1. Introduction
Owing to the growing importance of food preservation, the methods used for achieving safe food storage have increased significantly in variety. For example, use of a suitable material for food packaging can decrease the food’s deterioration rate, thereby extending its shelf life [
1]. Active food packaging is a novel concept, meaning that it can extend the shelf life of food through the interaction between the environment provided by packaging materials and the product during the packaging process [
2]. One of the most promising approaches is antimicrobial packaging, which inhibits the growth of microorganisms on food surfaces by releasing antimicrobial components. Food packaging material creates a low O
2 and high CO
2 gas environment between the external conditions and the food by forming a film on the surface of food, thereby reducing the exchange rate of the gas or substances used to extend the shelf life. Biodegradable packaging is a new material that replaces synthetic polymers with biopolymers to protect the environment [
3]. This biopolymer-based packaging is derived from natural and renewable agricultural and marine sources [
1] such as chitosan, starches, and carboxymethyl cellulose (CMC).
Derived from cellulose, CMC polysaccharide is an important agricultural product considered to be safe for human consumption [
4]. CMC is widely applied in industries such as food, cosmetics, and pharmaceuticals [
5]. In addition, CMC has excellent biodegradability and hydrophilic properties as well as the ability to form transparent films [
6,
7]. Several studies have shown that the addition of CMC to composite film significantly improves some of film’s properties [
8,
9]. Moreover, CMC has shown strong performance as a desirable matrix to form edible films. Perishable food products need to be protected during their preparation, storage, and distribution to extend their shelf lives [
10]. Because of long-term exposure to air, the food surface is easily infested with microorganisms that accelerate deterioration of the food. To solve this problem, CMC can be combined with an antimicrobial agent. Considering the consumer demand for natural and healthy products, essential oils have shown promise for this application.
In recent years, a novel cultivated grape variety has gradually entered the public eye-“Shine Muscat” grape, which was originated in Japan [
11]. Shine Muscat is breeded by crossing Akitsu-21 (
Vitis labruscana Baily × V. vinifera) and “Hakunan” (
V. vinifera) [
12]. The pericarp of Shine Muscat grapes is green and has a sweeter taste and they have a longer storage time compared with other grape varieties. They have thin skin and are seedless, so they can be eaten without peeling [
13,
14]. Therefore, it has been favored by consumers since it was first introduced into the Chinese market. Due to the limited planting area of Shine Muscat grapes in China, long-distance transportation is required, and the price is relatively high compared to similar products. Water loss [
15], mechanical injury [
16] and fungal decay [
17] of fruits are common conditions during transportation. The well-known long-distance storage methods are low-temperature storage, inflatable bag anti-collision packaging and spraying of chemical preservatives. The treatment methods of postharvest grapes include physical methods, such as UV irradiation and sonication; biological methods, such as using yeast species to inhibit
B. cinerea [
18]. Moreover, the coatings were prepared from natural polymers can protect grapes [
19,
20]. In various studies, SO
2 synthetic fungicide can also preserve grapes, but the residues of chemical preservatives can harm human health [
21], so the development of sustainable safe preservatives has become the mainstream of research.
Essential oils are volatile, natural products that are widely used for bacteriostatic, medicinal, and antiseptic purposes as well as for food preservation [
22]. These natural, antimicrobial agents are extracted from plants and are composed of numerous chemical compounds [
3]. As natural food preservatives, some essential oils can be added to edible film to prevent the growth of microorganisms [
23]. Moreover, several studies suggest that essential oils can be combined with CMC to extend the shelf life of fruit and to improve the product’s antimicrobial properties [
24,
25,
26].
Cunninghamia lanceolata, or Chinese fir, is widely planted in southern China [
27,
28] and plays an important role the country’s timber industry [
29]. However, the sawdust produced from Chinese fir is usually abandoned or burned, which does not make use of its benefits [
28]. To utilize this resource, essential oil can be extracted before this wood byproduct is discarded. Several studies have shown that Chinese fir essential oil (CFEO) has numerous biological effects including antibacterial, antifungal, anti-mite, and anti-mosquito properties [
30,
31]. In particular, its excellent antibacterial and antifungal properties give CFEO potential as a natural food preservative.
To our knowledge, no study has been conducted thus far on food preservation using CFEO. Therefore, the objectives of this study are to investigate the properties of edible film by combining various concentrations of CFEO with CMC and to evaluate the feasibility of CMC-CFEO edible film by analyzing its characteristics. In addition, the antimicrobial activity of this edible film is studied. This work is of great significance because it is the first to provide a theoretical basis for food preservation using CFEO.
2. Materials and Methods
2.1. Materials
The sawdust of Chinese fir used in this study was produced in Anhui Province (China). The CMC was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and Tween 80 and glycerol were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Staphylococcus aureus (ATCC6538), Penicillium citrinum (ATCC1109), Escherichia coli (ATCC25922), and Bacillus subtilis (ATCC6633) were obtained from Shanghai Luwei Bio-Technology Co. Ltd. (Shanghai, China). Lysogeny broth (LB) and potato dextrose agar (PDA) bouillon were prepared at the Biological Laboratory, Nanjing Forestry University (Nanjing, China). Shine Muscat grapes were purchased from the Yunnan Shine Muscat grapes planting base (Yunnan, China).
2.2. Chinese Fir Essential Oil Extraction
The CFEO was extracted by steam distillation. In this process, 105 g of Chinese fir sawdust with a moisture content of 99.8% was put in a two-necked flask similar to a breathable partition. Subsequently, 600 mL of distilled water was placed in a different flask. Both flasks were interlinked up and down and heated at 100 °C for 4 h of extraction until no more essential oil could be obtained. After volatilization, the CFEO product was collected by adding about 5 mL ether. The remaining solution was CFEO after the ether evaporated by using rotary evaporators at 40 °C. All of the data were confirmed by a preliminary experiment.
2.3. Preparation of Films
The films were prepared as described by Dashipour et al. with some modification [
25]. The CMC solution was prepared by dissolving 1 g CMC in 100 mL distilled water (1%
w/v) under constant magnetic stirring at 70 °C for 40 min until it was complete dissolution was achieved. Afterward, 0.5 mL glycerol (0.5%
w/v based on the CMC) was added, and the mixture was stirred continuously for 10 min. After cooling, the formed dispersion was cast in glass plates of about 64 cm
2 in area for subsequent use as control film. In addition, CFEO as an antimicrobial agent was added to the CMC solution to obtain final concentrations of 1%, 2%, and 3% (
v/v). Tween 80 was added to the CFEO at a ratio of 1:10, respectively, and the mixture stirred for 30 min. Afterward, an ultrasound machine (GBP-USC201L, CSIC715, Zhejiang, China) was used at 750 W for 5 min to create uniform mixtures. The blended emulsion was prepared.
Subsequently, the steps for preparing the edible films were described below. These solutions were then cast in the plates after air bubbles were removed. All of the films were dried at 35 °C for about 24 h and were then stored in a desiccator at 25 °C and 55% relative humidity (RH) for preservation.
2.4. Gas Chromatography–Mass Spectrometry Analysis Conditions
The composition was analyzed by using a gas chromatograph–mass spectrometer (GC-MS; ISQ, Thermo-Scientific, Waltham, MA, USA) equipped with a DB-5ms column. Helium was used as a carrier gas at 0.6 mL/min. The oven temperature was kept at 80 °C for 3 min; afterward, the temperature was increased to 280 °C at a rate of 15 °C/min and was kept constant for 3 min. The injection volume was 0.2 μL, and the injector and transfer line temperatures were 250 and 280 °C, respectively. For the MS conditions, the electron impact (EI) ion source temperature was 230 °C; the quadrupole temperature was 150 °C; the EI+ mode was 70 eV; and the mass scan range was 33–450 u.
2.5. Physical Properties of Prepared Films
The film thickness was measured by using a thickness meter (J-DHY03A, Changjiang Paper Instrument Co., Ltd., Sichuan, China) with 0.001 mm sensitivity. The results were obtained by selecting the average of at least five random locations for each film [
32].
The water solubility (WS) was determined following the method of Rincon [
33]. The films were placed into an oven at 110 °C to obtain the original constant weight (
) and were then immersed in 50 mL distilled water for 6 h at room temperature under constant magnetic stirring. Finally, the insoluble films were filtered and dried in an oven to obtain the final constant weight (
). The following formula was used to calculate the WS:
2.6. Color Properties of Prepared Films
The lightness (
L*), red/green coordinate (
a*), and yellow/blue coordinate (
b*) color parameters were obtained by using a white colorimeter (ZB-A, Paper State Automation Co., Ltd., Hangzhou, China). A white standard plate was used as the background (
L = 73.04,
a = −2.00,
b = 2.61). The film was removed from the desiccator, and at least three areas were selected for at least three measurements each. The following formula was used to calculate the total color difference (
:
where
);
;
;
L,
a, and
b are standard plate color parameter values; and
L*,
a*,
b*are film color parameter values.
2.7. Characterization of Prepared Films
The functional groups of the films were determined by Fourier transform infrared spectrometry (FTIR; VERTEX 80 V, Bruker, Ettlingen, Germany). The films were placed on attenuated total reflection (ATR) crystal material to absorb light directly for use with an ATR system. No sample was used for the test background. In total, 16 samples were scanned at a resolution of 4 cm−1 in a wavenumber range of 4000–500 cm−1.
The properties of thermal stability were identified by using a thermogravimetric analyzer (TGA; 209 F1, Netzsch, Selb, Germany). The temperature was increased from 25 to 700 °C at a constant rate of 25 °C/min, and N2 gas at a flow rate of 20 mL/min was used as protective gas.
The thermal parameters were measured by differential scanning calorimetry (DSC; 214, Netzsch, Selb, Germany). 6 mg of film pieces were put in a standard aluminum pan. The temperature was increased from 50 to 400 °C at a constant rate of 10 °C/min. The following formula was used to calculate the crystallinity index (Xc):
where
is the fusion enthalpy of the blended films,
is the fusion enthalpy of CMC film. The morphologies of the films were observed by environmental scanning electron microscopy (ESEM; Quanta 200, FEI, Hillsboro, OR, USA) at an accelerating voltage of 20 kV.
2.8. Mechanical Properties of Prepared Films
A universal tensile tester (SANS, MTS Co., Ltd., Minneapolis, MN, USA) was employed to measure the mechanical properties of the films, including the tensile strength (TS), elongation at break (EB) and elastic modulus (EM). Following the Plastics-Determination of Tensile Properties of Films test method (GB13022-1991), all the films were cut to dimensions of 10 cm in length × 1 cm in width. The two ends of the film strips were fixed to the tension machine with an initial separation of 40 mm, and the cross-head speed was 10 mm/min. It is worth noting that all of the film strips were previously equilibrated at conditions of about 50% RH and 25 °C for two days:
where
is the maximum tensile force when the film breaks,
A is the cross-sectional area of the film, ∆
L is the amount of change in film length when stretched,
L is the original length of the film.
2.9. Antimicrobial Effects of CFEO and Films
The antimicrobial effects were tested by applying disc diffusion following the method of Poaty with some modification [
34]. Specifically, the bacteria and fungus suspensions were mixed with LB agar and PDA media, respectively, at about 55 °C, and each mixture was poured into a Petri dish (
d = 90 mm). After solidification, filter paper discs (
d = 6 mm) impregnated with the sample were placed on the surface of the agar medium. The plates were incubated at temperatures of 37 and 28 °C for culturing the bacteria and fungi, respectively, for 24 or 48 h in the appropriate incubation chamber. The antimicrobial activities were evaluated by comparing the diameters of the inhibition zones.
2.10. Characterization of Shine Muscat Grape
The blended CMC-CFEO emulsion was coated on plastic wrap (10 cm × 10 cm) and air dried, forming a thin film on the surface. It was wrapped on Shine Muscat grapes, and then the fruits were stored at 25 °C and 40%–60% RH. Each treatment group was replicated five times.
The grapes were weighed with an analytical balance (BSA123S, Satorius Scientific Instruments Co., LTD, Beijing, China) and the weight was recorded every three days. There were 5 grapes in each group and the average value was calculated. The following formula was used to calculate the weight loss rate:
where
was initial weight;
was final weight.
Each group selected 10 grapes of uniform size and observed the rot on the surface of the grapes every day. The following formula was used to calculate the decay percentage:
where
was the number of decayed fruits;
was the number of total fruits.
2.11. Statistical Analysis
IBM SPSS software (version 26, SPSS Inc., Chicago, IL, USA) was used for all experimental data analysis. In addition, one-factor analysis of variance was performed on the experimental data. The measurement results were tested at least three times, all of which were shown as the mean value ± standard error. A p-value < 0.05 indicated a significant difference.