Effects of Different Fumigant Preservatives on Antioxidant Capacity of ‘Kyoho’ Grapes

Shuze LIU　Huiqin DAI　Nan WANG　Cheng WANG　Zhe SUN　Feng ZHANG　Chenxia LIU　Yongjin QIAO　Airong SHEN

AbstractWith ‘Kyoho’ grapes as the experimental material， the effects of different concentrations of chlorine dioxide （5， 10 and 15 mg/L） and ozone （30， 60 and 90 mg/m3） fumigation on the contents of total phenols， proanthocyanidins and malondialdehyde （MDA） and the activity of polyphenol oxidase （PPO）， peroxidase （POD）， catalase （CAT） and superoxide dismutase （SOD） in the grapes during low temperature storage were compared and analyzed， hoping to provide reference for the storage and preservation of ‘Kyoho’ grapes after harvest. The results showed that compared with the control， certain concentrations of fumigant preservatives inhibited the degradation of total phenols and proanthocyanidins in ‘Kyoho’ grapes to varying degrees， maintained better cell membrane integrity， and inhibited the increase of MDA content and PPO activity， and in the later stage， more stable POD and higher activity of CAT and SOD were maintained. Comprehensive analysis showed that among the six fumigation treatments， 10 mg/L ClO and 60 mg/m Ocould delay the aging process of grapes and maintain better antioxidant capacity and storage quality.

Key words‘Kyoho’ grape; Chlorine dioxide; Ozone; Antioxidant

‘Kyoho’ grape is one of the main fresh table grape varieties in China. The fruit has thick skin， more fruit powder， soft pulp， sweet and juicy taste， rich nutrition， good edible value and health benefits， and is deeply loved by consumers[1]. However， due to the high water content of fresh ‘Kyoho’ grapes， grain loss， wilting， softening， rotting and browning will occur when stored at room temperature for about 3 d， which seriously affects their sensory properties， quality and commercial value. Therefore， safe efficient ‘Kyoho’ grape storage and preservation technologies suitable for promotion are of great significance to the development of the grape industry. Chlorine dioxide and ozone are the latest generation of high-efficiency， broad-spectrum and safe bactericidal preservatives recognized internationally. They were approved by the U.S. Food and Drug Administration in the 1980s and 2001 as food additives and disinfectants. The two have the advantages of wide application， low dosage， fast response， good effect， long duration， no toxicity and side effects， no teratogenicity， carcinogenicity， and no harmful impact on the environment， and thus have been widely used in the storage and preservation of grains， oils， fruits and vegetables， and dried products[2-4]. Substances such as ethylene， acetaldehyde and methanol that promote the post-ripening of fruits and accelerate aging are produced in the post-harvest metabolism of fruits and vegetables. Chlorine dioxide can quickly and effectively prevent the oxidative decomposition of methionine into ethylene， CO and other substances that cause aging and maturation of fruits and vegetables. Ozone can oxidize and degrade harmful gases such as ethylene and ethanol， delay the aging and decay of fruits and vegetables， and effectively prolong the storage period of fruits and vegetables[5-6]. At present， many studies have shown that chlorine dioxide and ozone can reduce the rot rate of grapes and improve fruit quality. In this study， with ‘Kyoho’ grapes as the experimental material， the effects of different chlorine dioxide and ozone fumigant preservative treatments on the antioxidant substances and antioxidant enzyme activity in the grapes under the low temperature condition of （2±1） ℃ were investigated， and comparison and analysis were carried out while ensuring the fresh-keeping effects of the two fumigant preservatives on ‘Kyoho’ grapes， hoping to provide a reference for the storage and preservation of ‘Kyoho’ grapes after harvest.

Materials and Methods

Experimental materials

Test materials： ‘Kyoho’ grapes （Vitis vinifera×labrusca var. Kyoho） were collected from Shanghai Xingjian Fruit and Vegetable Professional Cooperative， and transported back to the refrigeration house of Agri-food Storage and Processing Research Center， Shanghai Academy of Agricultural Sciences on the same day of harvest. The ‘Kyoho’ grapes with no mechanical damage， no diseases and insect pests， similar particle size and color were selected as the raw materials. After pre-cooling in a 4 ℃ refrigerator for 24 h， they were screened again， grouped and numbered for experimental treatment.

Test reagents： Reagents including glacial acetic acid， anhydrous sodium acetate， polyethylene glycol （PEG）， polyvinylpyrrolidone （PVPP）， Triton X-100， 30% H2O2， catechol， guaiacol and stable chlorine dioxide solution （including chlorate solution and citric acid particles） were of analytical grade （AR） and purchased from Sinopharm Chemical Reagent Co.， Ltd. Total phenol， oligomeric proanthocyanidin （OPC）， malondialdehyde （MDA）， catalase （CAT） and superoxide dismutase （SOD） kits were purchased from Nanjing Jiancheng Technology Co.， Ltd.

Test instruments： SX-500 ozone generator， Ohnit Environmental Electron （shanghai） Co.， Ltd.; BP301S electronic balance， product of Sartorius， Germany; μQuant microplate reader， BioTek Instruments， Inc.， USA; JX-FSTPR-1， automatic sample freezer grinder， Shanghai Jingxin Technology; D37520O sterode high-speed refrigerated centrifuge， Biofuge company， Germany; water bath， Shanghai Yiheng Technology Co.， Ltd.; homogenizer， Guangdong Midea Life Appliance Manufacturing Co.， Ltd.

Experimental methods

Preparation of chlorine dioxide gasFirst， 10.0 ml of chlorate solution was added with 1.0 g of activator and diluted to 1 000 ml after 2 min of reaction with distilled water to obtain a chlorine dioxide mother liquid required for the test. The chlorine dioxide mother liquid was placed in a closed plastic box for a period of time， and when a large amount of gas was generated， a pipeline was connected to introduce the gas to a gas distribution instrument for adjusting the gas distribution ratio. Continuous and stable chlorine dioxide gas was output to the closed plastic boxes where the grapes were placed. During the process， the gas concentrations in the boxes were monitored by chlorine dioxide measuring instruments， and the gas distribution instrument was closed in time， and timers were started for fumigation treatment.

Preparation of ozone gasAn ozone generator was placed in a closed plastic box for a period of time. When a large amount of gas was generated， a pipeline was connected to introduce the gas to a gas distribution instrument， and other steps were the same as "Preparation of chlorine dioxide gas".

Experimental treatmentThe ‘Kyoho’ grapes screened after pre-cooling were divided into 7 groups. No fumigation treatment was used as the control （CK）， and other 6 groups were placed in sealed ClO gas boxes （with concentrations of 5， 10 and 15 mg/L， respectively） and sealed O gas boxes （30， 60， 90 mg/m3， respectively）， respectively， for 30 min of fumigation. The grapes were placed in PP plastic trays， packaged with fruit and vegetable plastic wrap having a thickness of 0.025 mm， tensile strength of 43.715 MPa， moisture permeability of 23 g/（m·24 h） and oxygen permeability of 9 370 cm/（m·24 h·0.1 MPa） and placed in a refrigeration house with a temperature of （2±1） ℃ for storage， and relevant indices were measured every 7 d.

Item determination

The polyphenol oxidase （PPO） activity was determined by the catechol colorimetric method[7]; the peroxidase （POD） activity was determined by the guaiacol method[7]; and kits were used to measure total phenol， oligomeric proanthocyanidins and MDA contents and CAT and SOD activity.

Data analysis

Excel 2013 software was used for data sorting and analysis， and Origin Pro 9.0 software was used for drawing. Statistical evaluation was determined by Duncans multiple range test in SPSS 17.0 （SPSS Inc.， Chicago IL， USA） software， and different letters indicated a significant difference at P<0.05. The test results were expressed as mean±standard deviation.

Results and Analysis

Effects of different fumigant preservatives on the contents of antioxidants in ‘Kyoho’ grapes

Total phenol contentPhenols are the main taste substances and key quality components of grapes. They have strong antioxidant activity and high capacity to eliminate oxygen free radicals. They can delay membrane lipid peroxidation and have a decisive effect on the color， taste and flavor of grapes， and are closely related to the storage， processing performance， nutritional value and health care effects of grapes[8]. It can be seen from Fig. 1 that the total phenolic content of ‘Kyoho’ grapes fluctuated during the post-harvest low-temperature storage period， showing a rising-falling-rising-falling trend. The total phenol content of grape berries at the beginning of storage was （6.08±0.08） μmol/g. With the prolongation of storage time， the total phenol content of the CK and various fumigant preservative treatment groups gradually increased， reaching a peak at 21 d. Among them， the 5 mg/L ClO treatment had a higher total phenol content at （15.00±0.15） μmol/g， indicating that a certain concentration of fumigation treatment could promote the synthesis and accumulation of phenolic substances. The total phenol content of ‘Kyoho’ grapes decreased to the end of storage （35 d）， mainly because the PPO activity was enhanced after the fruit was stored at low temperature， which in turn promoted the oxidation of phenolic substances[9]. Although the content decreased， it still maintained at 10.29-12.38 μmol/g. There were no significant differences between various fumigation treatments and the CK （P>0.05）.

OPC contentOPCs generally exist in grape fruits， seeds and skins. They are a new and efficient antioxidant and free radical scavenger. They have antioxidant capacity and can delay tissue aging by inhibiting lipid peroxidation[10-11]. It can be seen from Fig. 2 that the OPC content showed a rising-falling-rising trend. The OPC content of ‘Kyoho’ grapes was （0.04±0.03） mg/g at 0 d of storage， and the contents of the CK and various fumigation treatments increased greatly with the prolongation of storage time， and reached their peaks at 14 d， when there were no significant differences between the CK， the ClO groups and the O groups （P>0.05）. Then， the OPC contents of ‘Kyoho’ grapes in various groups decreased sharply. At the end of storage （35 d）， the contents of the 10 mg/L ClO， 15 mg/L ClO and 30 mg/m O treatments were （0.08±0.01） mg/g， significantly higher than the CK and other fumigation treatments （P<0.05）. In conclusion， the fumigant preservative treatments of certain concentrations of ClO and 30 mg/m O could maintain the OPC content and delay the aging of ‘Kyoho’ grapes.

Effects of different fumigant preservatives on MDA content and PPO activity of ‘Kyoho’ grapes

Malondialdehyde （MDA） contentMDA is the final product of membrane lipid peroxidation， and its content can indirectly reflect the degree of damage to the grape cell membrane system and the degree of grape aging[12]. It can be seen from Fig. 3 that the MDA content of ‘Kyoho’ grapes generally showed an upward trend during the storage period. The MDA content was only （10.00±2.47） nmol/g at 0 d， and it gradually increased with the increase of storage period. Among the various treatments， the CK increased the fastest， followed by the 15 mg/L ClO and 90 mg/m O treatments， and the 60 mg/m O treatment increased the slowest. At the end of storage （35 d）， the MDA content of ‘Kyoho’ grapes in the CK was 19.29 times of the initial content; the MDA contents of various ClO treatments were 10.29， 7.39 and 11.25 times of the initial contents， respectively; and the MDA contents of various O treatments were 8.57， 5.31 and 13.5 times of the initial contents， respectively. It indicated that certain concentrations of fumigation treatment could maintain the integrity of ‘Kyoho’ grape cell membrane， reduce the degree of membrane lipid damage during storage， and play an important role in fruit storage and preservation.

PPO activityBrowning is an important factor affecting the appearance， flavor and nutritional value of fruits and vegetables and their processed products. PPO is a metalloprotease that exists widely in nature and is the main cause of post-ripening senescence or enzymatic browning of fruits and vegetables during post-harvest storage and processing. It has been reported that enzymatic browning occurs during the loss of more than 50% of fruits[13]. It can be seen from Fig. 4 that the change trend of PPO activity of ‘Kyoho’ grapes during storage was basically the same as that of proanthocyanidin content， and both showed an overall increasing-decreasing-increasing trend. During 0-7 d of storage， in order to adapt to the low temperature environment stress， the oxidative defense of fruit and vegetable cells was enhanced， and the PPO activity increased significantly. When the berries were adapted to the low temperature environment， the PPO activity gradually decreased， but with the prolongation of storage time， the grape berry tissue was damaged， the degree of membrane lipid peroxidation was deepened， and the PPO activity was enhanced. At the end of storage， the PPO activity of the 30 mg/m O group was （1.40±0.05） △OD/（min·g）， which was significantly lower than those of the CK and other fumigation treatment groups （P<0.05）， indicating that certain concentration of ozone treatment could reduce the PPO activity of ‘Kyoho’ grapes and maintain good pulp quality.

Effects of Different Fumigant Preservatives on Antioxidant Capacity of ‘Kyoho’ GrapesEffects of different fumigant preservatives on antioxidant enzyme activity of ‘Kyoho’ grapes

When fruit and vegetable tissues age or are under stress， the intracellular metabolic balance is destroyed， which leads to the accumulation of active oxygen free radicals such as lipid free radicals （R·）， lipoxyl free radical （RO·）， lipid peroxyl radical （ROO·） and lipid hydroperoxide （ROOH）， which aggravates membrane lipid peroxidation. In order to protect the damage of the cytoplasmic membrane system of fruit and vegetable tissues， important antioxidant enzymes in the reactive oxygen species scavenging system play a defensive role， including peroxidase （POD）， superoxide dismutase （SOD） and catalase （CAT）， the activity of which is one of the physiological and biochemical indices to measure the aging degree of fruits and vegetables[14-15].

POD activityPOD activity can be used as a parameter of fruit post-ripening and senescence， and it is also one of the main enzymes associated with browning， which can act on phenolic substrates to oxidize fruits and cause browning pathological reactions[16]. It can be seen from Fig. 5 that at the beginning of storage， the fruit of ‘Kyoho’ grapes had normal metabolism， intact tissue， and POD activity maintained at a low level of only （0.19±0.04） △OD/（min·g）， but with the increase of storage time （0-7 d）， the aging process of grape fruit accelerated， and POD activity increased gradually in various treatments， among which the CK and 90 mg/m O treatment had the fastest increase rates. At the end of storage， the POD activity of the CK， the ClO treatments and the O treatments was 8.35， 8.05， 8.21， 3.89， 8.63， 5.42， and 8.68 times of the values in the initial storage period. Among them， the 5 mg/L ClO treatment， 10 mg/L ClO treatment and 30 mg/m O treatments reached their peaks at 35 d， 7 d later than other treatment groups， and the POD activity of the 15 mg/L ClO2 treatment and 60 mg/m O treatment was （0.74±0.02） △OD/（min·g） and （1.03±0.10） △OD/（min·g）， respectively， significantly lower than the CK and other preservation treatments （P<0.05）， indicating that certain concentrations of ClO and O could better maintain the antioxidant activity of grape fruit and delay the senescence of ‘Kyoho’ grape fruit.

CAT activityCAT is one of the antioxidant enzymes that scavenge reactive oxygen species in plant tissues. It can catalyze the decomposition of HO into HO and O， and eliminate the damage caused by the accumulation of HO in the aging process. The level of CAT activity reflects the resistance of plants to a certain extent[17-18]. It can be seen from Fig. 6 that the CAT activity of ‘Kyoho’ grapes was （2.95±0.23） U/g at the beginning of storage， and the CAT activity of the CK， the ClO treatments and the O3 treatments increased significantly after storage at low temperature for 7 d. On the 14th day， the adaptability of grape fruit to low temperature environment was enhanced. Except for the 10 mg/L ClO treatment， the CAT activity of the CK and other fumigation treatments remained at the normal metabolic level. With the prolongation of storage time， the aging degree of ‘Kyoho’ grapes accelerated， the CAT activity gradually increased， and the resistance of the fruit itself increased[19]. At the end of storage， the fruit of the CK deteriorated and the CAT activity dropped sharply， while the CAT activity remained at the highest level in the 60 mg/m O treatment at （4.58±0.19） U/g， which was 1.55 times of the initial value， indicating that the 60 mg/m O treatment could improve the antioxidant capacity of grapes， delay the aging process， and play an important role in the storage and preservation of ‘Kyoho’ grapes.

SOD activitySOD is an important antioxidant enzyme in plants， which can catalyze O· to generate non-toxic HO and O， prevent cell membrane lipid peroxidation and leakage of contents， and delay aging. It can be seen from Fig. 7 that the SOD activity of ‘Kyoho’ grapes increased first and then decreased with the storage time. At the beginning of storage， the SOD activity was （0.76±0.13） U/g. During the period from 0 to 7 d， grapes had normal metabolism and showed the capacities to generate and eliminate free radicals in a dynamic balance， and SOD activity in the CK and various fumigation treatments was stabile and not significantly different from those in the initial stage （P>0.05）. From 14 to 21 d， the SOD activity of the CK and various preservative treatments increased， and the value of the CK increased the most and was significantly higher than those of other preservative treatments （P<0.05）. With the prolongation of storage time， the senescence rate of grapes accelerated. At the end of storage， the SOD activity of the CK and various fumigation treatments decreased. Among the various treatments， the CK， the 15 mg/L ClO treatment and the 90 mg/m O treatment decreased more significantly， and compared with the initial time， the SOD activity decreased by 3.17， 2.38， and 2.71 times， respectively. Meanwhile， the SOD activity of the 10 mg/L ClO treatment was （0.78±0.03） U/g， which was not significantly different from the initial time （P>0.05）. It indicated that certain concentration of ClO2 fumigation could maintain the SOD activity of ‘Kyoho’ grapes and delay the senescence of grape fruit.

Discussion and Conclusions

ClO and O treatment are new types of efficient and safe fresh-keeping methods， which are widely used in the storage and transportation of fruits and vegetables after harvest. In this study， 5， 10， and 15 mg/L chlorine dioxide gas and 30， 60 and 90 mg/m ozone gas were used to fumigate ‘Kyoho’ grapes. During storage at （2±1） ℃ for 35 d， it was found that compared with the CK， certain concentrations of ClO and O could effectively maintain the content of grape proanthocyanidins， maintain the integrity of ‘Kyoho’ grape cell membrane， reduce the degree of grape membrane lipid damage during storage and prolong the fresh-keeping period. At the beginning of postharvest storage of ‘Kyoho’ grapes， the protective tissue on the peel surface was basically intact， and various life activities such as physiological metabolism were weak， which meant that under normal circumstances， phenolic substances and the activity of antioxidant enzymes were relatively stable. However， due to the increase of storage time， the aging of grape fruit accelerated， the content of MDA increased gradually， the oxidative defense effect of grape fruit cells was enhanced， and the activity of PPO increased significantly， which promoted the oxidation of total phenols. With the enhanced adaptability of grape berries to low temperature environment， the oxidation resistance and fruit stress resistance of fumigated ‘Kyoho’ grapes were improved. At the end of storage， the contents of total phenols and proanthocyanidins remained good， the activity of PPO and POD was weak， and the activity of CAT and SOD was maintained at a high level. Meanwhile， in the CK， the MDA content was （192.86±6.43） nmol/g， which was significantly higher than that of other fresh-keeping groups， the grape fruits deteriorated seriously， and the activity of antioxidant enzymes dropped sharply.

The comprehensive results showed that the 60 mg/m O fumigation had better overall fresh-keeping effect on ‘Kyoho’ grapes. After 35 d of storage， the grapes had stronger resistance to stress， slower fruit senescence， and lower degree of membrane lipid peroxidation， and the CAT and SOD activity were maintained at high levels.

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