Test reagents
Main reagents: chitosan (deacetylation degree > 90%), thyme essential oil (analytically pure), glycerin (analytically pure), acetic acid, polyvinyl pyrrolidone (PVPP), thiobarbituric acid (TBA), polyethylene glycol 6000 (PEG 6000), trichloroacetic acid (TCA), sodium hydroxide, guaiacol, glacial acetic acid, oxalic acid, anhydrous sodium acetate, traton X-100, hydrogen peroxide (30%) are all purchased from Tianjin Deen Chemical Reagent Co., Ltd. Company.
Instruments and equipment
Main instrument: FA1004 electronic balance, Shanghai Shunyu Hengping Scientific Instrument Co., Ltd. Research Plus handheld pipette from Germany’s Abend AG. DF-101 S Constant Temperature Heating Magnetic Stirrer Gongyi Yuhua Instrument Co., Ltd. JC-QXS Ultrasonic Cleaning Machine Qingdao Juchuang Environmental Protection Group Co., Ltd. 2020-0 desktop drying oven Beijing Yongguangming Medical Instrument Co., Ltd. SW-CJ-1D Ultra Clean Workbench Suzhou Fenjing Purification Equipment Co., Ltd. HD-100 Constant Temperature and Humidity Chamber Dongguan Haida Instrument Co., Ltd. YHT112924 Digital Thickness Gauge Shenzhen Yuanhengtong Technology Co., Ltd. YT-L intelligent electronic tensile testing machine from Jinan Zhongce Electromechanical Equipment Co., Ltd., GY-4 digital fruit hardness tester from Zhejiang Topyun Agriculture Technology Co., Ltd., LH-B55 refractometer from Hangzhou Luheng Biotechnology Co., Ltd., TGL-16 high-speed freeze centrifuge from Sichuan Shuke Instrument Co., Ltd., and UV759CRT UV visible spectrophotometer from Shanghai Youke Instrument Co., Ltd.
Preparation of Chitosan/thyme essential oil composite film
A certain mass fraction of chitosan was weighed and added into the acetic acid solution, a certain mass fraction of glycerin and thyme essential oil were added respectively, the DF-101 S constant temperature was maintained while heating magnetic stirrer to heat the solution to 60 ℃, and stir it for 2 h at a speed of 200 r/min to ensure that all solutes were evenly mixed and fully dissolved in the solvent, and then ultrasonic vibration was carried out for 30 min, finally the solution was spread on a polyethylene plate (culture dish 150 mm × 150 mm), and dried for 48 h at room temperature 25 ℃ to form a film. The environmental conditions of the constant temperature and humidity cabinet were 25 ℃, 55% relative humidity, and the obtained sample were stored under such condition.
Single factor experiment
Single factor test was carried out with thyme essential oil, chitosan and glycerin as the influencing parameters. Based on the influence of each parameter level on the tensile strength and elongation at break of the composite film, the relevant parameter mass fraction was determined. Table 4 displays the specific film liquid composition and mass fraction.
Response surface test design
Combined with the single factor test results, the tensile strength and elongation at break were selected as response values, and the response surface test was designed with three factors of chitosan mass fraction, glycerin mass fraction and thyme essential oil mass fraction. The response surface test factors and level design were shown in (Table 5).
Determination of properties of Chitosan/thyme essential oil composite film
Determination of mechanical properties
According to GB/T 1040.3–2006, the composite film was cut into rectangular specimens with a size of 100 mm × 20 mm, and subjected to equilibrium treatment for 24 h (equilibrium conditions were temperature of 25 ℃ and relative humidity of 26%). The intelligent electronic tensile testing machine was used for testing at a speed of 50 mm/min. Each group of specimens was subjected to 6 parallel tests. The tensile strength and elongation at break were calculated according to Eqs. 1 and 2, respectively:
$$\:TS=\frac{F}{dh}$$
(1)
In the formula: TS is the tensile strength of the film, MPa. F is the maximum tensile force experienced when the film breaks, N. d is the material thickness, mm. h is the material width, mm.
$$\:EB=\frac{{L}_{m}-{L}_{0}}{{L}_{0}}\times\:100\%$$
(2)
In the formula: EB is the elongation at break,%. Lm is the fracture distance of the specimen, mm. L0 is the initial distance of the specimen, mm.
Thickness
A digital thickness gauge was adopted and six points of the test sample were randomly selected for thickness measurement. The control rod of the digital thickness gauge was pressed, the test sample was placed on the lower measuring head, the control rod was released to make the upper measuring head press tightly against the surface of the sample, then the result was recorded, in millimeters.
Water vapor permeability
According to GB/T 1037–2021, the water vapor permeability of composite films was determined using the weight gain method. 10 g of color changing silicone gel was placed into a breathable cup, then the sample was cut into a circular piece with a diameter of 80 mm and covered in the middle of the cup. The distance between the silicone surface and the sample surface was 3 mm. Sealant was used to ensure that the circular piece was tightly bonded to the composite film10. Next, the initial weight of the circular piece was weighed. All sealed samples were placed in a chamber with constant temperature and humidity (temperature 38 ℃, relative humidity 90%) for 72 h, then taken out every 24 h, shaken well, and weighed. The relevant test data were recorded, and 3 parallel tests were conducted on each sample. The water vapor transmission rate was calculated according to Eq. (3), with the unit being g/h·m2.
$$\:WVT=\frac{\varDelta\:G}{\varDelta\:ts}$$
(3)
In the formula: WVT is the water vapor transmission rate, g/h·m2. ∆G is the increment within time t, g. ∆t is the increment time, h. s is the sample area, m2.
Solubility
The composite film was cut into square samples with dimensions of 20 mm × 20 mm, and placed in a drying oven at 50 ℃ for 24 h of drying treatment, then cooled to room temperature and the samples were weighed. The sample was dissolved in a beaker containing 50 mL of distilled water at a temperature of 23 ° C for 24 h, and dried in a 50 ° C drying oven for 24 h, then cooled to room temperature before weighing. The calculation of water-soluble Ws (%) is:
$$\:{W}_{s}=\frac{{m}_{1}-{m}_{2}}{{m}_{2}}\times\:100\%$$
(4)
In the formula: m1 is the mass of the film after drying in its initial state, g. m2 is the mass of the film after absorbing water and finally drying, g.
Antibacterial activity
Escherichia coli was selected as the experimental strain. About 20 mL of culture medium was poured into the sterilized culture dish, and remained horizontal for solidification. Then, a sterile coating rod was used to evenly spread the 0.1 mL bacterial suspension onto the surface of the culture medium. Composite films were prepared with appropriate sizes and placed on the surface of the culture medium. The cross method was adopted to measure the diameter of the antibacterial zone, and to obtain the strength of the antibacterial ability through the diameter size.
Antioxidant capacity
DPPH radical scavenging ability assay: Film was weighed at 100 mg and placed in a test tube. Then placed in 10 mL of 10 and 95% ethanol solution, respectively, and at room temperature and in the dark for 2 h. 1mL of soaking solution with 1mL of DPPH solution (0.2 mmol/L) were shaked and mixed, and standed in the dark for 30 min. 1 mL of 10 and 95% ethanol were mixed instead of the extraction solution with 1 mL of DPPH solution, and measured the absorbance value at 517 nm. The calculation formula was shown in Eq. (5).
$$\:DPPH\:\text{r}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{r}\text{a}\text{t}\text{e}=\frac{{A}_{1}-{A}_{0}}{{A}_{1}}\times\:100\%$$
(5)
In the formula: A1 is the absorbance value of the reference sample. A0 is the absorbance value of the sample.
Performance characterization of Chitosan/thyme essential oil composite film
Scanning electron microscope
The surface and cross-sectional structure of CTS/TEO composite film and CTS single film was observed by using a scanning electron microscope. The sample was cut into 10 mm × 10 mm, the surface and cross-section of the film was fixed with conductive tape, amplified with a 5 kV acceleration voltage to observe its surface and cross-section morphology.
Fourier transform infrared spectroscopy
FTIR spectrometer was used to measure the infrared spectra of CTS/TEO composite film and CTS single film. The thin film was tested by using ATR mode with a scanning wave number of (400–4000) cm−1, a scanning frequency of 32 times per second, and a resolution of 4 cm−1.
Characterization of preservation effect of Chitosan/thyme essential oil composite film on blueberry preservation
Blueberry fruits of the similar size, maturity, and without mechanical damage to the skin was selected as test samples. The samples were wrapped with chitosan/thyme essential oil composite film (CTS/TEO group) and chitosan single film (CTS group) respectively, and the fruits without the film were set as the blank control group (CK group). The preservation effect of blueberry fruits was evaluated by weight loss rate, hardness loss rate，content of soluble solids, anthocyanin content, Vitamin C content, Malondialdehyde content and Peroxidase activity. The blueberries used in this study were all purchased from a local fruit wholesale market.
Weight loss rate
The weighing method was used to study the weight loss rate of blueberry fruits under different storage conditions. Each group selected 6 samples, which were labeled before and after storage and weighed at intervals of 1 day to ensure the accuracy of the data. The weight loss rate was calculated, as shown in Eq. (6), the experiment were repeated three times, and the average value was recorded.
$$\:Weight\:loss\:rate=\frac{{m}_{1}-{m}_{2}}{{m}_{2}}\times\:100\%$$
(6)
In the formula, m1 is the initial weight of blueberries, g. m2 is the weight of blueberries after weight loss, g.
Hardness loss rate
The precise determination of blueberry hardness was carried out using a high-precision GY-4-J digital fruit hardness tester. The area exceeding 1 cm² on the blueberry peel was cut clean, and then an 11-mm-diameter probe was aimed at the blueberry to be tested. The blueberry was pressed vertically at a constant speed until it was inserted about 10 mm into the fruit, and the hardness data was recorded and processed. As shown in Eq. (7), the hardness loss rate of blueberries was calculated and 3 experiments was conducted to obtain the average value.
$$\:Hardness\:loss\:rate=\frac{{n}_{0}-{n}_{1}}{{n}_{0}}\times\:100\%$$
(7)
In the formula, n0 is the initial hardness, N/cm2. n1 is the hardness after storage, N/cm2.
Content of soluble solids
The peeled blueberry pulp was ground into juice, the measuring head was cleaned with distilled water, and a dropper was used to drip the juice into a handheld refractometer for measurement and data recording. The procedure was repeated 3 times to get the average.
Anthocyanin content
The pH difference method was used to determine the anthocyanin content (AYC) of blueberries. Blueberry fruit was quickly ground, weighed 1 and diluted. Solution was transferred to a 25 mL volumetric flask, added 75% acidified ethanol (1% HCl) to make up to the volume, soaked at room temperature for 2 h, and the filtrate used for measurement. 4.5 mL of potassium chloride/hydrogen chloride buffer (pH = 1.0) and citric acid/disodium hydrogen phosphate buffer (pH = 4.5) were added in centrifuge tubes, respectively. Then 0.5 mL of anthocyanin filtrate was added to the buffer and distilled water as a blank control. At last, absorbance values were measured at 510 and 700 nm, and calculated the anthocyanin content in blueberries according to formulas 8 and 9.
$$\:\varDelta\:\text{A}={({A}_{510nm}-{A}_{700nm})}_{\text{p}\text{H}1.0}-{({A}_{510nm}-{A}_{700nm})}_{\text{p}\text{H}4.5}$$
(8)
$$\:\text{A}\text{Y}\text{C}(\text{m}\text{g}/\text{g})=\varDelta\:\text{A}\times\:\text{M}\times\:\text{V}\times\:\text{F}/{\upepsilon\:}\times\:\text{m}$$
(9)
In the formula: A represents absorbance at different wavelengths in different buffer solutions. M is the relative molecular weight of cyanidin-3-glucose, 449.2 g/mol. V is the volume of the extraction solution, mL. F is the dilution factor of the extraction solution. The molar absorptivity of cyanidin-3-glucose is 26,900 L/moL·cm. M is the sample mass, g。.
Vitamin C (Vc) content
According to Khafid’s method60, standard and reference solutions were prepared to obtain the linear regression Eq. 10 g of blueberry pulp was peeled and added 10 mL of 5 g/L oxalic acid solution, followed by grinding it into a paste in an ice bath in a mortar. Then distilled water was taken to 100 mL, and set aside. 2 mL of filtrate was taken and added to a quartz colorimetric dish. Compared with 2 mL of 0.5 g/L oxalic acid solution, absorbance values were measured at a wavelength of 243 nm and substituted into the equation to obtain the ascorbic acid content. Calculate the Vc content in blueberries according to formula 10.
$$\:{V}_{C}(mg/100g)=\frac{V\times\:m}{{V}_{s}\times\:m\times\:1000}\times\:100$$
(10)
In the formula: Vc is the content of vitamin C, mg/100g. M is the mass of ascorbic acid, mg. M is the mass of the sample, g. V is the volume of the filtrate after constant volume, mL. Vs is the volume of the filtrate required for measurement, mL。.
Malondialdehyde (MDA) content
The thiobarbituric acid colorimetric method was used for determination. The peeled blueberry pulp was weighed and ground into a liquid, then centrifuged in a high-speed freeze centrifuge until turbidity was eliminated, and the supernatant was collected for enzyme extraction. The enzyme extract (1.0 mL) was mixed with 1.0 mL of 0.67% thiobarbituric acid (TBA) solution to form the test group, while the control group was prepared by adding 1.0 mL of 100 g/L trichloroacetic acid (TCA) solution to 1.0 mL of 0.67% TBA solution. The mixtures were heated at 95 ℃ for 15 min, then cooled in an ice water bath to room temperature, and the absorbance values were measured at 450 nm, 532 nm, and 600 nm, respectively. Based on the data obtained, the amount of MDA per gram of blueberries was calculated using formula 11.
$$\:MDA=\frac{\left[6.45\times\:({OD}_{600}-{OD}_{532}) -0.56\times\:{OD}_{450}\right]\times\:V}{{V}_{s}\times\:m}$$
(11)
In the formula: MDA represents the content of malondialdehyde, µmol/g. V is the total volume of the sample extract, mL. Vs is the volume of the sample extract required for measurement, mL. m is the sample mass, g。.
Peroxidase (POD) activity
Accurately weigh 2.5 g of ground sample, add 5 mL of 0.2 mol/L phosphate buffer solution (PBS, pH 6.5), freeze centrifuge at 10,000 r/min and 4 ℃ for 20 min, and extract the supernatant. Take 50 µ L of supernatant and add 0.15 mL of 10 g/L guaiacol, 0.15 mL of hydrogen peroxide (volume fraction 1%), and 2.66 mL of PBS solution (pH = 6.5). Shake well and quickly place it in a UV visible spectrophotometer. Record the changes in absorbance of the reaction system at 470 nm for 5 min, every 20 s. An increase of 1 in absorbance per minute per gram of sample is considered as one unit of activity (U). Replace the supernatant with distilled water for comparison. Calculate the POD enzyme activity according to formula 12.
A ground sample of 2.5 g was accurately weighed, and 5 mL of 0.2 mol/L phosphate buffer solution (PBS, pH 6.5) was added. The mixture was subjected to a freeze centrifuge at 10,000 r/min and 4 ℃ for 20 min to extract the supernatant. A volume of 50 µL of the supernatant was taken and combined with 0.15 mL of 10 g/L guaiacol, 0.15 mL of hydrogen peroxide (with a volume fraction of 1%), and 2.66 mL of PBS solution (pH = 6.5). The solution was thoroughly mixed and promptly placed into a UV-visible spectrophotometer. The changes in absorbance of the reaction system at 470 nm were recorded over a period of 5 min, with measurements taken every 20 s. An increase of 1 in absorbance per minute per gram of sample was defined as one unit of activity (U). For comparison, the supernatant was replaced with distilled water. The POD enzyme activity was calculated using formula 12.
$$\:U=\frac{{\varDelta\:A}_{470}\times\:V}{{V}_{S}\times\:m}$$
(12)
In the formula: \(\:{\varDelta\:A}_{470}\) represents the change in absorbance value per unit time. V is the total volume of enzyme extract, mL. Vs is the required volume of enzyme extract for measurement, mL. m is the sample mass, \(\:{g}_{o}\).
Statistical analysis
The experimental data was processed using Design Expert 13.0 software and one-way ANOVA. The experimental data processing was plotted using Origin 8.0 software.
