Methods
Preparation of coating solution and grape treatment
Nano zinc oxide suspension was prepared by adding 0.0, 0.5, and 0.10 w/w zinc oxide nanoparticles which had an average diameter of 70 nm and sterile deionized water in beaker. Subsequently, the resulting mixture was sonicated for a duration of 45 min at a temperature of 25 ºC and a frequency of 25 kHz in an ultrasound bath (TI-H-5, Elma GmbH, Germany). This process aimed to effectively disperse the zinc oxide nanoparticles within the suspension. The application of the coating was carried out through a three-stage spraying process, which encompassed the flowering stage, the unripe stage, and the ripening stage, occurring 20 days prior to the harvest. Subsequently, the harvested grapes samples were kept fresh, clean, uniform in size, without mechanical damage and insect pests (1 Kg) were carefully placed in polypropylene containers (10×20×20 cm) equipped with lids and stored at 4 ºC for 35 days. S0.0, S0.5, and S1.0 are grape sample coated by 0.0, 0.5, and 1.0% W/V nano zinc particles coatings, respectively.
pH and titratable acidity
pH was evaluated using a pH meter (744, Metrohm, Switzerland) at room temperature (Derradji-Benmeziane et al. 2014). The determination of titratable acidity was accomplished through the process of acid-base titration. In summary, a solution of 0.1 mol/L sodium hydroxide (NaOH) was prepared and calibrated using potassium hydrogen phthalate for future use. The standardized NaOH solution was then employed to titrate the acid content found in grapes. Phenolphthalein served as the indicator during this procedure. Ultimately, the volume of NaOH solution utilized in the titration allowed for the determination of the titratable acidity (Deng et al. 2019).
Weight loss and Brixº
The amount of mass loss was determined by weighing method. Quantitative measurement of weight loss was done with the Eq. 1 (Deng et al. 2019):
Weight loss (%) = [(Initial weight – Final weight)/ Initial weight] (1)
The Brixº of grape samples was determined with a refractometer (PR-101, Atago, Bellevue, WA) at room temperature (Derradji-Benmeziane et al. 2014).
Total phenolic, anthocyanin and ascorbic acid content
The initial step involved the removal of grape berries from each bunch, followed by their homogenization in an ice-cooled blender (Pars khazar, Iran). To obtain the extract from samples, 75 g of blended grape was macerated in 100 mL of methanol containing 0.1% HCl and left overnight in darkness. Subsequently, the extract was filtered using Whatman No. 1 paper under vacuum, and the residue was repeatedly extracted with the same solvent until it became colorless. This extraction process was continued until the solvents used became colorless, with a total solvent volume ranging between 500–1000 mL. The grape extracts obtained in methanol were diluted with buffer to achieve an absorbance reading between 0.4 and 0.6. The diluted grape extracts were then subjected to pH values of 1.0 (using 0.025 M potassium chloride buffer) and 4.5 (using 0.4 M sodium acetate buffer). The absorbance was measured at 520 nm using a spectrophotometer (Abou El-Nasr et al. 2021). The total phenolic content in the grape extracts was determined using the Folin-Ciocalteu reagent. 1 mL of the extract solution was mixed with 45 mL of distilled water. Then, 1 mL of Folin-Ciocalteu reagent was added and thoroughly mixed. After 3 min, 3 mL of Na2CO3 was added, and the mixture was allowed to stand for 2 hours. The absorbance was measured at 760 nm, and the concentration of TPC in the grape extracts was determined as microgram of gallic acid equivalent using an equation derived from the standard gallic acid graph (Orak 2007). To estimate the ascorbic acid content, 10 mL of grape extract was titrated with iodine (I2). Ascorbic acid content was expressed as mg of vitamin C/ 100 mL of grape extract (Derradji-Benmeziane et al. 2014).
Antioxidant activity
The antioxidant activity of the whole grape extracts was determined using DPPH radical scavenging method. A solution containing 24 mg/L of DPPH was prepared using methanol as the solvent. Subsequently, 0.05 mL of the sample was combined with 2 mL of the DPPH solution, and the absorbance was promptly measured at 517 nm in comparison to a methanol blank. Following a 16-min incubation period at room temperature, the absorbance was recorded once more. The inhibition of the DPPH radical induced by a grape sample was calculated using the formula: [(AC(0) - AA(t)) / AC(0)] × 100, where AC(0) represents the absorbance of the sample at t = 0 min, and AA(t) denotes the absorbance of the sample at t = 16 min (Du et al. 2012).
Color indexes
The color of the Hunter parameters L* (Lightness), a* (redness), and b* (blueness) was measured using a Hunter Lab spectrophotometer (Color Flex Reston, USA). The L* value ranges from 0 to 100, with 100 representing a perfectly reflecting diffuser and 0 indicating black. A positive value for a* corresponds to red, while a negative value represents green. Similarly, a positive value for b* indicates yellow, while a negative value signifies blue. The changes in the L*, a*, and b* values of stored grapes were assessed during storage (Venkatram et al. 2017).
Microbial analysis
The grape samples were analyzed to determine the total viable count, as well as the total yeast and mold count. In order to determine the total viable bacteria count, the pour plate method was utilized. Initially, 10 g of the sample was homogenized with 90 mL of phosphate buffer. Subsequently, the mixture was further diluted as necessary under aseptic conditions. For the spread plate method, 0.1 mL of appropriate dilutions was plated, while for the pour plate method, 1 mL was used. The plating process involved using Plate Count Agar for the total viable count, and Rose Bengal Chloramphenicol agar for the yeast and mold count. The plates were then incubated at 37°C for 48 h to determine the total viable bacteria count, and at 30°C for 96 h to determine the yeast and mold count. The resulting colony forming units were manually counted, and the yeast and mold count was determined using the standard spread plate method (Augustine et al. 2013).
Sensory analysis
The sensory evaluation of the grape samples involved a panel of 10 evaluators, ranging in age from 20 to 45 years, conducted within a sensory evaluation laboratory equipped with individual booths. Each grape was assigned a random 3-digit code and assessed at intervals of 0, 7, 14, 21, and 35 days during storage at 4°C, following a randomized, fair, and counterbalanced experimental design. The assessors rated the samples based on specific attributes such as odor and flavor, texture, color, and overall acceptance using a scale ranging from 1 to 5, where 1 = dislike extremely, 5 = like extremely. To ensure objectivity and accuracy in the evaluation process, water cups were provided for the panelists to rinse their palates between tasting different grape samples. The results obtained from the sensory evaluation were then averaged to provide a comprehensive assessment of the grapes' quality over the storage period (Kadi 2023).
Grape molasses properties
Grape molasses prepared according the method described by Heshmati, et al. (2019) and then the total sugar, Brixº, conductivity and specific gravity of different grape molasses were measured (Heshmati et al. 2019). Total sugar content was determined according to Başaran et al. (2021). Briefly, 5 g of different pekmez samples was dissolved in 40 mL of distilled water, and then, 25 mL of methanol was added. Sugar content were extracted with water, centrifuged, filtered, and injected into an HPLC device (LC 20 AT Prominence, Shimadzu, Japan) equipped with a refractive index detector. 20 µL of each sample was injected into a column. Determination were performed at 30 ºC column temperature, and 1.3 mL/min flow rate (Başaran et al. 2021).A digital refractometer (PAL-22S, Atago, Japan) was used to evaluate ºBrix values at 25 ºC (Güçlü et al. 2023). The electrical conductivity of samples was measured using a multi-conductivity measuring device (HQ40d, Colorado, USA) at 25 ºC and it was expressed in mS/cm (AOAC 2009). Specific gravity (SG) was determined using a pycnometer (Güçlü et al. 2023).