Effects of Combined Heat and Preservative Treatment on Storability of Ponkan Fruit ( Citrus reticulata Blanco cv . Ponkan ) during Postharvest Storage

1Laboratory of Fruit Quality Biology, The State Agriculture Ministry Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Zhejiang University, Zijingang Campus, Hangzhou 310058, China 2Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Key Opening Laboratory of Agricultural Products Processing and Quality Control, Ministry of Agriculture, Beijing 100193, China 3Quzhou Academy of Agricultural Science, Quzhou 324000, China 4Agricultural Bureau of Quzhou Kecheng, Quzhou 324000, China


Introduction
Ponkan (Citrus reticulata Blanco cv.Ponkan) is one of the most widely grown and economically important citrus fruit species in China.However, the fruit is often subjected to huge losses during postharvest storage and transportation, which presents a big challenge to the citrus industry.Biological diseases and physical damage are reported to be the most important reasons causing significant economic losses during storage [1].Many postharvest treatments have been used in industry to reduce the decay rate during storage and transport, including physical measures [2,3] and preservative applications [4,5].
Application of preservative is an effective way to reduce the decay rate, which can control the growth and spread of microorganisms.The most commonly used preservative mixture in the Ponkan industry contained 200 mg L −1 iminoctadine tris (albesilate), 100 mg L −1 2,4-dichlorophenoxyacetic acid, and 200 mg L −1 imazalil [6][7][8].However, the long-term application of preservative can generate pathogen resistance against many site-specific fungicides [9], and some preservatives even have bad effects on human health and the environment.Nowadays, people pay more attention to food safety; thus, to reduce or abandon the use of preservative becomes a new challenge for postharvest storage of fruit and vegetables and for preservation of other fresh foods.
Heat treatment (HT) is one of the most commonly used physical measures to reduce fruit decay, which has been reported to be effective in decreasing fly infestation, inducing resistance to chilling injury and enhancing the biocontrol effectiveness of antagonistic yeasts [10][11][12].Previous reports also showed that heat treatment enhanced the efficacy of preservative and decreased the amount of chemicals used for decay control in fruits [13,14].However, the effects of HT combined with iminoctadine tris (albesilate), 2,4-dichlorophenoxyacetic acid, and imazalil mixture on storability and quality in Ponkan fruit have rarely been studied.
In this study, the effect of HT combined with application of preservative mixture, including iminoctadine tris (albesilate), 2,4-dichlorophenoxyacetic acid, and imazalil, on storability and quality of Ponkan fruit was evaluated by analyzing the decay rate, weight loss, color, superficial microbial population, ethanol content, acetaldehyde content, pectin content, and fiber content during postharvest storage.The future applications of this method are discussed.

Fruit Materials and Treatments.
Fruits of uniform size and color were picked at economic harvest time from an orchard located in Quzhou, China, in 2012-2014.
Fruits used for heat treatments were picked in 2012.The control fruits were submerged in water at room temperature (RT), and the heat treatment groups were submerged in hot water at 55 ∘ C for 20 s, 30 s, and 40 s, respectively.After the treatments, fruits were stored in a ventilated warehouse at 10 ∘ C and were sampled at 0, 30, 60, 90, and 120 d storage.
Fruits used for combined HT and preservative treatments were picked in 2013 and 2014.The preservative mix includes iminoctadine tris (albesilate), 2,4-dichlorophenoxyacetic acid, and imazalil.0%, 25%, 50%, and 100% (w/v) of the preservative dosage used in production, which contained 200 mg L −1 iminoctadine tris (albesilate), 100 mg L −1 2,4dichlorophenoxyacetic acid, and 200 mg L −1 imazalil, were dissolved in both RT and 55 ∘ C water and were used for the HT combined preservative treatments.The CK, HT, 25% C ("C" represents the concentration of preservative used in production), HT + 25% C, 50% C, HT + 50% C, 100% C, and HT + 100% C fruits were submerged in corresponding preservative solutions for 20 s.After the treatments, fruits were stored for 0, 30, 60, 90, 120, and 150 d in a ventilated warehouse at 10 ∘ C and the fruits with 150 d storage were transferred to shelf storage for analysis at 7 and 15 d.These experiments were repeated twice over two years.

Decay Rate Analysis.
Each treatment contained 300 fruits, which were divided equally into three groups and were considered as three biological replicates.Fruits with obvious appearance of decay were recorded on each sampling day.Decay rate were calculated according to the following formula: decay rate (%) = /100 × 100%, where "" represents the total number of decayed fruits in each replicate.

Weight Loss Analysis.
Thirty fruits were randomly selected from each treatment and marked.The weight of each fruit was regularly recorded on each sampling day.The weight loss rate was calculated according to the following formula: weight loss rate (%) = ( 0 − )/ 0 × 100%, where " 0 " represents fruit weight on 0 d storage and "" represents fruit weight on each sampling day.

Color Analysis.
Color measurement was carried out with a Hunter Lab Mini Scan XE Plus colorimeter (Hunter Associates Laboratory, Inc., Reston, VA) at four evenly distributed equatorial sites of each fruit.The CIE 1976  *  *  * color scale was adopted.Citrus color index (CCI) was calculated according to the following formula: CCI = 1000 ×  * /( * ×  * ).
2.5.Ethanol and Acetaldehyde Contents Analysis.Ethanol and acetaldehyde production was determined with a gas chromatograph instrument (Agilent 6890N, Folsom, CA, USA) with an FID column (HP-INNOWAX, 0.25 mm, 30 m, 0.25 m, Agilent J&W, Folsom, CA, USA) according to the method described by Min et al. [15] with modifications.The injector, detector, and oven temperatures were 150 ∘ C, 160 ∘ C, and 100 ∘ C, respectively.Sec-butyl alcohol was added to each vial as an internal control.The results were calculated using standard curves for acetaldehyde and ethanol, respectively.

Microbial Population Analysis.
Microbial medium was obtained from peel of Ponkan fruit and cultured on potato dextrose agar (Difco, Detroit, MI).Twenty grams fruit peel was put into a blender and homogenized with 100 mL sterile water. 1 mL mixtures for each sample were taken out and homogenized with 9 mL sterile water.A volume of 100 L mix was cultured on PDA medium.Colony-forming units (CFU) were counted after incubation for 72 h at 28 ∘ C.

Pectin Content Analysis.
One gram fruit tissue samples were homogenized with 25 mL 95% ethanol, followed by 30 min boiling water bath.Then the homogenate was centrifuged at 8000 rpm for 30 min to remove the supernatant.Repeat the extraction step for three times.The resulting pellet was incubated with 20 mL distilled water at 50 ∘ C water bath for 30 min to dissolve the pectin.The homogenates were then centrifuged at 8000 rpm for 15 min.Transfer the supernatant into a 100 mL volumetric flask and wash the precipitate with distilled water.Move the supernatant into the volumetric flask and dilute with distilled water to volume.This is the soluble pectin.Add 25 mL 0.5 mol L −1 sulfuric acid into the precipitate and incubate it in boiling water for 1 h to dissolve the pectin.The homogenates were centrifuged at 8000 rpm for 15 min; then transfer the supernatant into a 100 mL volumetric flask and dilute it with distilled water to volume.This is the protopectin.Incubate 1 mL of the extracted solution with 6 mL concentrated sulfuric acid for 20 min in boiling water.Add 0.2 mL 1.5 g L −1 carbazoleethanol into the mixture, and keep it in dark for 30 min.The absorbance of the reaction mixture was measured at 530 nm to calculate the content of galacturonic acid by contrast with the standard curve.The total pectin content includes both the solution pectin and protopectin.
2.8.Fiber Content Analysis.Add 5 mL mixture of acetic acid and nitric acid into 100 mg drying samples, incubated with boiling water bath for 60 min; the homogenate was   1).Thus, heat treatment was effective in reducing decay during storage, which was similar to results from previous reports [16][17][18].In addition, heat treatment at 55 ∘ C for 20 s was the optimal condition to extend storage time of Ponkan fruit and was used for further research.

Effects of Combined Heat and Preservative Treatments on Decay Rate, Shelf Life, and Quality of Ponkan Fruit during Storage.
The decay rate of Ponkan fruit increased continuously in all the treatments during the whole storage period.Compared with the control, the decay rates in all the treated samples were significantly decreased during 90 to 150 d storage.At 150 d storage, the decay rate in the control reached a level of 30%, while all the treated samples exhibited decay rates lower than 18%, with a ranking order of HT, 25% C, 50% C, HT 50% C, HT + 25% C, HT + 100% C, and 100% C from high to low (Figure 1(a)).When transferred to shelf storage, the three combined treatments showed lower decay rates compared with the corresponding preservative treatments alone.Compared with 25% C treatment, the HT + 25% C treatment decreased the decay rate by about 75% and 50% after transferring to shelf storage for 7 and 15 d, respectively (Figure 1(b)).Weight loss and CCI in all the treatments increased continuously during the whole storage period, but no significant difference was found between the control and treated samples in either weight loss or CCI (Figures 1(c) and 1(d)).
From the above results, the combined heat plus 25% preservative treatment significantly decreased the decay rate of Ponkan fruit during storage, showing a similar effect to that with 100% preservative treatment.This may be because the heat treatment was effective in enhancing the efficacy of preservative [14] and was an effective measure to decrease the preservative content while still controlling fruit decay, which was similar with the effect of heat treatment combined with preservative on other fresh produce [19][20][21].Also, the heat treatment may promote resistance to microorganisms in Ponkan fruit during storage, which has been reported in other fruits [22][23][24].In addition, no significant difference in weight loss and CCI between the treatments was observed, indicating that the HT combined with preservative treatments had no adverse effects on fruit quality during storage.Thus, the optimal combination for long-term storage of Ponkan fruit is heat treatment at 55 ∘ C for 20 s and preservative with a concentration of 25% of the dosage used in production, and this method can be further applied during fruit storage in the citrus industry.

Effects of Combined Heat and Preservative Treatment on the Epiphytic Microbial Population of Ponkan Fruit during
Storage.The epiphytic microbial population increased in all the treatments during the whole storage period.No significant difference was investigated between the treated and untreated samples during the early storage period.Compared with the control, the 100% C and HT + 100% C treatments significantly decreased the epiphytic microbial population during the later storage period.At 120 d storage, the microbial population was 363, 290, 162, and 60 CFU L −1 in CK, HT + 25% C, HT + 50% C, and HT + 100% C treatment, respectively, showing a decreasing trend with increasing preservative concentration.At 150 d storage, the microbial population was decreased to 65% and 60% by 100% C and HT + 100% C treatments, respectively, while no significant difference was observed between the other treatments and the control (Figure 2).The observation that the HT + 25% C treatment showed a similar effect to the 100% preservative treatment in decreasing fruit decay rate suggests that the epiphytic microbial population is not the only determinative factor for fruit decay during storage.

Effects of Combined Heat and Preservative Treatments on Ethanol and Acetaldehyde Contents in the Fruit Pulp during
Storage.The content of ethanol in fruit pulp showed an increasing trend during storage in general, and it occurred more rapidly during the later storage period.A sharp increase was observed in HT treatments at 90 and 120 d storage compared with the control and other treatments.During the late storage period, the ethanol content of fruits in the HT + 25% C treatment was significantly lower than with the 25% C treatment alone.At 150 d storage, the ethanol contents in fruits of all the combined treatments were lower than those where preservative was used alone (Figure 3(a)).The variation of acetaldehyde content was more stable compared to ethanol.At 60 and 90 d storage, acetaldehyde content in fruits of HT + 50% C treatment was significantly lower than when preservative was used alone.No consistent variation was found between different treatments during storage (   Acetaldehyde and ethanol accumulated under anaerobic conditions during fruit development or storage, for example, through coating with films or in modified atmospheres or packages [25].Previous reports showed that fruits tend to accumulate more ethanol and acetaldehyde during the later storage stage, which contributed to dramatic change in the fruit flavor and appearance of browning [26,27].In addition, these molecules can reflect the fruit status or predict the quality characteristics [28].From our results, the ethanol content of fruits in HT combined with 25% preservative treatment was significantly lower than that of the 25% preservative treatment during the late storage period, which may indicate better internal quality and long-term storage ability of fruits in HT combined with 25% preservative treatment.

Effects of Combined Heat and Preservative Treatments on Pectin and Fiber Contents in the Fruit Peel during Storage.
The variation of fiber contents was relatively stable in all the treatments during storage.Compared with the control, the fiber content was lower in HT treatments but showed no regular trend during the whole period.No significant difference was observed between samples in 50% C and HT + 50% C treatments or between the 100% C and HT + 100% C treatments.However, the HT + 25% C treatment significantly increased the pectin content between 60 to 150 d storage when compared with the 25% C treatment alone (Figure 4).
The fiber contents showed irregular variation in all the treatments during storage.No significant difference was observed in fiber contents among different treatments at 30 and 60 d storage.Generally, the combined treatments increased the fiber content compared with the single preservative treatments from 90 to 150 d storage.Significant differences were investigated in all the combined treatments when compared with the corresponding single preservative treatments at 150 d storage, indicating that the HT treatment was effective in increasing the fiber content in fruit peel during long time storage (Figure 5).Pectin and fiber are important constitutes of the main structural support in the cell wall, playing important roles in material transportation and protection against adversity.Previous reports showed that the cell wall participated in perception of external signals by changing the components in response to various environmental stimuli or stresses [29].It has also been reported that heat shock treatment can inhibit the degradation of pectin and cellulose and lead to an increase in water soluble pectin, delaying the process of persimmon softening, alleviating chilling injury and extending the shelf life of fruits [30,31].The fiber contents were significantly increased by the combined HT and preservative treatments, suggesting that the structure and intensity of fruit cell wall were affected so as to prevent fungus and enhance fruit storage and resistance.

Conclusions
Heat treatment at 55 ∘ C for 20 s was effective in extending the storage life of Ponkan fruit.Furthermore, heat combined with 25% preservative treatment significantly decreased the decay rate of Ponkan fruit during storage, showing a similar effect to that observed with 100% preservative treatment.This may be because the heat treatment was effective in enhancing the efficacy of the preservative, permitting a decrease in the amount of chemicals used for decay control in Ponkan fruit.In addition, the increased pectin and fiber contents in fruit treated with HT combined with preservative suggested that the structure and intensity of cell wall were affected so as to prevent fungus infection and enhance fruit resistance during postharvest storage.

Figure 1 :
Figure 1: Effects of different combined heat and preservative treatments on the decay rate (a), shelf life (b), weight loss (c), and CCI (d) of Ponkan fruit during storage."C" represents the concentration of preservative used in production.The error bars represent the standard errors.Letters on columns represent significant differences at the 0.05 level.

Figure 2 :Figure 3 :
Figure 2: Effects of combined heat and preservative treatments on the epiphytic microbial population of Ponkan fruit during storage."C" represents the concentration of preservative used in production.The error bars represent the standard errors.Three repetitions were used in this analysis.

Figure 4 :− 1 )Figure 5 :
Figure 4: Effects of combined heat and preservative treatments on pectin content in the fruit peel during storage.The error bars represent the standard errors."C" represents the concentration of preservative used in production.Different superscripts between columns represent significant differences between samples at the 0.05 level.

Table 1 :
Effects of different heat treatments on decay rate of Ponkan fruit during storage.
2.9.Statistical Analysis.Standard errors and figures were drawn using Origin 8.0 (Microcal Software Inc., Northampton, MA, USA).The test of statistical significance was based on the total error criteria with a confidence level of 95.0%, calculated by SPSS Statistics 20.0 Software.