peach is one of the most well - liked fruits in the world because of their flavor , dietary value , attractive color , and medicinal worth .
it is enriched with ascorbic acid , flavonoid , and phenolic compounds , which were considered prime sources for antioxidants [ 1 , 2 ] .
however , peaches have a very short shelf life because they are highly susceptible to pathogenic infection and physiological deterioration during storage time under ambient temperature .
cold storage remains the main method to slow the product deterioration in terms of consumer perception and nutritional value .
however , low temperature results in chilling injury symptoms in some peach cultivars during or after cold storage [ 3 , 4 ] .
chemical treatments have been used to prevent insect attack and prolong shelf life of postharvest peach .
however , the use of chemicals has been minimized for food safety and environmental reasons .
many physical methods including modified atmosphere packaging , heat , and uv - c pretreatments are being extensively studied as substitutes for current chemical methods in the commercial applications of peach [ 58 ] .
carbon monoxide ( co ) is a simple diatomic gas molecule with low water solubility .
it easily combines hemoglobin , thus delaying oxygen transport and leading to death of organisms .
so in the past , it was considered a toxic gas to environment and biology .
however , recent researchers found that , similar to no and h2s , co might serve as a gaseous signal molecule to involve in stomata close regulated by plant guard cell and the formation of lateral root
. meanwhile , co may relieve plant tissue oxidation damage caused by a variety of abiotic stresses such as heavy metal , salt , and active oxygen donor [ 913 ] .
it has been reported that exogenous co donor treatment might postpone the senescence of cut flower through regulating active oxygen metabolism and inhibiting lipid peroxidation .
our previous study found that exogenous co treatment could restrain the browning of fresh cut lotus root and prolong the shelf life of postharvest jujube [ 15 , 16 ] . at present , there are few reports about the effect of co on plant senescence and fruit preservation , and the physiological mechanism of co on plant senescence stress is still unclear . in this work
, the postharvest peaches were fumigated with exogenous co gas at different concentrations , and the presentation quality , nutrients , and antioxidant activity of peaches were determined periodically during the storage time .
this research aims to investigate the effect of co treatment on postharvest peach and try to explore a novel method for fresh peach preservation .
yanhong ) were purchased from an orchard situated in yaodu district , linfen city , shanxi province .
peaches with uniform shape , size , and color , as well as no insect pest and mechanical damage , were selected and quickly transported to the laboratory of shanxi normal university in open cartons .
co gas with a purity of 99.99% was purchased from beijing huaneng special gases co. , ltd .
oxalic acid , gallic acid , rutin , potassium ferricyanide , trichloroacetic acid , and sodium hydroxide ( analytical grade ) were purchased from sinopharm chemical reagent co. , ltd .
analytical - grade 1,1-diphenyl-2-picrylhydrazyl radical , 2,2-diphenyl-1-(2,4,6-trinitrophenyl ) hydrazyl , and 2,6-dichloroindophenol were purchased from sigma ( usa ) .
other reagents of analytical grade were purchased from alfa aesar company ( tianjin , china ) .
the peaches were fumigated with co gas at different concentrations ( 0.5 , 5 , 10 , or 20 
about 10  kg of peaches were placed into a glass container , and then the container was sealed with lid . the diameter and height of used container are 40  cm .
peaches that had not been fumigated with co gas were also sealed in glass container for 2 hours , serving as control sample .
after treatment , all samples were then placed in plastic bags and stored under ambient temperature with 90% of relative humidity .
each sample was about 180 peaches and the relevant parameters for the analyses of peaches were measured periodically .
firmness was determined using a fruit digital sclerometer 8  mm in diameter for the head ( gy-4 , chendu bsida instrument co. , ltd . , chendu , china ) . for each fruit ,
two readings were taken in the equatorial region of the fruit after the skin was removed .
the firmness of six fruits during storage was regularly measured and expressed as kg / cm .
it was determined visually in the fruits from three trays and rated as 0 = absent ; 1 = very slight ( 1%  surface  10% ) ; 2 = slight ( 10%  surface  25% ) ; 3 = moderate ( 25%  surface  50% ) ; 4 = severe ( 50%  surface  100% ) .
the decay incidence for the treatment unit was calculated as follows : decay incidence = [ (  rank  quantity)/(4  
soluble solids content and titratable acidity were assayed according to the method of mignani with modifications .
tissues ( 50  g ) from ten fruits were homogenised and then centrifuged at 8000  g for 20  min using an eppendorf 5417r centrifuge ( germany ) .
the supernatant was collected to measure soluble solids content ( brix ) using a refractometer ( wyt - ii , qingyang optical instrument co. , ltd . , chendu , china ) .
titratable acidity expressed as citric acid on a fresh weight basis was determined by titration with 0.1  mol / l naoh to ph 8.2 .
the ph of the supernatant was measured using a ph meter ( phs-3tc , shanghai leici instrument inc . ,
pal activity was determined as per the method of hussain et al .   with modification .
5  g of sample from six fruits was homogenized in 5  ml of borate buffer ( 0.05  m , ph 8.0 ) containing 5  mm -mercaptoethanol and 1  mm edta .
about 1.0  ml of enzyme extract was incubated with an assay medium containing 2  ml of 200  mmol / l sodium borate buffer ( ph 8.0 ) , 1  ml of distilled water , and 1  ml of 50  mmol / l 1-phenylalanine as substrate at 30c for 1  h. the reaction was terminated by adding 0.2  ml of 6  mol  l  hcl .
one unit was defined as the change 0.01 absorbance at 290  nm per h. vitamin c content was measured through 2,6-dichloriondophenol titration .
briefly , tissues ( 5  g ) from 6 fruits were homogenised in 10  ml of 2% oxalic acid solution and then centrifuged at 8000  g for 15  min at 4c .
afterwards , 2  ml of the supernatant was titrated to a permanent pink colour using 0.1% of 2,6-dichlorpphenolindophenol .
vitamin c concentration was calculated according to the titration volume of 2,6-dichloriondophenol and expressed as g  g fresh weight .
total polyphenol content was determined using folin - ciocalteu 's phenol reagent via spectrophotometric analysis .
tissues ( 5  g ) from 10 fruits were homogenised in 20  ml of 50% aqueous methanol and then centrifuged at 3000  g for 20  min .
an aliquot ( 1  ml ) of a standard solution of gallic acid with 0 , 10 , 20 , 30 , 40 , and 50  mg / l aqueous methanol or supernatant was added to a 25  ml volumetric flask containing 9  ml of water . approximately 1  ml of folin - ciocalteu 's phenol reagent was added to the mixture and then shaken . after 8  min , 2  ml of 7.5% aqueous na2co3 solution was added .
the solution was immediately diluted with water to a final volume of 25  ml and thoroughly mixed .
after incubation for 30  min at 25c , the absorbance versus the prepared blanks was read at 765  nm .
tissues ( 5  g ) from 10 fruits were homogenised in 20  ml of 80% ethanol and then centrifuged at 3000  g for 20  min .
an aliquot ( 1  ml ) of a standard solution of rutin with different concentrations ( 0 , 10 , 20 , 30 , 40 , and 50  mg / l ) or supernatant was added to 10  ml volumetric flasks containing 4  ml of water . at the onset of each experiment
immediately , the solution was diluted with water to a final volume of 10  ml and thoroughly stirred .
the absorbance of the mixture was determined at 510  nm versus the prepared blanks .
total antioxidant activity ( taa ) was quantified according to the method of sayyari et al .   with modifications , which enables one to determine taa due to both hydrophilic and lipophilic compounds in the same extraction .
briefly , for each subsample , tissues ( 10  g ) from 10 fruits were homogenized in 10  ml of 50  mm phosphate buffer , ph 7.8 , and 5  ml of ethyl acetate and then centrifuged at 10000  g for 15  min at 4c .
the upper fraction was used for taa due to lipophilic compounds ( l - taa ) and the lower for taa due to hydrophilic compounds ( h - taa ) . in both cases
, taa was determined using the 2,2-diphenyl-1-(2,4,6-trinitrophenyl ) hydrazyl ( dpph ) radical scavenging activity and reducing power , respectively .
the reducing power of the fruit samples was determined using the method of jayaprakasha et al . .
a 0.2  ml aliquot of the supernatant was mixed with 2.5  ml of phosphate buffer ( 0.2  m , ph 6.6 ) and 2.5  ml of 1% potassium ferricyanide in 10  ml test tubes .
after incubation , 1  ml of 10% trichloroacetic acid was added to the mixtures , followed by centrifugation at 5000  g for 10  min .
the upper layer ( 2.5  ml ) was mixed with 2.5  ml of distilled water and 1  ml of 0.1% ferric chloride .
the increase in absorbance of the reaction mixture indicated the reducing power of the samples .
h - rp stands for hydrophilic compounds reducing power , and l - rp stands for lipophilic compounds reducing power .
dpph radical scavenging capacity was assayed as described by yang et al .   with slight modifications .
briefly , 0.2  ml of the supernatant was added to 3  ml of dpph ( 120 
shanghai , china ) was used , and the absorbance at 517  nm was after the reaction mixtures were incubated for 1  h at 30c in the dark .
the scavenging rate of dpph radicals was calculated as scavenging rate ( % ) = [ 1  ( a1  as)/a0 ]  100 , where a0 is the absorbance of the control solution ( 3  ml of phosphate - buffered saline in 3  ml of dpph solution ) , a1 is the absorbance of the supernatant in dpph solution , and as , which is used for error correction arising from unequal colour of the sample solutions , is the absorbance of the sample extract solution without dpph .
h - drsc represents the dpph radical scavenging capacity of hydrophilic compounds , and l - drsc represents the dpph radical scavenging capacity of lipophilic compounds .
each treatment was repeated three times , and the data were processed by analysis of variance using dps7.05 statistical software ( refine information tech .
the treatments were compared at p = 0.05 using tukey 's test , which indicates the multicomparison value in each case .
pearson correlations were used to determine the relationship among measured variables ( physical and physiological responses and antioxidant contents and activity ) .
the cultivar peach of yanhong belongs to melting - flesh fruit and its firmness decreased rapidly with fruit senescence . as shown in figure 1(a ) , the firmness of postharvest peach decreased quickly during the first 3 days of storage and then declined slowly from 3 to 6 days .
when peaches were fumigated with concentration of co ( 0.510  mol / l ) , the decrease of firmness became slow with the co concentration enhancement . whereas the peaches were treated with higher concentration co ( 20  mol / l ) ,
the firmness of peaches fumigated with 5 or 10  mol / l co was significantly higher than that of control samples .
particularly the peaches treated with 10  mol / l co demonstrated the slowest rate of firmness decline , and its firmness was 2.24 times of control fruit on day 6 .
as described in figure 1(b ) , peaches began to rot at day 2 , and its decay incidence increased rapidly after 3 days . on day 6 ,
fortunately , co fumigation might restrain the decay of postharvest peaches . during the whole storage time , the decay incidence of all treated samples was lower than that of control fruits .
of all these samples , peaches treated with 10  mol / l co showed the lowest increase of decay incidence , and there was significant difference compared to that of control samples ( p < 0.01 ) . at day 6 ,
the decay incidence of 10  mol / l co treated peaches was 40.50% , which was only 49.73% of control samples .
as shown in figure 2(a ) , the soluble solids content of postharvest peaches first gradually increased and then slowly decreased during the storage time . both control sample and 0.5 
mol / l co treated sample appeared to have the maximum value at day 3 , and 20  mol / l co treated peaches displayed the peak value at 2 day . while 5 or 10  mol / l co treated sample exhibited the maximum value on day 4 , postponing 1 day compared to that of control sample . from 4 to 6 days , the soluble solids content of 10  mol / l co treated fruits decreased the slowest . therefore ,
fumigating peaches with 10  mol / l co could effectively reduce the decrease of soluble solids content .
the titratable acid of postharvest peaches decreased during storage time ( figure 2(b ) ) .
/ l co treated peaches showed the fastest decrease followed by the control sample and 0.5  mol / l co treated sample . the titratable acid of 5 or 10  mol
/ l co treated sample decreased the least during storage time , which is higher than that of other samples from 3 to 6 days ( p < 0.05 ) . at day 6 ,
the titratable acid content of 10  mol / l co treated peaches was 72.8% higher than that of control samples .
the vitamin c of postharvest peaches showed increasing trend in the first two days and then decreased from 2 to 6 days ( figure 3(a ) ) .
the vitamin c content of 20  mol / l co treated peaches reached the peak value at day 1 and then decreased .
it was the lowest during the storage time compared to control and all other treated samples .
other samples appeared to have the maximum value of vitamin c at day 2 , and the vitamin c contents of treated samples with 0.510  mol / l co were higher than those of control samples .
furthermore , the peaches treated with 10  mol / l co showed the highest vitamin c content among all samples during the storage time , and significantly higher than that of control samples ( p < 0.01 ) .
as described in figure 3(b ) , the total flavonoid content of peaches first increased and then decreased during the whole storage time .
the peaches treated with 0.5 or 20  mol / l co and control sample appeared to have the maximum of total flavonoid content at day 2 , and there was no difference among them . 5 or 10 
mol / l co exhibited peak value of total flavonoid content at day 3 , but the total flavonoid content of peaches treated with 10  mol / l co was significantly higher than that of other samples from 4 to 6 days ( p < 0.05 ) .
similar to total flavonoid content , the total polyphenol content firstly increased and then decreased as well ( figure 3(c ) ) .
0.5 or 20  mol / l co treated peaches appeared to have the peak value of total flavonoid content on day 3 , but the total polyphenol content of peaches treated with 20  mol / l co was significantly lower than that of other samples .
the total polyphenol content of peaches treated with 5 or 10  mol / l co increased the slowest , and it reached the maximum at day 4 . obviously , treating peaches with 5 or 10  mol / l co might restrain the decrease of total polyphenol content of postharvest peaches .
as shown in figure 4 , the pal activity of postharvest peaches showed an obvious peak value during storage time .
the sample treated with 0.5 or 20  mol / l co and control sample appeared to have maximum of pal activity at day 3 , but the rising and falling speed of pal activity of peaches treated with 0.5 or 20  mol / l co was lower than that of control sample .
peaches treated with 5 and 10  mol / l co demonstrated peak value of pal activity at days 4 and 5 , and they were postponed at 1 and 2 days , respectively . apparently , peaches treated with 10  mol / l co maintained higher pal activity during the last storage time .
the h - rp and l - rp of postharvest peaches all firstly promptly decreased and then gradually increased during the storage time , but there were some differences between them ( figures 5(a ) and 5(b ) ) . at 0 day , the h - rp was slightly lower than l - rp .
however , it was significantly higher than l - rp during the storage time and was about 2.85.6 times compared to l - rp .
moreover , at the end of storage , the h - rp still maintained high level , while the l - rp decreased less 1/6 than that of original level .
compared to that of control sample , the h - rp or l - rp in all co treated samples was higher .
obviously , exogenous co fumigation was beneficial to maintaining the reducing power of postharvest peach , and 10  mol / l co treatment demonstrated the best effect . as shown in figure 5(c ) , the h - drsc of postharvest peach began to increase at day 2 and slightly decreased at day 6 .
all co treated samples showed significantly higher h - drsc between 2 and 6 days compared to that of control sample ( p < 0.05 ) .
the l - drsc of postharvest peach firstly decreased , then increased , and decreased again during the whole storage period ( figure 5(d ) ) .
after 2 days , the l - drsc of control sample was lower than that of co treated samples . at day 6
, 10  mol / l co treated sample showed the highest dpph radical scavenging activity , which was about 1.6 times of control samples .
compared with h - drsc of postharvest peach , the l - drsc was markedly lower , yet it could be significantly improved after the peaches were fumigated with co.
peach , a respiratory climacteric fruit , is easy to soften and rot under ambient temperature after harvest . therefore , firmness and decay incidence were important indexes of postharvest peaches .
the results showed that the firmness of postharvest peaches rapidly decreased under ambient temperature , from 14.18  kg / cm at day 0 to 2.74  kg / cm at day 3 .
so the peach used in our experiment belonged to the typical cultivar of melting - flesh type . after exogenous co fumigation ,
the firmness decrease and decay incidence increase of postharvest peaches were restrained during storage time .
furthermore , in low concentration ( 0.510  mol / l ) , the firmness of co treated fruit decreased slowly and decay incidence increased slowly as well with co concentration enhancement .
mol / l , the firmness of postharvest quickly decreased and accordingly the decay incidence fast increased .
as shown in table 1 , there were significant negative correlations between firmness variation and decay incidence of postharvest peaches during storage time .
the reason was probably that gradual fruit senescence led to the decomposition of intercellular pectin of flesh and cell separations with storage time extension .
thus , peaches softening occurred and the integrity of fruit cell wall structure was destroyed .
afterward , fruit physiological metabolism was disordered and the resistance to exogenous pathogen was gradually lost . as a result ,
postharvest peach eventually appeared to have some physiological changes owing to respiratory metabolism and ethylene production during senescence process .
these changes included flesh softening , starch degradation , flavor variation , and organic acid content decrease .
soluble solids and titratable acid content represented the quality of postharvest peaches , and their ratio was also an important index to reflect the maturity and senescence of postharvest fruit .
the soluble solids content of cultivar peach  yanhong  firstly increased and then decreased during storage period .
the amylase of postharvest peaches catalyzed the starch into soluble sugar and soluble solids content increased accordingly .
after storage for some time , the starch became less and the sugar production gradually lessened .
meanwhile , the physiological metabolism of postharvest peaches continued to consume sugar . as a result , the soluble solids content decreased with storage time extension .
0.510  mol / l exogenous co might inhibit the decrease of titratable acid content , postpone the variation of soluble solids and vitamin c content , and effectively maintain the qualities of postharvest peaches .
peach has good flavor and abundant nutrients , preventing many diseases of mankind induced owing to the accumulation of reactive oxygen compounds in organism [ 30 , 31 ] .
flavonoid and polyphenol , as main antioxidant substance originating from plant , are important functional phytochemicals of peach fruit .
the experimental result showed that co treating could postpone the flavonoid and polyphenol variation of postharvest peach and maintain them in higher level .
the correlation coefficients of pal activity relating with total flavonoid , total polyphenol , and total flavonoid + total polyphenol , were 0.48 , 0.72 , and 0.64 , respectively .
it catalyzes the deamination of l - phenylalanine to yield ammonia and transcinnamic acid , from which phenolic compounds including flavonoid , phenolic acid , and anthocyanin , are produced .
therefore , co probably increased the flavonoid and polyphenol content through heightening pal activity , maintaining the physiological function of postharvest peach .
these have hydrogen with activity that causes the hydrogen exchange reaction with free radical and structure of resonance stabilized .
research suggested that the antioxidant activity of postharvest peaches closely related with flavonoid and polyphenol content .
as shown in table 1 , the flavonoid content of postharvest peaches significantly correlated h - drsc ( r = 0.89 ) and moderately correlated t - drsc ( r = 0.70 ) .
polyphenol content closely correlated h - drsc , l - drsc , and t - drsc , and these correlation coefficients were 0.72 , 0.73 , and 0.81 , respectively .
in addition , the content of total flavonoid + total polyphenol also significantly positively correlated with h - drsc and t - drsc ( r = 0.87 and r = 0.81 ) .
obviously , co treating could increase the flavonoid and polyphenol content of postharvest peach , and accordingly the antioxidant activities of flesh were enhanced in terms of dpph radical scavenging capacity .
the experimental result also exhibited that the content of total flavonoid and polyphenol slightly correlated with reducing power including h - rp and l - rp ( |r | < 0.5 ) .
this probably related with the specific category and property of flavonoid and polyphenol contained in particular peach . 

table 1 suggested that the decay incidence of postharvest peaches had few correlation with total flavonoid content , total polyphenol content , and drsc , but it had significantly negative correlation with vitamin c content ( r = 0.73 ) and positive correlation with h - rp ( r = 0.85 ) .
though vitamin c contributed little to antioxidant activities in terms of dpph radical scavenging ability and reducing power , it was related with the enhancement to resist decay incidence . therefore , exogenous co might restrain the decay incidence through maintaining vitamin c content .
correlation analysis suggested that firmness was moderately negative correlation with total flavonoid , total polyphenol , and total flavonoid + total polyphenol content , and their correlation coefficients were 0.53 , 0.67 , and 0.64 , respectively ; it was high negative correlation with h - drsc , l - drsc , and t - drsc , and the correlation coefficients were 0.53 , 0.67 , and 0.64 , respectively .
therefore , co might maintain the firmness and postpone the shelf life through inducing pal activity of flesh .
this result is similar to zhang and li who treated jujube with exogenous co . with research depth
, people found that plant could generate co. heme oxygenase oxidation , lipid peroxidation , and ureide metabolism were probably the potential sources of plant co .
similar to no and h2s , co takes part in various abiotic stresses and physiological processes such as stomatal movement and lateral root . in plant postharvest physiology ,
co was used to protect the green of vegetable and inhibit the browning of lotus root slices .
our results exhibited that exogenous co might postpone the softening and decay incidence of postharvest peaches , maintain the nutrients and quality , and prolong the shelf life through increasing functional nutrients such as flavonoid , polyphenol , and vitamin c. without doubt , the exact mechanism should be further investigated in future . in general ,
oxidative regulation is a dynamic balancing process between systems that produce and scavenge ros ; the initial tissue response to stress leads to ros production ( step i ) , and this in turn , triggers antioxidant protection systems to ameliorate ros ( step ii ) but in case the tissue can no longer cope with stress then subcellular or cellular damage occurs ( step iii ) .
protection systems mainly include phenolics , ascorbate , glutathione , peroxidase , ascorbate peroxidase , glutathione peroxidase and reductase , and catalase .
in particular , there are many mechanisms by which phenolics can act either as antioxidants ( as agents for free radical scavenging , hydrogen donation , singlet oxygen quenching , and metal ion chelation ) or as substrates for attack by superoxide .
thus , we presumed that co probably indirectly adjusted the oxidative stress of tissue producing during fruit ripening and senescence process through regulating the flesh polyphenol content , thereby delaying fruit ripening and senescence .
the antioxidant defense system induced by co comprise of ascorbate peroxidase , glutathione reductase , superoxide dismutase , monodehydroascorbate reductase , ascorbic acid reductase , and so on , regulating reactive oxygen species and restraining oxidative stress through the transcription and expression of these enzymes . in our previous study ,
a similar result about the effect of co fumigation on the active oxygen metabolism of jujube was acquired ( unpublished ) .
therefore , co also probably maintained peach quality through regulating active oxygen [ 41 , 42 ] .
as a signal molecule , co of plant similar to no might play a physiological role through cgmp pathway .
xuan et al . found that the growth elongation of wheat root induced by co associated with cgmp signal pathway .
many evidences suggest that co controls the response process to abiotic stresses via molecular interaction with other signal moleculars , such as no .
currently , the research about co in plant postharvest physiology was limited . since no as a useful gas to preserve fruit had been widely recognized , whether it participates in co regulating plant senescence process and whether co plays a role on plant ripening and senescence via the cgmp pathway need to be further investigated in the future .
treating postharvest peaches with co demonstrated dual effect . in low concentration co ( 0.510  mol / l ) , exogenous co could delay the decrease of firmness and titrable acid content , restrain the increase of decay incidence , and postpone the variation of soluble solids content , and the positive effect was more obvious with co concentration enhancement .
however , treating peaches with high concentration of co ( 20  mol / l ) demonstrated adverse effect .
further researches exhibited that exogenous co might induce the pal activity , maintain nutrient contents such as vitamin c , flavonoid , and polyphenol , and enhance antioxidant activity according to reducing power and dpph scavenging ability .
this result suggested that treating peaches with appropriate concentration of co was beneficial to quality , nutrients , and antioxidant activity of postharvest peaches during storage period .
 peaches ( prunus persica cv . 
 yanhong ) were fumigated with carbon monoxide ( co ) at 0 , 0.5 , 5 , 10 , and 20  mol / l for 2 hours . 
 the result showed that low concentration co ( 0.510  
 mol / l ) might delay the decrease of firmness and titrable acid content , restrain the increase of decay incidence , and postpone the variation of soluble solids content , but treating peaches with high concentration co ( 20  mol / l ) demonstrated adverse effects . 
 further research exhibited that exogenous co could induce the phenylalnine ammonialyase activity , maintain nutrient contents such as vitamin c , total flavonoid , and polyphenol , and enhance antioxidant activity according to reducing power and 2,2-diphenyl-1-(2,4,6-trinitrophenyl ) hydrazyl radical scavenging activity . treating peaches with 
 appropriate concentration co was beneficial to the quality , nutrients , and antioxidant activity of postharvest peaches during storage time . 
 therefore , co fumigation might probably become a novel method to preserve postharvest peach and other fruits in the future . 
1. Introduction
2. Materials and Methods
3. Results and Analysis
4. Discussion
5. Conclusions