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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 universe’s 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 Earth’s 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 can’t 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 Parkinson’s 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 Alzheimer’s 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 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.
References
[1] DOLE M， WILSON FR， FIFE WP. Hyperbaric hydrogen therapy： A possible treatment for cancer[J]. Science， 1975， 190： 152-154.
[2] GHARIB B， HANNA S， ABDALLAHI OM， et al. Anti-inflammatory properties of molecular hydrogen： Investigation on parasite-induced liver inflammation[J]. Comptes Rendus de l’Academie des Sciences-Series III， 2001（324）： 719-724.
[3] OHSAWA I， ISHIKAWA M， TAKAHASHI K， et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals[J]. Nature Medicine， 2007（13）： 688-694. [4] FUKUDA KI， ASOH S， ISHIKAWA M， et al. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress[J]. Biochemical and Biophysical Research Communications， 2007（361）： 670-674.
[5] HAYASHIDA K， SANO M， OHSAWA I， et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury[J]. Biochemical and Biophysical Research Communications， 2008（373）： 30-35.
[6] BUCHHOLZ BM， KACZOROWSKI DJ， SUGIMOTO R， et al. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury[J]. American Journal of Transplantation， 2008（8）： 2015-2024.
[7] CAI J， KANG Z， LIU WW， et al. Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model[J]. Neuroscience Letters， 2008（441）： 167-172.
[8] KAJIYAMA S， HASEGAWA G， ASANO M， et al. Supplementation of hydrogen rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance[J]. Nutrition Research， 2008（28）： 137-143.
[9] FU Y， ITO M， FUJITA Y， et al. Molecular hydrogen is protective against 6-hydroxydopamine induced nigrostriatal degeneration in a rat model of Parkinson’s disease[J]. Neuroscience Letters， 2009（453）： 81-85.
[10] LIU SL， LIU K， SUN Q， et al. “Consumption of hydrogen water reduces paraquat-induced acute lung injury in rats”[J]. Journal of Biomedicine and Biotechnology， 2011（4）： 305086.
[11] CAI J， KANG Z， LIU K， et al. Neuroprotective effects of hydrogen saline in neonatal hypoxia-ischemia rat model[J]. Brain Research， 2009（1256）： 129-137.
[12] TAO B， LIU L， WANG N， et al. Effects of hydrogen-rich saline on aquaporin 5 in septic rat lungs[J]. Journal of Surgical Research， 2016， 202（2）： 291-298.
[13] WANG C， LI J， LIU Q， et al. Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-κB activation in a rat model of amyloid-beta-induced Alzheimer’s disease[J]. Neuroscience Letters， 2011（491）： 127-132.
[14] LAW M， CHARLES S， HALLIWELL B. Glutathione and ascorbic acid in spinach （Spinacia oleracea） choloroplasts， the effect of hydrogen peroxide and of Paraquat[J]. Biochemical Journal， 1983（210）： 899-930.
[15] WANG YS， TIAN SP， XU Y. Effects of high oxygen concentration on pro- and anti-oxidant enzymes in peach fruits during postharvest periods[J]. Food Chemistry， 2005（91）： 99-104.
[16] JAIN SK， MCVIE R， DUETT J， et al. Erythrocyte membrane lipid peroxidation and glycolylated hemoglobin in diabetes[J]. Diabetes， 1989（38）： 1539-1543. [17] YOKOZAWA T， DONG E， NATAGAWA T， et al. In vitro and in vivo studies on the radical 2 scavenging activity of tea[J]. Journal of Agriculture and Food Chemistry， 1998（46）： 2143-2150.
[18] GE YS， ZHANG QZ， LI H， et al. Hydrogen-rich saline protects against hepatic injury induced by ischemia-reperfusion and laparoscopic hepatectomy in swine[J]. Hepatobiliary & Pancreatic Diseases International， 2019， 18（1）： 58-71.
[19] LOW PS， MERIDA JR. The oxidative burst in plant defense： Function and signal transduction[J]. Physiologia Plantarum， 1996（96）： 533-542.
[20] SUNIL B， RAJSHEEL P， ASWANI V， et al. Photosynthesis is sensitive to nitric oxide and respiration sensitive to hydrogen peroxide： Studies with pea mesophyll protoplasts[J]. Journal of Plant Physiology， 2020（246-247）： 153133.
[21] HOWEVER. Hydrogen-rich saline protects against ischemia/reperfusion injury in grafts after pancreas transplantations by reducing oxidative stress in rats[J]. Mediators of Inflammation， 2016， 2015（31）： 281985.
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 universe’s 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 Earth’s 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 can’t 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 Parkinson’s 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 Alzheimer’s 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 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.
References
[1] DOLE M， WILSON FR， FIFE WP. Hyperbaric hydrogen therapy： A possible treatment for cancer[J]. Science， 1975， 190： 152-154.
[2] GHARIB B， HANNA S， ABDALLAHI OM， et al. Anti-inflammatory properties of molecular hydrogen： Investigation on parasite-induced liver inflammation[J]. Comptes Rendus de l’Academie des Sciences-Series III， 2001（324）： 719-724.
[3] OHSAWA I， ISHIKAWA M， TAKAHASHI K， et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals[J]. Nature Medicine， 2007（13）： 688-694. [4] FUKUDA KI， ASOH S， ISHIKAWA M， et al. Inhalation of hydrogen gas suppresses hepatic injury caused by ischemia/reperfusion through reducing oxidative stress[J]. Biochemical and Biophysical Research Communications， 2007（361）： 670-674.
[5] HAYASHIDA K， SANO M， OHSAWA I， et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury[J]. Biochemical and Biophysical Research Communications， 2008（373）： 30-35.
[6] BUCHHOLZ BM， KACZOROWSKI DJ， SUGIMOTO R， et al. Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury[J]. American Journal of Transplantation， 2008（8）： 2015-2024.
[7] CAI J， KANG Z， LIU WW， et al. Hydrogen therapy reduces apoptosis in neonatal hypoxia-ischemia rat model[J]. Neuroscience Letters， 2008（441）： 167-172.
[8] KAJIYAMA S， HASEGAWA G， ASANO M， et al. Supplementation of hydrogen rich water improves lipid and glucose metabolism in patients with type 2 diabetes or impaired glucose tolerance[J]. Nutrition Research， 2008（28）： 137-143.
[9] FU Y， ITO M， FUJITA Y， et al. Molecular hydrogen is protective against 6-hydroxydopamine induced nigrostriatal degeneration in a rat model of Parkinson’s disease[J]. Neuroscience Letters， 2009（453）： 81-85.
[10] LIU SL， LIU K， SUN Q， et al. “Consumption of hydrogen water reduces paraquat-induced acute lung injury in rats”[J]. Journal of Biomedicine and Biotechnology， 2011（4）： 305086.
[11] CAI J， KANG Z， LIU K， et al. Neuroprotective effects of hydrogen saline in neonatal hypoxia-ischemia rat model[J]. Brain Research， 2009（1256）： 129-137.
[12] TAO B， LIU L， WANG N， et al. Effects of hydrogen-rich saline on aquaporin 5 in septic rat lungs[J]. Journal of Surgical Research， 2016， 202（2）： 291-298.
[13] WANG C， LI J， LIU Q， et al. Hydrogen-rich saline reduces oxidative stress and inflammation by inhibit of JNK and NF-κB activation in a rat model of amyloid-beta-induced Alzheimer’s disease[J]. Neuroscience Letters， 2011（491）： 127-132.
[14] LAW M， CHARLES S， HALLIWELL B. Glutathione and ascorbic acid in spinach （Spinacia oleracea） choloroplasts， the effect of hydrogen peroxide and of Paraquat[J]. Biochemical Journal， 1983（210）： 899-930.
[15] WANG YS， TIAN SP， XU Y. Effects of high oxygen concentration on pro- and anti-oxidant enzymes in peach fruits during postharvest periods[J]. Food Chemistry， 2005（91）： 99-104.
[16] JAIN SK， MCVIE R， DUETT J， et al. Erythrocyte membrane lipid peroxidation and glycolylated hemoglobin in diabetes[J]. Diabetes， 1989（38）： 1539-1543. [17] YOKOZAWA T， DONG E， NATAGAWA T， et al. In vitro and in vivo studies on the radical 2 scavenging activity of tea[J]. Journal of Agriculture and Food Chemistry， 1998（46）： 2143-2150.
[18] GE YS， ZHANG QZ， LI H， et al. Hydrogen-rich saline protects against hepatic injury induced by ischemia-reperfusion and laparoscopic hepatectomy in swine[J]. Hepatobiliary & Pancreatic Diseases International， 2019， 18（1）： 58-71.
[19] LOW PS， MERIDA JR. The oxidative burst in plant defense： Function and signal transduction[J]. Physiologia Plantarum， 1996（96）： 533-542.
[20] SUNIL B， RAJSHEEL P， ASWANI V， et al. Photosynthesis is sensitive to nitric oxide and respiration sensitive to hydrogen peroxide： Studies with pea mesophyll protoplasts[J]. Journal of Plant Physiology， 2020（246-247）： 153133.
[21] HOWEVER. Hydrogen-rich saline protects against ischemia/reperfusion injury in grafts after pancreas transplantations by reducing oxidative stress in rats[J]. Mediators of Inflammation， 2016， 2015（31）： 281985.