Effects of Co-60 gamma-irradiation and refrigerated storage on the quality of Shatang mandarin

Ke Zhang,Yueye Deng ,Haohao Fu ,Qunfang Weng

Key Laboratory of Pesticide and Chemical Biology,Ministry of Education,Guangzhou 510642,PR China

Abstract The effectiveness of Co-60 gamma irradiation in controlling citrus red mite(Panonychus citri McGregor)had been proved in our earlier work.However,whether it could be used as an alternative method to replace the current way of quarantine treatment against citrus red mites depends on the performances of effective doses on citrus fruits.This study was conducted to explore the effects of Co-60 gamma irradiation on the nutrient composition of citrus(Shatang mandarin);selected fruits were divided into different groups and each group was irradiated at 0.0,0.2,0.3,0.4,0.5,and 0.6,respectively.And then the treated fruits were stored at 4°C and the nutrient composition was studied in the following days.The results showed that the shelf-life could be extended when fruits were irradiated in the dose range of 0.2–0.4 kGy,while most unirradiated citrus decayed by 15 d.It also turned out that the citrus irradiated at 0.5 and 0.6 kGy were fully decayed within 45 d of refrigerated storage.The content of total soluble solids(TSS),total sugar,ascorbic acid(AA),and titratable acidity had no significant differences compare to those of the control during the 15 d storage period.Nevertheless,the activities of peroxidase(POD)and superoxide dismutase(SOD)decreased after 15 d;the improvement of storage quality and shelf life may be explained by the change of the protective enzyme activity.In conclusion,the results of citrus fruit treated with irradiation at a certain dose indicated the potential use of Co-60 gamma irradiation as a safe quarantine treatment.

Keywords: Shatang mandarin;Irradiation;Cold storage;Nutrient composition

1.Introduction

Shatang mandarin (Citrus reticulateBlanco) is a kind of characteristic fruit in Southeast China,mainly grows in Guangdong Province,South China.It is one of the best commercial fruits of Guangdong Province.Since the fruit tastes as sweet as sugar,it is also called Mitangju.It is cultivated on a large area which accounts for about 80%of citrus planting area in Guangdong Province,due to its reasonably higher yield,better quality,greater taste and flavor than those of other citrus fruits.

Gamma radiation has been used as a post-harvest food preservation process for many years[1].Citrus is a seasonal product,which was consumed fresh or processed.Gamma irradiation emerged recently as a possible alternative technology for the citrus post-harvest processing,in order to fulfill the requirements of international phytosanitary trade laws.The goal of quarantine or phytosanitary treatments is to prevent the invasion and propagate of regulated pests[2].Phytosanitary treatments allow the destruction or removal of pests or making the pest reproductively sterile,to achieve the goal of control pest.There are various types of disinfestation treatments including cold,hot water immersion,heated air,methyl bromide fumigation and irradiation[3].In addition,some of these techniques could help to minimize the loss of quality in terms of flavor,color and nutritional value[4].Studies have showed that ‘Clemenules’ is a mandarin cultivar highly tolerant to X-ray irradiation and the commercial quality of the fruit was not adversely affected by post-harvest quarantine applications that effectively controlled the Medfly [5].Miller found that grapefruit irradiated at 0.3 kGy resulted in minimal injury to the fruit [6].Furthermore,studies have demonstrated that‘Rio Red’grapefruit exposed to irradiation doses of up to 0.5 kGy did not affect soluble solids,titratable acidity,appearance,and organoleptic quality compared to untreated fruit[7].Ascorbic acid is water-soluble vitamin,which is present in fresh fruit,especially citrus fruit and vegetable.In addition,ascorbic acid shows antioxidative effects and under certain conditions can protect against oxidatively induced DNA damage [8].Khalil reported that for citrus fruits with doses of 0.25 and 0.5 kGy alone packed in cellophane bags and stored at room temperature for 42 d,their acidity and ascorbic acid values were higher for the oranges irradiated at 0.5 kGy.Their weight loss decreased and total soluble solid (TSS) increased during storage period[9].Girennavar studied the influence of E-beam irradiation on bioactive compounds of grapefruits and found that the acidity decreased slightly with the increasing E-beam dose,whereas the TSS increased and irradiation did not affect the vitamin C content at doses up to 1.0 kGy[10].

Irradiation for post-harvest disinfestation has been investigated for various fruits and vegetables and shows great promise since it sterilizes insects at low doses that are not enough to be detrimental to most fruits and vegetables [11,12].The United States Department of Agriculture-Animal and Plant Health Inspection Services(USDA-APHIS),together with other international regulatory bodies,such as the International Atomic Energy Agency (IAEA) and the International Plant Protection Convention(IPPC),have issued guidelines for irradiation treatments to meet export and quarantine restrictions[13].

The benefits of using irradiation are that the cold treatment allows quality to be maintained without residue on the product,and reduces the use of fumigants and pallet loads can be treated at a time.Low dose gamma irradiation(1.0 kGy or less)has been shown to control insect pests with little quality loss of fresh produce[14];however,energy is imparted into metabolically alive tissues of the commodities and undesirable damage could occur[15].In 2006,the USDA-APHIS approved generic treatments of 0.15 kGy for fruit flies and 0.40 kGy for all insects except pupa and adult Lepidoptera[16].However,there are few reports on irradiation quarantine treatments in controlling pest mites.Similarly,the content of health-promoting compounds in citrus fruit may be altered by post-harvest treatments such as irradiation.For instance,recent studies showed that irradiation of citrus fruit significantly reduced the total ascorbic acid(TAA)content when irradiation doses were high[10,17].However,information is still scarce on the effect of Co60-γ irradiation on nutritional quality of many citrus cultivars.

The objectives of this research were to evaluate the dose response of quality factors of Shatang mandarin to irradiation at 0.2-0.6 kGy.Meanwhile,we found that the irradiation treatment can extend the shelf life and delay fruit senescence of the citrus fruits.

2.Materials and methods

2.1.Fruits and Reagents

Shatang mandarin samples were obtained from a certain supermarket and the citrus fruits were harvested in a commercial orchard in Shaoguan city,Guangdong Province.Fresh citrus with uniform size and maturity were selected.

Bovine albumin was purchased from Shanghai Fanke Biological Technology Co.Ltd.Guaiacol was purchased from Tianjin Kemiou Chemical reagent Co.Ltd.SOD enzyme kit was purchased from Nanjing Jiancheng Bioengineering Institute,and other chemicals and solvents were purchased from Guangzhou Chemical reagent Factory.Water was treated in a Unique +UV+UF (Research Scientific Instruments Co.Ltd)water purification system.

2.2.Exposure to Co60γ Irradiation

The experimental material was irradiated by Co60γ-rays at the Furui High-Energy Technology Co.Ltd.,Nansha District,Guangzhou Guangdong Province,China.The samples were divided into six groups to be exposed to different radiation doses(0,0.2,0.3,0.4,0.5 and 0.6 kGy,and the dose rate was 4 Gy/min,using Fricke dosimeter calibration)with 100 units per group.The groups 2–6 were placed into polyethylene plastic bags and irradiated with 0.2,0.3,0.4,0.5 and 0.6 kGy,respectively.Group 1 was the control sample.The control and the treated samples were then packed in plastic bags and stored at 4°C for 45 d.

2.3.Analysis of major quality indicators after Co60γ radiation

2.3.1.Analysis of weight loss and decay rates

Each treatment contained 10 fruits and their weight loss were tested.The percentage of weight loss was calculated by the following formula:

Similarly,the decay rate was calculated by(number of decayed fruits/total number of tested fruits)×100.

2.3.2.Total sugar

Total sugar concentration was determined by saccharimeter(LB32T,Guangzhou Mingrui Electronic Technology Co.,Ltd.)by measuring refractive index of fruit juices,and the concentration of sugar was calculated.

2.3.3.Titrable acidity

1.0 mL fresh juice was added into 10 mL double distilled water,and titrable acidity was determined according to the AOAC methods[18].

2.3.4.Ascorbic acid

Ascorbic acid concentration was determined by direct iodine titration.25 mL of the herbal fresh juice was transferred to a 250 mL Erlenmeyer flask.Twenty-five mL of 2 mol/L sulfuric acid was added and mixed.The mixture was diluted with 50 mL water and 3 mL Starch Soluble was added as an indicator.The solution was directly titrated with 0.1 mol/L iodine which was previously calibrated with primary standard arsenic trioxide.A blank titration was performed prior to titration of each sample(n=5).One mL of 0.1 mol/L iodine is equivalent to 8.806 mg ascorbic acid[19].

2.4.Analysis of protective enzyme activities

The pulp (1.00 g) of citrus tissues was mixed with 10 mL 5 mmol/L phosphate buffer(pH 7.8 or pH 6.8)in a cold mortar and grinded.The seriflux was then centrifuged at 12,000×gat 4°C for 15 min.The supernatant was used as the crude enzyme extract.Peroxidase (POD) activity was assayed by measuring the increase in absorbance at 470 nm using 4-methylcatechol as a substrate which was prepared in a buffer solution with pH 6.8.The reaction was carried out in a 10 mm light path quartz cell.One unit(U)of POD was defined as the amount of enzyme that caused the increase of one absorbance unit(AU)at 470 nm in 10 min.Superoxide dismutase(SOD)activity was assayed by SOD enzyme kit and was evaluated by xanthine oxidase method.

2.5.Statistical analysis

Statistical analysis was conducted for each of the measured traits by analysis of variance(ANOVA)and the means were separated by Duncan Multiple Range test using the SPSS software,version 18.0(SPSS,Inc.).In addition,a linear discriminant analysis(LDA)was used to assess the influence of different storage duration or irradiation doses on proximate composition profiles as well as on major quality indicators(weight loss,decay rate,total sugar,titrable acidity,ascorbic acid and protective enzyme activities).All statistical tests were performed at a 5% significance level.All the assays were carried out in triplicate.The results were expressed as mean values with standard deviation(SD).

3.Results and discussion

3.1.Physical properties

There were little influence on the index of Shatang mandarin when treated with the doses of 0.2–0.4 kGy.The shelf life of Shatang mandarin could be prolonged by treatment with doses of 0.2–0.4 kGy,and only slight changes of weight losses were observed during the whole experimental process.There was no significant difference between irradiated citrus and the control in weight loss after 7 d.Weight loss rates in groups treated with 0.2–0.4 kGy,which was no more than 0.01%,were significantly lower than that of the control after 15,30 and 45 d of storage(Table 1.),while groups treated with 0.5 and 0.6 kGy were almost decayed,indicating that the irradiation treatment with 0.2–0.4 kGy could prolong the shelf life of Shatang mandarin,and the high dose(0.5–0.6 kGy)would cause damage to fruit.

Decay rates of groups treated with 0.5 kGy and 0.6 kGy reached 21%and 35%after 7 d of storage,respectively(Table 2),while no rotted fruit was observed for the control and the citrus samples treated with 0.2–0.4 kGy,15 d after irradiation,both of the decay rates of citrus irradiated with 0.5 kGy and 0.6 kGy were more than 50%,while it was only 2.00%and 1.67%for the control group and 0.4 kGy treated group,respectively.There were no decayed fruits in 0.2 kGy and 0.3 kGy irradiated groups.30 d after irradiation,the decay rates of 0.5 kGy and 0.6 kGy irradiated groups were more than 90%,while the 0.2 kGy and 0.3 kGy treated groups still kept the lowest decay rates (less than 5%).And the decay rates of 0.4 kGy irradiated group was 5.3%,as compared to the control(6.7%).The decay rates reached 100%after 45 d irradiation in 0.5 kGy and 0.6 kGy groups,but they were less than 10% in 0.2 kGy and 0.3 kGy irradiated groups.And the decay rate of 0.4 kGy irradiated group was lower than that of the control.The irradiation dose below 0.4 kGy(including 0.4 kGy),had a preservative effect on Shatang mandarin,while high doses would cause serious damage to the Shatang mandarin.

The effect of gamma irradiation on total sugar with different doses was carried out in this study.The results showed that the total sugar content decreased with the extension of storage duration (Table 3).There were no significant differences between treated fruits and the control group in the content of total sugar after 30 d.Irradiation dose above 0.5 kGy caused great damage to the citrus fruits,while 0.2–0.4 kGy irradiation treatment had little effect on total sugar content.

It turned out that citrus fruits treated at doses of 0.2–0.4 kGy had no significant differences in weight loss,soluble solids content,total sugar content and titratable acid content compared with the control group.Fernandes [20]studied the effects of irradiation on chestnut fruits and found that the gamma irradiation doses ≤3 kGy did not affect the nutritional and chemical quality of chestnut fruits,which indicated that fruits irradiated at suitable doses would have little harm on its physical properties.According to Farkas[21],irradiation could reduce storage losses,extend shelf life and/or improve microbiological and parasitological safety of foods.In this study,similar phenomenon was observed,showing that irradiation doses at a proper level could prolong the shelf life of Shatang mandarin as well asenhance its appearance quality.We also found that the citrus fruits treated with 0.5 kGy and 0.6 kGy completely decayed in 45 d,suggesting that high dose irradiation treatment had a damaging effect on citrus fruits.However,at which exactly dose would this happen is still unknown and further studies would be needed to confirm the dosage.

Table 1 Weight loss rate(%)of the citrus fruits after different gamma irradiation treatments.

Table 2 Decay rate(%)of the citrus fruits after different gamma irradiation treatments.

No significant differences in ascorbic acid content between treated fruits and the control were observed in the first 7 d yet it was increased a little bit in the control group,which,then sharply decreased on the 30th day and increased again on the 45th day.Nevertheless,the ascorbic acid content of irradiation treatment groups kept decreasing during the whole experimental period,and this kind of change was much faster in the control than that in irradiation treatment group.There were no significant differences between treated groups and the control in ascorbic acid content after 15 d and 30 d.When tested on the 30th day,the ascorbic acid content of the treatment groups was slightly higher than that of the control but it then became lower in groups irradiated at 0.2–0.4 kGy than that in the control 45 d after treatment.The results illustrated that irradiation treatment doses of 0.2–0.4 kGy on citrus fruits could somehow affect its ascorbic acid content;however,the difference between irradiated samples and the control was not obvious after 30 d of storage at 4°C.

The results showed that titratable acid content of Shatang mandarin decreased as the storage duration extended.There was no significant difference between groups treated with 0.2 kGy and 0.3 kGy in titratable acid content when tested on the 7th day and 15th day,but the content of titratable acid significantly decreased 30 and 45 d after irradiation treatment in these two group compared with the control (Fig.1).And the content of titratable acid in groups treated with 0.3 kGy or above were less than a half of that in the control.It indicated that the irradiation effect on the titratable acid content of Shatang mandarin would become serious after 30 d.

Fig.2 shows that although irradiation treatment had a certain impact on the ascorbic acid content,it reduced much slower in irradiation treatment groups than that of the control.Kaewsuksaeng[22]reported that UV-B treatment induced a gradual increase in citric acid content and suppressed the increase of sugar contents during storage.In addition,the ascorbic acid content with or without UV-B treatment decreased during storage,but the decrease in the control was faster than that in the UV-B treatment group.Others also pointed out that irradiation not only affected seed formation in the fruit,but also reduced acidity[23].From Fig.1,we can conclude that the titratable acid content of Shatang mandarin decreased with the extension of storage duration,which revealed that irradiation treatments had significant effect on the titratable acid content of Shatang mandarin.

3.2.Enzyme activities

Results of the enzymatic analyses in Figs.3 and 4 illustrated that the SOD activity of citrus fruits with or without irradiation all increased greatly during the first 7 d,and the control showed the highest activity(Fig.3).This may attribute to free radicals induced by irradiation.The SOD activity of all groups dropped after 15 d probably because it was involved in the free radical clearance.After 30 d,SOD activity of all groups bounded back with great increase in the 0.4 kGy and 0.5 kGy treatment groups increased greatly.45 d after irradiation treatment,the SOD activity of 0.5 kGy and 0.6 kGy irradiated citrus fruits decayed completely,and the SOD activity of other groups decreased.And the SOD activity of irradiation treatment groups were lower than that of the control,showing that irradiated fruits produced more free radicals.However,the treated groups had alarger fluctuations in SOD activity than the control group during the whole experimental process;overall,low dose (≤0.4 kGy)irradiation could play a role in delayingC.reticulateBlanco fruit senescence as well as in prolonging the storage duration.

Table 3 Total sugar content(g/100 mL)of the citrus fruits after different gamma irradiation treatments.

Fig.1.Changes in the content of titrable acidity 7(A),15(B),30(C)and 45(D)d with different gamma irradiation in treated samples and the control compared to Day 0.Values with different letters from the same time point differ significantly(P<0.05).Similarly hereafter unless otherwise stated.CK(control).

SOD catalyzes the dismutation of superoxide anions to hydrogen peroxide,which is then removed by catalase.These two enzymes are thought to preserve food freshness by protecting the integrity of membranes[24].SOD activity in the citrus fruits increased with or without irradiation treatments,but its activity was significantly lower than that in the control throughout 7 d of storage at 4°C(Fig.3).This reduce of citrus enzymes may be caused by free radicals,which was induced by irradiation.SOD is a primary scavenger for superoxide free radicals,which plays a role in the dismutation of superoxide radicals,whereas catalase (CAT),ascorbate peroxidase (APX) and glutathione reductase (GR) activities would contribute,at least to some extent,to the elimination of hydrogen peroxide[25].

Studies have found a certain correlation between browning of fresh fruits and vegetables and tissue PODcontent.PODcan lead to deterioration fruits and vegetables through the oxidation reaction.The results of Fig.2 showed that POD activity of Shatang mandarin fruits increased at first,and then decreased as the storage duration extended.The POD activity of all experimental groups increased 7 d after irradiation in a dose-dependent manner.The POD enzyme activity of 0.2–0.4 kGy treatment groups were significantly lower than that of the control 45 d after irradiation,And the 0.5 kGy and 0.6 kGy irradiation groups showed the highest POD activity during the whole experiment process,indicated that high dose irradiation would cause damage to the citrus fruits.

The study by Falguera et al.[26]found a slight decrease in the activity of POD in fresh apple juices from Golden,Starking and Fuji after UV irradiation treatments and this value slightly decreased during the experiment.Meanwhile,the loss was 70.0%in the juice from King David.In this study,a decrease of POD activity was also observed(Fig.4)because the ascorbic acid of Shatang mandarin is not enough to inhibit the activity of POD for long time,and a large number of high active POD will lead to deterioration of the quality.The gamma irradiation may extend the shelf life of the fruit due to its influence on the basic metabolism of the fruit through suppressing respiratory enzyme activity,the release of CO2and ethylene production,resulting in delayed ripening and senescence;secondly,irradiation could prevent microbial spoilage in fruits,and play a role in the preservation.And it suggested that the SOD and POD enzymes played important roles in the citrus fruit senescence,while the mechanism needed to be further explored.

The results of effects of Co-60 gamma-irradiation on the quality indexes and the protective enzyme activities of the citrus fruit during storage showed that low dose irradiation (0.2–0.4 kGy)treatments could prolong the shelf life of the citrus fruits with slight but acceptable adverse effects on the quality of citrus fruits.These results showed that Co-60 gamma-irradiation below a certain dose could be a promising and safe method to control insect pests,extend the shelf life and delay senescence of fruits and vegetables.

Fig.2.Changes in the content of ascorbic acid of the citrus fruits 7(A),15(B),30(C)and 45(D)d after different gamma irradiation treated samples and control compared to Day 0.

Fig.3.Effect of Co60γ irradiation on the superoxide dismutase(SOD)activity of the citrus 7,15,30,and 45 d after treatment without or with different irradiation doses during storage at 4°C.

Fig.4.Effect of Co60γ irradiation on the peroxidase(POD)activity of the citrus 7,15,30,and 45 d after treatment without or with different irradiation doses irradiation during storage at 4°C.

4.Conclusions

Our results suggested that irradiation in the range of 0.2–0.4 kGy in combination with refrigerated storage is an effective post-harvest technique in mitigating the risk of pests and decay of quarantined fruits.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgment

The researchers gratefully acknowledge the grants from the International Atomic Energy Agency under Research Contract No.15630.