CN111925541A

CN111925541A - Antibacterial and fresh-keeping composite freeze-thaw edible film for cold fresh meat and preparation method thereof

Info

Publication number: CN111925541A
Application number: CN202010804058.7A
Authority: CN
Inventors: 张玉斌; 韩鋆; 王友玲; 张珍
Original assignee: Gansu Nong Chuang Food Technology Co ltd; Gansu Agricultural University
Current assignee: Suzhou Wisdom Agriculture Biotechnology Co ltd; Gansu Agricultural University
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-11-13
Anticipated expiration: 2040-08-12
Also published as: CN111925541B

Abstract

The invention relates to the technical field of food active packaging films, in particular to an antibacterial fresh-keeping composite freeze-thaw edible film for cold fresh meat, a preparation method thereof and an application method thereof in fresh keeping and color protection of the cold fresh meat. The main technological parameters are as follows: freeze thawing for 3 times, calcium lactate concentration of 3%, calcification of 6 min, and sodium alginate addition of 60%, wherein the tensile strength of the sodium alginate/hyaluronic acid composite membrane is 10.41 MPa. The main technical purpose of the invention is to use excellent renewable natural macromolecular sodium alginate and hyaluronic acid as film forming base materials, add calcium lactate as a cross-linking agent with a color protection effect to produce an active packaging film, improve the mechanical properties of the film through a physical freeze-thaw treatment method, prepare a green, safe and degradable composite edible film, effectively delay the quality deterioration process of chilled meat in a refrigeration process, and maintain the edible quality of the chilled meat.

Description

Antibacterial and fresh-keeping composite freeze-thaw edible film for cold fresh meat and preparation method thereof

Technical Field

The invention relates to the technical field of food active packaging films, in particular to an antibacterial fresh-keeping composite freeze-thaw edible film for chilled meat and a preparation method thereof.

Background

The researches on the fresh-keeping of the chilled meat at home and abroad mainly include tray packaging, vacuum packaging, modified atmosphere packaging and active packaging. Vacuum packaging can maintain the flesh bright red for a long time, reduce storage loss caused by dehydration on the surface of the meat, and delay the rate of fresh meat decay and fat oxidation. However, the contraction and extrusion of the vacuum treatment easily cause the deformation of the fresh meat and the outflow of juice, which affects the sale. The modified atmosphere packaging can better maintain the sensory quality and the pH value of the meat sample, and simultaneously delay the increase of the TVB-N value and the TBARS value. However, the controlled atmosphere packaging has the problems that the proportion of mixed gas is not easy to control, and the like.

Active packaging is an innovative packaging that protects the product from contamination, absorbs moisture from the food, or releases the active into the environment in which the food is located, thereby retarding deterioration of the product. Currently, the food packaging field is researched more mainly by two active packaging films, namely antibacterial and antioxidant. For meat and meat products, the active package of the antibacterial substance combined with the edible film or coating can maintain the color and luster of the meat product stable, avoid the influence of external bad smell, ensure the food quality safety and attract consumers at the same time.

Meat and meat products are the preferred high-quality protein source for consumers due to rich nutrition, and the protein provides essential amino acid and important micronutrient for human bodies, but also becomes a proper culture medium for the growth of pathogenic microorganisms and putrefactive microorganisms. Surface treatment and packaging have become substantial approaches to solving this problem, but most packaging involves the use of non-biodegradable, non-recyclable materials such as plastics, nylon, and polyester, and the worry of synthetic materials has been recognized worldwide. The plastic residues are not biodegradable, which causes serious environmental pollution. With the emphasis on environmental protection and food safety, researchers are striving to find alternative sources of packaging materials to reduce these environmental problems.

Edible Films (Edible Films) take natural Edible biopolymer as a base material, and form a protective thin layer for blocking moisture, oxygen, microorganisms and the like on the surface of food in the forms of spraying, soaking, wrapping and the like through intermolecular interaction. Can prevent food spoilage and water loss, maintain food quality, improve food appearance integrity and physical properties, and solve the problem of white pollution caused by plastic packaging to a certain extent. The film is completely made of renewable, edible components such as animal and vegetable proteins, lipids and polysaccharides, is completely biodegradable, and therefore safer than conventional synthetic materials, and can be consumed with packaged products without leaving any waste.

Among various edible films or coatings, polysaccharide biopolymers are widely used for film-forming substrates due to their good performance, safety and structural diversity. Sodium alginate is easy to dissolve in water, has strong antibacterial property, moisture retention and film forming property, is used as an excellent natural food preservative, and researches show that the sodium alginate, protein and carbohydrate are compounded and widely used in edible films and coatings. But the edible sodium alginate film used alone has the defect of poor mechanical property, and the performance of the edible sodium alginate film is improved by utilizing the fact that sodium alginate is easy to mix and react with other macromolecular substances in water. Hyaluronic acid is a high molecular chain polymer as a main component of an animal cell matrix. Has the characteristics of water retention, biocompatibility, gel property, film forming property and the like. The film structure can be densified by directly adding a cross-linking agent into the film-forming solution or by using the cross-linking agent in the subsequent treatment of film formation, and the mechanical property of the film is improved. Recent studies have shown the use of reversible ionic crosslinks with greater safety, such as Ca²⁺、Zn²⁺、Al³⁺The multivalent cation can crosslink sodium alginate, and is usually Ca²⁺Cross-linking sodium alginate to obtain alginate gels for use in food products, as with other hydrocolloid gelsRatio of Ca to Ca²⁺And sodium alginate, without heating, can generate electrostatic interactions and obtain irreversible gels. It has also been found that calcium lactate crosslinks to form a film having a tensile strength and elongation at break second to calcium chloride, which is the effect of crosslinking different calcium agents on film performance under otherwise identical conditions. Meanwhile, various lactate has the functions of antisepsis and bacteriostasis and is specified as a safety additive in meat processing industry by the United states department of agriculture, so that calcium lactate is selected for crosslinking the sodium alginate-based composite gel to prepare the gel film in the invention. In addition, hyaluronic acid and sodium alginate are hydrophilic polymers, have good compatibility and gel performance, and are physical methods for improving the mechanical performance of the sodium alginate film through freeze-thaw treatment, but research on the application of the freeze-thaw treatment method to edible films is not reported at present.

Therefore, the development of green, safe and degradable composite edible films has become a trend in the fields of preservation and food packaging, by using renewable natural macromolecules as film-forming substrates and adding a cross-linking agent with a color-protecting effect to produce active packaging films.

Disclosure of Invention

The invention provides a preparation method of a cold fresh meat antibacterial fresh-keeping composite freeze-thaw edible film and a beef refrigeration fresh-keeping application method.

The technical scheme adopted by the invention for solving the technical problems is that

A preparation method of an antibacterial fresh-keeping composite freeze-thaw edible film for cold fresh meat comprises the following steps

A. Preparing a hyaluronic acid solution: weighing hyaluronic acid powder, adding into distilled water, adding hyaluronic acid powder into 100 mL of distilled water per 1.00 g of hyaluronic acid powder, heating to 60 ℃, continuously stirring at 300 rpm/s by using an electric stirrer until the hyaluronic acid powder is completely dissolved, standing for 10-15min to remove bubbles, and preparing a hyaluronic acid solution with the solid-to-liquid ratio of 1%;

B. preparing a sodium alginate solution: weighing sodium alginate powder, adding into distilled water, adding into 100 mL of distilled water per 1.50 g of sodium alginate powder, heating to 60 deg.C, continuously stirring with an electric stirrer at 300 rpm/s until it is completely dissolved, standing for 10-15min to remove bubbles, and making into sodium alginate solution with solid-to-liquid ratio of 1.5%;

C. preparing a mixed solution: adding a sodium alginate solution with the volume ratio of 15-75% into the hyaluronic acid solution, mixing to obtain a mixed membrane solution, uniformly stirring, pouring into a beaker, and sealing with a sealing membrane to obtain a composite gel;

D. pretreatment: and (3) carrying out calcification treatment on the composite gel by using a calcium lactate solution, washing away calcium lactate on the surface by using distilled water, naturally drying, and stripping off an upper layer film to obtain a final product.

Preferably, in the step C, the gel is sealed by a sealing film, then is frozen for 24 hours at the temperature of-18 ℃, is unfrozen at room temperature for 12 times, and is subjected to freeze-thaw treatment for 1-4 times to obtain the composite gel.

Preferably, in the step C, the gel is frozen for 24 hours at the temperature of 18 ℃ below zero after being sealed by a sealing film, and is thawed at room temperature for 12 times to complete 1 freeze-thaw cycle, so that the composite gel is subjected to freeze-thaw treatment and freeze-thaw cycle for 2 times.

Preferably, 15%, 30%, 45%, 60% or 75% sodium alginate solution by volume is added into the hyaluronic acid solution in the step C.

Preferably, a 60% sodium alginate solution by volume is added into the hyaluronic acid solution in the step C.

Preferably, in the step D, the mass concentration of calcium lactate is 1-5%, and the calcification time is 6 min.

Preferably, in the step D, the calcium lactate mass concentration is 3%, and the calcification time is 6 min.

The composite membrane prepared by the preparation method of the sodium alginate/hyaluronic acid composite membrane is used for refrigerating and preserving beef.

Preferably, the skin fat and fascia are removed from the meat piece, the meat piece is cut into about 50g meat pieces with the same thickness, the composite gel prepared in the step A, B, C is evenly smeared on the surface of the meat piece, the composite gel is calcified by a calcium lactate solution in the step D, the calcium lactate on the surface is washed by distilled water, and the meat piece is naturally dried and stored at the temperature of 4 +/-1 ℃.

The invention has the beneficial effects that:

1. the optimal preparation process is determined through process optimization, and the result shows that the primary and secondary sequence of the influence of all factors on the tensile strength of the edible film is as follows: the freezing and thawing times is more than the adding amount of sodium alginate and more than the concentration of calcium lactate and more than the calcification time. The specific process parameters are as follows: freezing and thawing for 3 times, calcium lactate concentration of 3%, calcification of 6 min, and sodium alginate addition of 60%, wherein the tensile strength of the obtained SA/HA composite membrane is 10.41 MPa under the condition.

2. The basic physical properties of the composite membrane are researched by researching different freezing and thawing times, and the results show that: along with the increase of the freeze-thaw times, the composite membrane has a compact structure, and the transparency, the swelling degree and the solubility are all reduced. According to the analysis of infrared spectrum and SEM results, the film has no holes and becomes smooth and compact along with the increase of the number of times of freeze thawing, and the sodium alginate and the hyaluronic acid have better compatibility and are Ca-treated²⁺After the action, hydrogen bonds are generated among molecules, so that the mechanical property of the composite membrane is stable. The film prepared by the research basically meets the requirements of edible films, and provides a new idea for developing novel edible films.

3. The beef treated by the SA/HA composite membrane is used as a treatment group, the beef without any treatment is used as a control group, and the beef is stored at the temperature of 4 +/-1 ℃, and the result shows that: sensory beef scores of the treated groups compared with the control group,L*Value (c),a*The value and NADH concentration are obviously higher than those of a control group: (p<0.05），b*The value, the pH value, the TBARS value, the TVB-N value and the total number of bacterial colonies are obviously lower than those of a control group (p<0.05). Therefore, the SA/HA composite film can effectively delay the quality degradation process of the cooled Tibetan beef and keep the eating quality of the cooled Tibetan beef.

4. The composite edible film is green, safe and degradable, can effectively delay the quality deterioration process in the cold storage process of cooled meat and maintain the edible quality of the cooled meat.

Drawings

FIG. 1 is a schematic view of an interaction surface of a preparation process of an SA/HA composite membrane;

FIG. 2 dynamic rheological properties of composite gels versus freeze-thaw times;

FIG. 3 Infrared Spectrum study of different SA/HA composite membranes;

FIG. 4 is a scanning electron microscope image of SA/HA composite membrane under different freezing and thawing times.

Detailed Description

Preferably, the gel is sealed by a sealing film, then is frozen for 24 hours at the temperature of 18 ℃ below zero, is thawed at room temperature for 12 times, and is subjected to freeze-thaw cycle for 1 to 4 times to obtain the composite gel.

Preferably, the gel is frozen for 24 hours at the temperature of 18 ℃ below zero after being sealed by a sealing film, and is thawed at room temperature for 12 times to complete 1 freeze-thaw cycle, so that the composite gel is subjected to freeze-thaw treatment and freeze-thaw cycle for 2 times.

A preparation method of a composite freeze-thaw edible film for antibacterial preservation of chilled meat is used for refrigerating and preserving beef. Preferably, the skin fat and fascia are removed from the meat piece, the meat piece is cut into about 50g meat pieces with the same thickness, the composite gel prepared in the step A, B, C is evenly smeared on the surface of the meat piece, the composite gel is calcified by a calcium lactate solution in the step D, the calcium lactate on the surface is washed by distilled water, and the meat piece is naturally dried and stored at the temperature of 4 +/-1 ℃.

The preparation process and the performance research mode are as follows:

preparation process and performance research of sodium alginate/hyaluronic acid composite edible film

1.1 Experimental instruments and materials

1.1.1 Experimental materials and reagents

Hyaluronic acid: food grade, Shandong Furuida biomedical Co., Ltd; sodium alginate: food grade, Qingdao Mingyue seaweed group; calcium lactate: food grade, Zhengzhou Kai Yu food additives Co., Ltd.

1.1.2 Experimental instruments

TABLE 1 Main Instrument

1.2 Experimental methods

1.2.1 Single-factor experiment for preparation of sodium alginate/hyaluronic acid composite membranes

Preparing a hyaluronic acid solution: weighing 1.00 g of hyaluronic acid powder, adding into 100 mL of distilled water, heating to 60 ℃, continuously stirring at 300 rpm/s by using an electric stirrer until the hyaluronic acid powder is completely dissolved, standing to remove bubbles, and preparing a 1% (w/v) hyaluronic acid solution;

preparing a sodium alginate solution: weighing 1.50 g of sodium alginate powder, adding into 100 mL of distilled water, heating to 60 ℃, continuously stirring at 300 rpm/s by using an electric stirrer until the sodium alginate powder is completely dissolved, standing to remove bubbles, and preparing 1.5% (w/v) of sodium alginate solution;

and blending the two solutions according to a certain proportion to obtain a mixed membrane solution, uniformly stirring the mixed membrane solution, pouring the mixed membrane solution into a beaker, sealing the beaker by using a sealing membrane, and performing freeze-thaw treatment (freezing for 24 hours in a refrigerator at the temperature of minus 18 ℃ and thawing for 12 hours at room temperature for 1 freeze-thaw cycle) to obtain the composite gel. Respectively treating with calcium lactate solution, washing with distilled water to remove calcium lactate on the surface, air drying, peeling off the film, and measuring tensile strength and water vapor transmission rate of the film.

(1) Setting the concentration of calcium lactate to be 3%, the calcification time to be 6 min and the addition amount of sodium alginate to be 60%, respectively setting the freezing and thawing times to be 0 time, 1 time, 2 times, 3 times and 4 times, and investigating the influence of the freezing and thawing times on the tensile strength of the composite membrane.

(2) The freezing and thawing times are set to be 2 times, the calcification time is 6 min, the sodium alginate addition amount is 60%, the calcium lactate concentration is respectively set to be 1%, 2%, 3%, 4% and 5%, and the influence of the calcium lactate concentration on the tensile strength of the composite membrane is inspected.

(3) Setting the number of times of freeze thawing to be 2, the concentration of calcium lactate to be 3% and the addition amount of sodium alginate to be 60%, setting the calcification time to be 2 min, 4 min, 6 min, 8 min and 10 min respectively, and investigating the influence of the calcification time on the tensile strength of the composite membrane.

(4) Setting the number of times of freeze thawing to be 2, the concentration of calcium lactate to be 3% and the calcification time to be 6 min, respectively setting the addition amount of sodium alginate to be 15%, 30%, 45%, 60% or 75%, and investigating the influence of the addition amount of sodium alginate on the tensile strength of the composite membrane.

1.2.2 response surface optimization design of sodium alginate/hyaluronic acid composite membrane

On the basis of single factor, four factors of the number of times of freezing and thawing, calcium lactate concentration, calcification time and sodium alginate addition are selected to carry out a response surface optimization test, and a statistical analysis software Design-Expert 8.06 is used for carrying out regression analysis. The low, medium and high test levels of each independent variable are respectively coded by-1, 0 and 1, the model is used for fitting a quadratic polynomial equation by a least square method, and whether the influence of four factors on the tensile strength of the composite membrane has interaction or not is analyzed.

1.2.3 Effect of Freeze-thaw times on composite Membrane Properties

1.2.3.1 color measurement

The color of the composite film is measured by a color difference meter, and the standard plate is CX2604 (L*=52.94，a*=-0.50，b*= 4.85), for total color differenceE*It is shown that,

。

1.2.3.2 opacity

Reference is made to the method of Lu et al with minor modifications. Cutting the blended film into 4X 1 cm²The absorbance of the film at 600 nm was measured and a blank cuvette was used as a comparison.

Opacity=A₆₀₀/d

In the formula A₆₀₀: absorbance of the sample film at 600 nm; d: the thickness of the sample mold; opacity: opacity of the sample film.

1.2.3.3 determination of Water vapor Transmission Rate

Adopting a cup-like method. At 25 deg.C, the dried anhydrous CaCl is put into a weighing bottle (radius 3 cm is multiplied by 4.8 cm)₂And cutting the SA/HA edible film into a proper size at a position 5 mm away from the cup mouth, sealing and weighing the bottle mouth, and then placing the weighing bottle in a dryer filled with silica gel, and weighing every 2 h. The water vapor transmission coefficient (WVP) of the SA/HA composite membrane was calculated as follows:

WVP=m×d/h×A×p

in the formula, WVP: water vapor transmission rate g.mm/(m)²H kpa); m is the increase (g) in the weight of the weighing bottle; d is the thickness of the film (mm); h is a time interval (h); a is the membrane area (m)²) (ii) a p is the difference in water vapor pressure across the membrane (3.168 kpa at 25 ℃).

1.2.3.4 determination of the degree of swelling

The swelling capacity of the SA/HA composite membrane is measured by a gravimetric method. The film was first cut into 3X 3 cm pieces and its dry weight M was measured₀. Each sample was soaked in 15 mL phosphoric acidThe mixture was swelled in a buffer (pH 7.4) at 37 ℃ for 24 hours. The sample was removed, the water on the surface of the composite membrane was blotted dry with filter paper, weighed and M recorded₂₄。

Degree of swelling (%) = (M)₂₄-M₀）/M₀×100%

In the formula M₀: initial weight of composite film; m₂₄: weight of composite membrane after 24 h in swelling medium.

1.2.3.5 determination of solubility

Reference is made to Khoshgozaran et al with minor modifications. The composite membrane sample (1 cm multiplied by 4 cm sample strip) is balanced in an environment with the relative humidity of 75% for 48 h, taken out, put into a conical flask filled with 50 mL of distilled water, capped, shaken in a rotary oscillator of 100 r/min for 24 h, then dried in an oven at 105 +/-1 ℃ for 24 h, taken out and weighed.

WS（%）=（W₁-W₂）/W₂×100%

Wherein WS: solubility of the composite film; w₁: initial weight of composite film; w₂: weight of composite film after 24 h in oven.

1.2.3.6 rheology

Refer to methods such as Zhouyijia and so on and slightly improve. 0.6 mL of the composite film-forming solution was sucked and placed at the center of the rotational rheometer test platform, a 60 mm, 2 ℃ conical plate was selected, and the gap was set to 69 μm, for the experiments. Setting temperature 25 deg.C, shear rate increasing from 0.01 to 100 s^-1And decreases from 100 to 0.01 s^-1。

1.2.3.7 FTIR analysis

Reference is made to the method of Zhang et al with minor modifications. And (3) measuring by using a Fourier transform infrared spectrometer: 2 mg of the freeze-dried sample is added with a small amount of KBr powder (the mass ratio of the sample to the KBr powder is about 100: 1), and the mixture is uniformly ground and pressed into a transparent sheet. The measurement parameters were set as: scanning resolution 2 cm^-1At 400-4000 cm^-1The test is carried out within the wave band range.

1.2.3.8 scanning electron microscope observation

Cutting the film into 5X 1 mm²Size, placing in liquid nitrogen to obtain brittle fracture surface;and then, under the accelerating voltage of 1 kV, fixing the membrane sample by using conductive adhesive, and observing the section structures of different SA/HA composite membrane samples by adopting a scanning electron microscope.

1.2.4 data statistics and analysis

All treatments were repeated three times, the final data were averaged over three trials, the data were recorded and plotted using Excel 2010, and the measured data were analyzed for significance within and between groups using span 20.

1.3 results and analysis

1.3.1 sodium alginate/hyaluronic acid composite Membrane preparation Single factor test results

The effect of different process conditions on the tensile strength of the SA/HA composite membrane is shown in figure 1.

As can be seen from fig. 1, the tensile strength of the composite film increases and then decreases as the number of times of freezing and thawing increases, and reaches a maximum value of 10.87 MPa at 2 times of freezing and thawing. Then the times of freezing and thawing are continuously increased, and the change of the tensile strength of the composite membrane is not obvious until the change is reduced. The tensile strength of the composite membrane is obviously improved along with the increase of the concentration of calcium lactate (p< 0.05) reached a maximum of 11.98 MPa at a calcium lactate concentration of 3%. As calcium ions are contacted with the sodium alginate solution to form gel immediately, the concentration of calcium lactate is increased, the gelling reaction rate is too high, the generation of subsequent gel reaction is blocked, and uniform gel with good three-dimensional structure cannot be obtained. The effect of the calcification time on the composite film is similar to the concentration of calcium lactate, and the graph shows that the composite film has the maximum tensile strength when the calcification time is 6 min. When the added amount of sodium alginate is increased from 15 percent to 60 percent, the tensile strength of the composite film is remarkably increased from 5.08 MPa to 11.66 MPa (p＜0.05）More than twice as much as it was, and then the tensile strength began to decrease with the addition of sodium alginate. As the reaction with calcium lactate is more complete and the density of gel is increased along with the increase of the addition amount of the sodium alginate, the network structure becomes complete, and the stress is dispersed when the external force is applied, so that the tensile strength of the composite film is increased and then reduced.

1.3.2 sodium alginate/hyaluronic acid composite membrane preparation technique response surface test result

A horizontal table 2 of the SA/HA composite membrane preparation process response surface test design is designed according to the single-factor test result of the SA/HA composite membrane preparation process.

The results of the analysis of variance of regression models prepared from SA/HA composite membranes are shown in Table 2.

TABLE 2 regression analysis of variance for SA/HA composite membrane preparation process

Note: marked difference (p＜0.01) (ii) a Significant difference (0.01＜p＜0.05）

The response surface test result shows that the modelPThe tensile strength of the SA/HA composite membrane is extremely related to a regression equation of a preparation process of the composite membrane, a mismatching term P =0.0581 is more than 0.05, and R-Squared =0.9769 shows that the model HAs high reliability and can optimize the preparation process of the SA/HA composite membrane. The regression equation is:

y=10.22+0.68A-0.081B-0.069C+0.097D-0.015AB+0.092AC+0.052AD-0.25BC+0.29BC-0.35CD-0.67A²-0.45B²-0.88C²-0.30D²。

as can be seen from Table 2, the primary and secondary effects of the factors on the tensile strength of the SA/HA composite film are as follows: the freezing and thawing times is more than the adding amount of sodium alginate and more than the concentration of calcium lactate and more than the calcification time. In this model, the first term A has a very significant effect on the tensile strength of the composite film (pLess than 0.01), D has obvious influence on the tensile strength of the composite film (p< 0.05), B, C have no significant effect on the tensile strength of the composite film（p＞0.05）(ii) a Second order term A²、B²、C²、D²The tensile strength of the composite film is greatly influenced (p< 0.01); the interaction terms BC, BD and CD have very significant influence on the tensile strength of the composite film (pLess than 0.01), and the tensile strength of the composite film is not significantly influenced by AB, AC and AD. The optimal combination of SA/HA composite membrane preparation obtained according to the response surface test is that the freezing and thawing times are 2.51, the calcium lactate concentration is 2.99%, the calcification time is 5.88 min, and the sodium alginate addition is 63.55%. The tensile strength of the SA/HA composite membrane obtained under the condition is 10.41 MPa。

1.3.3 sodium alginate/hyaluronic acid composite membrane preparation process verification test

The optimal preparation process of the SA/HA composite membrane is determined through response surface analysis, and the actual operation sets the process conditions to be 3 times of freezing and thawing, 3% of calcium lactate concentration, 6 min of calcification time and 60% of sodium alginate addition. And (3) measuring the tensile strength of the composite membrane, and repeating the test for three times, wherein the tensile strength of the obtained SA/HA composite membrane is 10.02 +/-0.28 MPa, and is basically similar to the theoretical value of 10.41 MPa. The response surface method can accurately optimize the preparation of the composite membrane.

1.3.4 characterization of sodium alginate/hyaluronic acid composite membrane performance and structure

1.3.4.1 Effect of Freeze-thaw times on physical Properties of composite membranes

The color of the SA/HA composite film directly affects the sensory score of the consumer on the food product.L*、a*、b*Respectively representing the brightness, redness and yellowness values of the composite film. The brightness of the composite film is significantly reduced with the increase of the number of times of freeze thawing (p<0.05), 3 times of freeze-thaw was 32.63% lower than 1 time. The total color difference value increased significantly from 47.36 at the beginning to 68.96 (p<0.05); composite membranea*Value sumb*The difference in value is not significant (p>0.05), it is possible that the color of the composite film is not substantially affected except for brightness, because the selected substrate is a colorless transparent solution when dissolved in water.

Effect of solubility: the opacity directly affects other relevant indexes such as mechanical property, water vapor transmittance and the like of the composite film. The lower the water vapor transmission rate of the composite film is, the better the fresh-keeping effect of the edible film is. The swelling capacity is examined about the water absorption capacity of the composite membrane. After the freeze-thaw treatment, the opacity, water vapor permeability, swelling degree and solubility of the composite membrane all show significant changes with the increase of the freeze-thaw times (p<0.05). The opacity of the composite film is increased from 0.12 to 1.15, the water vapor transmission rate is reduced from 1.10 to 0.55, and the swelling degree and the solubility are reduced by more than one time at the end of freezing and thawing. The main reason is that the electrostatic compounding of sodium alginate and hyaluronic acid is promoted along with the increase of the number of times of freeze thawing, the stronger the intermolecular hydrogen bond is, the tighter the molecular chain arrangement is, and the more compact the network structure of the membrane is, so thatThe transmission and diffusion of water molecules in the composite membrane are reduced, the permeability is reduced, water vapor is less likely to permeate, and the solubility is reduced.

Rheological analysis of composite membranes for 1.3.4.2 Freeze-thaw times

The film-forming blend was subjected to frequency sweep at different freezing and thawing times, and the resulting curves of storage modulus (G') and loss modulus (G ") versus frequency are shown in fig. 2. As the frequency was increased, the elastic modulus and loss modulus of all deposition solutions showed an upward trend. Before freezing and thawing, the loss modulus G ″ is greater than the storage modulus G' over the entire scanning frequency range. The two have strong frequency dependence, the whole system shows that the viscous behavior is larger than the elastic behavior, and the viscous flow behavior is dominant, which belongs to the typical solution characteristics. Freeze-thaw 1 times with storage modulus G ' > loss modulus G ' ', the two lines essentially coincide with increasing frequency, and the rheological behavior gradually changes from a typical solution to a weak gel. G 'and G "increased significantly with increasing number of freeze-thaw cycles, with G' being much greater than G" for 3 freeze-thaw cycles, showing pronounced gel behavior and independent of frequency. As the times of freezing and thawing are more, a stable network structure is formed among macromolecular chain segments in the film-forming solution, so that the possibility of free movement of molecules is reduced, and the elastic behavior of the whole system is continuously enhanced.

1.3.4.3 Infrared Spectrometry of composite membranes based on Freeze and thaw times

The infrared spectrum of the SA/HA composite film is shown in FIG. 3. The two samples of sodium alginate and hyaluronic acid have basically the same infrared curve, and the sodium alginate is at 3438 cm^-1Hyaluronic acid at 3413 cm^-1The absorption peak is shifted to 3403-3411 cm after blending^-1Nearby, mainly N-H and O-H stretching vibration. The position of the absorption peak of the composite membrane may be slightly shifted with respect to the substrate. Mainly because the intermolecular interaction forces are different, the stretching vibration peak of the composite membrane slightly moves to the low-frequency direction, which shows that the sodium alginate and the hyaluronic acid have better compatibility. Sodium alginate is 1594 cm^-1At a distance of 1409.62 cm^-1A symmetric stretching vibration absorption peak and an anti-symmetric stretching vibration absorption peak of a-COO-bond are formed in the position. The hyaluronic acid is at 1413 cm^-1And 1385 cm^-1To (3). Indicating that both substances contain-COO-bonds and that the composite membrane also has these two vibration peaks. Adding Ca²⁺then-OH groups were destroyed, which shifted to 1328 cm^-1The smaller peak area is less intense, indicating a strong interaction between the components.

1.3.4.4 microscopic analysis of composite membrane by scanning electron microscope based on freeze-thaw times

In FIG. 4, a, b, c are scanning electron microscope images of the cross section of the SA/HA composite membrane frozen and thawed 1 time, 2 times, 3 times respectively. Cracks and ridges exist in the microstructure of the section of the SA/HA composite membrane after 1-time freeze thawing and 2-time freeze thawing, the cracks and the ridges are caused by artificial cutting, and the composite membrane after 3-time freeze thawing presents a smooth surface form. The reason is that with the increase of the number of times of freezing and thawing, the more compact the network structure of the gel, the microphase separation of the composite hydrogel molecules in the system occurs, the self-assembly is carried out by enthalpy-driven random nucleation to form crystalline fibers, the fibers grow in a uniaxial direction at low temperature, the branching is less likely to occur, and the fibers are interpenetrated and intertwined. A partially closed hole structure is formed, the smaller the gel pore diameter is along with the extension of the freezing time, the more regular the composite gel molecular network structure is, and the better compatibility of the sodium alginate and the hyaluronic acid is shown.

1.4 summary of this chapter

In this chapter, the sodium alginate/hyaluronic acid composite edible film is prepared for the first time, the preparation process of the edible film is determined by adopting a method of combining a single factor with a response surface experiment, the basic physical properties of the film are represented, and the microstructure is observed on the basis of analyzing the rheological property and the compatibility of the film. The main conclusions are:

(1) the method comprises the following steps of carrying out single-factor experiments by using sodium alginate and hyaluronic acid as main film forming base materials and calcium lactate as a cross-linking agent to obtain the ranges of the freezing-thawing times, the concentration of calcium lactate, the calcification time and the addition amount of sodium alginate, taking the tensile strength of the composite film as an investigation index, further determining the optimal conditions of all factors by using a response surface optimization method, and determining the process parameters as follows: freeze thawing for 3 times, calcium lactate concentration of 3%, calcification of 6 min, and sodium alginate addition of 60%. The main and secondary influence sequence of all factors on the tensile strength of the edible film is as follows: the freezing and thawing times is more than the adding amount of sodium alginate and more than the concentration of calcium lactate and more than the calcification time. The tensile strength of the SA/HA composite membrane obtained under the condition is 10.41 MPa.

(2) The influence of different freezing and thawing times on the chromaticity, the basic physical property, the rheology, the infrared ray and the microstructure of the composite membrane is researched, and the result shows that: as the number of freeze-thaw cycles increases, there is substantially no effect on the color of the composite film other than a decrease in brightness, which is consistent with the opacity study; the network structure of the film becomes compact with the increase of the freezing and thawing times, the surface of the edible film becomes uniform and regular, and the swelling degree and the solubility of the composite film are reduced through a scanning electron microscope picture. As can be seen from the frequency scanning graph, the blending liquid is changed into a weak gel state from a typical solution, and obvious gel behavior is shown after 3 times of freeze thawing; the infrared spectrum shows the common characteristic spectrum of sodium alginate and hyaluronic acid, the stretching vibration peak slightly moves to the low frequency direction, and the Ca content is measured by Ca²⁺After the action, hydrogen bonds are generated among molecules, which shows that the film-forming base material generates better compatibility. Therefore, the composite film prepared by the research basically meets the requirements of edible films, and provides a new idea for researching and developing novel edible films.

Research on preservation effect of sodium alginate/hyaluronic acid composite edible film on refrigerated beef

2.1 Experimental instruments and materials

2.1.1 Experimental materials and reagents

The meat sample used in the test is collected from Gansu Qilian Muge industry Co., Ltd, and beef cattle with 4-5 years old, good growth and development, health and no disease are selected.

TABLE 3 Main test reagents

Name (R)	Type (B)	Manufacturer of the product
			NADH	Analytical purity	Bangtai bioengineering Co Ltd
Trichloroacetic acid	Analytical purity	Tianjin City Guang Compound technology Co Ltd
			Diamine tetraacetic acid disodium salt	Analytical purity	Kaiton chemical reagent Co., Ltd, Tianjin City
Thiobabituric acid	Analytical purity	Kaixin chemical industries, Inc. of Tianjin
			Chloroform	Analytical purity	CHENGDU CHRON CHEMICALS Co.,Ltd.
Magnesium oxide	Analytical purity	CHENGDU CHRON CHEMICALS Co.,Ltd.

2.1.2 Experimental instruments

TABLE 4 Main Instrument

Name of instrument	Model number	Manufacturer of the product
			Color difference meter	Konica CR-10	Zhuhai Tianchuang Instrument Co Ltd
High-speed disperser	JY96-N	GRAND ANALYTICAL INSTRUMENT Co.,Ltd.
			High-speed table type refrigerated centrifuge	TGL-16M	Shanghai Peioo Analyzer Co Ltd
Ultraviolet visible light spectrophotometer	SP-756P	Beckman Co Ltd
			Electronic balance	PHS-3C	Vorte Co Ltd
Fluorescence spectrophotometer	RF-5301PC	Mono instruments Ltd
			Texture instrument	MSMTA-XT	Shanghai Tengting technologies, Inc
Ultrasonic cell crusher	CN10-JY92-2DA	Tomo root Biotechnology Ltd
			PH meter	MT-5000	Beijing Qiangsheng instrument manufacturing center

2.2 Experimental methods

2.2.1 preparation of sodium alginate/hyaluronic acid composite coating solution

blending the two solutions according to a certain proportion to obtain a blended solution (the addition of sodium alginate is 60%), uniformly stirring, pouring into a beaker, sealing with a sealing film, freezing and thawing in a refrigerator at (-18 ℃) for 24 hours, thawing at room temperature for 12 hours, and performing freeze-thaw cycle for 1 time), and freezing and thawing for 3 times to obtain the composite freeze-thaw gel.

2.2.2 sample treatment

The meat pieces were divided into two groups by removing epidermal fat and fascia, cutting into about 50g meat pieces with the same thickness. The surface of the treated meat pieces was evenly brushed with a layer of freeze-thaw gel, immediately soaked in 3% calcium lactate for 6 min, then fished out and drained in air, and the meat pieces were stored at 4 + -1 ℃ as control groups without treatment. The corresponding indices were determined at 0, 1, 3, 5, and 7 d of maturity, respectively.

2.2.3 exploring the influence of SA/HA composite membrane on beef refrigeration process

2.2.3.1 sensory evaluation

The sensory evaluation methods such as sensory index evaluation method and friendship are slightly modified according to GB/T22210-2008 sensory evaluation Specification for meat and meat products. An evaluation group is composed of 6 (3 boys and 3 girls) food professional students, evaluation indexes comprise four items of color, smell, viscosity and tissue elasticity, each item is full of 25 points, and the four items of index scores are added to form a total score of the meat sample. Fresh meat when the total score is more than or equal to 60; less than 40 percent of sub-fresh meat and less than 60 percent of sub-fresh meat; stale or spoiled meat < 40.

TABLE 5 index systems for sensory evaluation

Item	15 to 25 minutes	10 to 15 minutes	1 to 9 minutes
				Color and luster	Bright red and lustrous muscles	Red and lustrous muscles	Dark red and lusterless muscles
Smell(s)	Has special flavor of fresh beef	Slightly peculiar smell	Has putrefactive odor
				Viscosity of the oil	Slightly dry or dry film on surface, and is not sticky	Hair growing on the surfaceSticky, moist cut surface	The surface is sticky and soft
Elasticity of tissue	Immediate recovery of finger pressure	Recovery of finger pressure is slow and cannot be completely recovered	The finger pressure can not be recovered and has obvious indentation

2.2.3.2 color intensity

After the packaging bag is opened, the meat sample is cut, a fresh section is exposed, the meat sample is placed for 30 min at room temperature, the white board is used for calibration, and the flesh color of the sample is measured by a CR-410 colorimeter. The samples were placed under a light source and measured at 0, 1, 3, 5, 7 d, respectivelyL*、a*、b*And reading the value displayed by the color difference meter.

2.2.3.3 NADH concentration

Using a kit produced by Nanjing Jiancheng corporation, adding corresponding reagents according to the operation instructions: accurately weighing 0.1g of meat sample, centrifuging for 5min under the centrifugal force of 600 g, filtering supernatant, centrifuging for 10 min under the condition of 1100 g, taking precipitate, adding corresponding reagents according to the specification, crushing by ultrasonic waves with the power of 20%, the ultrasonic time of 3 s, the interval of 10 s, repeating for 25 times, and finishing all operations at 4 ℃. The absorbance values at 20 s and 80s were determined and calculated by zeroing with distilled water at 600 nm, adding the corresponding reagents according to the instructions.

2.2.3.4 pH value

Refer to the method of Kim et al and make slight modifications. A10 g meat sample was weighed, 40 mL of distilled water was added, and the sample was homogenized for 30 s, and the pH of the sample was measured with a pH meter.

2.2.3.5 TBARS

Reference is made to the method of Witte et al and a slight modification. 10 g of sample (exactly 0.0001 g) was weighed out and homogenized at 8000 r/min for 30 s by adding 50 mL of a buffer containing 0.1% EDTA and 7.5% trichloroacetic acid and continued for 30 min. Then using two layers of nylonFiltering with dragon cloth to obtain filtrate, sucking 5 mL of the filtrate, mixing with 5 mL of 0.02 mol/L thiobarbituric acid solution, and heating in boiling water bath for 30 min. Centrifuging the mixture at 10000 r/min for 5min, cooling to room temperature, adding 5 mL chloroform, standing for layering, and collecting supernatant, and measuring absorbance at 532 nm and 600 nm. Blank controls included 5 mL TBA and 5 mL trichloroacetic acid, TBARS values expressed as mg malondialdehyde per 100g meat, TBARS values (mg/100 g) = (A)₅₃₂-A₆₀₀）/155×（1/10）×72.6×100。

2.2.3.6 TVB-N

According to the national standard (GB/T5009.44.40), the TVB-N content in the cooled meat is measured by adopting a steam distillation method. Weigh 10.0 g (+ -0.1 g) of the meat sample and chop. Adding 27 mL of deionized water into the minced meat sample, fully soaking for 30 min at room temperature, taking 10 mL of sample liquid, putting into a glass nitration tube, adding 1g of magnesium oxide, shaking and mixing, and measuring the TVB-N content by using an automatic Kjeldahl apparatus. The distillation time was set to 3 min, 30 mL of boric acid was used as the receiving solution, calibrated with a standard sulfuric acid solution, pH =4.65 as titration endpoint, and TVB-N was calculated according to the following equation:

X=（V₁-V₂) X c × 14/m × 100, wherein X: units are milligrams per hundred grams (mg/100 g); v₁: the sample consumed the volume of sulfuric acid standard titration solution in milliliters (mL); v₂: the blank consumed the volume of sulfuric acid standard titration solution in milliliters (mL); m: meat-like quality.

2.2.3.7 Total number of colonies

The total number of bacterial colonies was determined according to the national standard GB 4789.2-2016.

2.2.3 data statistics and analysis

All treatments were repeated three times, the final data were averaged over three tests, the test data were recorded and plotted using Excel 2010, and the significance analysis of the measured data was performed using span 20.

2.3 results and analysis

2.3.1 Effect of sodium alginate/hyaluronic acid composite film on sensory evaluation of chilled beef

With delay of refrigeration timeThe sum of the sensory characteristic scores of the long beef gradually decreases, and the treatment group is significantly higher than the control group at any time point (except day 0) during the refrigeration period (thep<0.05). At the time of 0 d, the sensory score of the beef is as high as 94.66, the color of the muscle is bright red, and the surface is provided with dry films of wind and is not sticky. With the increase of the refrigeration time, the characteristics of the beef such as color, smell, viscosity and tissue elasticity are gradually deteriorated. When the meat is refrigerated to the 3 rd day, the sensory score of the control group is reduced to 47.3 points, the meat is changed into sub-fresh meat, the color of the muscle is deteriorated, and the peculiar smell is partially appeared, when the meat is refrigerated to the 5 th day, the meat of the control group is putrefactive, and the sensory score of the treatment group is 46.82 at the 7 th day, which is obviously higher than that of the control group (59.63%) (p<0.05). The SA/HA composite membrane can effectively keep the quality of the cold fresh meat and slow down the quality deterioration process of the cold fresh meat.

2.3.2 Effect of sodium alginate/hyaluronic acid composite membrane on color value of refrigerated beef

Treating the meat during the entire cold storage periodL*Control group with significantly high value: (p<0.05). Luminance values of processing groupsL*The temperature of the liquid tends to rise first and then fall as the refrigeration time is prolonged. Of control meat samplesL*The value at 5 d drops to 27.70 already below 30; for treating groups of meat samplesL*The value at 7 d was 34.93. Red value in cold storage perioda*Significantly reduce (p<0.05), yellowness indexb*Is significantly increased (p<0.05), and the control group was significantly higher than the treatment group at any time point during cold storage (except 0 d) ((ii)p<0.05). Since the meat-like sample of the control group was continuously oxidized by direct contact with oxygen, the content of the red-brown methemoglobin was increased, so thata*The reduction in value was not acceptable to the consumer, and was 39.68% and 47.98% for the treated group and the control group, respectively, at the end of refrigeration. The contact of the meat sample with oxygen also causes oxidation of the fat, the treatment group being at the end of the refrigerationb*Very significant value (p<0.01) lower than the control group, indicating that the sea SA/HA complex membrane inhibits the oxidation of intramuscular fat of beef.

2.3.3 Effect of sodium alginate/hyaluronic acid composite Membrane on NADH concentration of chilled beef

NADH is limiting the reduction of methemoglobinImportant coenzymes of (a). Ramanathan et al have shown that the addition of calcium lactate can enhance the antioxidant properties of beef, and promote the increase of reducing substrate and NADH in the reduction system of ferrimyoglobin, thereby improving the color stability of fresh meat. The NADH concentration of the treatment group and the control group shows a descending trend along with the extension of the refrigeration time, the treatment group slowly descends within 0-3 d, then the descending gradually becomes fast, and the control group shows a remarkable descending trend (the step (b) ((the step (b))p<0.05). Probably because the calcium lactate in the composite membrane of the treated group penetrated into the tissue via the muscle surface to NAD⁺On the basis of coenzyme, NADH reducing power is generated by the action of lactate dehydrogenase, thereby replenishing NADH. The control group is 53.56% lower than the treatment group after refrigeration is finished, and the NADH concentration of the treatment group is obviously higher than that of the control group (p<0.05）。

2.3.4 Effect of sodium alginate/hyaluronic acid composite membrane on refrigerated beef pH value

The pH value is an important index for judging the meat quality. In the cold storage period, the pH value of each group is in a descending trend at 0-3 d and then gradually rises. This is mainly due to the accumulation of lactic and phosphoric acids by anaerobic glycolysis after slaughter of animals, resulting in a decrease in pH, followed by the decomposition of the proteins in the meat into alkaline substances, such as ammonia and amine compounds, etc., under the action of bacteria and enzymes, which gradually increase the pH. From 3 d onwards, the pH value of the control group was significantly higher than that of the treatment group (p<0.05). The pH values of the treatment group and the control group are 6.17 and 6.84 respectively at the end of the cold storage period, and according to the regulation of the pH value in GB/T9695.5-2008, the primary freshness is 5.8-6.2, the secondary freshness is 6.3-6.6, and the deteriorated meat is more than 6.7. It can be seen that the control group had spoiled at 7 d. This shows that the SA/HA composite membrane can effectively control the pH value of the beef.

2.3.5 Effect of sodium alginate/hyaluronic acid composite membrane on TBARS value of refrigerated beef

During the storage of meat, lipids are easily oxidized by light, heat and enzymes, and finally, aldehydes, ketones and the like are generated. Therefore, the size of the thiobarbituric acid value represents the degree of oxidation of the lipids of the meat, and a larger value indicates a higher degree of oxidation, and the freshness of the meat can be assessed, as shown in detail: 0.20 mg/kg < fine meat < 0.66 mg/kg, deteriorated meat >1 mg/kg. The TBARS increased slowly in the treatment group and significantly in the control group with increasing refrigeration time: (p<0.05). And the control group was significantly higher than the treatment group at any time point during cold storage except 0 d: (p<0.05). The TBARs of the control group at the refrigerated stage 5 d exceeded the range of the good meat quality by 0.90 mg/100g, and the TBARS of the treatment group at the refrigerated stage 7 d exceeded the range of the good meat quality by 0.64 mg/100 g. The SA/HA composite membrane can effectively inhibit lipid oxidation of beef during refrigeration.

2.3.6 influence of sodium alginate/hyaluronic acid composite membrane on TVB-N value of chilled beef

In the cold storage process of the fresh meat, volatile basic nitrogen is one of the common important indexes for evaluating the freshness of the meat, and the food inspection method physicochemical part of the Ministry of health of the people's republic of China stipulates that the TVB-N of the fresh meat is less than or equal to 15 mg/100g, and the deteriorated meat is more than 20 mg/100 g. The TVB-N values of the treated group and the control group both showed an increasing trend with the increase of the refrigeration time, the treated group rose slowly within 0-3 days, and the control group showed a significant increasing trend (p<0.05). And the control group was significantly higher than the treatment group throughout the cold storage period (p<0.05). The TVB-N of the chilled beef is initially 4.17 mg/100g and is in a fresh state. The TVB-N value of the treated group at the 7 d cold storage time was 14.28 mg/100g and did not exceed the national standard of 15 mg/100g, while the TVB-N value of the control group was 27.65 mg/100g and exceeded the national standard of meat deterioration. At this time, the muscle color is dark red, the surface is sticky, and the muscle has serious putrefactive odor, which is consistent with the pH analysis result.

2.3.7 Effect of sodium alginate/hyaluronic acid composite film on the Total bacterial count of chilled beef

The stipulations of the total number of bacterial colonies according to the quality health indexes of the fresh meat are as follows: the first-level fresh meat is less than 4.0 lg (CFU/g), the second-level freshness is 4.0-6.0 lg (CFU/g), and the deteriorated meat is more than 6.0 lg (CFU/g). The initial bacterial colony count of the refrigerated beef is 3.48 lg (CFU/g), and the colony count of the control group is obviously increased in an exponential manner along with the extension of the refrigeration time (the total number of the bacterial colonies of the control group is obviously increased in an exponential manner: (p<0.05). The control group had deteriorated meat at the refrigerated stage 7 d, the total number of colonies increased 46.79% to 6.54 lg (CFU/g), while the treatment group had a slow increase in the total number of colonies during the refrigerated stage,at the end of the refrigeration 4.88 lg (CFU/g). Because the sodium alginate in the composite membrane utilizes-COONa and-OH active groups on molecules to destroy the structure of a bacterial cell membrane and influence the normal metabolism of bacteria, the growth of microorganisms is inhibited, and meanwhile, the composite membrane can limit oxygen permeation and inhibit the activity of the bacteria. The SA/HA composite edible film prepared by the test can inhibit the growth of microorganisms to achieve a good fresh-keeping effect.

2.4 technical summary

According to the method, sodium alginate and hyaluronic acid are compounded according to a certain proportion, composite freeze-thaw gel is obtained through freeze-thaw treatment for 3 times, the beef is coated on the surface of the beef and soaked in calcium lactate, a layer of air-dried film is formed on the surface of the beef after the beef is dried in the air, and the fresh-keeping effect of the beef in the cold storage period is researched. The influence of the composite membrane on the freshness of the beef is comprehensively evaluated by measuring sensory score, chromaticity, NADH concentration, pH value, TBARS value, TVB-N value and total bacterial colony number, and the result shows that:

the composite membrane can obviously inhibit the increase of beef sensory score, pH value, TBARS value, TVB-N value and total bacterial colony number in the refrigeration process, and delay the decrease of NADH concentration. Brightness value of treated group and control group compared with initial value in refrigeration 7 dL*Respectively reduced by 17.03% and 42.11%; the yellowness index b is respectively increased by 45.85 percent and 61.37 percent, and the meat color deterioration (b) is obviously improvedp<0.05). By measuring the change of each index, the preservation effect of the composite film is superior to that of a control group.

Claims

1. A preparation method of an antibacterial fresh-keeping composite freeze-thaw edible film for cold fresh meat is characterized by comprising the following steps

2. The preparation method of the antibacterial fresh-keeping composite freeze-thaw edible film for the cold fresh meat according to claim 1, wherein the step C is performed by sealing with a sealing film, freezing for 24 h at the temperature of-18 ℃, thawing for 12 times at room temperature, completing 1 freeze-thaw cycle, and performing freeze-thaw treatment on the obtained composite gel for 1-4 times.

3. The preparation method of the antibacterial fresh-keeping composite freeze-thaw edible film for the cold fresh meat as claimed in claim 2, wherein the step C comprises sealing with a sealing film, freezing for 24 h at-18 ℃, thawing at room temperature for 12 times for 1 freeze-thaw cycle, and subjecting the obtained composite gel to freeze-thaw treatment for 2 times.

4. The method for preparing the antibacterial and fresh-keeping composite freeze-thaw edible film for the cold fresh meat according to claim 1, wherein a sodium alginate solution with a volume ratio of 15%, 30%, 45%, 60% or 75% is added to the hyaluronic acid solution in the step C.

5. The method for preparing the antibacterial and refreshing composite freeze-thaw edible film for the cold fresh meat according to claim 4, wherein a sodium alginate solution with a volume ratio of 60% is added to the hyaluronic acid solution in the step C.

6. The method for preparing the antibacterial fresh-keeping composite freeze-thaw edible film for the cold fresh meat as claimed in claim 4, wherein the mass concentration of calcium lactate in the step D is 1-5%, and the calcification time is 6 min.

7. The method for preparing the antibacterial and refreshing composite freeze-thaw edible film for the cold fresh meat as claimed in claim 6, wherein the calcium lactate has a mass concentration of 3% and a calcification time of 6 min in the step D.

8. The method for preparing the composite freeze-thaw edible film for the antibacterial preservation of the chilled fresh meat according to any one of claims 1 to 7, which is used for the refrigeration preservation of beef.

9. The method as claimed in claim 8, wherein the method comprises removing skin fat and fascia from the meat, cutting into about 50g meat pieces, coating the meat pieces with the composite gel prepared in step A, B, C, calcifying the composite gel with calcium lactate solution in step D, washing with distilled water to remove calcium lactate, air drying, and storing at 4 ± 1 ℃.