Calcium alginate-gelatin-lysozyme composite antibacterial film as well as preparation method and application thereof

Abstract

The invention discloses a calcium alginate-gelatin-lysozyme composite antibacterial film and a preparation method and application thereof, and relates to the technical field of food antibacterial packaging.The method comprises the steps that sodium alginate is dissolved in water to obtain a solution containing 1%-3% (w / w) of sodium alginate, 0.5%-3% (w / w) of gelatin is added, the mixture is heated to 50-85 DEG C in a water bath till the solution is in a dissolved state, and the solution is cooled to the room temperature to obtain the calcium alginate-gelatin-lysozyme composite antibacterial film. Finally, glycerol with the total mass fraction being 0.2%-0.5% is added to serve as a plasticizer, and a sodium alginate-gelatin composite film is prepared through drying with a solution casting method; placing the sodium alginate-gelatin composite membrane in a 0.5%-3% (w / w) CaCl2 solution to prepare a calcium alginate-gelatin composite membrane; placing the calcium alginate-gelatin composite membrane in a lysozyme solution, oscillating, taking out, and drying at 25-50 DEG C to obtain the calcium alginate-gelatin composite membrane. The method has the beneficial effects that the method is simple and easy to implement, has a good antibacterial effect and better mechanical properties, is high in safety, is green and environment-friendly, and can inhibit growth and reproduction of common pathogenic bacteria and putrefying bacteria.

Description

Technical Field

[0001] The invention belongs to the field of antibacterial food packaging, and specifically relates to a calcium alginate-gelatin-lysozyme composite antibacterial film and a preparation method and application thereof. Background Art

[0002] Antimicrobial packaging systems are generally composed of a composite material with good film-forming properties and an active substance with antimicrobial function. When lysozyme is added to the film-forming matrix as an antimicrobial component, it is often affected by the complex application environment, and the actual effect is greatly reduced. In order to ensure the activity of lysozyme during application, it is necessary to use some technologies to improve its stability in complex environments; enzyme immobilization can solve the problem of reduced enzyme antimicrobial activity caused by environmental changes. Enzyme immobilization technology is one of the important means to improve its stability. Among them, some natural non-toxic and harmless macromolecules are considered to be a good immobilization medium in the food industry.

[0003] Sodium alginate is a linear heteropolysaccharide extracted from algae, composed of D-mannuronic acid and L-guluronic acid. As a natural polysaccharide, sodium alginate has attracted much attention in food packaging materials due to its non-toxicity, easy extraction, good film-forming properties and biocompatibility. L-guluronic acid of alginate is believed to be highly soluble in divalent cations (such as Ca 2+ ) can interact with the deprotonated carboxyl groups and produce a cross-linked "egg-box" structure, which is a good carrier for the delivery of active substances. However, the addition of divalent cations Ca 2+ After cross-linking, its mechanical properties will be affected. When used as a packaging material, the hardness and brittleness of the film will increase; this limits its application as a packaging material in food.

[0004] Most of the packaging films with antibacterial activity currently studied are colored or have high solubility, which limits their application in high humidity conditions. In addition, the colored packaging films will affect consumer acceptance to a certain extent when used.

[0005] Therefore, how to prepare a colorless, odorless, and highly transparent film with antibacterial function and good mechanical properties for use in food packaging is a technical problem that needs to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, to provide a safe, green and environmentally friendly food antibacterial packaging material, and specifically to provide a calcium alginate-gelatin-lysozyme composite antibacterial film and its preparation method and application.

[0007] As an animal-derived protein, gelatin has high biocompatibility and good film-forming properties. It is generally extracted from collagen through an acid-base extraction process, which is low in cost. After extensive research, the applicant found that gelatin-based films have good tensile properties. Sodium alginate and gelatin can combine with each other through electrostatic interactions and hydrogen bonds to form a stable complex, which can improve the problem of poor mechanical properties of a single component; at the same time, the composite formed by the combination of the two has a denser structure, which can also solve the problem of poor barrier properties of a single component.

[0008] The present invention uses gelatin as an improver of the mechanical properties of calcium alginate film, uses sodium alginate and gelatin as film-forming matrices, and uses calcium chloride as a cross-linking agent to prepare a calcium alginate-gelatin composite matrix film; and on this basis, lysozyme is further immobilized to prepare a calcium alginate-gelatin-lysozyme composite antibacterial film. The calcium alginate-gelatin-lysozyme composite antibacterial film obtained by the present invention has the advantages of being colorless, odorless, and highly transparent, and the raw material cost used is low, the safety is high, and it can be mass-produced and applied to food packaging.

[0009] The technical solution of the present invention is as follows: The first aspect of the present invention provides a method for preparing a calcium alginate-gelatin-lysozyme composite antibacterial film, comprising the following steps: S1, dissolving sodium alginate in water to obtain a sodium alginate solution; adding gelatin to the sodium alginate solution, heating and stirring until dissolved; then adding glycerol and stirring evenly to obtain a sodium alginate-gelatin composite liquid; casting the sodium alginate-gelatin composite liquid into a film-forming device, drying, and obtaining a sodium alginate-gelatin composite film; S2, adding the sodium alginate-gelatin composite film to a CaCl2 solution and stirring, then taking it out, washing the surface with water, and drying it to obtain a calcium alginate-gelatin composite film; S3, placing the calcium alginate-gelatin composite film in a lysozyme solution and shaking it, then taking it out and drying it to obtain a calcium alginate-gelatin-lysozyme composite antibacterial film.

[0010] Optionally, the mass percentage concentration of the sodium alginate solution is 1%-3%, and the amount of gelatin added is 0.5%-3% of the mass of the sodium alginate solution.

[0011] Optionally, the amount of glycerol added is 0.2%-0.5% of the total mass of the sodium alginate solution and gelatin.

[0012] Optionally, the heating temperature after adding gelatin is 50°C-85°C.

[0013] Optionally, the mass percentage concentration of the CaCl2 solution is 0.5%-3%.

[0014] Optionally, the stirring time of adding the sodium alginate-gelatin composite film to the CaCl2 solution is 5 min-120 min.

[0015] Optionally, the calcium alginate-gelatin composite film is placed in the lysozyme solution and shaken for 20 min-240 min.

[0016] Optionally, the concentration of the lysozyme solution is 5×10 -4 g / mL-5×10 -2 g / mL.

[0017] The second aspect of the present invention provides a calcium alginate-gelatin-lysozyme composite antibacterial film, which is obtained by the above-mentioned preparation method.

[0018] The third aspect of the present invention provides a use of the above-mentioned calcium alginate-gelatin-lysozyme composite antibacterial film in antibacterial food packaging materials.

[0019] The present invention has at least one of the following beneficial effects: The invention prepares a colorless, odorless, highly transparent composite film with antibacterial function and good mechanical properties. On the one hand, the invention uses CaCl2 as a cross-linking agent to cross-link it with sodium alginate, and the obtained calcium alginate has a uniform porous structure, which provides an effective matrix for the immobilization of the antibacterial agent lysozyme, ensures the stable performance of the lysozyme during application, thereby improving the antibacterial effect of the composite film, and solves the problem in the prior art that the actual performance of lysozyme as an antibacterial component is greatly reduced due to the influence of the complex application environment; on the other hand, the invention improves the problem of increased hardness of the composite film caused by the introduction of calcium ions in CaCl2 by adding gelatin, and gelatin and sodium alginate are combined with each other through electrostatic interaction and hydrogen bonds to form a stable composite, thereby improving the mechanical properties of the composite antibacterial film during application, so that the composite film of the invention has both good antibacterial effect and better mechanical properties.

[0020] The preparation method of the invention is simple and easy, and the obtained composite antibacterial film has certain practical value, has the advantages of being colorless, odorless, highly transparent, etc., has an inhibitory effect on common pathogenic bacteria and spoilage bacteria, and can be used for food packaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown is a flow chart of the preparation of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention; Figure 2 The figure shows the appearance of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention, wherein a corresponds to Example 1, b corresponds to Example 3, and c corresponds to Example 1; Figure 3The microstructure diagram of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention is shown, wherein a and c are the surface and cross-sectional microstructure diagrams of the sodium alginate-gelatin composite film prepared in Comparative Example 1, b and e are the surface and cross-sectional microstructure diagrams of the calcium alginate-gelatin composite film prepared in Comparative Example 3, and c and f are the surface and cross-sectional microstructure diagrams of the calcium alginate-gelatin-lysozyme composite film prepared in Example 1; Figure 4 The figure shows the appearance of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention when applied to the surface of beef, wherein a corresponds to Example 1, b corresponds to Example 3, and c corresponds to Example 1; Figure 5 The figure shows the appearance of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention after being applied to the beef surface and peeled off, wherein a corresponds to Example 2, and b corresponds to Example 1; Figure 6 The graph shows the antibacterial results of the calcium alginate-gelatin-lysozyme composite antibacterial film of the present invention against four common spoilage bacteria and foodborne pathogens, wherein the Control group is a negative control, the ALG-Gel group corresponds to the corresponding proportion 1, the Ca-ALG-Gel group corresponds to the corresponding proportion 3, and the Ca-ALG-Gel-LYS group corresponds to Example 1.

[0022] Figure 7 Shown is the calcium alginate precipitate formed by adding CaCl2 to the sodium alginate solution in Comparative Example 4. DETAILED DESCRIPTION

[0023] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Example 1 A method for preparing a calcium alginate-gelatin-lysozyme composite antibacterial film, referring to Figure 1 , including the following steps: (1) Preparation of sodium alginate-gelatin composite film: Weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add 1.515 g of gelatin to the dissolved sodium alginate solution and stir it under heating conditions at 70°C until it is dissolved, then add 0.513 g of glycerol and stir evenly to obtain a sodium alginate-gelatin composite solution. Cast the composite solution into a culture dish and dry it at 30°C to obtain a sodium alginate-gelatin composite film.

[0025] (2) Preparation of calcium alginate-gelatin composite film: The obtained sodium alginate-gelatin composite film was added into a 2% CaCl2 solution and stirred for 40 min. The CaCl2 solution on the surface was washed with distilled water and then dried at 30°C to obtain the calcium alginate-gelatin composite film.

[0026] (3) Preparation of calcium alginate-gelatin-lysozyme composite antibacterial film: The calcium alginate-gelatin composite film was placed in a solution with a concentration of 1×10 -3 The mixture was placed in a lysozyme solution of 100 g / mL and shaken for 60 min. After being taken out, it was dried at 30 °C to obtain a calcium alginate-gelatin-lysozyme composite antibacterial film.

[0027] The performance of the calcium alginate-gelatin-lysozyme composite antibacterial film obtained in this example was tested according to the following method: (1) Tensile strength and elongation at break: The mechanical properties of the composite film were determined according to the method of Zhong Chan, et al. (2021). The film with a smooth surface and complete morphology was cut into a rectangle of 3 cm × 5 cm. Five points were randomly selected with a micrometer to measure the thickness of the film and calculate the average value. The tensile strength (TS) and elongation at break (EAB) of the film were measured using a texture analyzer with the initial spacing of the clamps set to 30 mm and the stretching speed set to 1 mm / s. Before the measurement, the film was placed in a constant temperature and humidity incubator at 25°C and 50% relative humidity for 48 h. The measurement of each film was repeated three times. The TS and EAB of the film were calculated according to formulas (1) and (2).

[0028] (1) (2) Where: F is the maximum tension when the membrane breaks (N); A is the cross-sectional area of ​​the membrane (m 2 ). L0 is the original distance between the clamps (30 mm); L1 is the distance between the clamps after the membrane is stretched and broken (mm).

[0029] (2) Water solubility (WS): The prepared membrane was placed in an oven and dried to a constant weight (W1). The dried composite membrane was immersed in distilled water at 25°C for 24 h, taken out and placed in an oven again to dry to a constant weight (W2). The calculation formula is as follows: (3) (3) Lysozyme activity: The activity of the enzyme after immobilization was determined according to the method described by Wang Lechuan, et al. (2020). The Ca-ALG-Gel-LYS composite membrane loaded with lysozyme and the uncross-linked ALG-Gel-LYS composite membrane were stored at room temperature (25°C). The enzyme activity was determined based on the turbidity method. The Ca-ALG-Gel-LYS composite membrane loaded with lysozyme and the uncross-linked ALG-Gel-LYS composite membrane samples with different storage times were added to a suspension of Micrococcus lysodeikticus with an absorbance of 1.300 at 450 nm. After reacting in a 25°C water bath for 1 min, the absorbance value A1 of the bacterial suspension at 450 nm was recorded. After reacting for 2 min, the absorbance value A2 at 450 nm was recorded. The lysozyme activity was estimated by the change in the absorbance of the bacterial suspension at 450 nm per minute. Each group of samples was measured in parallel 3 times. Activity calculation formula (4): (4) Where: A1-A2: Absorbance change of bacterial solution containing lysozyme composite membrane at 450 nm for 1 min; 0.001: one unit of lysozyme activity causes the absorbance at 450 nm to decrease by 0.001 per minute; S: area of ​​composite membrane contained in the test solution, cm 2 .

[0030] (4) Antibacterial performance: The antibacterial rate of the membrane was determined according to the method of Wei Zhengxun, et al. (2021). Using the viable cell colony counting method, 100 mg of the membrane was added to 5 mL and cultured for 10 6 CFU / mL of logarithmic bacterial suspension was mixed and cultured at 37℃ and 180 r / min for 8 h, and the bacterial suspension without membrane was used as negative blank control. The ten-fold gradient dilution method was used to detect Escherichia coli ( E. coli ), Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ) and Salmonella ( Salmonella ) to quantitatively analyze the antibacterial properties of the membrane.

[0031] After testing, the tensile strength of the calcium alginate-gelatin-lysozyme composite antibacterial film obtained in this embodiment is (46.03±0.54) MPa; the elongation at break is (38.51±0.25)%; the water solubility is (3.53±0.04)%; the transmittance is close to zero in the ultraviolet range (200-300 nm); after one month, there is no significant difference between the lysozyme activity in the composite film and the initial activity; within 8 hours, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella can be reduced by approximately 2.66 log CFU / mL, 3.42 log CFU / mL, 3.73 log CFU / mL and 2.9 log CFU / mL, respectively.

[0032] Example 2 A method for preparing a calcium alginate-gelatin-lysozyme composite antibacterial film comprises the following steps: (1) Preparation of sodium alginate-gelatin composite film: Accurately weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add 0.505 g of gelatin to the dissolved sodium alginate solution and stir it at 70°C until it is dissolved; add 0.508 g of glycerol and stir evenly to obtain a sodium alginate-gelatin composite solution. Cast the composite solution into a culture dish and dry it at 30°C to obtain a sodium alginate-gelatin composite film.

[0033] (2) Preparation of calcium alginate-gelatin composite film: The obtained sodium alginate-gelatin composite film was added into a 2% CaCl2 solution and stirred for 40 min. The CaCl2 solution on the surface was washed with distilled water and then dried at 30°C to obtain the calcium alginate-gelatin composite film.

[0034] (3) Preparation of calcium alginate-gelatin-lysozyme composite antibacterial film: The calcium alginate-gelatin composite film was placed in a solution with a concentration of 1×10 -3 The mixture was placed in a lysozyme solution of 100 g / mL and shaken for 60 min. After being taken out, it was dried at 30 °C to obtain a calcium alginate-gelatin-lysozyme composite antibacterial film.

[0035] The detection method is the same as that in Example 1. After testing, the tensile strength of the calcium alginate-gelatin-lysozyme composite antibacterial film obtained in this example is (52.15±0.92) MPa; the elongation at break is (24.63±0.25)%; the water solubility is (2.32±0.52)%; the transmittance is close to zero in the ultraviolet range (200-300 nm); after one month, there is no significant difference between the lysozyme activity in the composite film and the initial activity; within 8 hours, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella can be reduced by approximately 2.63 log CFU / mL, 3.45 log CFU / mL, 3.75 log CFU / mL and 2.8 log CFU / mL, respectively.

[0036] Compared with Example 1, the water solubility of the composite film obtained in Example 2 is reduced; the mechanical properties are reduced, and the hardness and brittleness of the film are increased. This is mainly because the amount of gelatin added will affect the mechanical properties of the film. When the amount of gelatin added is low, the content of sodium alginate used for cross-linking with CaCl2 will increase, and the content of calcium alginate finally formed will increase, which will increase the tensile strength of the film and reduce the elongation at break of the film, resulting in an increase in the brittleness of the film and a decrease in elasticity.

[0037] Example 3 A method for preparing a calcium alginate-gelatin-lysozyme composite antibacterial film comprises the following steps: (1) Preparation of sodium alginate-gelatin composite film: Accurately weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add 1.515 g of gelatin to the dissolved sodium alginate solution and stir it under heating conditions at 70°C until it is dissolved, then add 0.513 g of glycerol and stir evenly to obtain a sodium alginate-gelatin composite solution. Cast the composite solution into a culture dish and dry it at 30°C to obtain a sodium alginate-gelatin composite film.

[0038] (2) Preparation of calcium alginate-gelatin composite film: The obtained sodium alginate-gelatin composite film was added to 0.5% CaCl2 solution and stirred for 40 min. The CaCl2 solution on the surface was washed with distilled water and then dried at 30°C to obtain the calcium alginate-gelatin composite film.

[0039] (3) Preparation of calcium alginate-gelatin-lysozyme composite antibacterial film: The calcium alginate-gelatin composite film was placed in a solution with a concentration of 1×10 -3 The mixture was placed in a lysozyme solution of 100 g / mL and shaken for 60 min. After being taken out, it was dried at 30 °C to obtain a calcium alginate-gelatin-lysozyme composite antibacterial film.

[0040] The detection method is the same as that in Example 1. After testing, the calcium alginate-gelatin-lysozyme composite antibacterial tensile strength obtained in this example is (38.43±2.58) MPa; the elongation at break is (45.22±1.37)%; the water solubility is (5.5±0.93)%; the transmittance is close to zero in the ultraviolet range (200-300 nm); after one month, there is no significant difference between the lysozyme activity in the composite film and the initial activity; within 8 hours, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella can be reduced by approximately 1.54 log CFU / mL, 1.92 log CFU / mL, 2.01 log CFU / mL and 1.35 log CFU / mL, respectively, which is weaker than that in Example 1.

[0041] Compared with Example 1, the composite film obtained in Example 3 has a higher elongation at break, which proves that the elasticity of the film is better. This may be because the added CaCl2 content is lower, the amount of calcium ions introduced is less, and the mechanical properties of the film are better than those of Example 1; however, the formed calcium alginate cross-linked structure is less, the fixed lysozyme content is reduced, and the inhibitory effect of the composite film on bacteria is reduced.

[0042] Comparative Example 1 A method for preparing a sodium alginate-gelatin composite film comprises the following steps: Weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add 1.515 g of gelatin to the dissolved sodium alginate solution and stir at 70°C until dissolved; add 0.513 g of glycerol and stir evenly to obtain a sodium alginate-gelatin composite solution. Cast the composite solution into a culture dish and dry it at 30°C to obtain a sodium alginate-gelatin composite film.

[0043] Compared with Example 1, Comparative Example 1 did not add the CaCl2 cross-linking agent and the lysozyme antibacterial agent, and adopted ordinary compounding to mix sodium alginate and gelatin together to prepare a composite film. The composite film obtained by this method has a high solubility, and the integrity of the film cannot be guaranteed under high humidity conditions. It has limitations in application and has no antibacterial activity.

[0044] Comparative Example 2 A method for preparing a sodium alginate-gelatin-lysozyme composite antibacterial film comprises the following steps: (1) Preparation of membrane-forming solution: Accurately weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water. Add 1.515 g of gelatin to the dissolved sodium alginate solution and stir at 70°C until dissolved. Add 0.513 g of glycerol and stir evenly. After cooling, add 100 mg of lysozyme to the composite solution to obtain a sodium alginate-gelatin-lysozyme composite solution.

[0045] (2) Casting into membrane: The composite solution was cast into a culture dish and dried at 30°C to obtain a sodium alginate-gelatin-lysozyme composite membrane.

[0046] Compared with Example 1, in Comparative Example 2, no CaCl2 crosslinking agent was added, and sodium alginate, gelatin and lysozyme were mixed together to prepare a composite membrane by ordinary compounding. The stability of the lysozyme obtained by this method decreased during application. After one month, the enzyme activity of the composite membrane obtained in Comparative Example 2 decreased significantly compared with the initial enzyme activity. After 31 days, the enzyme activity of the composite membrane decreased by 532 U / cm 2 ; And this method does not add CaCl2 cross-linking agent, the solubility is relatively high, the solubility in water is almost 100%, and its application is hindered under high humidity conditions.

[0047] Comparative Example 3 A method for preparing a calcium alginate-gelatin composite film comprises the following steps: (1) Preparation of sodium alginate-gelatin composite film: Weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add 1.515 g of gelatin to the dissolved sodium alginate solution and stir at 70°C until dissolved; add 0.513 g of glycerol and stir evenly to obtain a sodium alginate-gelatin composite solution. Cast the composite solution into a culture dish and dry it at 30°C to obtain a sodium alginate-gelatin composite film.

[0048] (2) Preparation of calcium alginate-gelatin composite film: The obtained sodium alginate-gelatin composite film was added into a 2% CaCl2 solution and stirred for 40 min. The CaCl2 solution on the surface was washed with distilled water and then dried at 30°C to obtain the calcium alginate-gelatin composite film.

[0049] Compared with Example 1, Comparative Example 3 did not add lysozyme. Although the solubility of the composite membrane obtained by this method was reduced and the integrity of the membrane was guaranteed when used under high humidity conditions, it did not have antibacterial activity and had certain limitations in application.

[0050] Comparative Example 4 A method for preparing a calcium alginate-gelatin-lysozyme mixture comprises the following steps: Weigh 1 g of sodium alginate and dissolve it in 100 mL of deionized water; add the dissolved sodium alginate solution into 2% CaCl2 solution and stir for 40 min to form calcium alginate; 1.515 g of gelatin was then added and stirred under heating at 70° C. 0.513 g of glycerol was added and stirred evenly to obtain a mixture.

[0051] 100 mg of lysozyme was added to the mixture and shaken for 60 min.

[0052] Since calcium alginate is formed first in this comparative example, and the water solubility of the formed calcium alginate solid is extremely weak, gelatin cannot be mixed with calcium alginate, so gelatin cannot play a role in improving the mechanical properties of the calcium alginate film; and since the formed calcium alginate will quickly form a precipitate (see Figure 7 ), no film can be formed, so a complete antibacterial film cannot be prepared under the conditions of Comparative Example 4, and the conditions for packaging application cannot be met.

[0053] Figure 2 a is a physical picture of the sodium alginate-gelatin composite film prepared in comparative example 1 adhered to paper, Figure 2 b is a physical picture of the calcium alginate-gelatin composite film prepared in comparative example 3 adhered to paper, Figure 2 c is a physical picture of the calcium alginate-gelatin-lysozyme composite antibacterial film prepared in Example 1 adhered to paper; Figure 2 It can be seen that the packaging film obtained by the present invention has the characteristics of being colorless and nearly transparent.

[0054] Figure 3 a. Figure 3 d are the surface and cross-sectional microstructures of the sodium alginate-gelatin composite film prepared in Comparative Example 1, Figure 3 b. Figure 3 e are the surface and cross-sectional microstructures of the calcium alginate-gelatin composite film prepared in Comparative Example 3, Figure 3 c. Figure 3 f are the surface and cross-sectional microstructures of the calcium alginate-gelatin-lysozyme composite membrane prepared in Example 1; Figure 3 This indicates that lysozyme can be immobilized in the microscopic network structure formed by cross-linking.

[0055] Figure 4 a is a physical picture of beef wrapped in sodium alginate-gelatin composite film prepared in comparative example 1, Figure 4 b is a physical picture of beef wrapped in calcium alginate-gelatin composite film prepared in comparative example 3, Figure 4 c is a physical picture of beef wrapped in the calcium alginate-gelatin-lysozyme composite antibacterial film prepared in Example 1; Figure 4 It can be shown that the packaging film prepared by the present invention will not affect the appearance of the product when used.

[0056] Figure 5 a is a physical picture of beef wrapped in the sodium alginate-gelatin-lysozyme composite antibacterial film prepared in Comparative Example 2, Figure 5 b is a physical picture of beef wrapped in the calcium alginate-gelatin-lysozyme composite antibacterial film prepared in Example 1; Figure 5 It can be explained that adding CaCl2 cross-linking can ensure the integrity of the membrane during application.

[0057] Figure 6 The membrane pairs prepared for each group E. coli :Escherichia coli, S. aureus :Staphylococcus aureus, L. monocytogene : Listeria monocytogenes and Salmonella : Antibacterial effect on Salmonella, wherein the Control group is a negative control, the ALG-Gel group is the sodium alginate-gelatin prepared in Comparative Example 1, the Ca-ALG-Gel group is the calcium alginate-gelatin prepared in Comparative Example 3, and the Ca-ALG-Gel-LYS group is the calcium alginate-gelatin-lysozyme composite film prepared in Example 1; Figure 6 It can be seen that the antibacterial effect of the calcium alginate-gelatin-lysozyme composite film prepared in Example 1 on Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella is significantly better than that in Comparative Example 1 and Comparative Example 3, which shows that whether to add CaCl2 cross-linking agent and lysozyme antibacterial agent will affect the antibacterial performance.

[0058] Figure 7 This is the calcium alginate mixture prepared in Comparative Example 4, which shows that directly adding CaCl2 to the sodium alginate solution will form a water-insoluble precipitate and cannot prepare a complete packaging film.

[0059] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a calcium alginate-gelatin-lysozyme composite antibacterial film, characterized in that: The following steps are involved: S1, dissolving sodium alginate in water to obtain a sodium alginate solution; adding gelatin to the sodium alginate solution, heating and stirring until dissolved; continuing to add glycerol and stirring evenly to obtain a sodium alginate-gelatin composite liquid; casting the sodium alginate-gelatin composite liquid into a film-forming device, drying, and obtaining a sodium alginate-gelatin composite film; S2, adding the sodium alginate-gelatin composite film to a CaCl2 solution and stirring, then taking it out, washing the surface with water, and drying it to obtain a calcium alginate-gelatin composite film; S3, placing the calcium alginate-gelatin composite film in a lysozyme solution and shaking it, then taking it out and drying it to obtain a calcium alginate-gelatin-lysozyme composite antibacterial film.

2. The preparation method according to claim 1, characterized in that: The mass percentage concentration of the sodium alginate solution is 1%-3%, and the amount of gelatin added is 0.5%-3% of the mass of the sodium alginate solution.

3. The preparation method according to claim 1, characterized in that: The added amount of the glycerol is 0.2%-0.5% of the total mass of the sodium alginate solution and gelatin.

4. The preparation method according to claim 1, characterized in that: The heating temperature after adding gelatin is 50°C-85°C.

5. The preparation method according to claim 1, characterized in that: The mass percentage concentration of the CaCl2 solution is 0.5%-3%.

6. The preparation method according to claim 1, characterized in that: The stirring time of adding the sodium alginate-gelatin composite film into the CaCl2 solution is 5 min-120 min.

7. The preparation method according to claim 1, characterized in that: The calcium alginate-gelatin composite film is placed in the lysozyme solution and shaken for 20 min-240 min.

8. The preparation method according to claim 1, characterized in that: The concentration of the lysozyme solution is 5×10 -4 g / mL-5×10 -2 g / mL.

9. A calcium alginate-gelatin-lysozyme composite antibacterial film, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the calcium alginate-gelatin-lysozyme composite antibacterial film as claimed in claim 9 in antibacterial food packaging materials.

Images