Iron cobalt oxide body / two-dimensional nitride / chitosan composite antibacterial preservative film and preparation method and application thereof

Abstract

The application provides an iron cobalt oxide body / two-dimensional nitride / chitosan composite bacteriostatic preservative film and a preparation method and application thereof, and belongs to the technical field of chitosan preservative film material preparation.The two-dimensional nitride and the iron cobalt oxide body are combined with CS by adopting an ice bath ultrasonic method to prepare CM, and then a solvent casting method is adopted to prepare a CS / CM composite film with good performance.CS and CM have good compatibility, and the internal structure of the composite film is compact.The CS / CM composite film has excellent mechanical properties, ultraviolet blocking performance and thermal stability.In addition, the inhibition rate of the CS / CM composite film on staphylococcus aureus, escherichia coli and aspergillus niger is extremely high.In the preservative experiment of preserving qiufeng grapes by using the CS / CM composite film, the grapes still have high quality after being stored for 12 days.Therefore, the CS / CM is a good packaging material and can be widely applied to food preservation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preparation of chitosan preservative film materials, and particularly relates to an iron-cobalt oxide body / two-dimensional nitride / chitosan composite bacteriostatic preservative film and a preparation method and application thereof. BACKGROUND

[0002] With the change of dietary structure, people's demand for vitamins is increasing. Fruits and vegetables contain relatively rich vitamins, but fruits and vegetables are prone to rot, and fresh fruits and vegetables are easily contaminated by microorganisms during harvesting, transportation and storage, so the inhibition of bacteria and preservation has always been the pursuit of people.

[0003] Kyoho grapes are often used to supplement the vitamins needed by the human body because they are rich in vitamin A, vitamin B6 and vitamin C. In addition, Kyoho grapes are also rich in resveratrol, polymeric proanthocyanidins and catechins, which can play an antioxidant and cardiovascular disease prevention effect. However, at the same time, the rich nutrients of Kyoho grapes make them a good place for the growth and reproduction of many microorganisms (such as Staphylococcus aureus, Escherichia coli and Aspergillus niger), which can cause serious economic losses and food safety problems if not stored properly.

[0004] At present, the commonly used preservation methods for grapes include air conditioning preservation, heat treatment preservation and natural extract preservation, etc. These methods all have certain shortcomings. For example, the high carbon dioxide and low oxygen used in air conditioning preservation may cause abnormal metabolism and tissue damage of the fruit; heat treatment preservation is easy to cause fruit dehydration, discoloration and damage; the substances extracted by natural extract preservation usually have a strong odor, which affects the fruit's sensory properties. Therefore, in the field of grape preservation, there is an urgent need to develop a new type of time-saving, fast, low-cost and efficient preservation method.

[0005] The cost of the film preservation method is lower than that of other storage and preservation methods, and the production method is simple and stable, which is more suitable for popularization and application. Plastic film is currently the most common, but long-term use will cause serious harm to the human body and the environment due to its low degradability and harmful substances generated during decomposition. Therefore, the development of more green and environmentally friendly films is imperative.

[0006] Chitosan (CS) is the only alkaline polysaccharide in natural polysaccharides, and has good biodegradability, biocompatibility, non-toxicity and antibacterial properties, and is widely used in food, textile and agricultural fields. Studies have shown that the film prepared from CS as raw material has more excellent mechanical strength, barrier properties and structural stability compared with other films such as protein film and fat film. Although CS can interfere with the permeability of microbial cell membranes, its antibacterial performance needs to be improved.

[0007] At present, the antibacterial property of CS is often improved by combining it with other bioactive substances (polyphenolic substances, flavonoid substances, aromatic substances, etc.) to obtain better food preservation effect. For tea polyphenols and other polyphenolic substances, its stability is greatly affected by environmental factors, and after adding, the antibacterial property of the composite film is unstable. Catechin, as a commonly used flavonoid substance, has strong irritability and heavy bitter taste, which also affects its preservation of food. Commonly used aromatic substances such as plant essential oils have a strong odor, which can bring bad sensory evaluation to food when used for food preservation. Therefore, new composite materials need to be further developed.

[0008] Nanoplasma is a kind of artificially synthesized material with excellent stability and enzyme-like activity, which is an inorganic nanoparticle with enzyme-like activity in oxidation-reduction reaction. Therefore, the nanoplasma obtained by processing has unique performance and catalytic function. Nanoplasma has the advantages of convenient synthesis, high catalytic activity, high stability and low cost. It has been reported that nanoplasma can be used for food preservation. At present, the nanoplasma applied to food preservation composite film mainly includes metal nanoplasma (Ag and Fe, etc.) and metal oxide nanoplasma (TiO2, Fe3O4, CuO, etc.), but both of them have the problems of migration of packaging material and antibacterial property to be improved.

[0009] Chinese patent document CN 106883465 A (publication date: June 23, 2017) discloses a kind of chitosan composite preservative film, which is prepared by compounding modified nanocarbon crystal and chitosan to prepare preservative film, which improves the antibacterial property of chitosan preservative film, but its bactericidal performance still needs to be improved, and its antibacterial rate to escherichia coli and staphylococcus aureus only reaches more than 90%, at the same time, it does not have the function of free radical removal of enzyme, and the mechanical property is not high enough.

[0010] On the other hand, although nanoplasma has photocatalytic performance, peroxide mimic enzyme activity and hydrogen peroxide mimic enzyme activity, it can sterilize more efficiently, but because nanoplasma is a nanoparticle, it is easy to agglomerate, which leads to poor dispersion and affects the performance of the composite film.

[0011] Therefore, how to compound nanoplasma with chitosan film to provide a composite film material with good dispersion performance, excellent mechanical property and significant antibacterial effect has become a technical problem to be solved. SUMMARY

[0012] The present application is to solve the above technical problems, and provides a kind of iron cobalt oxide body / two-dimensional nitride / chitosan composite antibacterial preservative film and its preparation method and application. The technical purpose of the present application is to solve the problems of easy agglomeration of nanoparticles, poor dispersion and insufficient improvement of mechanical property in the process of compounding nanoplasma with chitosan to form film, and the performance of the composite film after film forming is not good, and the antibacterial property needs to be improved.

[0013] In order to achieve the above technical purposes, the present application adopts the following scheme to achieve:

[0014] The present application first provides a preparation method of a FeCo2O4 / MXene / chitosan composite antibacterial preservative film, comprising the following steps:

[0015] (1) lithium fluoride is added to hydrochloric acid, and two-dimensional nitride is synthesized by in-situ etching method with titanium aluminum carbide;

[0016] (2) FeCo2O4 is synthesized by hydrothermal method with iron chloride hexahydrate, cobalt chloride hexahydrate, sodium acetate and sodium hydroxide;

[0017] (3) the two-dimensional nitride and the FeCo2O4 are mixed in a weight ratio of 1:1, then dissolved in water, and ultrasonic treated under ice bath to obtain a FeCo2O4 / MXene composite nano-enzyme;

[0018] (4) chitosan and glycerol are dissolved in a 1% acetic acid solution by volume percentage to prepare a chitosan solution, wherein the mass percentage of chitosan is 3%; then the nano-enzyme obtained in step (3) is mixed with the chitosan solution to obtain a composite solution, and the volume ratio of the nano-enzyme is 5%. Then the composite solution after ultrasonic treatment is poured into a mold, dried to obtain a film, and cooled to room temperature to remove the film, thereby obtaining the composite antibacterial preservative film.

[0019] The above synthesis method provided by the present application uses FeCo2O4 (CFO) and two-dimensional nitride (MXene) as precursor substances to synthesize FeCo2O4 / MXene nano-enzyme (CM) with multiple simulated enzyme activities, and then successfully prepares a CS / CM composite antibacterial film by solvent casting method. The present inventors have found through a large number of explorations on the preparation process of the composite film that only when the concentration of the chitosan solution is prepared to be 3wt% and the concentration of the nano-enzyme solution is controlled to be 5vol%, the composite film with excellent mechanical properties, excellent dispersion performance, good film forming effect and significant antibacterial performance can be obtained.

[0020] As shown in the results of the embodiments of the present application, when the concentration of the nano-enzyme solution is controlled to be higher than 5vol%, the mechanical properties of the composite film decrease significantly, and only when the concentration of the nano-enzyme solution is controlled to be 5vol%, the film forming performance of the composite film is best.

[0021] In addition, the bacteriostasis performance of the composite film obtained by the scheme is extremely significant, compared with the CS film, the tensile strength, elongation at break and DPPH clearance rate of the composite bacteriostasis film are increased by 23.06%, 54.38%, 356.31%. The bacteriostasis rate of the composite bacteriostasis film to E. coli, S. aureus and A. niger in the dark condition is 96.2%, 96.8% and 92.2%; under the condition of light for 6h, the bacteriostasis rate is 99.7%, 99.8% and 97.1% respectively. The hardness, weight loss rate, titratable acidity (TA), soluble solids content, Vc content, POD activity and CAT activity of the CS / 5%CM composite bacteriostasis film group on the 12th day of preservation are 0.61 Kg / cm 2 , 13.5%, 0.332%, 13.3%, 4.47 mg / 100 g, 29 Ug -1 min -1 and 19 Ug -1 min -1 , the sensory and physicochemical properties change less than other groups, indicating that the composite bacteriostasis film can effectively reduce the loss of nutrients in food and achieve the purpose of prolonging the preservation period of food.

[0022] The application of CM to food preservation not only expands the application market of CM, but also fills the gap in food packaging. However, single nano-enzyme cannot be directly attached to the surface of food, and its dispersion with CS is extremely difficult, and the application solves the problem of dispersion difficulty and poor performance after film formation.

[0023] Further, the volume ratio of the chitosan solution to the nano-enzyme solution in step (4) is 19:1.

[0024] The volume ratio of the chitosan solution to the nano-enzyme solution in the application is also extremely critical for the performance of the composite film, as shown in the comparative example of the application, when the volume ratio of the two is less than 19:1, the film cannot be completed, and when the volume ratio of the two is greater than 19:1, the mechanical properties of the film after preparation will decrease significantly.

[0025] Further, the weight ratio of lithium fluoride to titanium carbide in step (1) is 1:1; the synthesis reaction condition is to react at 35℃ for 24h.

[0026] Further, the weight ratio of the iron chloride hexahydrate, cobalt chloride hexahydrate, sodium acetate and sodium hydroxide in step (2) is 18:19:90:25.

[0027] Further, the synthesis reaction condition in step (2) is to react at 180℃ for 12 hours.

[0028] Further, the hydrothermal method in step (2) is carried out in an ethylene glycol solution.

[0029] Further, the time of the ultrasonic in step (3) is 0.5 hours.

[0030] Further, the condition of the ultrasonic treatment in step (4) is ultrasonic treatment at 40℃ for 4 hours.

[0031] The second object of the present application provides the iron cobalt oxide body / two-dimensional nitride / chitosan composite bacteriostatic preservative film prepared by the method.

[0032] The third object of the present application provides the application of the iron cobalt oxide body / chitosan composite bacteriostatic preservative film in preparing fruit and vegetable bacteriostatic preservative materials.

[0033] The beneficial effects of the present application are as follows:

[0034] (1) The present application provides a method for preparing a CS / CM composite film by chitosan and nano-enzyme. The characterization results show that the CS / CM composite film has excellent mechanical properties, ultraviolet blocking performance and thermal stability.

[0035] (2) The CS / CM composite film prepared by the present application has extremely high inhibition rate on Staphylococcus aureus, Escherichia coli and Aspergillus niger. In the preservation experiment of Jufeng grapes, the grapes still have high quality after 12 days of storage. Therefore, CS / CM is a good packaging material and can be widely used in food preservation. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 UV-Vis absorption changes of different samples on MB (A) and RhB (B);

[0037] Figure 2 TEM images of CM (A-B); surface and cross-section SEM images of CS / 5%CM (C-D) and CS film (E-F);

[0038] Figure 3 AFM images (A); Ra and Rq parameters of different films (B);

[0039] Figure 4 XRD (A) and FTIR (B) images of different films;

[0040] Figure 5 UV-Vis absorption spectra (A), water contact angle (B) and DSC curves (C) of different films;

[0041] Figure 6 DPPH scavenging rate of different films;

[0042] Figure 7The effect of light and dark treatment on the growth of E. coli (A), S. aureus (B) and A. niger (C) for different films;

[0043] Figure 8 The changes of sensory score (A), hardness (B), weight loss rate (C), TA (D), TSS (E), Vc (F), POD (G) and CAT (H) during the preservation of grapes;

[0044] Figure 9 The images of grapes stored in CS / 5%CM (A), blank (B), CS (C) and PE (D);

[0045] Figure 10 The dispersion picture between CM and CS;

[0046] Figure 11 The case of CS and CM in the volume ratio of 24:1;

[0047] Figure 12 The antibacterial performance of the composite film;

[0048] Figure 13 The thermal stability performance of the composite film;

[0049] Figure 14 The ultraviolet light absorption performance of the composite film. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is described in detail below in combination with examples. It is necessary to point out that the following examples are only used to explain and illustrate the present application, and do not limit the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above description still fall within the protection scope of the present application.

[0051] Example 1

[0052] I. Experimental materials and methods

[0053] 1. Materials

[0054] Jumbo grapes were collected from a family farm in Hanyuan, LB broth and potato dextrose agar medium were purchased from Qingdao Hope Biotechnology Co., Ltd., trypsin soybean agar medium was purchased from Sigma-Aldrich, methyl red, bromocresol green and 1,1-diphenyl-2-trinitrobenzene hydrazine were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., E. coli (10536), S. aureus (29213) and A. niger used in this experiment were provided by ATCC, and other materials were purchased from the market.

[0055] 2. Preparation of CM nanozyme

[0056] (1) Synthesis of two-dimensional nitride (MXene) by in-situ etching method: 1 g of lithium fluoride is added to 20 mL of 9 mol / mL hydrochloric acid, and stirred in ice bath for 1 h; then 1 g of titanium aluminum carbide is added, and reacted at 35°C for 24 h, and the MXene powder is collected.

[0057] (2) Synthesis of iron-cobalt oxide (CFO) by hydrothermal method: 0.36 g of iron chloride hexahydrate, 0.38 g of cobalt chloride hexahydrate, 1.8 g of sodium acetate and 0.5 g of sodium hydroxide are dissolved in 40 mL of ethylene glycol solution, and then transferred into an autoclave, and reacted at 180°C for 12 hours. After washing with deionized water to remove impurities, the CFO powder is collected.

[0058] (3) Synthesis of nanoenzyme (CM) by ice bath ultrasonic method: 5 mg of MXene and 5 mg of CFO are dissolved in 10 mL of deionized water, and ice bath ultrasonic treatment is performed for 0.5 h to obtain nanoenzyme (CM).

[0059] 3. Photodynamic properties of MXene, CFO and CM

[0060] Methylene blue (MB) and rhodamine B (RhB) are used as ·OH trapping agents to detect the generation of ·OH. MB and RhB are mixed with the sample respectively, and treated with xenon lamp and dark respectively for 10 min. The absorbance is measured by ultraviolet-visible spectrophotometer (wavelength = 664 nm, 554 nm).

[0061] 4. Antibacterial activity of MXene, CFO and CM

[0062] Escherichia coli (ATCC 10536) and Staphylococcus aureus (ATCC 29213) are used as typical gram-negative bacteria and gram-positive bacteria. 0.5 mL of bacterial suspension is mixed with 0.5 mL of sample, and then irradiated under xenon lamp for 10 min. The control group is placed in the dark. The treated mixture is inoculated on agar medium plate by spread plate method. Incubate in a 37°C incubator for 24 h, and count the number of bacterial colonies.

[0063] 5. Preparation of CS / CM composite film

[0064] Chitosan (CS) and nanoenzyme (CM) are used as raw materials to prepare composite antibacterial film by solvent casting method, as follows:

[0065] CS (3 wt%) was dissolved in 70 mL of acetic acid solution (1 vol%) and stirred at room temperature for 4 h. Then, glycerol (1 wt%) was added, and stirring continued for another 4 h to obtain the CS solution. Different concentrations of CM solutions (1%, 3%, 5%, or 7%, vol%) were added to the CS solution, and the solution was sonicated at 40 °C for 4 h to remove foam. The composite solutions were then sequentially poured onto transparent glass plates (70 mL) and placed in an oven at 55 °C for 4 h to obtain a film. After cooling to room temperature, the film was peeled off the mold and stored at 25 °C and 50% relative humidity.

[0066] The preparation method of the single CS membrane is the same as the method described above, except that the CM solution is not added. The single CS membrane and the prepared CS / CM composite membranes with 1%, 3%, 5%, and 7% CM are named as follows: CS, CS / 1%CM, CS / 3%CM, CS / 5%CM, and CS / 7%CM, respectively.

[0067] 6. Morphological and structural analysis

[0068] The morphology and structure of CM were investigated using high-resolution transmission electron microscopy (HRTEM) at an accelerating voltage of 200 kV. The surface and fracture cross-section of CS and CS / 5%CM were observed using scanning electron microscopy. The composite films were cut into 3 mm × 3 mm pieces, fixed on a metal sample stage, and their X-ray diffraction (XRD) patterns were analyzed using a polycrystalline X-ray diffractometer equipped with Cu-Kα radiation. Spectra were recorded at 40 kV, 40 mA. Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 380 FTIR spectrometer equipped with a horizontally attenuated total reflectance (ATR) crystal. The range was 650–4000 cm⁻¹. 1 The film was scanned 32 times at the wavenumber and the spectrum was recorded. The surface structure of the film was characterized using atomic force microscopy, and the roughness of the film was measured. A transverse cross-section of the film was plotted using Gwyddion to observe its thickness distribution. The average surface roughness (Ra) and root mean square surface roughness (Rq) of the film were calculated using NanonScope. A comprehensive evaluation of the surface morphology of the film was then performed.

[0069] 7. Solubility, swelling rate, and water content

[0070] Cut the film to a size of (2cm × 2cm) and weigh it (M1). Dry the film at 105°C to a constant mass (M2). Determine the moisture content using the following formula (1):

[0071] Moisture content = (M1 - M2) ÷ M1 × 100% (1)

[0072] The film was cut into a size of (2 cm x 2 cm), and the film was dried at 105°C to a constant mass (M1). They were placed in a 50 mL beaker with distilled water, covered with plastic wrap, and stored at 25°C for 24 h. Next, it was dried at 105°C to a constant weight to obtain the final dry mass (M2). The solubility was calculated using the following equation (2):

[0073] Solubility = (M1 - M2) ÷ M1 x 100% (2)

[0074] After weighing the film, the film was placed in a 50 mL beaker with distilled water at 25°C for 24 h and weighed (M1). The surface of the wet film was dried with filter paper, and the wet film was weighed (M2). The swelling ratio was calculated using the following equation (3):

[0075] Swelling ratio = (M1 - M2) ÷ M1 x 100% (3) 8. Thickness and mechanical properties

[0076] The thickness of the film was measured using a micrometer with an accuracy of 0.01 mm, and the average value of 10 random points on each film was taken as the thickness of the film. The film was cut into a size of 6 cm x 2 cm and equilibrated at 25°C and 50 ± 1% RH for 48 h. The tensile strength (TS) and elongation at break (EB) of the film were measured using a digital analyzer. The results were taken as the average value of 10 random points on each film.

[0077] 9. UV blocking properties

[0078] The film was cut into a size of 6 cm x 2 cm and tightly attached to the inner wall of a test tube. The UV blocking properties of the film were determined by measuring the light absorption of the film in the wavelength range of 200-800 nm.

[0079] 10. Water vapor transmission rate and water contact angle

[0080] The water vapor transmission rate (WVT) of the film sample was tested as follows: the test temperature was 38°C and the relative humidity was 90%, and the WVT was calculated using the following equation (4):

[0081] WVT = (AM1 - AM2) ÷ A x t (4)

[0082] In the formula:

[0083] WVT - water vapor transmission rate, in units of grams per square meter per 24 hours [g / (m 2 ·24h)];

[0084] AM1 - difference between two measurements of the same test group, in units of grams (g);

[0085] AM2 - difference between two measurements of the same test combination of the blank sample, in grams (g);

[0086] A - active test area, in square meters (m 2 );

[0087] t - test time, in hours (h).

[0088] The water contact angle of the film surface was measured using an optical contact angle measuring device. 5 μL of distilled water was placed on the film surface to easily measure the hydrophobicity; the average value was taken after repeating three times.

[0089] 11. Thermal analysis

[0090] Differential scanning calorimetry (DSC) experiments were performed using a SIINT instrument. The film (approximately 5 mg) was placed in a crucible with an empty crucible as a reference. Inert gas (nitrogen) was added and the temperature was raised from 20 °C to 200 °C at a rate of 10 °C / min to measure the thermal stability of the film.

[0091] 12. DPPH scavenging rate

[0092] DPPH was dissolved in ethanol to prepare a 1 mg / ml solution. The absorbance of the solution without the film (A0) and the solution with the film (A t ) were measured at a wavelength of 516 nm, and the DPPH was calculated using the following equation (5):

[0093] DPPH = (A0- A t ) ÷ A0 x 100% (5)

[0094] 13. Antibacterial properties of CM / CS composite antibacterial film

[0095] The antibacterial activity of the film sample on bacteria, i.e. gram-negative pathogenic bacteria (Escherichia coli) and gram-positive pathogenic bacteria (Staphylococcus aureus), was evaluated. After the pure culture of the bacteria to be tested was streaked on a TSA plate and incubated at 37 °C for 18-24 h, 3-5 single colonies were taken with a inoculating loop and mixed well in 150 mL of LB broth, and the bacterial suspension concentration was measured (approximately 10 9 CFU·mL -1 ). 50 mg of the film was divided into centrifuge tubes containing 5 mL of the bacterial solution, and was subjected to light and dark treatment using a xenon lamp. After the bacterial solution was diluted to a certain concentration, 100 μL of the bacterial solution was spread on a TSA plate, and the culture dish was placed in a self-sealing bag and incubated in a constant temperature incubator for 18-24 h. The growth condition of the culture dish was observed and recorded. The growth inhibition rate of the bacteria by light and dark treatment was calculated using the following equation (6):

[0096] Growth inhibition rate = (N0- N) ÷ N0x 100% (6)

[0097] wherein:

[0098] N0—total number of colonies in dark treatment;

[0099] N t total number of colonies in light treatment.

[0100] The antifungal activity of the film samples against mold, i.e., Aspergillus niger, was evaluated as follows: Aspergillus niger was inoculated onto potato agar (PDA) slant medium and incubated at 30°C for 5 days. The spores on the medium were then washed down with sterile water and transferred to a sterile flask, where the spore clusters were broken up by shaking. After the mycelium was removed by sterile gauze filtration, the concentration of the bacterial solution was adjusted to 10 6 individuals / mL using a hemocytometer and sterile water. 50 mg of the film was divided into a centrifuge tube containing 5 mL of the bacterial solution, and the film was subjected to light and dark treatment using a xenon lamp. After the bacterial solution was diluted to a certain concentration, 100 uL of the bacterial solution was spread on a PDA plate, and the plate was placed in a self-sealing bag and incubated in an incubator for 5 days. The growth of the plate was observed and recorded. The growth inhibition rate of Aspergillus niger under light and dark treatment was calculated using formula (6).

[0101] 14. Fresh-keeping experiment of Kyoho grapes

[0102] Grapes of similar maturity were selected and randomly divided into 4 groups. The grapes were packaged with PE film, single CS film, CS / 5% CM, and no film, respectively. The grapes were stored at room temperature, and the experiment was conducted every two days.

[0103] (1) Hardness

[0104] The hardness of the grapes was measured using a fruit hardness tester.

[0105] (2) Weight loss rate

[0106] The grape samples were weighed at the beginning of the experiment and marked as (M1). At different times, the grape samples were weighed again after removing the packaging and marked as (M2). The weight loss rate was calculated using the following formula (7):

[0107] Weight loss rate = (M1-M2) ÷ M1 x 100% (7)

[0108] (3) Titration acid (TA)

[0109] The TA was measured by titration: 5 g of grape pulp was ground into a homogenate and diluted with distilled water to 50 mL. After mixing well, 20 mL of the mixed solution was titrated with 0.01 mol / L NaOH, with 1% phenolphthalein as an indicator.

[0110] The titration acid was calculated using the following formula (8):

[0111] Titration acid = V 氢氧化钠 X 0.1 X 0.064 X V0 ÷ m X VI (8)

[0112] In the formula:

[0113] V 氢氧化钠 V - the volume of NaOH consumed in titration, unit: mL;

[0114] VI - the molar concentration of NaOH, unit: mol / L;

[0115] 0064 - citric acid conversion factor;

[0116] V0 - the constant volume, unit: mL;

[0117] m - the volume of green field, unit: mL.

[0118] (4) Soluble solids

[0119] The soluble solids content of grape samples was determined by using a handheld refractometer. 20 g of grape pulp was thoroughly ground with 20 g of oxalic acid solution (20 g / L) under light-proof conditions. After adding a certain amount of white clay, it was diluted to 100 mL with oxalic acid solution. After filtration, 10 mL of filtrate was titrated with 2,6-dichloroindolyl oxyl alcohol solution. The following formula (9) was used to calculate the soluble solids:

[0120] X = [(V - V0) X T X A] ÷ m X 100% (9)

[0121] In the formula:

[0122] V - the volume of solution consumed in titration, unit: mL;

[0123] V0 - the volume of the blank test, unit: mL;

[0124] T - the titration degree, unit: mg / mL;

[0125] A - the dilution factor;

[0126] m - the sample weight, unit: g.

[0127] (5) Vc content

[0128] The Vc content in the sample was determined according to the 2,6-dichloroindophenol titration method in GB / T 5009.86-2016 standard.

[0129] (6) POD activity

[0130] Mix 3 g of grape pulp with 5 mL of phosphate buffer for 30 min, and collect the supernatant by freezing centrifugation. Mix 0.5 mL of the supernatant with 2.5 mL of 0.3% catechol solution and 0.2 mL of 2% H2O2. Measure the absorbance of the mixed solution (wavelength = 470 nm) within 3 min. The blank control is pure distilled water. Calculate the POD activity using the following formula (10):

[0131] POD = ΔOD 470 × V ÷ (0.01 × V s × m) (10)

[0132] In the formula:

[0133] POD - peroxidase activity, unit: U·g- 1 ·min- 1 ;

[0134] V - total volume of sample extract, unit: mL;

[0135] V S - total volume of sample extract taken during the measurement, unit: mL;

[0136] m - sample weight, unit: g.

[0137] (7) CAT activity

[0138] Mix 3 g of grape pulp with 5 mL of phosphate buffer for 30 min, and collect the supernatant by freezing centrifugation. Mix 0.1 mL of the supernatant with 2.9 mL of H2O2 (20 mmol / L), and measure the absorbance of the mixed solution (wavelength = 240 nm). Calculate the CAT activity using the following formula (11):

[0139] CAT = ΔOD 240 × V s1 ÷ (0.01 × V s2 × m2) (11)

[0140] In the formula:

[0141] CAT - catalase activity, unit: U·g- 1 ·min- 1 ;

[0142] V S1 - total volume of sample extract, unit: mL;

[0143] V S2 - total volume of sample extract taken during the measurement, unit: mL;

[0144] m2 - sample weight, unit: g.

[0145] II. Experimental Results

[0146] 1. Photodynamic performance of CM

[0147] To determine the photodynamic properties of different materials, MB and RhB were used as ·OH trapping agents. For example... Figure 1 As shown in Figures A and B, the absorption peaks of CFO and Mxene at 664 nm and 554 nm did not change significantly before and after illumination, indicating that CFO and Mxene did not generate sufficient ·OH to degrade MB and RhB after illumination. However, the absorption peaks of CM at 664 nm and 554 nm decreased significantly before and after illumination, indicating that CM has a significant ability to generate ·OH and excellent photodynamic properties.

[0148] 2. Morphological structure

[0149] TEM images of CM, such as Figure 2 As shown in Figures A and B, the synthesized CM exhibits good dispersion. Combined with high- and low-magnification TEM images, it can be seen that the spherical CFO nanoparticles are tightly adhered to the monolayer MXene. The high-magnification TEM image reveals clear lattice fringes; the lattice spacing of CFO is 0.256 nm, while that of MXene is 0.473 nm. The deposition of spherical CFO nanoparticles on the MXene surface indicates successful CM synthesis.

[0150] Cross-sectional and surface scanning electron microscope images of CS membrane and CS / 5%CM composite membrane are shown below. Figure 2 As shown in Figure CF, the two films exhibit similar morphologies and have no surface cracks. The CS film has a smooth and dense surface, with a rich porous structure in its cross-section. The addition of CM leads to an increase in the surface roughness of the film. The reduction of the original structural voids in the CS matrix can improve the resistance to water molecule diffusion on the film surface, thereby enhancing the hydrophobicity of the film.

[0151] AFM images of the thin film as follows Figure 3 As shown in Figure A, from left to right, the two-dimensional morphology, cross-sectional profile, and three-dimensional morphology of the film are presented. Within a scanning range of 5 μm × 5 μm, it can be seen from the two-dimensional and three-dimensional images that the film roughness increases with increasing CM content. The cross-sectional profile also shows that the film thickness gradually increases with increasing CM content. Furthermore, the average surface roughness (Ra) and root mean square roughness (Rq) parameters of the film were analyzed. Figure 3 As can be seen from Figure B, the single CS film is relatively smooth. With the increase of CM addition, both Ra and Rq gradually increase, and the roughness of the film increases.

[0152] XRD images of the thin film as follows Figure 4A, the single CS film showed a broad and weak diffraction peak at 20°, indicating that the molecules were arranged in an amorphous state. The CM reached peaks at 10.1°, 18.3°, 30.0°, and 43.1°. The films with different amounts of CM also showed the same diffraction peak at 20°. With the increase of the amount of CM, strong characteristic peaks appeared at 10.1° and 18.3°, and gradually increased. With the increase of the amount of CM, its crystallinity and diffraction characteristic peaks gradually appeared.

[0153] The FTIR spectra of the films are shown in Figure B. Figure 4 As shown in Figure B, the crosslinking effect caused by the interaction between CS and CM affected the position and intensity of the peaks. The peak at 3300 cm -1 was caused by the hydrogen bond between CS and glycerol. The peak at 2880 cm -1 was caused by the alkyl group C-H of CS. The peak at 1636 cm -1 was caused by the hydrogen bond of CM, which also confirmed the presence of CM. The peak at 1550 cm -1 was related to the bending vibration of -NH2. The peak at 1410 cm -1 was caused by the deformation band of C-H. The peak at 1150 cm -1 was due to the O-H bending vibration caused by the structure of CS. The peak at 1030 cm -1 was related to the vibration of C-O-C. In summary, after adding CM, the functional groups in the CS film did not change significantly, proving that CS and CM had good compatibility.

[0154] 3. Solubility, swelling rate and water content

[0155] The solubility, swelling rate and water content of different films are shown in Table 1. Compared with the single CS film, after adding CM, the solubility, swelling rate and water content of the film were reduced. This may be due to the hydrogen bond formed between CS and CM, which hinders the interaction between the chitosan matrix and water molecules, or due to the hydrophobic influence of CFO on the diffusion of water molecules in the film.

[0156] Table 1 Solubility, swelling rate and water content of different films

[0157]

[0158] 4. Thickness and mechanical properties

[0159] Table 2 shows the thicknesses of single CS films and composite films with different CM addition amounts. Compared with single CS films, the film thickness increases with increasing CM addition. This may be due to the increased intermolecular interactions within the film, resulting in a more compact film structure and thus an increase in thickness. TS and EB are both indicators for measuring the flexibility and strength of the film. With the addition of CM, the elongation at break and tensile strength of the film both show a trend of first increasing and then decreasing.

[0160] As shown in Table 2, the addition of CM significantly improved the mechanical properties of the film. The film exhibited the highest elongation at break and tensile strength when the addition amount was 5%. This is likely due to the unique lamellar structure of CM, which enhances its cross-linking with the chitosan matrix, thereby strengthening the film's mechanical properties. However, when the addition amount was 7%, the mechanical properties of the film decreased, possibly due to the increased water content.

[0161] Table 2 Thickness, TS, EB, and WVT of different films

[0162]

[0163]

[0164] 5. Ultraviolet blocking properties

[0165] The ultraviolet (UV) blocking properties of thin films are one of the important indicators for evaluating their quality. The absorbance of different thin films under UV radiation in the range of 200 nm to 800 nm was recorded experimentally. For example... Figure 5 As shown in Figure A, the film's ability to block ultraviolet light gradually increases with the increase of CM content. This indicates that the addition of CM results in a film with excellent ultraviolet light shielding properties, effectively preventing ultraviolet radiation from reaching food. Furthermore, irradiation by a light source can increase the photodynamic properties of CM, enhancing the film's inhibitory effect on microorganisms.

[0166] 6. Water vapor transmission rate and water contact angle

[0167] Water vapor transmission rate (WVT) is an important indicator for measuring the water vapor permeability of thin films. To ensure food quality, the WVT value should be kept as low as possible. The WVT values ​​of different films are shown in Table 2. After adding CM, the WVT of the film decreased significantly, indicating that increasing the amount of CM can enhance the hydrophobicity of the film. Figure 5 The same results were obtained in B. With the addition of CM, the water contact angle of the film increased from 87.7° to 107°, transforming it from a hydrophilic film to a hydrophobic film. The increase in the hydrophobicity of the film may be due to the addition of CM increasing the film thickness or the ability of CM to form intermolecular hydrogen bonds with CS, both of which hinder the diffusion of water molecules.

[0168] 7. Thermal Analysis

[0169] Thermal stability is an important index for the application of films in food preservation. The DSC curves of different films are shown in Fig. 4. All films have a single glass transition temperature (Tg) in the DSC curves. The maximum endothermic peak of the single CS film appears at 121℃, and the initial, peak and final temperatures change slightly with the addition of CM. The ΔH value of the film increases with the increase of CM. The results show that the film has good thermal stability and compatibility between components. Figure 5

[0170] 8、DPPH scavenging rate

[0171] The DPPH scavenging rates of different films are shown in Fig. 5. With the increase of CM addition, the DPPH scavenging rate of the film shows an upward trend, with the highest level of 47% (CS / 7%CM). This may be due to the fact that CM has peroxidase and catalase mimetic activity, which can improve the antioxidant capacity of the film and prevent the oxidative degradation of nutrients in food. Figure 6

[0172] 9、Antibacterial performance

[0173] The antibacterial effects of different films on E. coli, S. aureus and A. niger after dark and light treatment are shown in Fig. 6 A, B and C. With the addition of CM, the antibacterial performance of the film gradually increases. This may be due to the fact that CM contains multivalent Co and Fe ions, which have good photodynamic performance and excellent enzyme catalytic activity. The ions released by CM can contact the cell wall of microorganisms, thereby destroying the surface of microorganisms and causing the contents to leak out, eventually leading to the death of microorganisms. The inhibitory effect of the film on gram-positive bacteria is better than that on gram-negative bacteria, which can be attributed to the difference in the composition of the cell wall of the two types of bacteria. The cell wall of gram-negative bacteria is composed of hydrophilic lipopolysaccharides, which can hinder the entry of hydrophobic substances. In comparison Figure 7 Figure 7 With light catalysis (6h) and light (0h), it can be confirmed that the inhibitory effect on microorganisms after light catalysis is significantly improved. The inhibition rate of CS / 7%CM film on S. aureus, E. coli and A. niger is as high as 99.7%, 99.8% and 97.1%. This can be attributed to the ·OH produced by CM as a photosensitizer, which induces intracellular material peroxidation, eventually leading to microbial death.

[0174] Table 3 Antibacterial performance of different films

[0175]

[0176] 10、Jufeng grape preservation experiment

[0177] (1) Grape appearance

[0178] As​​​Figure 9 As shown, the appearance changes of grapes stored under different packaging conditions for 12 days were recorded. For the control group, i.e., grapes without any packaging, obvious wrinkles were observed on day 12. This is likely due to moisture loss caused by prolonged exposure to air. The single CS film, due to its water-resistant properties, slowed down the moisture loss of the grapes, but some wrinkles were still observable. The CS / 5% CM film maintained its intact appearance on day 12, attributed to its strong water molecule barrier properties. The PE film, due to its limited antibacterial properties, led to microbial contamination of the grapes by day 12, rendering it unsuitable for preservation.

[0179] (2) Hardness and weight loss

[0180] Changes in grape firmness under different packaging conditions, such as Figure 8 As shown in Figure B, the hardness of the air group and the CS film decreased faster than that of the CS / 5%CM film, confirming the superior preservation performance of the CS / 5%CM film. The change in weight loss rate of grapes during storage is shown in Figure B. Figure 8 As shown in Figure C, the air group exhibited the highest weight loss rate among the grapes during storage, while the weight loss rate decreased from 18.9% to 13.5% after using the CS / 5%CM membrane. This structure indicates that the CS / 5%CM membrane effectively prevents the permeation of water molecules, thus preserving the moisture in the fruit.

[0181] (3) Titratable acid, soluble solids and vitamin C

[0182] The acidity changes of grapes during storage can be titrated as follows: Figure 8 As shown in Figure D, the lower TA reduction rate exhibited by the CS / 5%CM film is likely due to the addition of CM improving the film's gas barrier properties (reducing O2 content and increasing CO2 content), inhibiting ethylene production, and thus reducing the consumption of organic acids. The excellent barrier properties of the CS / 5%CM film can inhibit fruit respiration and metabolism, reducing nutrient loss. Therefore, during storage, the reduction rates of soluble solids and vitamin C in the CS / 5%CM film are lower than those of other components, such as... Figure 8 As shown in EF, the content was 13.5% and 4.47 mg / 100g, respectively, on day 12 of storage. Furthermore, the excellent UV shielding capability of the CS / 5%CM film further reduced the loss of Vitamin C.

[0183] (4) POD activity and CAT activity

[0184] Changes in POD and CAT activity in grapes during storage, as follows Figure 8 As shown in GH. After the addition of the CS / 5% CM membrane, the POD and CAT activities of grapes increased by 23 and 15 U·g, respectively. -1 ·min -1Rise to 29 and 19 U·g -1 ·min -1 This is due to the POD and CAT simulated enzyme activity of CM has a certain degree of oxidation, can inhibit the oxidation reaction in the process of grape preservation, slow down the fruit softening, maintain fruit quality.

[0185] Comparative Example 1

[0186] The complex process between CM and CS was studied, and the picture of single CM is shown in Figure 10 A, CM was directly mixed with CS solution, and the results are shown in Figure 10 B, it can be seen that it cannot be well dispersed. CM was first mixed with ultrapure water, and then mixed with CS aqueous solution, and the results are shown in Figure 10 C, it shows that the dispersion of the two is still poor. Ultrasonic mixing was carried out by the method of Example 1 of the application, and the results are shown in Figure 10 D, it shows that the two can be well dispersed.

[0187] Comparative Example 2

[0188] As shown in the research results of Table 3, when the concentration of CM is 5%, the solubility, water content and swelling rate of the composite film can be kept at the lowest level, which can better guarantee the film forming performance of the composite film, therefore, the concentration of CM is selected as 5%, and the effect is better.

[0189] Comparative Example 3

[0190] As shown in the research results of Table 4, when the concentration of CM is too high, the performance of the composite film will decrease obviously, therefore, when the concentration of CM in the composite film is 5%, the effect is better.

[0191] Comparative Example 4

[0192] As shown in the research results of Table 3, when the concentration of CM is 5%, the inhibition rate of the obtained composite film on Staphylococcus aureus, Escherichia coli and Aspergillus niger is as high as 98.1%, 99.6% and 96.4%, and the bacteriostatic effect is extremely significant.

[0193] Comparative Example 5

[0194] The volume ratio of CS and CM was investigated, when the ratio of CS to CM is less than 19:1 (reduce the volume of CS while keeping the volume of CM unchanged, when 66.5 mL of CS solution and 3.5 mL of CM are mixed, the volume ratio is 19:1), CM cannot be completely dissolved in the CS solution, at this time, film making cannot be completed, and the dispersion of the mixture of the two is shown in Figure 11 .

[0195] When the ratio of CS to CM is greater than 19:1 (increasing the volume of CS while keeping the volume of CM constant), the CM can be completely dissolved in the CS, but can result in a decrease in the performance of the film after it is made, and the mechanical properties of the film are shown in Table 4 below:

[0196] Table 4 Mechanical properties of films with different ratios

[0197]

[0198] The antibacterial properties of the composite films are shown in Figure 12 It can be seen that when the ratio of CS to CM is greater than 19:1, the antibacterial properties of the composite film decrease.

[0199] The thermal stability properties of the composite films are shown in Figure 13 It can be seen that when the ratio of CS to CM is greater than 19:1, the thermal stability properties of the composite film decrease.

[0200] The ultraviolet light absorption properties of the composite films are shown in Figure 14 It can be seen that when the ratio of CS to CM is greater than 19:1, the ultraviolet light absorption properties of the composite film decrease.

Claims

1. A preparation method of iron-cobalt oxide body / two-dimensional nitride / chitosan composite antibacterial preservative film, characterized in that, Comprising the following steps: (1) adding lithium fluoride into hydrochloric acid, and synthesizing two-dimensional nitride by in-situ etching method with titanium aluminum carbide; (2) synthesizing iron cobalt oxide body by hydrothermal method with ferric chloride hexahydrate, cobalt chloride hexahydrate, sodium acetate and sodium hydroxide; (3) mixing two-dimensional nitride and iron cobalt oxide body according to weight ratio of 1:1, dissolving in water, and obtaining nanometer enzyme solution of iron cobalt oxide body and two-dimensional nitride composite under ultrasonic treatment in ice bath; (4) dissolving chitosan and glycerol in acetic acid solution with volume percentage of 1%, and preparing chitosan solution with mass percentage of chitosan of 3%; then mixing nanometer enzyme solution obtained in step (3) and chitosan solution to obtain composite solution, and making volume percentage of nanometer enzyme solution 5%, and volume ratio of chitosan solution to nanometer enzyme solution 19:1; subsequently pouring the composite solution after ultrasonic treatment into a mold, drying to obtain a film, and demolding after cooling to room temperature, thereby obtaining the composite antibacterial preservative film.

2. The production method according to claim 1, characterized by, The weight ratio of lithium fluoride to titanium aluminum carbide in step (1) is 1:1; and the synthesis condition is to react at 35℃ for 24 h.

3. The preparation method according to claim 1, characterized in that, The weight ratio of ferric chloride hexahydrate, cobalt chloride hexahydrate, sodium acetate and sodium hydroxide in step (2) is 18:19:90:

25.

4. The method of claim 1, wherein, The synthesis condition in step (2) is to react at 180℃ for 12 h.

5. The preparation method according to claim 1, characterized in that, The hydrothermal method in step (2) is carried out in ethylene glycol solution.

6. The method of claim 1, wherein, The ultrasonic treatment time in step (3) is 0.5 h.

7. The preparation method according to claim 1, characterized in that, The ultrasonic treatment condition in step (4) is to ultrasonically treat at 40℃ for 4 h.

8. The iron cobalt oxide body / two-dimensional nitride / chitosan composite antibacterial preservative film prepared by the method of any one of claims 1-7.

9. The use of the iron cobalt oxide body / two-dimensional nitride / chitosan composite antibacterial preservative film of claim 8 in preparing fruit and vegetable antibacterial preservative materials.

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