Chitosan coupled polypeptide and preservative film prepared from chitosan coupled polypeptide

Abstract

The invention provides chitosan coupled polypeptide and a preservative film prepared from the chitosan coupled polypeptide, active peptide used for preparing the preservative film has good antibacterial activity on escherichia coli, salmonella, staphylococcus aureus and drug-resistant escherichia coli, and the amino acid sequence of the active peptide is SEQ ID NO: 1. The preservative film prepared by coupling the active peptide and the chitosan provided by the invention has remarkable antibacterial activity, also has good resistance to drug-resistant strain drug-resistant escherichia coli, is good in mechanical property, air permeability, moisture penetrability and stability, and is suitable for being used in the fields of food preservation, medical dressing and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological activity preparation, and particularly relates to a chitosan-coupled polypeptide and a fresh-keeping film prepared therefrom. Background Art

[0002] As a multifunctional material, plastic wrap has been widely used in the food industry in recent years. Its main function is to extend the shelf life of products and prevent bacterial contamination. As consumers pay more and more attention to food safety, traditional plastic wrap faces the challenge of how to cope with the increasing threat of drug-resistant strains.

[0003] At present, the common cling film on the market mainly relies on chemical additives for functionalization. Although this method can provide a certain antiseptic effect, it has safety and environmental friendliness issues. For example, some plastic materials containing chloromethyl or bromoethyl are not only harmful to the human body, but may also pollute the environment. Therefore, finding a natural, safe and highly effective antibacterial cling film material has become a hot topic in current research.

[0004] In recent years, scientists have gradually paid attention to the application of natural polysaccharide materials (such as chitosan and maltose) and their conductive properties and self-healing mechanisms in plastic wrap. These materials not only provide good mechanical strength and waterproofness, but also have low toxicity and biocompatibility, making them suitable for food packaging. In addition, antibacterial substances combined with natural polysaccharides can effectively inhibit bacterial reproduction, providing new ideas for the development of plastic wrap.

[0005] However, most existing research focuses on optimizing a single function, while the dual functions of cling film (i.e., having both antiseptic and antibacterial capabilities) remain to be explored in depth. Therefore, how to achieve the efficiency, safety, and sustainability of cling film through the rational design and combination of natural materials has become a key issue at present. Summary of the invention

[0006] The purpose of the present invention is to provide a chitosan-coupled polypeptide and a fresh-keeping film prepared therefrom, which not only has the physical isolation function of a traditional fresh-keeping film, but also has a broad-spectrum antibacterial property, especially has a significant inhibitory effect on drug-resistant strains.

[0007] The present invention first provides an active peptide, which has good antibacterial properties against Escherichia coli, Salmonella, Staphylococcus aureus and drug-resistant Escherichia coli, and its amino acid sequence is AEDFKCPIGA (SEQ ID NO: 1);

[0008] In another aspect, the present invention also provides a use of the active peptide, which is used for preparing a product with antibacterial activity;

[0009] The product, as a specific record of the embodiment, is a fresh-keeping film.

[0010] In another aspect, the present invention provides a fresh-keeping film, which is prepared by coupling the above active peptide to chitosan and adding glycerol.

[0011] The preservative film is prepared by coupling the active peptide to chitosan, adding glycerol after dissolving and mixing, removing bubbles, spreading the film-forming solution, and drying.

[0012] The dissolution is to dissolve the active peptide and chitosan conjugate in 1% acetic acid solution to prepare a 2% (w / v) solution;

[0013] The mass of the glycerol added is 25% of the mass of the chitosan;

[0014] Furthermore, the thickness of the film is 0.1 mm.

[0015] The fresh-keeping film prepared by coupling the active peptide and chitosan provided by the present invention has significant antibacterial properties, and also has good resistance to drug-resistant strains and drug-resistant Escherichia coli. It has good mechanical properties, air permeability, moisture permeability and stability, and is suitable for use in the fields of food preservation and medical dressings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Reversed phase high performance liquid chromatography liquid phase distribution diagram;

[0017] Figure 2 :Mass spectrometry identification of active peptides;

[0018] Figure 3 : Structural diagram of polypeptide;

[0019] Figure 4 : Mechanical properties diagram of cling film;

[0020] Figure 5 :Air permeability of cling film (cm 3 / m 2 ·day·atm) and moisture permeability (g / m 2 day) chart;

[0021] Figure 6 : Light transmittance and fogging properties of plastic wrap. DETAILED DESCRIPTION

[0022] The present invention obtains an active peptide having antibodies against drug-resistant Escherichia coli through screening, and couples it with chitosan to prepare a fresh-keeping film. The prepared fresh-keeping film can not only realize the fresh-keeping function through its natural mechanical properties and waterproof properties, but also effectively inhibit the growth of bacteria and drug-resistant strains through the action of the active peptide, thereby providing a full range of food preservation needs.

[0023] The present invention is described in detail below in conjunction with embodiments and drawings.

[0024] Example 1: Preparation and screening of active peptides capable of resisting drug-resistant strains

[0025] 1) Preparation of active peptides

[0026] 100g frog skin was cut into pieces and mixed with 1L 1% acetic acid solution, homogenized at 4°C for 5min and allowed to stand for extraction for 3h, then centrifuged (8000rpm, 15min) to collect the supernatant; then, it was preliminarily purified using an ultrafiltration membrane with a molecular weight cutoff of 5kDa, and further separated by reversed-phase high performance liquid chromatography (RP-HPLC), using a C18 column and 0.1% TFA aqueous solution / acetonitrile gradient elution (5% to 60% acetonitrile, 30min), gradient elution: 0 to 5 minutes, 5% B; 5 to 30 minutes, 5% to 60% B; 30 to 35 minutes, 60% to 100% B. The flow rate was 1mL / min, and the target peak ( Figure 1 ), and the eluate collected from the target peak was analyzed by MALDI-TOF-MS. The mass spectrometry results showed that ( Figure 2 ), the molecular weight of the target peptide was consistent with the theoretical molecular weight, further verifying its purity. The target peptide was analyzed by MS / MS (tandem mass spectrometry) and its amino acid sequence was confirmed to be AEDFKCPIGA (SEQ ID NO: 1). The peptide structure is shown in Figure 3 The purified solution was concentrated and freeze-dried (pre-freezing at -80°C for 4 h, vacuum drying at 0.1 mbar for 24 h) to obtain 50 mg of active peptide in the form of white powder.

[0027] 2) Antibacterial properties of active peptides against food spoilage bacteria

[0028] Common food spoilage bacteria (Escherichia coli, Salmonella, Staphylococcus aureus) and drug-resistant strains of drug-resistant Escherichia coli were streaked onto LB agar plates and incubated in a 37°C constant temperature incubator for 24 hours. Subsequently, single colonies were picked and inoculated into 100 ml of LB liquid culture medium, and cultured overnight in a shaker at 37°C and 150 r / min to prepare bacterial suspensions. The bacterial concentration of each bacterial suspension was adjusted to 5×10 6 CFU / mL.

[0029] The peptide samples were diluted in a gradient manner, and 10 μL was added to each well of a 96-well plate. Then 90 μL of the culture medium of each strain with adjusted concentration was added to make the total volume of each well 100 μL. The final peptide concentrations in the wells were 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.12 μg / mL. The 96-well plate was placed in a shaking culture at 37°C for 24 hours, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader and the data was recorded. In the experiment, an Escherichia coli suspension (5×10 6 CFU / mL) was used as a negative control, and sterile LB liquid culture medium was used as a positive control, representing 0% and 100% inhibition rates, respectively. The results are shown in Table 1.

[0030] Table 1 Antibacterial effect MIC of active peptides (μg / mL)

[0031]

[0032] The results in Table 1 show that the minimum inhibitory concentration of the prepared active peptides against common food spoilage bacteria Escherichia coli, Salmonella and Staphylococcus aureus is no more than 12.5 μg / mL, and the minimum inhibitory concentration against drug-resistant Escherichia coli is no more than 25 μg / mL, indicating that the prepared active peptides have good antibacterial properties.

[0033] Example 2: Chitosan-coupled active peptides and preparation of fresh-keeping film

[0034] 1) Active peptide coupled to chitosan

[0035] Chitosan with a deacetylation degree of ≥90% was dissolved in 1% acetic acid solution to prepare a 1% (w / v) chitosan solution, and the pH was adjusted to 5.5 with NaOH and stirred until completely dissolved. At the same time, the purified active peptide was dissolved in PBS buffer (pH7.4) to prepare a 1 mg / mL solution. Subsequently, the chitosan solution and the active peptide solution were mixed in a volume ratio of 1:1, and EDC was added and stirred at room temperature for 2.5 hours. After the reaction was completed, glycine was added to terminate the cross-linking reaction and stirring was continued for 30 minutes. Finally, the reaction solution was dialyzed using a 3.5 kDa dialysis bag to remove unreacted reagents and small molecule impurities, and freeze-dried after dialysis to obtain an active peptide-chitosan complex.

[0036] 2) Preparation of fresh-keeping film by chitosan coupled with active peptides

[0037] The active peptide-chitosan conjugate was dissolved in 1% acetic acid solution to prepare a 2% (w / v) solution, and stirred at 500 rpm on a magnetic stirrer until completely dissolved. Glycerol 25% of the mass of chitosan was added and continued to stir for 30 minutes, and then the solution was allowed to stand for 30 minutes to remove bubbles in the solution, and the film-forming solution was poured into a flat glass plate and spread, and the film thickness was controlled to be 0.1 mm. The mold was placed in an oven at 45°C until the film was completely dry, and a fresh-keeping film containing chitosan-active peptide was obtained.

[0038] Embodiment 3: Determination of the physicochemical properties of cling film

[0039] 1) Mechanical properties test

[0040] The prepared active peptide-chitosan cling film was cut into standard size (10 mm×50 mm), and 5 samples were prepared for each group. The tensile tester was used for testing at a tensile speed of 10 mm / min, and the tensile strength (TS) and elongation at break (EAB) were observed and recorded.

[0041] 2) Air permeability and moisture permeability test

[0042] At 25°C and 50% relative humidity, use an air permeability tester according to the instructions to measure the air permeability of the cling film. At 25°C and 90% relative humidity, use a moisture permeability tester according to the instructions to measure the moisture permeability of the cling film.

[0043] 3) Optical performance test

[0044] Use a UV-visible spectrophotometer to scan at 620 nm and record the transmittance (%) and haze (%) of the plastic wrap.

[0045] 4) Stability test

[0046] (1) Temperature stability

[0047] The film samples were placed in constant temperature boxes at 4°C, 25°C and 37°C for 7 days, and the appearance changes (color, texture) of the films were observed. The mechanical properties (tensile strength) and antibacterial properties against Escherichia coli after storage were tested.

[0048] (2) Humidity stability

[0049] The film samples were placed in a constant humidity chamber at relative humidity of 30%, 50% and 90% respectively and stored for 7 days. The appearance changes of the films were observed, and the mechanical properties (tensile strength) and antibacterial properties against Escherichia coli after storage were tested.

[0050] Table 2: Stability results of plastic wrap

[0051]

[0052]

[0053] Depend on Figure 4-Figure 6 It can be seen that the tensile strength (TS) of the cling film is 25.06MPa, the elongation at break (EAB) is 217.8%, and the air permeability is 50.17cm 3 / m 2 ·day·atm, moisture permeability 10.5g / m 2 day, light transmittance 83%, fog 8%. The results in Table 2 show that the cling film exhibits good stability at 4°C and 25°C, and may only soften slightly at 37°C; in a high humidity (90%) environment, it absorbs moisture slightly, but its performance does not decrease significantly.

[0054] Example 4: Determination of antibacterial properties of chitosan cling film coupled with active peptides

[0055] The film samples were dissolved in PBS buffer, and the antibacterial properties of chitosan cling film coupled with active peptide (film A) and ordinary chitosan cling film (film B) against Enterobacteriaceae, Salmonella, Staphylococcus aureus and drug-resistant strains of Escherichia coli were determined according to the method in Example 1. Film B was a cling film prepared according to the description in Example 2, except that it was made of chitosan and glycerol.

[0056] Table 3: MIC (μg / mL) of two plastic wraps

[0057]

[0058] As shown in Table 3, the MIC value of membrane A is 25 μg / mL, and the antibacterial activity of membrane B against Escherichia coli, Salmonella and Staphylococcus aureus is 100 μg / mL, but it has no antibacterial activity against drug-resistant Escherichia coli. This shows that the addition of active peptides enhances the antibacterial effect of the plastic wrap, and it also has good inhibitory activity against drug-resistant strains of Escherichia coli.

[0059] In summary, the cling film prepared with the active peptide-chitosan conjugate provided by the present invention has good mechanical and moisture barrier properties, and has good antagonism to drug-resistant Escherichia coli, providing a new solution for food preservation and food safety, and has broad application prospects.

Claims

1. An active peptide, characterized in that The amino acid sequence of the active peptide is SEQ ID NO:

1.

2. Use of the active peptide according to claim 1 in the preparation of products with antibacterial activity.

3. The use according to claim 2, characterized in that The product is a fresh-keeping film.

4. A fresh-keeping film, characterized in that: The fresh-keeping film is prepared by coupling the active peptide described in claim 1 to chitosan to prepare an active peptide-chitosan coupling, and adding glycerol.

5. The cling film according to claim 4, characterized in that: The preservative film is prepared by coupling the active peptide described in claim 1 to chitosan to prepare an active peptide and chitosan conjugate, dissolving the active peptide and chitosan conjugate, adding glycerol and mixing, removing bubbles, spreading the film-forming solution, and then drying.

6. The cling film according to claim 5, characterized in that: The dissolution is to dissolve the active peptide and chitosan conjugate in 1% acetic acid solution.

7. The cling film according to claim 5, characterized in that: The active peptide and chitosan conjugate are prepared into a 2% solution.

8. The cling film according to claim 5, characterized in that: The mass of the added glycerol is 25% of the mass of the chitosan.

9. The cling film according to claim 5, characterized in that: The thickness of the film is 0.1 mm.

10. Use of the fresh-keeping film according to claim 4 in preserving food.

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