Effects of 1-methylcyclopropene and modified atmosphere packaging on fruit quality and superficial scald in Yali pears during storage

Journal of Integrative Agriculture 2018, 17(7): 1667–1675 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

Effects of 1-methylcyclopropene and modified atmosphere packaging on fruit quality and superficial scald in Yali pears during storage FENG Yun-xiao1, 2, CHENG Yu-dou1, 2, HE Jin-gang1, 2, LI Li-mei1, 2, GUAN Jun-feng1, 2 1 2

Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051, P.R.China Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, P.R.China

Abstract The Yali pear (Pyrus bretschneideri Rehd.) is susceptible to superficial scald during prolonged cold storage and at shelf life. This study investigated the effects of 1-methylcyclopropene (1-MCP) and modified atmosphere packaging (MAP) on changes of fruit quality and superficial scald during cold storage and at shelf life in Yali pear. Compared with MAP, the combination of MAP and 1-MCP (MAP+1-MCP) treatment reduced the carbon dioxide and ethylene content inside the packaging bag. The 1-MCP, MAP, and MAP+1-MCP treatments reduced the superficial scald index, malondialdehyde content, O2–· production rate and relative conductivity and inhibited the accumulation of α-farnesene and conjugated trienes in the peel. 1-MCP and MAP+1-MCP treatments maintained a higher phenolic content and enhanced the catalase and superoxide dismutase activities in the fruit, while reduced activities of lipoxygenase and polyphenol oxidase in the peel preceding the onset of superficial scald. Comprehensive analysis indicated that the MAP+1-MCP treatment is the most effective method tested for improving the quality of Yali pears during cold storage and at shelf life. Keywords: pear, 1-methycyclopropene, modified atmosphere packaging, superficial scald, browning

superficial scald in pears is similar to that in apples. Studies

1. Introduction The Yali pear (Pyrus bretschneideri Rehd. cv. Yali) is a famous Chinese pear variety with delicious and juicy flesh. However, it is predisposed to browning scald in the peel during prolonged cold storage. This phenomenon is commonly known as superficial scald. Pathogenesis of

have shown that superficial scald is closely associated with the accumulation of α-farnesene and conjugated trienes (CTols) in the peel as well as the reduction of antioxidative enzyme activity and an increase of reactive oxygen in fruits (Ahn et al. 2001; Rupasinghe et al. 2001; Whitaker 2007; Sabban-Amin et al. 2011; Bordonaba et al. 2013; Gao et al. 2015). Many postharvest methods have been established to inhibit the development of superficial scald; however, the mechanism through which it occurs is not completely understood (Lurie and Watkins 2012). In recent years, increasing applications of

Received 27 October, 2017 Accepted 1 February, 2018 FENG Yun-xiao, E-mail: [email protected]; Correspondence GUAN Jun-feng, Tel: +86-311-87652118, Fax: +86-311-87652119, E-mail: [email protected]

1-methycyclopropene (1-MCP) in the preservation of

© 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: 10.1016/S2095-3119(18)61940-9

and inhibits α-farnesene biosynthesis and the subsequent

freshness for harvested fruits have been reported. Studies have shown that 1-MCP effectively delays fruit senescence accumulation of its oxidation products (CTols), reducing

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the incidence of superficial scald in pears (Ekman et al. 2004; Isidoro and Almeida 2006; Xie et al. 2014). Modified atmosphere packaging (MAP) can keep fruit fresh, suppress respiration and delay the aging process (Li et al. 2013; Wang and Sugar 2013; Cheng et al. 2015). In addition, the combination of 1-MCP and MAP treatment has been shown to be more effective in the preservation of some fruits compared to either treatment alone (Reuck et al. 2009; Sabir and Agar 2011; Li et al. 2013). However, to date, no report has shown the effects of combined MAP and 1-MCP treatment on the storage quality of Yali pears. The objective of this study was to investigate the effects of 1-MCP, MAP, and their combined treatment (MAP+1-MCP) on fruit quality as well as superficial scald during cold storage and at shelf life, especially with respect to changes in superficial scaldrelated physiological and biochemical indices in Yali pears.

2. Materials and methods 2.1. Materials Yali pears (Pyrus bretschneideri Rehd. cv. Yali) were harvested at 163 days after full bloom from an orchard in Zhao County, Hebei Province, China, and delivered to the laboratory within 2 h. Pears (average single-fruit weight: (280.49±8.06) g; soluble solids content: (12.47±0.80)%) of similar size and no signs of pest damage or disease were selected and evenly divided into two groups. The first group was sealed with 1.0 μL L–1 1-MCP (SmartFreshTM Agro Fresh Inc., Dow Agro Sciences, Philadelphia, PA, USA) for 24 h at room temperature ((20±2)°C). The second group was placed in sealed containers with air at the same temperature for 24 h. Afterwards, each of the two groups was further divided into two subgroups. The first subgroup was directly put into an ordinary paper box, and the second subgroup was first packed with microporous plastic film (with O2 and CO2 permeabilities of 10 955 and 75 200 mL m–2 d 24 h–1 0.1 Mpa–1 at 20°C, respectively, made by the National Engineering Technology Research Center for Preservation of Agricultural Products, Tianjin, China) and subsequently placed into a paper box. The treated subgroups were labeled control (no 1-MCP treatment or packaging), 1-MCP (1-MCP treatment alone), MAP (microporous plastic film packaging alone), and MAP+1-MCP (MAP combined with 1-MCP treatment). Each box contained 30 pears and was stored at 0°C. All fruits were removed after 210 days of cold storage, and packaging bags were unwrapped for MAP treatments and then stored at (20±2)°C for seven days over the shelf life period. The fruit quality indices were tested on days 0, 60, 120, 180, and 210 during cold storage and at shelf life. The peel samples were immediately frozen in liquid nitrogen and subsequently

stored at –80°C for further experiments. Three replicates of 10 fruit each were measured for each treatment at every sampling time.

2.2. Analysis the O2, CO2 and ethylene contents inside the packaging films The O 2 and CO 2 contents inside the packaging films were measured with a handheld O2/CO2 analyzer (Model: OXYBABY M+ O2/CO2, WITT-Gasetechnik GmbH & Co KG, Witten, Germany). For ethylene measurement, 1 mL of gas inside the packaging film was withdrawn with a gas-tight syringe, which was then injected into a gas chromatograph (Model: GC9790II, Fuli Instruments Technology Co., Ltd., Wenling, China) equipped with a GDX-502 column and a flame ionization detector (FID). The O2 and CO2 contents were expressed as %, while the ethylene content was expressed as μL L–1. Three replicates were measured for each treatment.

2.3. Fruit quality evaluation Fruit firmness was determined using a digital fruit hardness meter (Model: GY-4, Top Instruments Co., Ltd., Hangzhou, China). The soluble solids content (SSC) was determined with a PAL-1 pocket digital refractometer (Atago Co., Ltd., Tokyo, Japan).

2.4. Determination of the superficial scald index and the contents of α-farnesene and CTols The severity of superficial scald was evaluated according to an index based on the percentage of the fruit surface area affected: 0, 1, 2, and 3 suggested no superficial scald and superficial scald covering <25%, 25–50%, and >50%, respectively (Zanella 2003), with the superficial scald index calculated using the following formula: Superficial scald Index=∑(Scald level×Number of fruits at the level)/(3×Total number of fruits) The α-farnesene and CTols contents were analyzed by the hexane-extraction method (Xie et al. 2014; Gao et al. 2015). Four discs (1 cm in diameter) of peel tissue were removed from the equatorial portion of each pear and were placed in tubes containing 20 mL of hexane for 2 h. After incubation, 2 mL of the extract was filtered through a Florisil column and eluted using 3 mL of hexane. The absorbances at 232, 281 and 290 nm were recorded using a spectrophotometer (Model: UV-2100, UNICO Instrument, Dayton, USA). The α-farnesene and conjugated trienols were calculated using the molar extinction coefficients ε232=27 740 for α-farnesene and ε 281–290=25 000 for the conjugated trienols, expressed as nmol cm–2.

FENG Yun-xiao et al. Journal of Integrative Agriculture 2018, 17(7): 1667–1675

2.5. Determination of the relative conductivity, O2–· production rate and the malondialdehyde (MDA) and phenolic compound contents To assess relative conductivity, fifteen peel discs (1 cm in diameter) were immersed in 20 mL of mannitol (100 mmol L–1) and gently shaken at 20°C for 2 h (Zhao et al. 2009). The conductivity of the solution (L1) was measured with a conductivity meter (Model: DDS-307, INESA Auto Electronics System Co., Ltd., Shanghai, China). Solutions were boiled for 10 min and then cooled to 20°C, and the conductivity of dead tissues (L2) was measured. Relative conductivity was calculated as the ratio of L1 to L2. To measure the rate of O2–· production, 0.5 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended in 5 mL of 50 mmol L–1 phosphate buffer (pH 7.8), containing 1 mmol L–1 EDTA, 0.3% (v/v) Triton X-100, and 2% (w/v) polyvinylpyrrolidone, and then the extract was centrifuged at 12 000×g for 20 min at 4°C. After centrifuging, 1 mL of the supernatant was blended thoroughly with 1 mL of 50 mmol L–1 phosphate buffer (pH 7.8) and 1 mL of 1 mol L–1 hydroxylamine hydrochloride and then was incubated at 4°C for 1 h. After incubating, the extract was added to 1 mL of 17 mmol L–1 p-aminobenzene sulfonic acid and 1 mL of 7 mmol L–1 naphthylamine, after which it was incubated at 25°C for 20 min (Wang and Luo 1990). The absorbance at 530 nm was recorded using a spectrophotometer (Model: UV-2100, UNICO Instrument, Dayton, USA). A standard curve for KNO2 was used to quantify the O2–· content, and the production ratio of O2–· was represented as µmol min–1 g–1 frozen weight (FW). To measure the MDA content, 2 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended with 10 mL of 10% (w/v) trichloroacetic acid, which was then centrifuged at 12 000×g for 15 min at 4°C. After centrifuging, 2 mL of the supernatant was added to 3 mL of 10% (w/v) trichloroacetic acid aqueous containing 0.67% (w/v) thiobarbituric acid. The mixture was boiled at 100°C for 15 min. After rapid cooling, the mixture was centrifuged at 5 000×g for 10 min at 4°C. The absorbance of supernatant was measured at 450, 532, and 600 nm. The MDA content was calculated as 6.45×(OD532–OD600)–0.56×OD450 (Xu et al. 2012) and was represented as µmol g –1 FW. To measure the total phenolic content, 0.5 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended in 5 mL 70% ethanol (v/v) and extracted with an ultrasonic instrument at 40°C for 1 h. Next, the mixture was centrifuged at 10 000×g for 15 min at 4°C, and 0.5 mL of the supernatant was withdrawn and diluted eight fold. Afterwards, 0.1% (w/v) Fast Blue RR salt (4-benzoylamino2,5-dimethoxybenzenediazonium chloride hemi-[zinc chloride] salt) and 5% (w/v) NaOH were added to the

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diluted supernatant (4 mL) at a ratio of 1:10, after which the mixture was incubated at 25°C for 90 min. The absorbance of reaction was determined at 420 nm (Lester et al. 2012). A standard curve for gallic acid was used to quantify the total phenolic content, and the results were expressed as mg g–1 FW.

2.6. Measurement of enzyme activity To extract and determine the activity of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), 2 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended in 5 mL of extract solution containing 100 mmol L–1 phosphate buffer (pH 7.8), 2 mmol L–1 DTT, 5% (w/v) polyvinylpolypyrrolidone (PVPP), 0.1 mmol L–1 EDTA and 1.25 mmol L–1 polyethylene glycol, after which the extraction was centrifuged at 12 000×g for 30 min at 4°C. The supernatant was collected as the crude enzyme extract to measure SOD and CAT activity (Gao et al. 2015). For the SOD activity assay, a reaction mixture containing 1.5 mL of 50 mmol L–1 phosphate buffer (pH 7.0), 0.3 mL of 130 μmol L–1 L-methionine, 0.3 mL of 750 μmol L–1 nitroblue tetrazolium (NBT), 0.3 mL of 100 μmol L–1 EDTA-Na2, 0.3 mL of 20 μmol L–1 riboflavin, 200 μL of deionized H2O, and 100 μL of the crude enzyme extract was initiated by illumination with 60 mol m–2 s–1 white fluorescent light at 25°C. After 8 min, the reaction was stopped by removing the light source. A control tube with the reaction mixture and enzyme was maintained in the dark, while another tube without the enzyme was maintained in light conditions to serve as the control for the reduction of NBT by light. The absorbance of the reaction was read at 560 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited the photoreduction of NBT by 50% under the assay conditions and was represented as U g–1 FW. For the CAT activity assay, a 3-mL reaction mixture containing 50 mmol L–1 phosphate buffer (pH 7.0) and 100 μL of the crude enzyme extract was initiated by the addition of 100 μL of 100 mmol L–1 H2O2. The decomposition was directly assessed by observing the decrease in absorbance at 240 nm every 30 s for 2 min at 25°C. The CAT activity was defined as the amount of enzyme that reduced the absorbance and was represented as ΔOD240 min–1 g–1 FW. To extract and measure lipoxygenase (LOX, EC 1.13.11.12) activity, 1 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended in 10 mL of 100 mmol L–1 phosphate buffer (pH 6.8) containing 1.5% polyvinylpyrrolidone (PVP). Next, the mixture was centrifuged at 12 000×g for 20 min at 4°C, after which the supernatant was collected as the crude enzyme extract. The 3 mL reaction mixture contained 25 µL of 10 mmol L–1 sodium linoleic and 2.775 mL of 100 mmol L–1 sodium

FENG Yun-xiao et al. Journal of Integrative Agriculture 2018, 17(7): 1667–1675

2.7. Statistical analysis The results were statistically analyzed using SPSS 18 (SPSS Inc., Chicago, IL, USA). Least significant differences (LSD) at a significance level of 0.05 were generated by ANOVA.

3. Results 3.1. Change in gas composition within the fruit packaging The gas composition within the packaging bags varied dynamically during cold storage. There was no significant difference in the oxygen content inside the packaging bags between the MAP and MAP+1-MCP treatments except for day 60 (Fig. 1-A). However, compared with MAP alone, the carbon dioxide content at days 60 and 120, and the ethylene content during whole storage period for the MAP+1-MCP treatment were lower (Fig. 1-B and C). This observation suggested that 1-MCP reduced the contents of carbon dioxide and ethylene within the packaging bag and more significantly inhibited the accumulation of ethylene.

3.2. Change in fruit quality The firmness declined gradually during storage, which was significantly higher in different treatments than that in control at later storage time (Fig. 2-A). The SSC in the MAP treatment was lower than in the control after day 60, while it had no significant difference between the 1-MCP treatment and the control. Moreover, the SSC in the MAP+1-MCP

MAP

A O2 content (%)

25 22

B

LSD0.05=1.15

16 13 0

60

120 180 Storage time (d)

210

120 180 Storage time (d)

210

120 180 Storage time (d)

210

1.8 1.5

LSD0.05=0.13

1.2 0.9 0.6 0.3 0

C

MAP+1-MCP

19

10

CO2 content (%)

acetate buffer (pH 4.4) and was initiated by the addition of 200 µL of the crude enzyme extract. The decomposition was directly assessed by observing the increase in absorbance at 234 nm for 2 min at 25°C (Axelrod et al. 1981). The LOX activity was defined as the amount of enzyme that raised the absorbance in 1 min and was expressed as ΔOD234 min–1 g–1 FW. For polyphenol oxidase (PPO, EC 1.14.18.1) extraction and activity measurements, 0.5 g of frozen peel tissue was powdered in liquid nitrogen with a mortar and blended in 5 mL of 100 mmol L–1 phosphate buffer (pH 7.0) containing 0.3 g PVP. Next, the solution was centrifuged at 20 000×g for 15 min at 4°C, after which the supernatant was collected as the crude enzyme extract. The reaction mixture contained 100 mmol L–1 catechol in 50 mmol L–1 phosphate buffer (pH 6.0) (Cheng et al. 2015). Changes in the absorbance at 420 nm were measured by using a spectrophotometer (Model: UV-2100, UNICO Instrument, Dayton, USA). The PPO activity was expressed as ΔOD420 min–1 g–1 FW.

300

C2H4 content (μL L–1)

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250

0

60

LSD0.05=11.39

200 150 100 50 0

0

60

Fig. 1 Oxygen (A), carbon dioxide (B), and ethylene (C) contents inside the packaging films. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. LSD, least significant differences. Error bars represent ±SE from three replicates.

treatment was lower than in the control on day 210 and at shelf life (Fig. 2-B).

3.3. Changes of the superficial scald index, the contents of α-farnesene and CTols in peels Superficial scald was first observed at day 210 of cold storage and was more obvious over the shelf life period in Yali pear. Compared with the control, 1-MCP, MAP and MAP+1-MCP significantly reduced the superficial scald index, while the MAP+1-MCP treatment showed the lowest index (Fig. 3-A). The α-farnesene content first increased to maximum value at day 180 and subsequently decreased, and the values were significantly lowered by the 1-MCP and MAP+1-MCP treatments (Fig. 3-B). The CTols content increased to maximum value at day 210 during cold storage and decreased over the shelf life period. Compared with the control, different treatments significantly reduced the CTols content of the peel, with the MAP+1-MCP treatment

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90

1-MCP

MAP+1-MCP

LSD0.05=3.3

75 Firmness (N)

MAP

60 45 30 15 0

0

60

120

180

210

210+7

A Superficial scald index

CK A

CK 0.5 0.4

15

B LSD0.05=0.71

α-Farnesene content (nmol cm–2)

Soluble solids content (%)

18 12 9 6 3 0

0

60

120 180 Storage time (d)

210

Fig. 2 Changes in fruit firmness (A) and soluble solids content (SSC, B) in Yali pears during storage. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. 210+7 indicates 7 days at shelf life after 210 days of cold storage. Error bars represent ±SE from three replicates.

showing the lowest CTols content (Fig. 3-C). –

3.4. Changes in the rate of O2 · production and the activities of CAT and SOD in peels The rate of O2–· production in the peel was increased with prolonged storage, which was significantly lower in the MAP, 1-MCP, and MAP+1-MCP treatments after day 180 compared with the control (Fig. 4-A). The CAT activity first declined and subsequently increased in all treatments, showing significantly higher CAT activities in the 1-MCP and MAP+1-MCP treatments than in control after day 120 (Fig. 4-B). Compared with control, the MAP, 1-MCP and MAP+1-MCP treatments had significantly higher SOD activities after day 180 (Fig. 4-C).

3.5. Changes in LOX activity, MDA content, and relative conductivity of the peels In all treatments, the LOX activity first increased, reaching maximal activity at day 180, and then subsequently declining, showing significantly lower in the 1-MCP and MAP+1-MCP treatments than that in control after 180 days of cold storage, while no significant difference was observed between the MAP and control (Fig. 5-A). The

Conjugated trienes content (nmol cm–2)

C

MAP+1-MCP

LSD0.05=0.014

0.2 0.1 0

0

60

120 180 210 Storage time (d)

210+7

120 100

LSD0.05=3.43

80 60 40 20 0

210+7

1-MCP

0.3

Storage time (d) B

MAP

0

60

120

180

210

210+7

210

210+7

Storage time (d) 12 10

LSD0.05=0.55

8 6 4 2 0

0

60

120 180 Storage time (d)

Fig. 3 Changes in the superficial scald index (A), α-farnesene (B) and conjugated trienes (CTols, C) contents of Yali pear peels during storage. 210+7 indicates 7 days at shelf life after 210 days of cold storage. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. LSD, least significant differences. Error bars represent ±SE from three replicates.

MDA content increased during storage. Compared with the control, all treatments significantly inhibited the increase in MDA content in the peel after 120 days of cold storage, and the MAP+1-MCP treatment had the lowest MDA content at shelf life (Fig. 5-B). The relative conductivity also increased during storage and was significantly lower in the 1-MCP, MAP and MAP+1-MCP treatments than that in control after day 210 (Fig. 5-C).

3.6. Changes of total phenolic content and PPO activity of the peels Total phenolic content decreased during storage, which was significantly higher in the MAP+1-MCP treatment than that in the control after 120 days of cold storage. However,

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CK

SOD activity (U g FW)

MAP+1-MCP

LSD0.05=0.56

4.5 3.0 1.5 0

0

60

120 180 Storage time (d)

210

210+7 B

5 LSD0.05=0.41

MDA content (μmol g–1 FW)

4 3 2 1 0

0

10

60

120 180 Storage time (d)

210

C

6 4 2 0

0

60

120 180 Storage time (d)

5

210

210+7

–

Fig. 4 Changes in the production rate of O2 · (A), activities of catalase (CAT, B) and superoxide dismutase (SOD, C) in pear peels during storage. 210+7 indicates 7 days at shelf life after 210 days of cold storage. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. LSD, least significant differences. Error bars represent ±SE from three replicates.

no significant difference was observed between the MAP treatment and the control (Fig. 6-A). The change in the PPO activity was rather low during cold storage but significantly increased at the shelf life. Compared with the control, the 1-MCP and MAP+1-MCP treatments exhibited significantly lower PPO activities on day 180 of cold storage and at the shelf life, but MAP had no significant effect on PPO activity (Fig. 6-B).

3.7. Correlation between the superficial scald index and physiological and biochemical indices of the peels of Yali pears Correlation analysis of different indices was conducted during fruit storage. By linear stepwise regression, we concluded that the 5 indices (PPO activity, CAT activity,

4

MAP

1-MCP

MAP+1-MCP

LSD0.05=0.50

3 2 1 0

0

60

120 180 Storage time (d)

210

210+7

120 180 Storage time (d)

210

210+7

210

210+7

1.5 1.2

LSD0.05=0.12

0.9 0.6 0.3 0

210+7

LSD0.05=0.85

8

CK

A LOX activity (ΔOD234 min–1 g–1 FW)

6.0

CAT activity (ΔOD240 min–1 g–1 FW) –1

1-MCP

7.5

B

C

MAP

Relative conductivity (%)

Production rate of O2–· (μmol g–1 min–1 FW)

A

0

60

48 LSD0.05=2.55

40 32 24 16 8 0

0

60

120 180 Storage time (d)

Fig. 5 Changes in lipoxygenase (LOX) activity (A), malondialdehyde (MDA) content (B), and relative conductivity (C) in Yali pear peels during storage. 210+7 indicates 7 days at shelf life after 210 days of cold storage. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. LSD, least significant differences. Error bars represent ±SE from three replicates.

relative conductivity, α-farnesene content, and MDA content) are closely related to the superficial scald index, and the scald index can be well predicted through the following regression equation: Scald index=–0.360+0.749×PPO activity+0.228×CAT activity+0.190×Relative conductivity–0.148×α-farnesene content+0.161×MDA content, r=0.9607 (P<0.01) Furthermore, path analysis showed that the direct path coefficient of PPO to the scald index was the largest (0.749). Thus, PPO was an important direct determinant, and the direct path coefficient of the α-farnesene content to the scald index was negative (–0.148), showing that the substance has a slightly negative correlation with the scald index. The indirect path coefficient showed that PPO activity, MDA content and relative conductivity were important indices

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for influencing the scald index (Table 1). This suggested that the PPO activity, MDA content, and cell membrane permeability were closely correlated with the superficial scald index for Yali pears.

4. Discussion In this study, the MAP and MAP+1-MCP treatments maintained higher firmness (Fig. 2-A), which was similar to a previous study of the Laiyang pear (Li et al. 2013). However, the MAP, 1-MCP, and MAP+1-MCP treatments had a relatively lower effect on the SSC in Yali pear (Fig. 2-B), and the 1-MCP treatment had inconsistent effects on SSC in apples, peaches, plums, and other fruits (Watkins 2006).

Total phenolic content (mg g–1 FW)

A

PPO activity ( ΔOD420 min–1 g–1 FW)

B

CK

MAP

1-MCP

MAP+1-MCP

7.5 LSD0.05=0.46

6.0 4.5 3.0 1.5 0

0

60

120 180 210 Storage time (d)

210+7

18 LSD0.05=0.77

15 12 9 6 3 0

0

60

120 180 210 Storage time (d)

210+7

Fig. 6 Changes in total phenolic content (A) and polyphenol oxidase (PPO) activity (B) in Yali pear peels during storage. 210+7 indicates 7 days at shelf life after 210 days of cold storage. 1-MCP, 1-methylcyclopropene; MAP, modified atmosphere packaging. LSD, least significant differences. Error bars represent ±SE from three replicates.

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The 1-MCP treatment reduced the ethylene content in the packaging to a much lower level than that in the MAP alone (Fig. 1), which may be due to 1-MCP inhibiting the ethylene production of the pears. It is generally believed that the accumulation of α-farnesene, and more significantly, CTols, is important for the induction of superficial scald in apples and pears. Some studies have shown that 1-MCP significantly reduced the concentrations of α-farnesene and CTols in the peels of pear (Ekman et al. 2004; Gapper et al. 2006; Isidoro and Almeida 2006; Xie et al. 2014; Gao et al. 2015). In this study, the 1-MCP and MAP+1-MCP treatments reduced the contents of α-farnesene and CTols (Fig. 3-B and C) and lowered the superficial scald index (Fig. 3-A). These results further indicated that the accumulation of α-farnesene and CTols in the peel had a positive effect on the onset of superficial scald. However, the α-farnesene and CTols contents in the peel were reduced at shelf life (Fig. 3-B and C), this might be related to the metabolic exhaustion of α-farnesene and CTols during the development of superficial scald. Therefore, the promoting roles of α-farnesene and CTols on scald development were weakened at the shelf life period in Yali pear. In addition, based on path analysis results (Table 1), α-farnesene content was shown to have a slightly negative correlation with the scald index. It is thought that O2–· together with H2O2 could form the radical hydroxyl ·OH, after which the ·OH might cause lipid peroxidation and result in damage to the cell membrane and physiological disorders, which could induce the onset of superficial scald (Lu et al. 2011; Sabban-Amin et al. 2011; Tian et al. 2013). SOD and CAT could enhance the resistance to various abiotic stresses by scavenging the reactive oxygen species (ROS) in cells (Gao et al. 2015). In this work, lower production of O2–· and higher activities of CAT and SOD were observed in the 1-MCP and 1-MCP+MAP treatments (Fig. 4), in agreement with previous studies in the Blanquilla and Wujiuxiang pears and in Granny Smith apples (Larrigaudière et al. 2004; Sabban-Amin et al. 2011; Gao et al. 2015). These results suggested that the 1-MCP and 1-MCP+MAP treatment could reduce ROS accumulation by improving the activities of SOD and CAT,

Table 1 Path coefficient of the scald index and related indices Independent variable1) X1 (PPO) X2 (CAT) X3 (relative conductivity) X4 (α-farnesene) X5 (MDA) 1)

Simple correlation coefficient (r) with y (superficial scald index) 0.907 0.083 0.610 0.017 0.732

Direct path coefficient (P) 0.749 0.228 0.190 –0.148 0.161

PPO, polyphenol oxidase; CAT, catalase; MDA, malondialdehyde.

X1 (PPO) –0.137 0.453 0.163 0.544

X2 (CAT) –0.042 –0.089 –0.167 –0.060

Indirect path coefficient X3 X4 X5 (relative conductivity) (α-farnesene) (MDA) 0.115 –0.032 0.117 –0.074 0.109 –0.043 –0.075 0.131 0.096 0.073 0.154 –0.067

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helping to maintain the cell membrane integrity. MDA is one of the primary products of membrane lipid peroxidation. Accumulation of MDA causes specific damage to cell membranes and is important in the development of superficial scald in pears (Hui et al. 2011). LOX can activate membrane lipid peroxidation, and its activity was closely correlated with lipid peroxidation during fruit ripening (Li and Wang 2009; Singh et al. 2012). In this study, the 1-MCP and MAP+1-MCP treatments significantly reduced LOX activity, MDA content and relative conductivity preceding the onset of superficial scald (Fig. 5), suggesting that 1-MCP and MAP+1-MCP treatments could protect cell membrane integrity by inhibiting LOX activities and MDA accumulation. PPO catalyzes the oxidation reaction of phenolic compounds to form quinones, resulting in tissue browning (Franck et al. 2007). In this study, the 1-MCP and MAP+ 1-MCP treatments markedly delayed the decrease in phenolic content and inhibited the increase in PPO activity (Fig. 6). Linking the reduced superficial scald indices in 1-MCP and MAP+1-MCP treatments, and the result of correlation analysis of different indices (Table 1), it is proposed that PPO might play an important role in the development of superficial scald, and the 1-MCP and MAP+1-MCP treatments may inhibit this process by decreasing PPO activity of peel in Yali pears.

5. Conclusion The 1-MCP, MAP, and MAP+1-MCP treatments delayed fruit softening but had a low effect on the SSC and significantly reduced the superficial scald index in Yali pears. The overall positive effect of the MAP+1-MCP treatment was more effective. A possible mechanism of MAP+1-MCP treatment on inhibiting superficial scald development might include delaying α-farnesene and CTols accumulation before the onset of superficial scald and suppressing the increase in the MDA and O2–· content. This would maintain the cell membrane integrity and higher SOD and CAT activities and decrease the LOX and PPO activities of the peel, thereby reducing peel browning and superficial scald severity in Yali pears.

Acknowledgements This research was supported by the emarked fund for the China Agriculture Research System for National Technology System for Pear Industry (CARS-28-22), the Innovation Project of Modern Agricultural Sciences and Technology of Hebei Province, China (494-0402-YSN-C8RA), the Youth Fund of Hebei Academy of Agriculture and Forestry Sciences, China (A2015110106), and the Finance Special Foundation of Hebei Province, China (494-0402-YBN-0G4L).

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