stages; DL1–DL5, double-layer bag treatment harvested at S1 to S5 stages; TL1–TL5, three-layer bag treatment harvested at S1 to S5 stages; C1–C14, unidentified carotenoids 1–14; C15, violaxanthin; C16–C17, unidentified carotenoids 15–16; C18, luteoxanthin; C19, unidentified carotenoid 17; C20, auroxanthin; C21, β-citraurin; C22, 9-cis-violaxanthin; C23–C25, unidentified carotenoids 18–20; C26, xanthophyll; C27–C29, unidentified carotenoids 21–23; C30, zeaxanthin; C31–C33, unidentified carotenoids 24–26; C34–C35, phytofluene; C36–37, unidentified carotenoids 27–28; C38, phytofluene; C39–C40, unidentified carotenoids 29–30; C41, β-cryptoxanthin; C42, phytofluene; C43–C44, unidentified carotenoids 31–32; C45, α-carotene; C46–C47, unidentified carotenoids 33–34; C48, β-carotene; C49–C50, unidentified carotenoids 35–36; DW, dry weight.

Discussion

As a commonly used cultivation technology in fruit production, bagging showed positive effects on improving the marketability of citrus fruit. However, it is noteworthy that the bags completely or partially shut out the light, which is an important factor for the accumulation of flavor components, pigments and various secondary metabolites. Thus, the bagging treatment will potentially change the chemical composition of the fruit, and affect the quality of taste and appearance.

Rind color was the most obviously changed phenotypic trait by bagging. With ripening, most citrus fruit turn yellow or orange. Bagging accelerates this process, as reported in a study on Citrus paradisi cv. ‘Star Ruby’ (Cao et al., 2020) and C. sinensis cv. ‘Beibei 447 Jincheng’ (Wang et al., 2009). The reduction of chlorophyll content caused by bagging is one of the main reasons for this change. In studies on pomelo (Promkaew et al., 2020), Nules Clementine mandarin and Navelina orange (Lado et al., 2019), bagging significantly reduced the chlorophyll contents throughout the whole development period. Our result was slightly different from those of the referred studies in that the decrease of chlorophylls (especially chlorophyll b) in the late mature stage was significantly inhibited by bagging, and even showed a certain increase at the full ripe stage. This was similar to the results of Hiratsuka et al. (2012) on the satsuma mandarin. Although bagging accelerated the rind color turning of grapefruit, we found that the color was much paler than that of the control at the full ripe stage, due to the reduction of carotenoid content. This phenomenon is also reported in pomelo (Promkaew et al., 2020), Nules Clementine mandarin and Navelina orange (Lado et al., 2019). In Nules Clementine mandarin and Navelina orange (Lado et al., 2019), shading significantly reduces most colored carotenoids, such as lutein, β-cryptoxanthin, α-carotene, and β-carotene, while inducing the accumulation of colorless carotenoids, such as phytofluene. The downregulation of key carotenoid biosynthesis genes after bagging (PSY, PDS, ZDS1, LCY2a, LCY2b, and CHX) might be the reason for the reduction of carotenoids. However, some of the carotenoids, such as β-citraurin, are not affected. We evaluated the changes in carotenoids on a larger scale, and found that in the 45 carotenoid compounds, about 40%–76% of the carotenoid species were not significantly affected in different development stages. But all the chief carotenoids were downregulated. In the single-layer bag, as there was partial light transmitted through the bag, the effects of bagging were weakened. As reported in previous studies, UV-A (290–320 nm), UV-B (320–400 nm) and visible light (400–700 nm) of appropriate strength could promote the synthesis of several carotenoids, such as α-carotene, β-carotene, xanthophyll and zeaxanthin (Ngamwonglumlert et al., 2020). Thus, the tiny proportion of light transmission can significantly induce carotenoid synthesis in the single-layer bag. However, it is noteworthy that, in S5, the total carotenoid contents showed a certain degree of increase in the bagged treatments, indicating that light is not the only determining factor for the carotenoid accumulation in the peel.

The effects of bagging on total carotenoid contents and certain carotenoid components might vary between the peel and the juice sac. However, there have not been many studies on the effects of bagging on carotenoids in the juice sac. In red pulp pomelo, although bagging reduces the total carotenoid content in the peel, it induces the accumulation of lycopene content and β-carotene content in the pulp, which might be due to the bagging maintaining the fruit temperature at a level beneficial to lycopene synthesis (Promkaew et al., 2020; Qiu et al., 2020). However, in the study of Wang et al. (2007), bagging reduced the content of β-carotene and lycopene. In the present study, 2%–32% of the 50 carotenoid species were significantly changed in different development stages, with some being upregulated and some being downregulated. In general, compared to the peel, the effects of bagging on carotenoids in the juice sac were less significant and tended to be ruleless.

Soluble sugars, organic acids and amino acids are the chief contributors to the taste quality of citrus. There have been several reports on the effects of bagging on soluble sugars and organic acids, and the results have varied. In several previous studies, bagging has been reported to reduce the TSS and titratable acid contents in different citrus species, such as blood orange (Sun et al., 2014), Newhall orange (Chun et al., 2008) and ‘Huangjinmiyou’ (Lin et al., 2014). In ‘Nules Clementine’ mandarin (C. reticulata Blanco), bagging was reported to reduce the soluble sugar content and increase ascorbic acid content, but has little effect on the other organic acids (Magwaza et al., 2013). However, in several studies on pomelo (Lin et al., 2014; Feng et al., 2017; Promkaew et al., 2020), bagging elevated the soluble sugar contents. The effect of bagging on amino acids has rarely been studied. In our study, bagging treatments exerted little effect on sugars, organic acids and amino acids. These different results might be attributed to the bagging time and bag material used in different studies. In the study of ‘Nules Clementine’ mandarin, the fruit are bagged with brown non-transparent bags, at the time immediately after the physiological fruit drop (Magwaza et al., 2013), while in the pomelo study (Promkaew et al., 2020), the fruit are bagged one month before the mature green. According to the study of Hiratsuka et al. (2012) on the satsuma mandarin, the net photosynthetic rate and the phosphoenolpyruvate carboxylase activity of the fruit peaked at 112 d after flowering. It can be concluded that bagging at a time earlier than this time point might significantly affect the dry matter accumulation. Thus, choosing an appropriate bagging time was important to minimize the negative effects of bagging on the accumulation and metabolism of sugars, acids and amino acids.

In addition to sugars, acids and amino acids, bitter components such as limonoids, flavonoids and coumarins also affect the taste quality of citrus. There have been several studies on the effects of bagging on flavonoids contents. In blood orange, bagging increases the total flavonoid content in the pulp, especially the total anthocyanin content (Sun et al., 2014). In ‘Nules Clementine’ mandarin, the rind of the shaded fruit contained higher hesperidin content at harvest (Magwaza et al., 2013). In pomelo ‘Tubtim Siam’, bagging decreases the total flavonoid content in the pulp throughout the whole development stage (Promkaew et al., 2020). Thus, the results of previous studies varied. Our study provides a comprehensive understanding of the effects of bagging on secondary metabolites. The changes in most of the compounds were insignificant and ruleless both in the peel and the pulp. These results indicated that the limonoids, flavonoids and coumarins remained stable after the bagging time point, and were minimally affected by environmental changes caused by bagging.

Conclusions

The present study provided a comprehensive understanding of the effects of bagging on grapefruit. The bagging treatment that completely shut out the light reduced the chlorophyll and the chief carotenoid content in the rind, and made the rind turn yellow earlier, while it was paler than the unbagged control. However, the bagging treatments in the present study had little effect on the content of flavonoids, limonoids and coumarins in the peel and juice sac, or on the content of carotenoids, sugars, acids, and amino acids in the juice sac. Thus, we concluded that bagging at approximately 110–120 d after anthesis exerted an influence mainly on the pigments in the peel, but less on the sugars, acids and amino acids, flavonoids, limonoids, and coumarins of grapefruit.

Acknowledgements

We thank Zhiwei Ge and Ya’er Zhu from the Analysis Center of Agrobiology and Environmental Sciences of Zhejiang University for support in the UPLC-HRMS assay and GCMS assay, respectively. We also thank Lang Cheng from ICAS for support in the light transmission ratio assay.

Author Contributions

Anze Jiang contributed to investigation, data collection and analysis, and writing the original draft; Lizhen Zheng, Dengliang Wang, and Peilin Fang contributed to samples collection; Chen Kang and Jue Wu contributed to reviewing and editing; Jinping Cao contributed to supervision, data analysis, writing, reviewing and editing; Chongde Sun contributed to supervision, reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Basic Public Welfare Research Program of Zhejiang Province (LGN19C200022) and the Fundamental Research Funds for the Central Universities, Science and Technology Innovation Team of the Ministry of Agriculture and Rural Affairs, China.

Conflict of Interest

The authors declare no conflict of interest.