*2.2. Extraction of HFE*

The fruits of hairy fig (origin: Guangdong, China) were purchased from Huafeng herbs store in Zhangshu (Jiangxi, China) and powdered in a grinder (less than 20 mesh) after drying below 40 ◦C. The HFE was obtained using an ultrasonic-assisted method, as described below [21]. The air-dried mixture of 100 g powder sample was suspended in 3.1 L 90% ethanol (*v*/*v*) at 51 ◦C with ultrasonic-assisted extraction (40 kHz) for 65 min. The HFE was filtered and concentrated by vacuum distillation at 45 ◦C, using a Buchi rotary evaporator (R210, Buchi, Labortechnik AG, Flawil, Switzerland). The remaining solution was dissolved in sterile water and made up to 100 mL at a concentration of 1 g mL−<sup>1</sup> (raw herb/solvent, *w*/*v*), then stored at 4 ◦C for further use.

### *2.3. Preparation of CH-HFE and Chitosan Coatings*

Chitosan solution (1.5%, *w*/*v*) was prepared by dissolving 15.0 g of chitosan (the degree of deacetylation of 90%, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in 800 mL of acetic acid solution (0.5%, *v*/*v*). Crude HFE extract (35 mL) was put into the coating, with agitation for 1 h. The pH of the solution was adjusted with 1.0 M NaOH to pH 5.4, and the total volume of the solution was made up to 1000 mL. Chitosan coating of the same concentration (1.5%, *w*/*v*) was prepared in the same way.

#### *2.4. Navel Orange Treatment and Storage*

The selected fruits were washed with tap water and air-dried at room temperature (25 ± 1 ◦C), then coated by dipping in chitosan coating and/or HFE for 1 min (CH-HFE and 1.5% chitosan coating, respectively), while the control group was dipped in 0.5% acetic acid solution (pH 5.4). After drying, the coated, as well as control, fruits were individually film (18 cm × 15 cm, Lingqu fresh packaging products Co. Ltd., Guilin, China)-packaged and pre-cooled (10–12 ◦C, 12 h). Finally, all fruits were stored at 5 ± 0.5 ◦C, and 85%–90% relative humidity (RH) for 120 days. The procedures for the coating and control group were performed three times of total 560 navel oranges per treatment. At each sampling point (15, 30, 45, 60, 75, 90, 105, and 120 days), each replicates of 10 navel oranges were randomly picked out from the coating and control group for analyzing total soluble solid (TSS) content, titratable acid (TA) content, vitamin C (VC) content, total sugar content, respiration rate, MDA content, as well as protective enzyme activities.

#### *2.5. Measurement of Physicochemical Indexes*

Fruit decay rate was visually evaluated using the same 80 navel oranges per treatment per replicate, during the storage period of semi-monthly, and expressed as the percentage of rotted fruits. Navel oranges with apparent disease spots were considered to be decayed. Weight loss was measured by weighing the same 20 fruits during storage every 15 days, and the data are means of 20 samples ± SE.

TSS, TA, VC, and total sugar were analyzed after completely mixing the orange juice from 10 fruits in the coated and control groups. TSS was measured using a V RA-250 WE digital brix-meter (KYOTO, Tokyo, Japan) and expressed as a percentage. TA and VC were determined by titration with 0.1 M NaOH, and 2,6-dichlorophenol indophenol, respectively. Total sugar content was measured using the anthrone colorimetric method [22].

#### *2.6. Assay of Respiration Rate and MDA Content*

The respiration rate was determined based on a method described by our previous study [23]. Six fruits, per treatment per replicate, were weighed before being sealed in an airtight plastic container (internal diameter of 27.5 cm, 30.0 cm high) at 25 ◦C. The increased CO2 concentration in the container was monitored by using a GHX-3051H infrared CO2 fruit and vegetable breathing apparatus (Jingmi Scientific LLC., Shanghai, China). Respiration rate, measured by CO2 production, was expressed as mg kg−<sup>1</sup> h<sup>−</sup>1.

The MDA content in coating and control groups was measured according to the method of Hodges et al. [24]. Pericarp tissues from 10 fruits were ground in a MM 400 frozen grinder (Retsch GmbH., Arzberg, Germany), and 2.0 g of powder were homogenized in 25 mL of ice-cold 50 mM phosphate buffer (pH 7.8) containing 1 mM EDTA and 2% (*w*/*v*) PVP, and centrifuged at 12,000 g (5804R, Eppendorf, Hamburg, Germany) for 20 min at 4 ◦C. Afterwards, 2 mL of the collected supernatant was mixed with 2 mL of 0.5% (*w*/*v*) thiobarbituric acid (TBA), and further incubated in boiling water for 30 min. After being cooled and centrifuged at 6000 g (5804R, Eppendorf) for 10 min, the absorbance of supernatant was measured at three different wavelengths (450, 532, and 600 nm) using a M5 Multiscan Spectrum microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA). The MDA content

was calculated according to the formula (6.452 × (*A*<sup>532</sup> − *A*600) − 0.559 × *A*450), and expressed as mmol g−<sup>1</sup> frozen weight (FW).

#### *2.7. Determination of Protective Enzymes Activities*

Aliquots of powder (2.0 g) were homogenized with various ice-cold extraction buffers to prepare extracts for assay of the following protective enzymes: 10 mL of 50 mM ice-cold phosphate buffer (pH 7.8) containing 1 mM EDTA, 5 mM DTT, and 2% (*w*/*v*) PVP for superoxide dismutase (SOD, EC 1.15.1.1); 8 mL of 100 mM ice-cold phosphate buffer (pH 7.5) containing 1 mM polyethylene glycol (PEG), 4% (*w*/*v*) PVP, and 1% (*w*/*v*) Triton X-100 for peroxidase (POD, EC 1.11.1.7); 10 mL of 100 mM ice-cold sodium acetate buffer (pH 5.2) containing 5 mM β-mercaptoethanol, 1 mM PEG, 1 mM EDTA, 4% (*w*/*v*) PVP, and 0.5% (*w*/*v*) Triton X-100 for chitinase (CHI, EC 3.2.1.14) and β-1,3-glucanase (GLU, EC 3.2.1.73). All homogenates were centrifuged at 12,000 g (5804R, Eppendorf) for 30 min at 4 ◦C. The supernatants were then collected and used for the enzyme activity assays.

SOD activity was assayed by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) according to the method of Sala and Lafuente, with slight modifications [25]. The reaction mixture consisted of 1.5 mL PBS (50 mM), 0.3 mL Met (130 mM), 0.3 mL NBT (0.75 mM), 0.3 mL EDTA-Na2 (0.1 mM), 0.3 mL riboflavin (20 μM), 0.1 mL enzyme extract, and 0.5 mL distilled water in a total volume of 3.3 mL. The mixtures were illuminated by light (4000 Lx) for 20 min at 28 ◦C, and the absorbance was then determined at 560 nm (UV-1800, Shimadzu, Tokyo, Japan). One unit of SOD activity was defined as the amount of enzyme that would inhibit 50% of NBT photoreduction, and expressed as U min−<sup>1</sup> g−<sup>1</sup> FW.

POD activity was based on the determination of guaiacol oxidation at 470 nm in the presence of H2O2. Collected supernatant (100 μL) was mixed with 3.0 mL of 25 mM guaiacol and 200 μL of 50 mM H2O2. Oxidation of guaiacol was determined at 470 nm for 3 min at 25 ◦C. One unit of POD activity was defined as an increment of 0.01 in absorbance per minute at 470 nm, and expressed as U min−<sup>1</sup> g−<sup>1</sup> FW.

CHI and GLU activities were assayed by the method described by Abeles et al. [26] using chitinase and laminarin as substrate. One unit of CHI activity was defined as the amount of enzyme that catalyzed the produce of 1 nmol of *N*-acetyl-D-glucosamine per hour at 585 nm, and expressed as U h−<sup>1</sup> g−<sup>1</sup> FW. One unit of GLU activity was defined as the amount of enzyme that produced a reducing sugar equivalent to 1 nmol glucose equivalents per hour at 540 nm, and expressed as U h−<sup>1</sup> g−<sup>1</sup> FW.

#### *2.8. Statistical Analysis*

Three biological replicates per treatments were done, and the effect of CH-HFE coating on "Newhall" navel orange preservation was analyzed using variance analysis (*p* < 0.05). Duncan's multiple range test was used to determine the mean differences. The data are represented as the mean with standard error (SE); these were calculated from physical and chemical experiments, which were performed in three replications.

#### **3. Results and Discussion**
