Effects of Hydrogen on Storage Decay and Antioxidant System of Strawberry

Lihua ZHANG Zhiwei WEI Wei TAN Zhongguan SUN Yuanhu ZHANG

Abstract In order to determine whether hydrogen acts as antioxidant in plant tissues， fresh strawberry was put in sealed bags filled with 4% H2 atmosphere （T1）， 100% H2 （T2）， 100% air （CK1） and 100% N2 （CK2）， respectively. The decay rate， soluble solids content （SSC）， titration acid （TA）， ascorbic acid （Vc）， MDA content， SOD and antioxidant activity in strawberry were measured during storage periods. Results showed that the decay rate and MDA content were reduced in H2 treatments; and H2 treatments gave lower level of TA and higher SSC， Vc， SOD and antioxidant activity， and 100% H2 had the best effect. It is suggested that strawberry fruit quality， antioxidant system and membrane lipid peroxidation were affected by H2 treatments.

Key words Hydrogen; Strawberry; Decay; Antioxidant system

Received： March 1 2021  Accepted： May 13， 2021

Supported by Key Research and Development Projects of Shandong Province （2019GNC20401）.

Lihua ZHANG （1968-）， male， P. R. China， professor， devoted to research about resource development.

*Corresponding author. E-mail： yhzhang9@163.com.

Hydrogen is the lightest and most abundant chemical element， constitutes nearly 75% of the universes elemental mass. Hydrogen gas is a colorless， odorless， tasteless， non-metallic and highly combustible diatomic gas with the molecular formula H and was produced firstly by Robert Boyle （1671） when he dissolved iron in diluted hydrochloric acid. On earth， free hydrogen is comparatively rare， as Earths atmosphere contains less than 1 part per million of hydrogen. The majority of hydrogen atoms are in water and organic compounds. It is highly reactive to oxygen and other oxidants in the presence of specific catalysts and/or heat. One of the more spectacular examples of its reactivity was shown in 1937 by the Zeppelin Hindenburg disaster.

It has long been known that organisms cant absorb and produce hydrogen in a large amount. Hydrogen does not participate in the life metabolic activity of organisms like oxygen. Therefore， most biologists have thought that hydrogen is a physiological inert gas. But the potential exploitation of hydrogen as a therapeutic medical gas has only recently been explored in animal models and in the clinic. The first study reported that there was significant cancer regression in patients with squamous cell carcinoma exposed to hyperbaric hydrogen for 14 d[1]. In 200 another study reported that hyperbaric hydrogen could therapy schistosomiasis-associated chronic liver inflammation[2]. However， these studies were not extended by other researchers. It was in 2007 that hydrogen medical value got the attention of scholars. The study in Natural Medicine reported that brain ischemia reperfusion injury of animal models was significantly improved by 2% hydrogen inhalation[3]. The same research group also studied the effects of hydrogen on liver and heart ischemia animal models and suggested that ischemia-reperfusion injury of liver and heart can be cured by 2% hydrogen inhalation， too[4-5]. Later， some studies showed that 2% hydrogen inhalation still can therapy inflammation damage of the small intestine transplantation[6] and neonatal hypoxia-ischemia rat model[7]. Oral ingestion of H2-saturated water can improve lipid and glucose metabolism in patients with type 2 diabetes[8]， be protective against 6-hydroxydopamine induced nigrostriatal degeneration in a rat model of Parkinsons disease[9] and reduce paraquat-induced acute lung injury in rats[10]. In addition， injection of H2 rich saline also reduces organ ischemia/reperfusion injury[11-12] and Amyloid-beta-induced Alzheimers Disease in rat models[13].

The therapeutic properties of hydrogen were ascribed to selectively scavenging hydroxyl radicals （·OH） and peroxynitrite （ONOO-）， which are very strong oxidants that react indiscriminately with nucleic acids， lipids and proteins resulting in DNA fragmentation， lipid peroxidation and protein inactivation[3]. Then， does hydrogen play the same role of antioxidant activities in plant tissues？ So far， no study on antioxidant and protection of hydrogen against oxidative injury in plant tissues has been reported. Thus， in the present paper， effects of hydrogen on postharvest physiology and antioxidant system of strawberry have been studied to reveal the scientific problem whether hydrogen acts as antioxidant in plant tissues.

Materials and Methods

Plant materials and treatments

The cv. Tianbao strawberries were purchased from local market in Taian City， China. The strawberries with defects （damaged， shriveled and unriped） were rejected. Sound strawberries of a homogeneous size （mean weight±SD were 23-25 g） and appearance were selected and divided into 4 lots of 80 strawberries each at random. Each lot was placed in a sealable plastic bag of 10 L volume under the condition at 25 ℃ and 80%-85% relative humidity （RH）. Treatment 1 （T1）： The bag was filled with 0.4 L H2 firstly， and then the bag was filled with 9.6 L air to keep H2 /air is about 4%. Treatment 2 （T2）： The bag was full filled with 10 L H2 （100% H2）. Control 1 （CK1）： The bag was full filled with 10 L air as the blank control. Control 2 （CK2）： The bag was full filled with 10 L N2 as the positive control （100% N2）. All the gas atmospheres were humidified to approximately 90% RH by bubbling through water. Some strawberries for analysis were taken every 24 h. After this， the four treatments were treated according to the above methods again.

Five strawberries from each replicate were wrapped in cheesecloth and squeezed with a hand press， and the juice was centrifuged at 10 000 g （Eppendorf 5804R， Germany） for 10 min at 4 ℃. Then the juice supernatant was used for measurements of SSC， TA， ascorbic acid and DPPH·scavenging activity.

Chemicals

2，2-Diphenyl-1-picryl hydrazyl （DPPH·）， lactoflavin and Vc standard were purchased from Sigma Chemical Co. （St. Louis， MO， USA）. 2-Thiobarbituric acid （TBA）， nitroblue tetrazolium （NBT）， ethylenediaminetetraacetic acid （EDTA）， trichloroacetic acid （TCA）， H2SO4 and NaOH were of analytical grade and were bought from Tianjin Yongda Chemical Reagent Development Center， Tianjin， China. 100% H2 and 100% N2 were obtained from Jinan Deyang Special Gas Co. Ltd.， Jinan， China.

Estimation of fruit quality

The entire sample of fruit was visually inspected for decay and physiological disorders. Strawberries were scored for external physiological disorders， namely skin surface pitting and softening， according to the following scale： 0=none visible; 1=slight （≤10% of the skin）; 2=moderate （11%-30% of the skin）; and 3=severe （>30% of the skin）. The softening/decay index was calculated using the following formula： ∑ （softening scale×percentage of corresponding fruit within each class）.

Measurements of soluble solids content （SSC）， titrable acidity （TA） and ascorbic acid （Vc）

SSC was determined at 20 ℃ with an Atago RX-1000 digital refractometer （Atago Co. Ltd.， Tokyo， Japan） and expressed as a percentage. TA was determined by diluting each 5 ml aliquot of strawberry juice in 50 ml distilled water and then titrating to pH 8.2 using 0.1 N NaOH. Titrable acidity was expressed as grammes of citric acid per 100 g of strawberry weight. A modification of the method of Law et al.[14] was used to assay Vc. Briefly， A sample （800 μ1） of the strawberry juice was added to 400 μ1 of 10% （w/v） trichloroacetic acid. After vortex-mixing， it was allowed to stand in ice for 5 min. NaOH （20 μ 5 M） was added， followed by mixing， and the mixture was centrifuged for 2 min in a Microfuge. To a 400 μl of the supernatant sample was added 400 μ1 of 150 mM-NaH2PO4 buffer， pH 7.4， and 400 μ1 of water. To another 400 μl sample of supernatant was added 400 μ1 of buffer， 200 μ1 of l0 mM-dithiothreitol and， after thorough mixing and being left at 25 ℃ for 15 min， 200 μ1 of 0.5% （w/v） N-ethylmaleimide. Both samples were vortex-mixed and incubated at 25 ℃ for >30 s. To each was then added 800 μ1 of 100% （w/v） trichloroacetic acid， 800μ1 of 44% （v/v） H3PO4， 800 μ1 of 4% （w/v） bipyridyl in 70% （v/v） ethanol and 400 μl of 3% （w/v） FeCl3. After vortex-mixing， samples were incubated at 37 ℃ for 60 min and the A525 was recorded. A standard curve in the range 0-40 nmol of Vc was used for calibration.

Superoxide dismutase （SOD） assay

Fruit flesh （5 g） from five strawberries was homogenized in 20 ml of ice-cold extraction buffer containing 0.5 g polyvinyl polypyrrolidone （PVPP）. The 50 mmol/L sodium borate solution （pH 8.8， containing 5 mol/L β-mercaptoethanol）， and 100 mmol/L potassium phosphate （pH 7.8） was used as the buffer. Homogenates were centrifuged at 12 000 g （Eppendorf 5804R， Germany） for 20 min at 4 ℃ and the resulting supernatants were used for SOD assay. The activity of SOD was analyzed according to the method of Wang et al.[15]. The reaction mixture （3 ml） contained 50 mmol/L  sodium phosphate buffer of pH 7.8 （1.7 ml）， 6.5 mmol/L methionine （0.3 ml）， 0.5 mmol/L NBT （0.3 ml）， 0.1 mmol/L EDTA （0.3 ml）， 0.2 mmol/L riboflavin （0.3 ml） and 0.1 ml crude enzyme extraction solution. The mixtures were illuminated by fluorescent lamp （4 000 Lx） for 20 min and then the absorbance was determined at 560 nm. Identical solutions held in the dark served as blanks. One unit （U） of SOD activity was defined as the amount of enzyme that caused a 50% decrease of the SOD-inhibitable NBT reduction.

Malondialdehyde （MDA） determination

The fruit flesh MDA concentration was determined according to the method described by Jain et al.[16] based on TBA reactivity. Briefly， flesh （2 g） from five strawberries was taken and kept at -78 ℃ during the analysis. The flesh was homogenized in 20 ml of 10% ice-cold TCA solution using a glass-porcelain homogenizer （20 KHz frequency ultrasonic， Jencons Scientific Co.）， and then centrifuged at 4 000 g （Eppendorf 5804R， Germany） for 10 min. All processes were carried out at 4 ℃. After this， 2 ml of supernatant was taken and added to each tube， and then 1 ml of 0.6 % TBA was added. These tubes with Teflon-lined screw caps were incubated at 90 ℃ in a water bath for 15 min and cooled to room temperature. The optical density was measured at 450， 532 and 600 nm in a spectrophotometer for fruit flesh MDA （Shimadzu 2550 UV-Vis spectrophotometer， Japan）. The formula for calculating the content of MDA is： MDA content （μmol/g） =C （μmol/L）×V （L）/W （g） where V is the volume of MDA extraction solution， and W is the fresh weight of fruit flesh and C is from the next formula. That is： C （μmol/L）=6.45（D532-D600）-0.56D450， in this formula， D450， D53 and D600 represent the optical density at 450， 532 and 600 nm， respectively.

Antioxidant capacity assay

Antioxidant capacity was assessed using the free radical DPPH· scavenging activity according to the method of Yokozawa et al.[17]， with modifications. Briefly， the stock solution （0.1 mM DPPH· in methanol） was diluted with methanol to the absorbance of 1.5 at 517 nm before using. 3 ml of methanolic solution of DPPH· was mixed with 1 ml of the juice supernatant and the mixture was vortexed. The radical scavenging activity of DPPH· was measured spectrophotometrically （Shimadzu 2 550 UV-Vis spectrophotometer， Japan） at 517 nm after 30 min incubation in the dark at room temperature. The control samples contained all reagents except the extract. DPPH· radical scavenging activity was expressed using the formula： % DPPH· radical scavenging activity=[（A0-A1）/A0]×100 where A0 was the absorbance of the control and A1 was the absorbance of the sample.

Statistical analysis

All assays were carried out in triplicates. Data were analyzed using Microsoft Excel 2003. In this software， variance was calculated manually by one-way analysis of variance method. Significant differences were detected at P<0.05.

Results

Effects of H2 on softening/decay

As shown in Fig.  softening/decay index increased as the time of storage increased. After 48 h of storage， the initial bright color of CK1 and CK2 fruit had largely disappeared. As the storage time was extended to 96 h， the softening/decay indexes of CK1 and CK2 fruit had increased up to 100%. Compared with CK1 and CK the softening/decay of H2 treated strawberries was reduced significantly （P<0.05.）， especially 100% H2 treatment （T2）. After 96 h storage at 25 ℃， the softening/decay indexes of T1 and T2 were 45.6% and 21.8%， respectively. It is suggested that H2 treatment is beneficial to protecting integrity of plant tissues and organs.

Effects of H2 on SSC and TA

Changes in the SSC of strawberries during storage are shown in Fig. 2A. A decline in the SSC of all strawberry samples was detected along with the prolonged storage. It can be expected that SSC decreases in mature fruit due to respiration. Both air treated （CK1） and N2 treated （CK2） strawberries decreased obviously. However， the decrease in SSC was inhibited by hydrogen treatments （T1 and T2）， especially， the 100% hydrogen treatment （T2） with the most significant effect （P<0.05）. After 24 h of storage at 25 ℃， TA content in both CK1 and CK2 strawberries was significantly （P<0.05） higher than the initial value （Fig. 2B）. This increase in TA could be related to some acidic metabolites produced from some biological degradation of macromolecules. On the other hand， a decrease in TA was found for H2 treated strawberries （T1 and T2）; and especially the decrease in TA of 100% H2 treated strawberries （T2） was the most significant （P<0.05）. However， after 48， 72 or 96 h of storage at 25 ℃， TA for all samples did not significantly change （P<0.05） from the 24 h value. In addition， no significant differences in SSC and TA were observed in CK1 and CK2. It is indicated that H2 is able to inhibit the decrease in SSC and the increase in TA of strawberry fruit after harvest. The different change patterns of TA and TSS seem to be associated with the different effects of H2 on commodity respiratory rate.

Ascorbic acid is not only one of the nutritional content， but also an important antioxidant to scavenge reactive oxygen species in fruit. Then ascorbic acid plays the role of anti-aging after harvest. But the ascorbic acid in fruit and vegetables is susceptible to oxidation and loss of activity. Therefore， it is important to prevent oxidation and maintain a high content of ascorbic acid in fruit. It is shown in Fig. 3 that the ascorbic acid contents of all strawberry samples appeared a downward trend along with the extension of storage time. And the most significant decrease for CK1 and CK2 was observed， whereas no significant difference between CK1 and CK2. But the decline in ascorbic acid contents was reduced by hydrogen treatments （T1 and T2）， especially， the 100% hydrogen treatment （T2） with the most significant effect （P<0.05）. It is indicated that the hydrogen is able to inhibit the decomposition of ascorbic acid in strawberry fruit. Hydrogen is a kind of antioxidant， too， and may play a role of antioxidant to protect ascorbic acid during the storage period.

Superoxide Dismutase （SOD） is an antioxidant enzyme that catalyzes O- the most common free radical， to generate O2 and H2O2 in plant tissue. So it is the first line of defense in plant reactive oxygen scavenging system. Strawberry fruit becomes aging during storage， which results in the increase of free radical， and the SOD is consumed. Therefore， SOD activity will continue to decline. The results in Fig. 4 indicate that SOD activity of all samples declined， among them， CK1 and CK2 declined most sharply， T1 declined more smoothly than the controls and the smoothest decline of T2 was found. After 72 h of storage at 25 ℃， the SOD activity of T1 and T2 was higher than that of CK 46.6% and 108.8%， respectively. It is indicated that H2 protects SOD in strawberry fruit; and the higher the concentration of H2 is， the more effective the protection becomes.

The results in Fig. 5 showed that an upward trend of the MDA contents for all strawberry samples was observed along with the prolonged storage period. And the most significant increase for CK1 and CK2 was found， whereas no significant difference between CK1 and CK2. But the increase in MDA contents was significantly inhibited by hydrogen treatments （T1 and T2）， especially， the 100% hydrogen treatment （T2） （P<0.05）. In comparison to CK1 after 72 h of storage at 25 ℃， the inhibition rate was about 31.9% and 44.8% for T1 and T respectively， showing a dose-depended effect. It is indicated that the hydrogen may play the role of inhibiting membrane lipid peroxidation during strawberry storage period.

2-Diphenyl-1-picryl hydrazyl （DPPH·）， an organic free radical， is often used to evaluate the antioxidant capacity of some plant materials. Under the same reaction conditions， the higher the rate of scavenging DPPH free radical is， the stronger the antioxidant capacity becomes. It is shown in Fig. 6 that the scavenging capacity on DPPH· of all strawberry samples appeared a downward trend along with the prolonged storage period. And the most significant decrease for CK1 and CK2 was observed， whereas no significant difference between and CK2. But the decline in scavenging capacity was inhibited by hydrogen treatments （T1 and T2）， especially， the 100% hydrogen treatment （T2） with the most significant effect （P<0.05）. After 72 h of storage at 25 ℃， the scavenging capacities of T1 and T2 were higher than CK1 about 8% and 19%， respectively. It is indicated that the hydrogen is able to maintain the antioxidant capacity of strawberry fruit during storage period， and the dose-depended effect is observed.

Discussions

The accumulated evidence in a variety of biomedical fields using clinical and experimental models for many diseases proves that hydrogen， administered either through gas inhalation or oral ingestion of H2-saturated water， can act as a therapeutic agent. More than 100 papers about hydrogen medical have been published all over the world. These studies have targeted a diverse range of disorders and organ systems including the nervous， digestive， and cardiovascular and respiratory systems[18]. As far as we know， the effect of H2 on plant material has been studied for the first time in this paper.

In this paper， air treated （CK1） and 100% N2 treated （CK2） strawberries were used as controls. No obvious differences were observed in those indicators determined in this study between the two controls. Because safe hydrogen concentrations in air and in pure oxygen gas are<4.6% and 4.1%， >74.5% and 94% by volume， respectively[2]. Then 4% and 100% H2 by volume were selected to treat the strawberry fruit. Although 100% H2 treatment （T2） will cause no oxygen conditions as same as 100% N2 treatment （CK2）， significant differences in experimental data were found between T2 and CK2. The result indicates that H2 can play the special role to keep the strawberry fruit fresh after harvest， and obviously affect the post-harvest physiology of strawberry. Treatments with 4% and 100% H2 obviously reduced fruit decay， SSC loss， TA and MDA produce， and obviously maintained ascorbic acid content， SOD activity and antioxidant capacity. The 100% H2 treatment was the most effective one， and may provide a potential alternative way to control postharvest quality of strawberry fruit.

Free radicals are atoms， molecules or ions with unpaired electrons in an open shell configuration. These unpaired electrons make the radical species highly chemically reactive. Reactive oxygen species （ROS）， includes superoxide anion （O-2·）， hydroxyl radical （·OH）， hydrogen peroxide （H2O2） and singlet oxygen （1O2）. These different species interconvert via cascade reactions[18]. Although generally regarded as toxic byproducts， ROS has been shown to play an important role as signaling molecules. Then， it is necessary to maintain certain degree of ROS in plant tissues. Nevertheless， excessive production of ROS resulting from oxidative stress has been associated with a range of cytotoxic effects， such as membrane lipid peroxidation， protein denaturation or degradation and DNA fragmentation et al.， resulting in the decrease of cell membrane stability， the increase of ion leakage and plant body damage[19]. The accumulation of ROS is generally counterbalanced by a sophisticated endogenous antioxidant defense system that comprises enzymes-such as superoxide dismutase （SOD）， catalase and glutathione peroxidase-and non-enzymes， such as vitamin A， vitamin C， carotene and bilirubin. SOD is the enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. Thus， it is the first line of antioxidant defense to remove active oxygen system in nearly all cells exposed to oxygen[20]. Vitamin C is a kind of water-soluble small molecular antioxidants existing widely in plant body. The results of this paper proved that the decline of ascorbic acid content， SOD activity and antioxidant capacity of the strawberry fruit were obviously inhibited by 4% and 100% H2 treatment. It is suggested that H2 can protect the antioxidant system of strawberry fruit. MDA is the membrane lipid peroxidation product， and often used as an indicator of oxidative injury. MDA content of strawberry fruit was decreased in this study， which suggests that H2 can also protect the membrane system.

The mechanisms of the protection for antioxidant system and membrane system afforded by hydrogen exposure have been proposed， the role of hydrogen as an antioxidant has been advocated. The antioxidant capabilities of hydrogen include activities as a scavenger of free radicals. Hydrogen selectively reduces peroxynitrite （ONOO-） and hydroxyl radicals （·OH）， which are very strong oxidants that react indiscriminately with proteins， lipids and nucleic acids resulting in protein inactivation， lipid peroxidation and DNA fragmentation. Biochemical experiments， using fluorescent probes and electron resonance spectroscopy spin traps， suggest that the effects of hydrogen against ONOO- are less potent than those against ·OH[3]. Subsequent studies confirmed that the beneficial effects of H2 are principally mediated by ·OH scavenging[4，21]. Hydrogen may have several potential advantages. First， hydrogen is highly diffusible and could potentially reach subcellular compartments， such as mitochondria and nuclei， which are the primary site of ROS generation and DNA damage. Secondly， hydrogen selectively reduces detrimental ·OH and ONOO-， but does not decrease the steady-state levels of ROS[3]. Finally， H2O is the product of reaction between H2 and ROS. Therefore， hydrogen has almost no adverse effect on plant tissue. So， it is proposed that hydrogen eliminated some free radicals in strawberry fruit tissue and reduced the oxidative damage by free radicals， which protected the antioxidant enzymes such as SOD， and antioxidants such as Vc， and membrane system. And then the strawberry fruit decay was decreased， the fruit quality was kept. But the molecular mechanism of protection role of hydrogen needs further research.

Conclusions

In summary， the data presented in this paper indicates that 100% H2 significantly affect strawberry fruit decay， fruit quality， antioxidant system and membrane lipid peroxidation which were mildly affected by 4% H2 during storage. Therefore， H2 has great application potential in the fruit and vegetable fresh preservation.

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