2.1. Materials
Corn starch, Heilongjiang Yufeng Corn Development Co., Ltd (Qiqihar, China). Grape seeds, pig lard, Qiqihar City Liuyuan market. Yeast Extract, Trypsin (biochemical reagent), Beijing Aoboxing Biotech Co., LTD. S. aureus strains (ATCC29213), Escherichia coli (ATCC25922), School of Food and Biological Engineering, Qiqihar University (Qiqihar, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Other chemical reagents (analytical grade), Tianjin Kaitong Chemical Reagents Co., LTD.
2.2. Extraction of GSE
Extraction of GSE was performed according to the method of Gao et al. (2021) with some modifications. Break the grape seeds after a multi-function (mill GX-220 multi-function crusher, Zhejiang Gaoxin Industry and Trade Co., LTD), add 70% ethanol to the crushed grape seeds at a ratio of 1:20 (w:v). After 2h of extraction at 25℃, centrifuges were centrifuged at 4000g, and take the supernatant and spare. The sediments were repeatedly dipped and centrifuged, and the two centrifugation supernatants were combined. The supernatant was transferred to a rotary evaporator (2 L-ARE Rotation evaporator, Shanghai Haozhuang Instrument Co., Ltd) at 50℃ for concentrate, then Lyophilize the GSE (The extraction rate was 9.24%) in a vacuum freezer dryer (2.5 L-freezePrysystem, Labconco Co., USA) and stored in a 4℃ refrigerator.
2.3. Prepara0tion of films
CS/кC -GSE films were prepared by solvent pouring method. Corn starch was dissolved into distilled water to obtain a 3% corn starch suspension, mixing at 90℃ for 0.5 h. Then, add 1.8 g carrageenan(w/w)(Food grade, Qingdao Dehui Marine Biotechnology Co., Ltd), and 1.8 g glycerol(w/w). Continue stirring for 0.5 h. GSE at concentrations of 0%, 1%, 3%, and 5% were added to the CS solution(w/w), continue to stir for 0.5 h. Add 1.5 g glycerol(w/w) to the CS/кC film-forming solution, continue to stir for 0.5 h, and use hot water to replenish the evaporated water. The composite film solution was prepared by sonication of the solution to 80℃ for 30 min to remove the bubbles. A poly ethanol ring with a diameter of 120 nm was fixed on a glass plate laid with release paper. Pour 25 g of CS solution onto the glass plate. It was dried at 40℃ for 12 h. Finally, the prepared films containing 0wt% GSE, 1wt% GSE, 3wt% GSE, and 5wt% GSE were named CS/кC, 1%CS/кC -GSE, 3%CS/кC -GSE, and 5%CS/кC -GSE films, and placed in a dryer containing a saturated sodium bromide solution(25℃, 56.8 RH) for 72 h.
2.4. Characterization of the CS/кC -GSE films
2.4.1. Mechanical property
The mechanical properties of films are referred to in the method of Huang et al. (2020) with some modifications. This experiment used a texture analyzer (TA. XT plus C, Stable Micro System Co., UK) to measure the physical properties of the film. The film is trimmed to a 6 cm × 2 cm rectangle, and the test speed was 2 mm / s, Initial clip distance was 20 mm. The tensile strength (MPa)and fracture elongation (%) of the film was measured. It is averaged after three measurements of each sample. The mechanical properties (Ts) and elongation at break (EB) of the film are calculated as follows: L1-L0
$$Ts=\frac{{F}_{MAX}}{\text{S}}$$
1
$$EB\%=\frac{{L}_{1}-{L}_{0}}{{L}_{0}}\times 100$$
2
where Fmax is the maximum load at film fracture (N), S is the cross-sectional area of the film (mm2), L1 is the length of the film after stretching (mm), L0 is the initial length of the film(mm).
2.4.2. Water vapor permeability
The WVP was determined by weight, referring to the method of Roy et al. (2021b), and made a slight modification. Anhydrous calcium chloride particles (0% RH) were placed in a weighing bottle(35 mm×90 mm), covered with the film sample of the weighed bottle port, and sealed, then put into a desiccator with distilled water at the bottom. The quality of the weighed bottles is weighed at 24 h intervals between 8 successive days. WVP was calculated as follows Eq.
$$WVP=\frac{W}{t\times A}+\frac{X}{\varDelta P}$$
3
Where W was the total quality of the bottle was weighed after the film sealing (g), x was the film thickness (m), t was the testing time (s), A was the permeable film area (m2), and ΔP was water vapor pressure difference on both sides of the film (1583 Pa at 25℃).
2.4.3. Analysis of film color and transparency
Verify the reflection of the color of the film. The lightness (L*), red (a*) and yellow (b*) values of the film samples were determined using a chromameter (UPG-722 Visible spectrophotometer, Beijing Uber General Technology Co., Ltd, China). Before the measurement, the standard plate is calibrated (L* = 103.98, a* = -5.80, b* = 9.25), each sample was measured three times, and the results were averaged.
The film samples were cut into rectangles (10 mm×45 mm) and posted on the inner wall of the petri dish. Empty dishes were used as a reference, and sample absorbance values were measured at 600 nm. Each sample was measured three times, and the results were averaged. Opacity was calculated as following Eq.
$$Opacity=\frac{{A}_{600}}{\text{X}}$$
4
where A600 is the absorbance at 600 nm, and x is the film thickness (mm).
2.4.4. Scanning electron microscopy
Cut the composite film into small rectangles (4 cm × 6 cm), and use liquid nitrogen to break it. Micro scanning was performed using a type Gemini 300 scanning electron microscope (S-4300, Hitachi Co., Japan), sweep voltage is 2.00 kV, electricity is 64.0 µA. The surface and cross-sections of the film were observed and recorded.
2.4.5. Fourier transform infrared (FT-IR) spectroscopy
In this experiment, to study the chemical structure of the film, FT-IR spectra were conducted using an FT-IR/NIR (Spotlight 400, Perkin Elmer Co., USA). Infrared spectroscopy of films using the method of Wang et al. (2018) After placing the balanced dried film onto the ATR attachment, the test temperature was 25℃, in the wavenumber range from 4000 to 650 cm− 1 at room temperature, the resolution was 4cm− 1 and the number of scans 32.
2.4.6. X-ray diffraction
The XRD (SmartLab, Rigaku Co., Japan) analysis of the films was carried out using the method of Ilyas et al. (2018) using CuKα radiation, the XRD scan ranged from 5–80°(2θ), and the scan rate was 2° / min.
2.4.7. Differential scanning calorimetry (DSC) analysis
Thermal stability analysis was performed using differential scanning calorimetry. The 4mg dried film samples were sealed in an aluminum crucible. The empty aluminum crucible acts as a control group and takes the nitrogen gas as the protective gas. The protective gas flow rate is 20 mL/min. The heat-up rate was 10℃ / min. The differential scanning calorimetry analysis is performed in a temperature range of 20℃ to 250℃.
2.5. Assay of the antioxidant and antibacterial activity of films
2.5.1 Total phenol content
The total phenol content of the film was determined by the Folin phenol method with slight modifications (Chilczuk et al., 2020). Place 125 mg of film sample in 15 mL distilled water for 24h to get film immersion, then take 0.1 mL of film immersion, 7 mL of distilled water, 0.5mL of formaldehyde and add it to 50 mL conical bottle and shake it gently for 8 min. The composite solution was subjected to darkness for 2 h by adding 1.5 mL Na2CO3 (10% wt) and 0.9mL distilled water. The absorbance of the composite mixture was measured by UV spectrophotometer at 765 nm. Configure an aqueous gallic acid solution with a concentration of between 0–15 µg/ mL, and at the absorbance at 765 nm, and draw the standard curve, the standard curve was obtained (y = 0.117x + 0.0171, R2 = 0.9995). Each sample was measured in triplicate and averaging. The total phenol content of the model is indicated as mg gallic acid equivalent (GAE) per gram of dry substance (GAE mg/DWg).
2.5.2 DPPH free radical scavenging activity of films
The film sample (10 mg) was mixed with a methanol DPPH solution (1.5 mL, 0.2 mM). After that, the mixture was placed into a dark chamber for the reaction to avoid light for 0.5 h at room temperature. The absorbance of the mixture was determined at 517 nm (UV), and the DPPH methanol solution was used as a control. Each sample was measured three times, and the results were averaged. The formula for calculating the DPPH free radical scavenging rate is:
$${k}_{DPPH}=\frac{{A}_{C}-{A}_{s}}{{A}_{C}}\times 100\%$$
5
where kDPPH is the DPPH free-radical scavenging ability at 517 nm, Ac is the absorbance of the control group at 517 nm, and As is the absorbance of samples at 517 nm.
2.5.3 ABTS free radical scavenging activity of films
The film ABTS radical performance assay was slightly modified using the method of Riaz et al. (2019). The film sample (10 mg) was mixed with the ABTS free radical working solution, and the light is avoided for 6 min at room temperature. The absorbance of the mixture was determined at 734 nm, and the absorbance of the mixture was determined at 734 nm. Afterwards, the absorbance of the mixture was determined at 734 nm. Acetic acid buffer solution was used as a blank control. Each sample was measured three times, and the results were averaged. The formula for calculating the DPPH free radical scavenging rate is:
$${k}_{ABTS}=\frac{{A}_{C}-{A}_{s}}{{A}_{C}}\times 100\%$$
6
where kABTS is the ABTS free-radical scavenging ability at 734 nm, Ac is the absorbance of the control group at 734 nm, and As is the absorbance of samples at 734 nm.
2.5.4 Antimicrobial activity
The antimicrobial activity of the films is determined by disk diffusion experiments. The expanded E. coli and S. aureus strains were diluted tenfold in sterile saline as the initial bacterial solution. The film samples were made into a 7 mm diameter wafer, and pieces were placed in a culture medium containing 0.1 mL of bacteria. The medium was inverted in a 37 ± 1℃ incubator and fostered for 24 h. The diameter of the film antibacterial coil was measured with a cursor caliper, each sample was measured three times, and the results were averaged. The antimicrobial properties of the film were indicated by measuring the antibacterial coil size.
2.5.5 Determination of peroxide value
The film samples were coated with 5 g of solid lard, and the film was heat-sealed, accelerating oxidation in a 60℃ drum air drying box. Piece were sampled at every 24h interval and measured continuously for 7 d. And each piece was measured three times, and the results were averaged. The POV value was determined as described in GB5009.227- -2016.
2.6 Statistical Analysis
All experiments were performed in triplicate independently, and the results are expressed as the mean ± standard deviation. Differential significance analysis is performed using Duncan's in the software SPSS 25 system. Drawing was conducted using the Origin 8.5 software. Differences were considered statistically significant if p <0.05.