Preparation method and application of lactoferrin-nisin nanoparticles

Abstract

The invention relates to a preparation method and application of lactoferrin-nisin nanoparticles, L-N nanoparticles with antibacterial activity are synthesized through self-assembly of lactoferrin and nisin, gelatin is used as a film substrate material, the L-N nanoparticles are cast in the film substrate material to prepare a film with functional characteristics of oxidation resistance, bacteria resistance and the like, and the film is used for preparing the lactoferrin-nisin nanoparticles. The refrigerator is used for refrigerating and keeping food fresh. According to the method provided by the invention, the technical problems that the stability of the nisin is poor and the self antibacterial ability is easily weakened by the external environment are solved, and the lactoferrin-nisin is further loaded into the gelatin-based film, so that the growth of microorganisms in food can be effectively inhibited, the spoilage of the food can be delayed, and the shelf life of the food can be prolonged; good biodegradability and mechanical properties are also realized.

Description

Technical Field

[0001] The invention relates to the technical field of food preservation, and in particular to a preparation method and application of lactoferrin-nisin nanoparticles. Background Art

[0002] Food safety is a core issue of global public health, and the contamination of foodborne pathogens is a focal issue. In order to ensure the safety of food, effective strategies must be adopted to control and prevent the contamination of foodborne pathogens. Nisin is a natural antimicrobial peptide that has a good inhibitory effect on foodborne pathogens. It has the advantages of no harm to human health, no bacterial resistance, and no change in the sensory properties of food, and is widely used in food-related industries. However, due to its poor stability, changes in the external environment, endogenous proteases, divalent cations, fats and glutathione in the food matrix can affect the activity of Nisin. Nanotechnology is often used to encapsulate Nisin to overcome this defect. In addition, the safety issues caused by the application of nanoparticles in films should also be considered in the study. For example, the potential health risks of nanoparticles in gelatin films that are in direct contact with food migrating from packaging to food. Summary of the invention

[0003] The invention provides a preparation method of lactoferrin-nisin nanoparticles and application thereof. LN nanoparticles with antibacterial activity are synthesized by self-assembly of lactoferrin and nisin, and gelatin is used as a film matrix material to cast the LN nanoparticles into the film matrix to prepare active packaging with functional properties such as anti-oxidation and antibacterial properties, which is used for refrigeration and preservation of food, solves the problem that nisin has poor stability and its antibacterial ability is weakened by environmental influences in actual application, and provides a theoretical basis for actual food packaging technology.

[0004] The present invention solves the above technical problems as follows: A method for preparing lactoferrin-nisin nanoparticles comprises the following steps:

[0005] 1) adding the nisin solution dropwise to the lactoferrin solution, continuously stirring to form a mixed solution, adjusting the pH of the mixed solution to 4-6, heating the mixed solution in a water bath, and storing the mixed solution in an ice bath for 8-12 hours to form a composite solution;

[0006] 2) The composite liquid is subjected to ultrasonic crushing to obtain lactoferrin-nisin nanoparticles.

[0007] Preferably, in step 1), the concentration of the nisin solution is 2-6 g / L and the concentration of the lactoferrin solution is 4-5 g / L.

[0008] Preferably, in step 1), the volume ratio of the nisin solution to the lactoferrin solution is 1:1.

[0009] Preferably, in step 1), the water bath heating temperature is 40-60° C. and the duration is 30-45 min.

[0010] Preferably, in step 2), the ultrasonic power is 200-280 W, and the ultrasonic time is 3-10 min.

[0011] A method for preparing a lactoferrin-nisin nanoparticle film comprises the following steps:

[0012] 1) Dissolve gelatin in distilled water, heat and stir, and after the solution cools, add glycerol and stir to prepare a membrane-forming solution;

[0013] 2) The lactoferrin-nisin nanoparticles prepared as described above are added to the film-forming solution, and after air drying, a film loaded with lactoferrin-nisin nanoparticles is formed.

[0014] Preferably, in step 1), the concentration of gelatin in the membrane-forming solution is 0.02-0.05 g / ml, and the mass ratio of glycerol to gelatin is 1:2-4.

[0015] Preferably, in step 2), the concentration of the loaded lactoferrin-nisin nanoparticles in the film-forming solution is 0.001-0.005 g / ml.

[0016] Preferably, in step 2), the drying temperature is 35-40° C. and the drying time is 12-14 hours.

[0017] Application of lactoferrin-nisin loaded nanoparticle film prepared by the above method in food packaging.

[0018] The invention has the following beneficial effects: the invention provides a gelatin-based composite film loaded with lactoferrin-nisin nanoparticles, firstly realizes the encapsulation of Nisin by a self-assembly method, and improves the antibacterial performance and stability of Nisin. The gelatin-based composite film prepared in this way still maintains good antibacterial performance, and also has good ductility, UV resistance, oxidation resistance and biodegradability.

[0019] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0021] Figure 1 is the SEM image of Nisin and LN nanoparticles in Example 1;

[0022] Figure 2 is the 24h L. monocytogenes antibacterial activity of Nisin and LN nanoparticles in Example 1;

[0023] Figure 3 is the 24h S. aureus antibacterial activity of Nisin and LN nanoparticles in Example 1;

[0024] Figure 4 is the 24h B.cereus antibacterial activity of Nisin and LN nanoparticles in Example 1;

[0025] Figure 5 The appearance and light transmittance of the LN nanoparticle film in Examples 2 to 4 and the film in Comparative Example 1; A is the macroscopic morphology of the film; B is the light transmittance of the film at 200 to 800 nm;

[0026] Figure 6 is the water compatibility of the LN nanoparticle film in Examples 2 to 4 and the film in Comparative Example 1; A is the water vapor permeability of the film; B is the moisture content of the film after immersion in water; C is the swelling degree of the film in Examples 2 to 4 after immersion in water; D is the water solubility of the film in Examples 2 to 4 after immersion in water;

[0027] Figure 7 The oxygen permeability of the LN nanoparticle films in Examples 2 to 4 and the film in Comparative Example 1;

[0028] Figure 8 The LN nanoparticle films in Examples 2 to 4 and the film in Comparative Example 1 were tested for DPPH and ABTS + Free radical scavenging ability;

[0029] Fig. 9 These are images of the LN nanoparticle films in Examples 2 to 4 and the film in Comparative Example 1 after being incubated in natural soil for different periods of time;

[0030] Fig.10The antibacterial properties of the LN nanoparticle films in Examples 2 to 4 and the film in Comparative Example 1 when applied to food; A is the antibacterial property of the LN nanoparticle film against Listeria monocytogenes in cheese; B is the antibacterial property of the LN nanoparticle film against Staphylococcus aureus in cheese; C is the antibacterial property of the LN nanoparticle film against Listeria monocytogenes in chicken; D is the antibacterial property of the LN nanoparticle film against Staphylococcus aureus in chicken;

[0031] Fig.11 The barrier properties of the LN nanoparticle films in Examples 2 to 4L and the film in Comparative Example 1 when applied to food; A is the barrier property of the LN nanoparticle film against Listeria monocytogenes in cheese; B is the barrier property of the LN nanoparticle film against Staphylococcus aureus in cheese; C is the barrier property of the LN nanoparticle film against Listeria monocytogenes in chicken; D is the barrier property of the LN nanoparticle film against Staphylococcus aureus in chicken;

[0032] Fig.12 The graph shows the change in the total number of colonies in cheese and chicken before and after storage after encapsulation with the LN nanoparticle film in Examples 2 to 4L and the film in Comparative Example 1; A is the total number of colonies in cheese; B is the total number of colonies in chicken. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] Example 1

[0035] This embodiment provides a method for preparing lactoferrin-nisin nanoparticles, and the specific steps are as follows:

[0036] 1) adding a 5 g / L nisin solution dropwise to a 4 g / L lactoferrin solution at a volume ratio of 1:1, stirring continuously for 2 hours to form a mixed solution, adjusting the pH of the mixed solution to 5, heating the mixed solution in a water bath at 50° C. for 30 minutes, and then immediately placing the mixed solution in an ice bath at 4° C. for 8-12 hours to fully hydrate the mixed solution to form a transparent or opaque composite solution;

[0037] 2) The composite solution which had been placed in an ice bath overnight was further treated with an ultrasonic cell disruptor at an ultrasonic power of 240 W for 5 min. After freeze drying, lactoferrin-nisin loaded nanoparticles (denoted as LN nanoparticles) were obtained.

[0038] The LN nanoparticles prepared in Example 1 were characterized as follows:

[0039] A. Stability test of LN nanoparticles

[0040] The particle size, polydispersity index (PDI) and Zeta potential of lactoferrin (LF), Nisin and LN nanoparticles in the solution were measured using a nanoparticle size potentiometer, and the results are shown in Table 1. The test temperature was 25°C, the refractive index of water was set to 1.33, and the refractive index of the sample was set to 1.45.

[0041] Table 1. Particle size, PDI and Zeta potential of LF, NIisn and LN nanoparticles

[0042]

[0043] Particle size is one of the important characteristics of nanoparticles, which will affect many properties of nanoparticles, such as physicochemical properties, storage stability and bioavailability. Generally, nanoparticles with small particle sizes have more unique functional properties than particles with larger particle sizes. The particle size of LN nanoparticles is significantly smaller than that of Nisin, with an average particle size of 29.83±2.42nm. PDI is mainly used to characterize the dispersion of samples. The smaller the PDI, the more uniform the dispersion of the sample. The PDI of LN nanoparticles is significantly smaller than that of the individual components, indicating that the dispersion of the nanoparticles is improved. It can be seen from the Zeta potential that after lactoferrin binds to Nisin, the surface potential of the nanoparticles changes, which indicates that there is a potential interaction between Nisin and lactoferrin, which promotes the binding of the two.

[0044] B. Morphology test of LN nanoparticles

[0045] Scanning electron microscopy (SEM) was used to observe the morphology of Nisin and LN nanoparticles. Figure 1 As shown. Nisin is irregular in shape and has a rough surface, with voids of varying sizes. The SEM image of LN nanoparticles shows that LF and Nisin are in an orderly cross-linked state, forming a dense network structure with a fuller and rounder surface and fewer voids.

[0046] C. Antibacterial activity test of LN nanoparticles

[0047] Dilute the activated bacterial solution with LB liquid medium to about 10 6CFU / mL. Take 1mL of diluted bacterial solution, add 1mL of LN nanoparticle complex solution, mix well, and culture at 37℃, 180r / min shaking for 24h. Take samples at 0h, 0.5h, 1h, 2h, 4h, 6h, 9h, 12h, 16h, 20h, and 24h, and count the colonies. Set up a control group, and perform the same operation with Nisin solution of the same concentration and sterile PBS to observe the 24h short-term antibacterial activity of LN nanoparticles. The results are as follows: Figures 2 to 4 shown.

[0048] LN nanoparticles effectively inhibited the growth of L.monocytogenes to an undetectable level in 0.5h, while Nisin achieved this effect in 4h, and no growth of L.monocytogenes was observed until 24h. This shows that the nanoparticles and Nisin have a fast bactericidal rate against L.monocytogenes and a good antibacterial effect. For S.aureus, LN nanoparticles effectively inhibited its growth in 2h, and the LN nanoparticles had a fast bactericidal rate, and no S.aureus growth was observed afterwards. Nisin quickly reduced the S.aureus colony count to approximately 2.94Log CFU / mL in 2h, and the colony count remained stable thereafter until S.aureus began to grow slowly 12h later. Figure 4 It was observed that Nisin sterilized rapidly in the first hour and then slowly until the colony count was detected at around 1.91Log CFU / mL at 24 hours. However, under the action of LN nanoparticles, no colony growth was detected in the second hour, and the sterilization speed of LN nanoparticles was faster.

[0049] Example 2

[0050] This embodiment provides a method for preparing a lactoferrin-nisin nanoparticle film, and the specific steps are as follows:

[0051] 1) Weigh gelatin and dissolve it in distilled water, and stir vigorously at 60°C for 1 hour. After the solution cools to room temperature, add 30% glycerol (w / w, based on the mass of gelatin) and stir for 15 minutes to obtain a membrane-forming solution with a gelatin concentration of 3%.

[0052] 2) Add the LN nanoparticles prepared in Example 1 to the film-forming solution and stir for 15 minutes. The concentration of LN nanoparticles in the film-forming solution is 0.1% w / v to form a mixed solution. If there are bubbles in the mixed solution, ultrasonicate it in an ultrasonic cleaner for 5 minutes to remove the bubbles. Pipette 15mL of the mixed solution into a disposable plastic culture dish (d = 9cm) for casting into a film, and air dry it at 37°C for 12-14h, which is recorded as G / LN 0.1%. After the film is formed, place it in a drying dish at 25±0.5°C and relative humidity (RH) of 50±5% (saturated magnesium nitrate solution) until analysis.

[0053] Example 3

[0054] This embodiment provides a lactoferrin-nisin nanoparticle film and a preparation method thereof, wherein the steps are substantially the same as those of embodiment 2, except that in step 2), the concentration of LN nanoparticles in the film-forming solution is 0.2% w / v, denoted as G / LN 0.2%.

[0055] Example 4

[0056] This embodiment provides a lactoferrin-nisin nanoparticle film and a preparation method thereof, wherein the steps are substantially the same as those of embodiment 2, except that in step 2), the concentration of LN nanoparticles in the film-forming solution is 0.4% w / v, denoted as G / LN 0.4%.

[0057] Comparative Example 1

[0058] In this comparative example, the film-forming solution prepared in Example 2 was dried in the same manner to form a film and tested (without adding LN nanoparticles), which was denoted as G.

[0059] The G / LN nanoparticle films prepared in Examples 2 to 4 were subjected to the following characterization tests:

[0060] A. Mechanical properties test of G / LN nanoparticle film

[0061] The LN nanoparticle films prepared in Examples 2 to 4 were first placed in an environment of 25±0.5°C and 50±5% RH for 48 hours. The films provided in each example were cut into strips of 20mm×50mm, and their tensile strength (TS) and elongation at break (EAB) were measured using a texture analyzer. The test probe was A / TG, the weight of the weighing sensor was 5kg, the test speed was 1mm / s, and the initial clamping distance was 30mm. The calculation formulas for TS and EAB are as follows:

[0062]

[0063] Where: F: maximum tension when the membrane breaks, N;

[0064] d: measured film thickness, mm;

[0065] W: membrane width, mm.

[0066]

[0067] Where: L: distance between marking lines when the membrane breaks, mm;

[0068] L0: Distance of original marking lines on the film, mm.

[0069] Table 2 Thickness and mechanical properties of films with different contents of LN nanoparticles

[0070]

[0071] Note: The values ​​with different superscript letters in the same column of the table indicate significant differences between the data in the same group (p<0.05).

[0072] The results are shown in Table 2. The pure gelatin film is thin, with a thickness of 0.089±0.016mm. With the addition of LN nanoparticles, the thickness of the film gradually increases. The thickness of the gelatin-based film loaded with LN nanoparticles is about 0.100mm, with no significant difference. Observing the mechanical properties, it was found that the TS of the pure gelatin film reached 24.46±1.12MPa, but the addition of LN nanoparticles gradually reduced it. This may be because the addition of LN nanoparticles weakened the hydrogen bonds of the gelatin molecules and destroyed the cohesion of the gelatin film, resulting in a decrease in TS. With the continuous addition of LN nanoparticles, the EAB of the film gradually increased, reaching the highest in G / L-N0.4%, which was 131.94±7.38%, an increase of 61.90% compared with the pure gelatin film. This may be because the addition of LN nanoparticles makes the structure between the gelatin matrix more compact, and the softness and elasticity are improved, thereby increasing the EAB.

[0073] B. Test of light transmittance of G / LN nanoparticle film

[0074] The films prepared in Examples 2 to 4 were cut into strips of 10 mm × 40 mm and attached to one side of a cuvette, with an empty cuvette as a control. The transmittance of the film between 200 and 800 nm was measured using a UV-visible spectrophotometer, with the same settings. The transmittance at 280 nm can be used to evaluate the ultraviolet light blocking performance of the film, and the transmittance at 600 nm can be used to evaluate the visible light blocking performance of the film.

[0075] like Figure 5As shown in Figure A, the four gelatin-based composite films are all transparent. As the concentration of LN nanoparticles increases, the glossiness of the film gradually decreases and the color becomes slightly yellow, which is the same as the chromaticity change trend of the film. Figure 5 B. The transmittance of pure gelatin film in the ultraviolet region (200-400nm) is high, but after the introduction of LN nanoparticles, the transmittance of the film in the ultraviolet region gradually decreases. The transmittance of G / LN 0.1%, G / LN 0.2% and G / LN 0.4% at 280nm is 1.30 times, 2.55 times and 4.00 times lower than that of pure gelatin film, respectively. This means that the ultraviolet blocking performance of the film is improved, which is beneficial to delay the lipid oxidation caused by ultraviolet rays in food. In addition, after adding LN nanoparticles, the transmittance of the film in the visible light region has no obvious change, indicating that LN nanoparticles have no effect on the transparency of the film, and all films remain transparent.

[0076] C. Water permeability test of G / LN nanoparticle film

[0077] The film obtained in each embodiment was covered on a dry weighing bottle filled with anhydrous calcium chloride, and the edge of the film was sealed with vaseline and tied with a rubber band to prevent moisture from passing through. The weighing bottle was placed in a dry dish (25±0.5°C, RH 75±2%) with a saturated NaCl solution, and the weight of the weighing bottle was measured at the beginning and every 2 hours using an analytical balance (accuracy of 0.0001g) for a total of 12 hours. The calculation formula of WVP is as follows:

[0078]

[0079] Where: Δm / Δt: the increase in water transferred through the membrane surface per unit time, g / s;

[0080] d: average thickness of the film, m;

[0081] A: Water vapor permeability area, m 2 ;

[0082] ΔP: The difference in water vapor pressure between the inner and outer surfaces of the membrane.

[0083] Cut the film into 20 mm × 20 mm pieces and weigh them, then place them in a 105°C oven to dry to constant weight. Soak the dried film in 15 mL of distilled water for 24 hours, then use filter paper to absorb excess water on the film surface and weigh it. Finally, place the film in a 105°C oven again to dry to constant weight.

[0084] The calculation formulas for WC, SD and WS are as follows:

[0085]

[0086] Where: m1: initial mass of the film, g;

[0087] m2: mass of the film after the first drying, g;

[0088] m3: the mass of the film after absorbing water, g;

[0089] m4: mass of the film after the second drying, g.

[0090] like Figure 6 As shown in Figure A, due to the hydrophilic groups contained in gelatin, the water vapor permeability (WVP) of the film is relatively high, which is 2.72±0.18 (×10 -11 g·m -1 ·s -1 ·Pa -1 After adding 0.1% LN nanoparticles, the WVP of the film decreased to 2.48±0.33 (×10 -11 g·m -1 ·s -1 ·Pa -1 ), but the effect is not significant. As the concentration of LN nanoparticles further increases, the WVP of the film gradually increases, and the water vapor barrier performance weakens. This shows that LN nanoparticles can help reduce the WVP of the film within a certain concentration range, but a higher concentration will cause the film to produce a higher WVP.

[0091] like Figure 6 As shown in Figure B, the film moisture content (WC) of each film is around 14%, with no significant difference, indicating that the water holding capacity of each film is the same. Figure 6 C shows that the swelling degree (SD) of pure gelatin film is 409.11±66.21%. After adding LN nanoparticles, its SD first increases and then decreases, but there is no significant difference. This may be because the addition of LN nanoparticles increases the hydrophilicity of gelatin film. Figure 6 As shown in D, the increase in LN nanoparticle content leads to an increase in the WS of the film, reaching the highest value (24.63±1.36%) in G / LN 0.4%, which is significant compared with the control group and other samples.

[0092] D. Oxygen permeability test of G / LN nanoparticle film

[0093] Add 15mL soybean oil to the weighing bottle, cover the test film on the bottle mouth, seal the edge of the test film with vaseline and tie it with a rubber band, and place it at 50℃ for 7 days. Use a weighing bottle without covering any substance as a control. Use sodium thiosulfate titration to determine the peroxide value (PV) of soybean oil after 7 days. The PV value of soybean oil indirectly reflects the oxygen permeability of the film. The calculation formula of PV is as follows:

[0094]

[0095] Where: V: volume of sodium thiosulfate solution consumed by the sample, mL;

[0096] V0: volume of sodium thiosulfate solution consumed in the blank test, mL;

[0097] c: concentration of sodium thiosulfate solution, 0.1 mol / L;

[0098] 0.1269: The mass of iodine equivalent to 1.00 mL of sodium thiosulfate solution, g / mmol;

[0099] m: mass of the sample, g;

[0100] 100: Conversion factor for 100g sample.

[0101] The results are as follows Figure 7 As shown in the figure, the peroxide value (PV) of soybean oil in the blank control group (no film treatment) was the highest, reaching 0.41±0.01g / 100g, which was already a deteriorated oil. After being packaged with gelatin-based composite films, the PV values ​​decreased to varying degrees, among which the addition of LN nanoparticles significantly decreased the PV value, with the highest decrease of about 0.12g / 100g compared with the blank control group. This shows that the addition of LN nanoparticles enhances the oxygen barrier ability of the film and improves the gas barrier properties of the film. This improvement can be attributed to the introduction of LN nanoparticles, which makes the film structure compact, thereby effectively cutting off the gas permeation path.

[0102] E. Effect of G / LN nanoparticle film on DPPH and ABTS + Free radical scavenging rate test (1) Determination of DPPH free radical scavenging rate

[0103] 0.1 g of the film prepared in each example was mixed with 10 mL of distilled water and stirred until dissolved to obtain a film extract. 100 μL of the film extract was mixed with 900 μL of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution as the test group (A s ); 100 μL of film extract was mixed with 900 μL of anhydrous ethanol solution as the control group (A c); 100 μL of distilled water was mixed with 900 μL of DPPH solution as the blank group (A b ). After being fully mixed, react at room temperature in the dark for 30 minutes, and measure the absorbance at 517 nm using a multifunctional microplate reader. The calculation formula for DPPH radical scavenging activity is as follows:

[0104]

[0105] Where: A s : absorbance of the mixture of film extract and DPPH solution;

[0106] A c : absorbance of the mixture of film extract and anhydrous ethanol solution;

[0107] A b : Absorbance of the mixture of distilled water and DPPH solution.

[0108] (2)ABTS + Determination of free radical scavenging rate

[0109] 0.1 g of the film prepared in each example was mixed with 10 mL of distilled water and stirred until dissolved to obtain a film extract. 100 μL of the film extract was mixed with 900 μL of ABTS (2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay solution as the experimental group (A s ); 100 μL of distilled water was mixed with 900 μL of ABTS assay solution as a blank group (A b ). Incubate at room temperature in the dark for 5 min, and measure the absorbance at 734 nm using a multifunctional microplate reader. + Free radical scavenging rate (ABTS + The calculation formula of radicalscavenging activity is as follows:

[0110]

[0111] Where: A b : absorbance of the mixture of distilled water and ABTS solution;

[0112] A s : Absorbance of the mixture of film extract and ABTS solution.

[0113] The results are as follows Figure 8 As shown in the figure, the scavenging rate of pure gelatin film for DPPH free radical was 8.01±1.16%, and that for ABTS +The free radical scavenging rate was 6.81±2.48%, which was below 10%, indicating that the antioxidant capacity of pure gelatin film was weak. The gelatin-based composite films loaded with LN nanoparticles all showed significant antioxidant capacity, and with the increase of LN nanoparticles, the antioxidant capacity gradually increased, indicating that the antioxidant capacity of the film was attributed to the LN nanoparticles. When 0.4% of LN nanoparticles were added, the antioxidant capacity of the film reached the highest level. At this time, the DPPH and ABTS of the film + The free radical scavenging rates were 22.30±2.33% and 45.72±4.67%, respectively.

[0114] F. Biodegradability test of G / LN nanoparticle film

[0115] Select suitable soil and pile it in a flat-bottomed container. The container needs to be pierced with holes to maintain air circulation. Place the film samples (20mm×20mm) of each embodiment on the soil surface. Water the soil twice a day to maintain a certain humidity. Set up a polyethylene film as a control group and measure the speed in the same way. Observe the appearance changes of the film every 5 days and take photos to record.

[0116] The results are as follows Fig. 9 As shown in the figure, the initial film surface was flat and smooth with good glossiness. As the incubation time increased, the film began to gradually shrink and holes appeared on the 15th day until it was completely degraded on the 20th day. The results show that the gelatin-based composite film loaded with LN nanoparticles prepared in this experiment has excellent biodegradability and can be completely degraded within 20 days without causing pollution to the environment. It is a green and environmentally friendly packaging material.

[0117] Application of G and G / LN nanoparticle films in food refrigeration and preservation

[0118] (1) Antibacterial performance study: Under sterile conditions, cut cheese and chicken (samples) into small pieces of 2 cm × 2 cm × 0.5 cm and irradiate under ultraviolet light for 30 min. Take 20 μL of about 10 6 CFU / mL of L.monocytogenes and S.aureus bacterial suspensions were applied to the sample surface and dried. The film (2cm×2cm) sterilized by UV in each example was placed on the contaminated surface of the sample to fit tightly and placed in a disposable culture dish. At the same time, the unfilmed sample with bacteria was used as a blank group and cultured at 4°C for 7 days. The total number of colonies of the sample and the film was measured at the beginning and end of the test.

[0119] The results are as follows Fig.10As shown in the figure, after storage at 4℃ for 7 days, there was no significant difference in the colony count between the samples treated with pure gelatin film and the blank control group, indicating that pure gelatin film had no antibacterial effect. However, after packaging with gelatin-based composite film loaded with LN nanoparticles, the colony count on the surface of the samples decreased to a certain extent compared with the blank control group after 7 days. Among them, although G / L-N0.1% reduced the number of bacteria in the samples, the difference was not significant. This is because the concentration of LN nanoparticles contained in G / LN 0.1% was low and the antibacterial effect was weak. However, after 7 days, G / L-N0.2% and G / LN 0.4% both significantly reduced the number of bacteria on the samples. G / LN 0.2% reduced the number of L.monocytogenes colonies on the surface of cheese and chicken by 0.82Log CFU / g and 0.99Log CFU / g, respectively, compared with the blank control group, and reduced the number of S.aureus colonies by 0.35Log CFU / g and 0.60Log CFU / g, respectively. G / LN 0.4% reduced the number of L.monocytogenes colonies on the surface of cheese and chicken by 0.86Log CFU / g and 0.92Log CFU / g, respectively, and reduced the number of S.aureus colonies by 0.69Log CFU / g and 0.19Log CFU / g, respectively, compared with the blank control group. It can be found that the antibacterial performance of the gelatin-based composite film loaded with LN nanoparticles on chicken is slightly better than that of cheese. It is speculated that this may be because the moisture content on the surface of chicken is higher than that of cheese, and the active substances in the film are dissolved in it, so the antibacterial performance is slightly stronger. The above results show that the gelatin-based composite film loaded with LN nanoparticles has a good antibacterial effect in cheese and chicken, and can play a long-term antibacterial role.

[0120] (2) Barrier performance study: Under sterile conditions, cheese and chicken were cut into small pieces of 2 cm × 2 cm × 0.5 cm and irradiated under ultraviolet light for 30 min. A film (2 cm × 2 cm) sterilized by ultraviolet light was placed on the surface of the sample to make the film fit the cheese. Then 20 μL of about 10 6 CFU / mL of L.monocytogenes and S.aureus bacterial suspensions were smeared on the film surface, dried, and placed in a disposable culture dish. At the same time, the bacterial sample without film coverage was used as a blank group and cultured at 4°C for 7 days. The number of colonies on the sample (to verify the barrier performance of the film) and the film (to verify the antibacterial performance of the film) were measured at the beginning and end of the test.

[0121] The results are as follows Fig.11As shown in the figure, the cheese without film packaging had a very high colony count of 3.97±0.06Log CFU / g after 7 days due to direct contact with L.monocytogenes. However, after film packaging, no L.monocytogenes was detected on the cheese surface after 7 days, indicating that the gelatin-based composite film loaded with LN nanoparticles can effectively prevent L.monocytogenes from entering the cheese surface. Comparing the colony counts on the films of the same period, it was found that G / LN 0.2% and G / L-N0.4% significantly reduced the colony counts of L.monocytogenes after 7 days, which were reduced by about 1.93Log CFU / mL and 3.15Log CFU / mL respectively compared with the colony counts on the initial films. The film's barrier effect on S. aureus on cheese was observed, and it was found that it could also effectively block S. aureus from entering the cheese surface, but its inhibitory effect on S. aureus was lower than that on L. monocytogenes. G / LN 0.2% and G / LN 0.4% could only reduce about 0.61Log CFU / mL and 0.96Log CFU / mL after 7 days. The film's barrier effect on L. monocytogenes in chicken was observed, and it was found that the number of colonies on the surface of chicken wrapped with pure gelatin film was consistent with that of the blank control group after 7 days, indicating that pure gelatin film has no barrier ability to L. monocytogenes in chicken. For G / LN 0.2% and G / LN 0.4%, no L.monocytogenes was detected on either the chicken surface or the film, indicating that G / LN 0.2% and G / LN 0.4% have excellent barrier and antibacterial capabilities against L.monocytogenes on chicken. The barrier effect of the film on S.aureus on chicken was observed, and it was found that the number of S.aureus on the chicken samples increased significantly after 7 days. This may be due to the presence of about 1.69±0.12Log CFU / g of S.aureus in the initial chicken, which grew and reproduced using chicken as a nutrient matrix during storage. Among them, the number of colonies in the chicken treated with G / LN 0.2% was the lowest, indicating that S.aureus in this group grew the slowest, and the number of colonies on the G / LN 0.2% film was also the lowest. This shows that G / LN 0.2% has a good barrier ability in chicken, which can not only prevent the invasion of external pollution, but also slow down the growth of microorganisms in chicken samples. The above results indicate that gelatin-based composite films loaded with LN nanoparticles have excellent barrier properties in cheese and chicken, and have obvious effects on both L. monocytogenes and S. aureus.

[0122] (3) Determination of total colony count: Determine the total colony count in the sample according to the national standard GB 4789.2-2022. Accurately weigh 1g of cheese and chicken samples, add 9mL of sterile saline, and grind thoroughly to prepare sample homogenate. Dilute the homogenate appropriately, and then spread the colony count. After incubation at 37°C for 24h, determine the total colony count in the sample.

[0123] The results are as follows Fig.12 As shown in the figure, no bacteria were detected in fresh cheese. After storage, except for the cheese in the G / LN 0.2% group, no bacteria were detected. The total colony counts of the cheeses in the other groups increased significantly, among which the total colony counts of the cheeses in the pure gelatin film and the blank control group increased rapidly, while the total colony counts of the cheeses in the G / LN 0.1% and G / LN 0.4% groups increased slowly, and only increased by 1.53Log CFU / g and 1.15Log CFU / g after 7 days, respectively. The total colony count of fresh chicken was 2.56±0.11Log CFU / g. After 7 days of storage, the total colony count increased to above 5.00Log CFU / g. The total colony counts of pure gelatin film, G / LN 0.1% and blank control groups reached about 5.43Log CFU / g, with no significant difference among them. The total colony counts of G / LN 0.2% and G / LN 0.4% were lower, at 5.10±0.16Log CFU / g and 5.08±0.08Log CFU / g, respectively. This indicates that G / LN 0.2% and G / LN 0.4% can reduce the growth and reproduction of bacteria on chicken, which is beneficial to the refrigeration preservation of chicken.

[0124] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and described above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing lactoferrin-nisin nanoparticles, characterized in that: The following steps are involved: 1) adding the nisin solution dropwise to the lactoferrin solution, continuously stirring to form a mixed solution, adjusting the pH value of the mixed solution to 4-6, heating the mixed solution in a water bath, and storing the mixed solution in an ice bath for 8-12 hours to form a composite solution; 2) The composite liquid is subjected to ultrasonic crushing and freeze-dried to obtain lactoferrin-nisin nanoparticles.

2. A method for preparing lactoferrin-nisin nanoparticles according to claim 1, characterized in that: In the step 1), the concentration of the nisin solution is 2-6 g / L; the concentration of the lactoferrin solution is 4-5 g / L.

3. A method for preparing lactoferrin-nisin nanoparticles according to claim 2, characterized in that: In the step 1), the volume ratio of the nisin solution to the lactoferrin solution is 1:

1.

4. The method for preparing lactoferrin-nisin nanoparticles according to claim 1, characterized in that: In the step 1), the water bath heating temperature is 40-60° C. and the duration is 30-45 minutes.

5. The method for preparing lactoferrin-nisin nanoparticles according to claim 1, characterized in that: In the step 2), the ultrasonic power is 200-280W, and the ultrasonic time is 3-10min.

6. A method for preparing a lactoferrin-nisin nanoparticle film, characterized in that: The following steps are involved: 1) Dissolve gelatin in distilled water, heat and stir, and after the solution cools, add glycerol and stir to prepare a membrane-forming solution; 2) adding the lactoferrin-nisin nanoparticles prepared by the method of claim 1 into the film-forming solution and mixing to obtain a mixed solution; 3) The mixed solution is dried by air blast to form a film of lactoferrin-nisin nanoparticles.

7. A method for preparing a lactoferrin-nisin nanoparticle film as claimed in claim 6, wherein in step 1), the concentration of gelatin in the film-forming solution is 0.02-0.05 g / ml, and the mass ratio of glycerol to gelatin is 1:2-4.

8. The method for preparing a lactoferrin-nisin nanoparticle film as claimed in claim 6, characterized in that: In the step 2), the concentration of lactoferrin-nisin nanoparticles in the mixed solution is 0.001-0.005 g / ml.

9. The method for preparing a lactoferrin-nisin nanoparticle film according to claim 6, characterized in that: In the step 3), the drying temperature is 35-40° C. and the drying time is 12-14 hours.

10. Use of a film loaded with lactoferrin-nisin nanoparticles prepared by the method according to any one of claims 6 to 9 in food packaging.

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