Effect of Protocatechuic Acid-Grafted-Chitosan Coating on the

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Effect of Protocatechuic Acid-Grafted-Chitosan Coating on the Postharvest Quality of Pleurotus eryngii Jun Liu, Chen-guang Meng, Xing-chi Wang, Yao Chen, Juan Kan, and Chang-Hai Jin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02468 • Publication Date (Web): 05 Sep 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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Journal of Agricultural and Food Chemistry

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Effect of Protocatechuic Acid-Grafted-Chitosan Coating on the Postharvest

2

Quality of Pleurotus eryngii

3

Jun Liu*, Chen-guang Meng, Xing-chi Wang, Yao Chen, Juan Kan, Chang-hai Jin*

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College of Food Science and Engineering, Yangzhou University, Yangzhou 225127,

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Jiangsu, China

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* Corresponding authors. Tel: 86-514-87978158, E-mail: [email protected] (Jun Liu),

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[email protected] (Chang-hai Jin)

1

ACS Paragon Plus Environment


Protocatechuic

acid-grafted-chitosan

(PA-g-CS)

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solution

with

8

ABSTRACT:

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antioxidant activity was developed as a novel edible coating material for Pleurotus

10

eryngii postharvest storage. The effect of PA-g-CS coating on the postharvest quality of

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P. eryngii was investigated by determination of various physico-chemical parameters

12

and enzyme activities. Results showed that the antioxidant capacity and viscosity of

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PA-g-CS solutions were closely related to the grafting degree, and were much higher

14

than that of chitosan (CS) solution. At the end of 15 days of storage, serious mushroom

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browning was observed in the control and CS coating groups. By contrast, PA-g-CS

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coating groups with medium and high grafting degrees maintained better physical

17

appearance. Among all the treatment groups, P. eryngii in PA-g-CS III coating group

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exhibited the highest firmness; however, the lowest weight loss, browning degree,

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respiration rate, malondialdehyde content, electrolyte leakage rate, superoxide anion

20

production rate and hydrogen peroxide content. Moreover, P. eryngii in PA-g-CS III

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coating group maintained relatively higher antioxidant enzyme activities but lower

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polyphenol oxidase activity than other treatment groups. Therefore, PA-g-CS III is a

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promising preservation agent for P. eryngii.

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KEYWORDS: chitosan;

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protocatechuic acid

grafted;

Pleurotus

eryngii;

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postharvest

quality;

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■ INTRODUCTION

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Mushrooms are generally considered as functional foods due to their valuable

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nutritional and pharmaceutical properties.1–3 Among various cultivated species of

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mushrooms, the king oyster mushroom (Pleurotus eryngii) is rich in minerals, amino

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acids,

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polysaccharides.4 Because of these nutritional and bioactive ingredients, P. eryngii has

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lots of useful biological activities, such as antitumor, antihypercholesterolemic,

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antihypertensive, antimutagenic, antidiabetic and antioxidant activities etc.4–6 However,

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the fresh P. eryngii possesses high respiration and water loss rates, and is easily attacked

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by microorganisms.7–8 For these reasons, P. eryngii usually exerts a short shelf-life,

36

which greatly restricts its distribution and marketing. Therefore, it is essential to

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develop effective preservation techniques for P. eryngii.

essential

fatty

acids,

vitamins,

peptides,

proteins,

polyphenols

and

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Till now, several methods including cold storage, gamma irradiation, modified

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atmosphere packaging as well as edible coating have been used for the postharvest

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storage of P. eryngii.7–11 Among these methods, edible coating has received increasing

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attention because it can effectively prevent P. eryngii from water loss and gas exchange

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with outside environment. Besides, edible coating can also serve as the carrier of

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functional components with antimicrobial and antioxidant properties.12 In recent years,

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many different kinds of edible coatings have been successfully developed and further

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applied for the postharvest storage of fruits and vegetables.13–14 Chitosan (CS) is such a

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natural biodegradable polymer that can be used as edible coating to suppress mushroom

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quality changes during storage.11, 15, 16 However, the effect of CS coating on P. eryngii

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preservation is not very ideal, which is probably due to the low solubility, antioxidant

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and antimicrobial activities of CS.11

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Phenolic compounds are essential in human diet due to their potent antioxidant,

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antimicrobial, antidiabetic and anticancer activities.17 In recently years, many phenolic

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compounds have been conjugated with CS through graft copolymerization reaction in

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order to enhance the biological activities of CS.18–19 The graft copolymerization

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techniques, such as the carbodiimide cross-linking, enzyme catalyzation and hydroxyl

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radical graft are regularly adopted.20–24 It has been demonstrated that phenolic grafted

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CS can be used as antioxidant, antimicrobial, antitumor, antidiabetic, adsorptive,

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encapsulation and packing materials.25–28 However, the effect of phenolic grafted CS

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coating on mushroom preservation is still unclear till now.

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In our previous work, a potent antioxidant agent (protocatechuic acid, PA) was

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covalently linked with CS chains via the 1-ethyl-3-(3-dimethylaminopropyl)

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carbodiimide hydrochloride (EDC) cross-linking reaction. The structure and

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physicochemical characteristics of PA-grafted-CS (PA-g-CS) were analyzed in details.29

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Results showed that the conjugation between PA and CS were achieved through amide

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and ester linkages. Antioxidant assay further revealed that PA-g-CS possessed stronger

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2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity than CS, indicating

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PA-g-CS could be a promising antioxidant agent. In the present study, we further

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developed PA-g-CS as a novel edible coating material. The effect of PA-g-CS solution

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coating on the postharvest quality of P. eryngii during storage was investigated. Several

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physico-chemical parameters and enzyme activities of P. eryngii were evaluated

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throughout the storage period. The preservation effect of PA-g-CS on P. eryngii was also

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compared with that of CS. Our study provides novel information about the effect of

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phenolic grafted CS coating on mushroom preservation.

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■ MATERIALS AND METHODS

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Reagents. PA, EDC, N-hydroxysuccinimide (NHS), methionine, riboflavin and

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thiobarbituric acid (TBA) were purchased from Sigma-Aldrich Co. (St. Louis, MO,

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USA). CS with average-molecular weight 2.5 × 105 Da and 71% deacetylated degree,

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trichloroacetic acid (TCA), hydroxylamine hydrochloride, polyvinylpolypyrrolidone

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(PVPP), p-aminophenylsulfonic acid, α-naphthylamine, catechol and glutathione

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oxidized (GSSG) were purchased from Sangon Biotechnology Co. Ltd. (Shanghai,

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China). All other reagents used were of analytical grade.

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Synthesis of PA-g-CS with different grafting degrees. Three PA-g-CS products

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with different grafting degrees were synthesized by an EDC/NHS cross-linking method

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as described previously.29–30 The grafting degrees of PA-g-CS products were controlled

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via altering the molar ratios of reactants (e.g. CS, EDC, NHS and PA). As a result,

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PA-g-CS products with low, medium and high grafting degrees were obtained and

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named as PA-g-CS I (61.64 mg/g), PA-g-CS II (190.11 mg/g) and PA-g-CS III (279.69

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mg/g), respectively.

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Preparation of PA-g-CS and CS coating solutions. To prepare PA-g-CS coating

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solution, 5 g of lyophilized PA-g-CS sample was completely dissolved in 500 mL of

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acetic acid solution (0.5%, v/v) by magnetic stirring and then stored at 4 °C. CS coating

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solution of the same concentration (1%, w/v) was prepared in the same way.

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Determination of antioxidant capacity of PA-g-CS coating solutions.

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Superoxide anion (O2• ) radical scavenging capacity of PA-g-CS coating solutions was

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quantified according to the method of Jing and Zhao.31 Firstly, 0.4 mL of pyrogallol

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(0.025 M) was mixed with 1 mL of coating solution (0.2–1 mg/mL) and 4.5 mL of

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Tris–HCl buffer (0.05M, pH 8.2). Subsequently, 1 mL of HCl (8 mM) was added into

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mixture to initiate the formation of O2•－. Finally, O2•－ scavenging capacity of the

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coating solutions could be calculated from the absorbance of reaction mixture at 420

99

nm.

－

100

Hydrogen peroxide (H2O2) scavenging capacity of PA-g-CS coating solutions was

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measured using the reported method.32 Firstly, 0.6 mL of H2O2 (0.04 M) was mixed with

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1 mL of coating solution (0.2–1 mg/mL) and 2.4 mL of sodium phosphate buffer (0.1 M,

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pH 7.4). Afterwards, the acquired mixture was further reacted at 20 °C for 10 min.

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Finally, H2O2 scavenging capacity of PA-g-CS coating solutions was calculated from

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the absorbance of mixture at 230 nm.

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Lipid peroxidation inhibition effect of PA-g-CS coating solutions was assayed

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following the reported method.33 Firstly, 1.5 mL of ICR mice’s liver homogenate (1%,

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w/v) was mixed with equal volume of coating solution (0.2–1 mg/mL) followed by

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addition of 75 µL of H2O2 (0.5 mM) and 75 µL of FeCl2 (0.5 mM). The resultant

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mixture was allowed to react at 37 °C for 1 h. Afterwards, the reaction was quenched by

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addition of 2.25 mL of TBA (0.8%, w/v) along with 2.25 mL of TCA (20%, w/v),

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followed by incubation for 15 min at 100 °C and centrifugation for 30 min at 5000 g.

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Finally, lipid peroxidation inhibition effect of the coating solutions could be calculated

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from the absorbance of supernatant at 532 nm.

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Determination of rheological behavior of PA-g-CS coating solutions.

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Rheological behavior of PA-g-CS coating solution was measured on a Kinexus Pro

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Rheometer (Malvern Instruments Ltd., UK) at 20 °C. The viscosity was recorded by

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continuously increasing the shear rate ranging from 2 to 256 s−1.

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Mushrooms treatment and storage. P. eryngii were kindly supplied by Maosheng

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Edible Mushroom Co. Ltd. (Xuzhou, China). Fresh harvested P. eryngii were

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immediately transferred to our laboratory in 2 h under refrigerated conditions. P. eryngii

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with uniform size, weight (105–127 g) and color, but without mechanical damage were

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selected out. Subsequently, the mushroom coating procedure was applied according to

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the literature.15 Selected P. eryngii were equally divided into five groups: (1) control

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(distilled water coating) group, (2) CS coating group, (3) PA-g-CS I (low grafting

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degree) coating group, (4) PA-g-CS II (medium grafting degree) coating group, and (5)

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PA-g-CS III (high grafting degree) coating group. For each treatment, P. eryngii were

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soaked into corresponding coating solution for 30 s and dried on a plastic sieve for 30

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min. The excess coating solution on the surface of P. eryngii was adsorbed by filter

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paper. Afterwards, coated P. eryngii were placed in 28 cm × 30 cm polyethylene bags

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(0.04 mm in thickness) and further stored in a cold room (maintained at 4 ± 1 °C with

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95% relative humidity) for 15 days. During the storage period, five replicates of P.

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eryngii were randomly picked out from each coating group every 3 days for analyzing

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their physico-chemical parameters as well as enzyme activities.

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Firmness and weight loss measurement. TMS-PRO texture analyzer (Food

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Technology Corporation, USA) equipped with a cylindrical probe (diameter of 6 mm)

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was used to measure the firmness of P. eryngii mushroom cap. The penetration speed of

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2.0 mm/s along with the penetration depth of 5 mm was applied. Firmness was acquired

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from the curve of force vs time and expressed as Newton (N). Weight loss of P. eryngii

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was determined by measuring the weight of mushroom every 3 days throughout the

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storage period.

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Respiration rate measurement. To determine the respiration rate, P. eryngii were

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picked out of polyethylene bags and allowed to expose in room condition for 1 h.

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Afterwards, P. eryngii were placed together with a Petri dish containing 10 mL of

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NaOH solution (0.4 M) into an air-tight jar. After 30 min of adsorbance, NaOH solution

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was titrated by using diluted oxalic acid aqueous solution (0.2 M).33 The respiration rate

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of P. eryngii was calculated from the change in the amount of adsorbed CO2.

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Browning degree measurement. To measure the browning degree, P. eryngii (4 g)

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was ground in an ice bath by addition of 20 mL sodium phosphate buffer (0.2 M, pH 6.8)

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which contained PVPP (2.5%, w/v) and NaCl (0.15 M). The obtained mixture was then

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centrifuged at 10000 g and 4 °C for 10 min.34 The absorbance of supernatant was

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measured at 420 nm and represented the browning degree of P. eryngii.

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Malondialdehyde (MDA) content measurement. MDA content in P. eryngii was

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determined by the method of Liu and Wang.35 P. eryngii (1 g) was ground in an ice bath

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by addition of 5 mL TCA (5%, w/v). The obtained mixture was centrifuged at 10000 g

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and 4 °C for 20 min. Afterwards, 1 mL of TCA (10%, w/v) that contained TBA (0.67%,

157

w/v) was added into 1 mL of supernatant, and further incubated in boiling water for 20

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min. After being cooled to room temperature, reaction mixture was finally centrifuged at

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10000 g and 4 °C for 10 min. The MDA content in P. eryngii was calculated by

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measuring the absorbance of supernatant at three different wavelengths (450, 532 and

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600 nm, respectively).

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Electrolyte leakage rate measurement. The membrane permeability of P. eryngii

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was checked by the electrolyte leakage rate.36 P. eryngii stipe (5 mm×5 mm×5 mm) was

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repeatedly washed using distilled water followed by drying with filter paper. The dried

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sample was further suspended in 25 mL of distilled water. A DDSJ-308A electrical

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conductivity meter (Leici Instrument Co., China) was used to measure the electrical

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conductivity of sample solution (Co) immediately. Afterwards, sample was placed in

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boiling water for 10 min and the electrical conductivity of cooled sample solution was

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measured and recorded as Ct. Following equation was used to calculated electrolyte

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leakage rate of P. eryngii: electrolyte leakage rate (%) ＝ Co/Ct × 100.

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O2• － production rate measurement. O2•－ production rate of P. eryngii was

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measured following the reported method.35 P. eryngii (2 g) was ground in an ice bath by

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addition of 5 mL sodium phosphate buffer (50 mM, pH 7.8). The resultant mixture was

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centrifuged at 10000 g and 4 °C for 15 min. Then, 2 mL of hydroxylamine

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hydrochloride (10 mM) along with 1 mL of sodium phosphate buffer (50 mM, pH 7.8)

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was added into 1 mL supernatant, and further reacted for 1 h at room temperature.

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Finally, the obtained reaction mixture was blended with 1 mL of α-naphthylamine (7

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mM) and 1 mL of p-aminophenylsulfonic acid (50 mM) by incubating at room

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temperature for 20 min. O2•－ production rate of P. eryngii was obtained by recording

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the absorbance of reaction mixture at 530 nm.

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H2O2 content measurement. H2O2 content in P. eryngii was assessed by the

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method of Khan et al.37 P. eryngii (1 g) was ground in an ice bath by addition of 5 mL

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TCA (0.1%, w/v). The homogenate was then centrifuged at 10000 g and 4 °C for 15 min.

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Subsequently, 1 mL of potassium iodide (1 M) along with 1 mL of sodium phosphate

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buffer (0.1 M, pH 7.0) was added into 1 mL of supernatant, and further reacted at room

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temperature for 1 h in dark conditions. H2O2 content in P. eryngii was obtained by

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recording the absorbance of reaction mixture at 390 nm.

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Determination of enzyme activity. In order to prepare crude enzyme extract, P.

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eryngii (2 g) was ground in an ice bath by addition of 5 mL sodium phosphate buffer

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(50 mM, pH 7.8) which contained PVPP (1%, w/v), Tween-20 (0.3%, v/v) and EDTA (1

191

µM). The homogenate was centrifuged at 10000 g and 4 °C for 20 min to obtain

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supernatant that served as crude enzyme extract.

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Superoxide dismutase (SOD) activity of P. eryngii was assayed by the method of Li

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et al.9 Firstly, 0.2 mL of P. eryngii enzyme extract was homogenized with 0.4 mL of

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EDTA (100 µM), 0.4 mL of nitroblue tetrazolium (0.75 mM), 0.4 mL of methionine

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(0.13 M) and 4.8 mL of sodium phosphate buffer (50 mM, pH 7.8). Afterwards, 0.2 mL

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of riboflavin (20 µM) was added into the homogenate to initial the reaction at 25◦C for

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60 min by 4000 lx of irradiance. SOD activity of P. eryngii was calculated by recording

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Catalase (CAT) activity of P. eryngii was measured following the reported

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method.38 Firstly, 0.1 mL of P. eryngii enzyme extract was homogenized with 2.5 mL of

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sodium phosphate buffer (50 mM, pH 7.8). Subsequently, 1 mL of H2O2 (0.1 M) was

203

added into the homogenate to initial the reaction. CAT activity of P. eryngii was

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calculated by recording the absorbance of reaction mixture at 240 nm.

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Ascorbate peroxidase (APX) activity of P. eryngii was assayed as reported

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previously.37 P. eryngii enzyme extract (0.1 mL) was homogenized with 2.9 mL of

207

sodium phosphate buffer (50 mM, pH 7.8) which contained H2O2 (2 mM), ascorbic acid

208

(0.5 mM) and EDTA (0.1 mM). APX activity of P. eryngii was calculated by recording

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Glutathione reductase (GR) activity of P. eryngii was determined according to the

211

method of Jiang et al.39 P. eryngii enzyme extract (0.2 mL) was homogenized with 2.7

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mL of sodium phosphate buffer (0.1 M, pH 7.5) along with 40 µL of nicotinamide

213

adenine dinucleotide phosphate (4 mM) and 0.1 mL of GSSG (5 mM). GR activity of P.

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eryngii was calculated by recording the absorbance of reaction mixture at 340 nm.

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Polyphenol oxidase (PPO) activity of P. eryngii was assayed as reported

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previously.40 P. eryngii enzyme extract (0.15 mL) was homogenized with 1.5 mL of

217

catechol (50 mM) and 6 mL of sodium phosphate buffer (0.1 M, pH 7.0). PPO activity

218

of P. eryngii was obtained by recording the absorbance of reaction mixture at 420 nm.

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Scanning electron microscopy (SEM) observation. Micro-structure of P. eryngii

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cap tissue was observed by SEM according to the method of Ogawa et al.41 Small

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pieces of P. eryngii cap tissue were prepared by using the surgical blade. Samples were

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chemically fixed by soaking in formaldehyde solution (10%, v/v) overnight. Afterwards,

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the resultant fixed tissue pieces were then sequentially dehydrated in ethanol aqueous

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solutions with different concentrations (70%, 80%, 90%, 95% and 100%). The obtained

225

dehydrated samples were freeze-dried and sputtered with gold. XL-30 ESEM scanning

226

electron microscope (Philips Electron Optics, Netherlands) was used for observing the

227

micro-structure of tissue piece.

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Statistical analysis. Data were expressed as mean ± standard deviation (SD), and

229

analyzed by one-way analysis of variance (ANOVA) as well as Duncan’s multiple-range

230

comparisons. Difference was considered to be statistically significant in the case of p
0.05).

326

Effect of PA-g-CS coating on MDA content of P. eryngii. MDA level is a good

327

indicator to reflect the degree of lipid peroxidation.51 As shown in Figure 6A, MDA

328

contents of P. eryngii in different coating groups increased to 0.46−1.11 µmol/g when

329

storage period reached 15 days. The highest MDA content of P. eryngii was observed in

330

distilled water coating group (p < 0.05). P. eryngii in CS coating group showed

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relatively lower MDA content than the control group, but higher MDA content than

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PA-g-CS coating groups (p < 0.05). Notably, there was no statistical difference of MDA

333

content among three PA-g-CS coating groups (p > 0.05). The relatively lower MDA

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contents of P. eryngii in PA-g-CS coating groups could be due to the lipid peroxidation

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inhibition effect of PA-g-CS.

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Effect of PA-g-CS coating on electrolyte leakage rate of P. eryngii. Membrane

337

permeability of mushroom is often reflected by the electrolyte leakage rate. Figure 6B

338

showed that the electrolyte leakage rate of P. eryngii in the control and CS coating

339

groups rapidly increased during the storage period. Similar phenomenon was also

340

observed by Li et al.7 By contrast, P. eryngii in PA-g-CS coating groups showed

341

relatively lower electrolyte leakage rate than mushroom in distilled water and CS

342

coating groups (p < 0.05). However, PA-g-CS II coating group exhibited no significant

343

difference in electrolyte leakage rate as compared with PA-g-CS III coating group (p >

344

0.05). When storage period reached 15 days, electrolyte leakage rates of P. eryngii in

345

different coating groups increased to 14.2−48.8%. Results suggested that the membrane

346

integrity of P. eryngii could be maintained by PA-g-CS with medium and high grafting

347

degrees.

348

Effect of PA-g-CS coating on O2•－ production rate and H2O2 content of P.

349

eryngii. ROS productions, such as O2•－, OH• and H2O2, are mainly produced in the

350

cellular metabolism process. High level of ROS can cause damage in several cellular

351

components (e.g. DNA, proteins and lipids), which results in mushroom senescence.34

352

As shown in Figure 7A, O2•－ production rate of P. eryngii significantly increased after 9

353

days of storage in all the treatment groups, indicating the degree of mushroom

354

senescence accelerated. Among all the treatment groups, P. eryngii in PA-g-CS III

355

coating group showed the slowest O2•－ production rate (p < 0.05), suggesting PA-g-CS

17



－

356

III could greatly reduce the increase of O2• production rate in P. eryngii. Figure 7B

357

showed that H2O2 content of P. eryngii in all the treatment groups significantly

358

increased after 6 days of storage. The highest H2O2 content in P. eryngii was observed in

359

distilled water coating group (p < 0.05). By contrast, P. eryngii in PA-g-CS coating

360

groups exhibited relatively lower H2O2 content (p < 0.05). The relatively slower O2•－

361

production rate and lower H2O2 content in PA-g-CS coating groups should be attributed

362

to the potent antioxidant activity of PA-g-CS, which could scavenge free radicals

363

formed in mushroom.

364

Effect of PA-g-CS coating on antioxidant enzyme activity of P. eryngii. In

365

general, antioxidant enzymes are crucial defense systems in mushrooms.15–16 As shown

366

in Figure 8A, SOD activity in all the treatment groups gradually decreased throughout

367

the storage period. This result was in agreement with the finding of many other

368

reportors.15, 51 The lowest SOD activity of P. eryngii was observed in distilled water

369

coating group (p < 0.05). By contrast, CS and PA-g-CS coatings could significantly

370

delay the decrease of SOD activity. It has been reported that SOD could scavenge most

371

of the O2• － at the early storage stage.34 Thus, O2• － production rate increased

372

significantly at the late storage stage (Figure 7A). CAT plays an important role in

373

H2O2 degradation. Significant increase of CAT activity in P. eryngii was observed after

374

6 days of storage (Figure 8B). The highest CAT activity of P. eryngii was shown in

375

PA-g-CS III coating group (p < 0.05). When storage period reached 15 days, CAT

376

activities of P. eryngii in different treatment groups increased to 1.66−2.36 times of the

377

initial value. As shown in Figure 8C and Figure 8D, remarkable decrease of APX and

18


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378

GR activity in P. eryngii was observed in distilled water coating group. CS and PA-g-CS

379

coatings could significantly prohibit the decrease in APX and GR activity. Notably,

380

PA-g-CS III coating was more efficient than any other treatment groups (p < 0.05).

381

Effect of PA-g-CS coating on PPO activity of P. eryngii. PPO plays a crucial role

382

in mushroom postharvest browning. When storage period reached 9 days, PPO activity

383

of P. eryngii significantly increased in both distilled water and CS coating groups

384

(Figure 9). By contrast, PA-g-CS coatings could remarkably reduce the increase in PPO

385

activity. However, PA-g-CS II coating group exhibited no significant difference in PPO

386

activity as compared with PA-g-CS III coating group (p > 0.05). Notably, a positive

387

correlation was found between browning degree and PPO activity, indicating that P.

388

eryngii browning was probably caused by PPO.

389

Effect of PA-g-CS coating on micro-structure of P. eryngii. Effect of different

390

coating solutions on the micro-structure of P. eryngii cap tissue was observed by SEM.

391

As shown in Figure 10, the fibrous arrangement of fresh P. eryngii cap tissue was

392

compact. By contrast, distilled water and CS coatings caused tangled or random fibrous

393

structure with enlarged microporous in P. eryngii cap tissue. The micro-structure change

394

should be attributed to the physical deformation of structural polysaccharides, such as

395

chitin and glucan, which could cause severe tissue shrinkage and collapse during

396

storage.41 In PA-g-CS coating groups, less tangled fibrous structure and smaller

397

microporous were observed as compared to distilled water and CS coating groups.

398

These results further confirmed the superior preservation effect of PA-g-CS coating.

399

Overall, PA-g-CS has been demonstrated as a good candidate of edible coating for

19



400

its high antioxidant capacity and viscosity. P. eryngii in the control group showed

401

significant higher ROS production rate and MDA level, as well as lower antioxidant

402

enzyme activity than other treatment groups. The overproduction of ROS and reduced

403

enzyme activity are intrinsic features of mushroom senescence. Although CS coating

404

could improve the postharvest quality of P. eryngii, its effect was not that good as

405

PA-g-CS coating. It should be noted that PA-g-CS III has been demonstrated as the best

406

coating material for the postharvest storage of P. eryngii. The superior preservation

407

effect of PA-g-CS III could be mainly attributed to its highest antioxidant activity and

408

proper viscosity. Thus, PA-g-CS III is a very potential coating material for mushroom

409

postharvest storage.

410

■AUTHOR INFORMATION

411

Corresponding Authors

412

*(Jun Liu) Tel: 86-514-87978158. Email: [email protected].

413

*(Chang-hai Jin) Tel: 86-514-87978158. Email:[email protected].

414

Funding

415

This work was supported by grants of National Natural Science Foundation of China

416

(31571788 and 31101216), Natural Science Foundation of Jiangsu Province

417

(BK20151310), Jiangsu Provincial Government Scholarship for Overseas Studies, High

418

Level Talent Support Program of Yangzhou University, and Qing Lan Project of Jiangsu

419

Province.

420

Notes

421

The authors declare no competing financial interest. 20


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422

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FIGURE LEGENDS Figure 1. The superoxide radical scavenging activity (A), H2O2 scavenging activity (B) and lipid peroxidation inhibition effect (C) of CS, PA, PA-g-CS I, PA-g-CS II, PA-g-CS III and Vc. Data are presented as means ± SD of triplicates. Figure 2. Viscosity behavior of CS, PA-g-CS I, PA-g-CS II and PA-g-CS III coating solutions. Figure 3. Effect of CS and PA-g-CS coating on the physical appearance of P. eryngii. A: fresh harvested mushroom; B, C, D, E and F: mushroom coated with distilled water, 1% of CS, PA-g-CS I, II and III solutions respectively, and stored at 4 ± 1 °C with 95% relative humidity for 15 days. Figure 4. Effect of CS and PA-g-CS coating on the firmness (A) and weight loss (B) of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates. Figure 5. Effect of CS and PA-g-CS coating on the respiration rate (A) and browning degree (B) of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates. Figure 6. Effect of CS and PA-g-CS coating on the MDA content (A) and electrolyte leakage rate (B) of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates. Figure 7. Effect of CS and PA-g-CS coating on the O2•－ production rate (A) and H2O2 content (B) of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates.

28


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Figure 8. Effect of CS and PA-g-CS coating on the SOD (A), CAT (B), APX (C) and GR (D) activities of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates. Figure 9. Effect of CS and PA-g-CS coating on the PPO activity of P. eryngii during storage at 4 °C for 15 days. Data are presented as means ± SD of five replicates. Figure 10. Micro-structure of P. eryngii cap tissue observed by SEM. A: fresh harvested mushroom; B, C, D, E and F: mushroom coated with distilled water, 1% of CS, PA-g-CS I, II and III solutions respectively, and stored at 4 ± 1 °C with 95% relative humidity for 15 days.

29



(A)

Page 30 of 41

Scavenging activity (%)

100 80

Vc PA CS PA-g-CS I PA-g-CS II PA-g-CS III

60 40 20 0 0

0.2

0.4

0.6

0.8

1

Concentration (mg/mL)

Scavenging activity (%)

(B)

100 80


60 40 20 0 0

0.2

0.4 0.6 0.8 Concentration (mg/mL)

1

(C) 100 Inhibition effect (%)

80


60 40 20 0 0

0.2

0.4 0.6 0.8 Concentration (mg/mL)

1

Figure 1 30


Page 31 of 41


Viscosity (Pa·s)

1.00 CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

0.10

0.01

0.00 1

10

100

Shear rate (1/s) Figure 2

31


1000


(A)

(B)

(C)

(D)

(E)

(F)

Figure 3

32


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(A)

5.5

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

4.8

Firmness (N)

Control

4.1 3.4 2.7 2.0 0

3

6

9

12

15

12

15

Storage time (days)

Weight loss (%)

(B)

2.5

Control

CS

PA-g-CS I

PA-g-CS II

2 PA-g-CS III

1.5 1 0.5 0 0

3

6

9

Storage time (days)

Figure 4

33



Respiration rate (mg CO2/kg/h)

(A)

420 360 300 240 180

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

120 0

3

6

9

12

15

Storage time (days)

Browning degree (A 450)

(B) 0.8 0.6

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

0.4 0.2 0.0 0

3

6

9

12

15

Storage time (days)

Figure 5

34


Page 34 of 41

Page 35 of 41


MDA content (μmol/g)

(A)

1.2

Control

CS

1.0

PA-g-CS I

PA-g-CS II

PA-g-CS III

0.8 0.6 0.4 0.2 0.0 0

3

6

9

12

15

12

15

Storage time (days) (B) Relative leakage rate (%)

55 45

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

35 25 15 5 0

3

6 9 Storage time (days)

Figure 6

35



(nmol/min/g)

－

O2• production rate

(A)

8

Control

CS

7

PA-g-CS I

PA-g-CS II

PA-g-CS III

6 5 4 3 2 0

3

6

9

12

15

12

15

Storage time (days)

H2O2 content (μmol/g)

(B)

420

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

340 260 180 100 0

3

6

9

Storage time (days)

Figure 7

36


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SOD activity (U/g)

(A)

0.15 0.12

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

0.09 0.06 0.03 0 0

3

6

9

12

15

12

15

Storage time (days)

CAT activity（U/g）

(B) 6.0 5.0

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

4.0 3.0 2.0 0

3

6

9

Storage time (days)

37



APX activity (U/g)

(C)

60 50

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

40 30 20 10 0 0

3

6

9

12

15

Storage time (days) Control PA-g-CS I PA-g-CS III

(D) 3.0

GR activity(U/g)

2.5

CS PA-g-CS II

2.0 1.5 1.0 0.5 0.0 0

3

6 9 Storage time (days)

12

15

Figure 8

38


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PPO activity (U/g)

130 110

Control

CS

PA-g-CS I

PA-g-CS II

PA-g-CS III

90 70 50 30 0

3

6

9

12

15

Storage time (days)

Figure 9

39



(A)

(B)

(C)

(D)

(E)

(F)

Figure 10

40


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OH

OH

O

O HO

NH2

O HO

Pleurotus eryngii

O NHCOCH 3

CS O R= C

OH

PA EDC/NHS

OH

ROS production rate (O2 •－ and H2O2 )

OR

OR

O

O RO

Physico-chemical parameters (Firmness, weight loss, respiration rate, browning degree, MDA content and electrolyte leakage rate)

NHR

O RO

O NHCOCH3

PA-g-CS (Enhanced antioxidant activity and rheological property)

Enzyme activities CS and PA-g-CS coating (4 °C storage for 15 days)

(SOD, CAT, APX, GR and PPO)

TOC Graphic

41


Effect of Protocatechuic Acid-Grafted-Chitosan Coating on the

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