ROS-responsive edible antibacterial preservative film and preparation method thereof

Abstract

The invention discloses an ROS-responsive edible antibacterial preservative film and a preparation method thereof. The invention discloses a ROS (reactive oxygen species)-responsive antibacterial agent which is composed of sodium alginate (SA), carboxymethyl chitosan (CS), an ROS response group (TK-NH2) and antibacterial micromolecules (AB), TK-SA with ROS response characteristics is obtained through acylation reaction of TK-NH2 and SA through EDC, the antibacterial micromolecules (AB) are loaded into TK-SA through acylation reaction of EDC, and AB-TK-SA with antibacterial activity is obtained; aB-TK-SA and CS are mixed, sprayed to a clean glass plate and dried at the room temperature. The ROS-responsive edible antibacterial preservative film (AB-TK-SA / CS) provided by the invention has the characteristics of safety, low toxicity and ROS-responsive release, and can effectively inhibit the growth of staphylococcus aureus; the method is simple and convenient in preparation process, low in cost and good in reproducibility, and has a good application prospect in the field of related food preservation.

Description

Technical Field

[0001] The invention relates to an edible antibacterial fresh-keeping film responsive to ROS and a preparation method thereof, belonging to the technical field of food fresh-keeping films. Background Art

[0002] Edible cling film is a new food storage and preservation technology in recent years. It can effectively reduce the transfer of water vapor, oxygen and carbon dioxide, provide a barrier to prevent the movement of water, oxygen and solutes, maintain the gloss of food to a certain extent, inhibit the growth of microorganisms, thereby extending the shelf life and improving the commercial value of food. These cling films can form a complete food coating on the surface of food by dipping or spraying, or form a thin film independently by drying in a mold. They are simple and convenient to operate, inexpensive to make and promote, and environmentally friendly. They have high economic benefits and broad development prospects in food preservation.

[0003] Sodium alginate (SA) is a natural polysaccharide isolated from brown algae. It has the characteristics of wide source, low price, biodegradability, good water solubility and excellent film-forming properties. At the same time, because the molecule contains a large number of carboxyl and hydroxyl groups and has high chemical activity, it can quickly form a film under a relatively mild environment. Many studies have shown that edible films based on sodium alginate can effectively improve the quality changes of food during storage. It is a safe, environmentally friendly and natural food preservative film that has been widely used in the storage and preservation of fruits, vegetables, meat and seafood. However, the mechanical strength and antibacterial activity of SA films are still relatively poor, which limits its application in food packaging. In order to broaden the application of sodium alginate, the method of blending modification is usually used to improve its performance.

[0004] Chitosan (CS) is a common natural cationic polysaccharide with good antibacterial properties, biocompatibility, degradability and film-forming properties, and low cost. When SA is mixed with CS, the negatively charged carboxyl groups in SA can form a polyelectrolyte composite membrane with the amino groups in CS due to electrostatic interaction and hydrogen bonding, which not only exerts the performance of a single material in the composite membrane, but also improves the mechanical properties, stability and adsorption capacity of SA.

[0005] In order to further enhance the antibacterial activity of cling film, sodium alginate with reactive oxygen species (ROS) response was prepared by in-situ grafting method to load antibacterial small molecules. ROS is a class of oxygen-containing substances with strong oxidizing ability. Studies have shown that food spoilage is an oxidative phenomenon, and its quality deterioration is closely related to the level of ROS. Based on this, antibacterial small molecules were grafted into SA through ROS responsive groups (TK-NH2) to obtain AB-TK-SA, and CS was compounded to prepare a ROS-responsive edible antibacterial cling film AB-TK-SA / CS, which can effectively block the migration of moisture and oxygen, inhibit the growth of microorganisms, and thus extend the shelf life. It has good application potential in the field of related food preservation. Summary of the invention

[0006] The invention aims to provide an edible antibacterial cling film responsive to ROS. The cling film is a hydrophilic composite film and has the advantages of good antibacterial activity, ROS responsiveness, low toxicity, excellent stability and the like.

[0007] Correspondingly, another object of the present invention is to provide a method for preparing a ROS-responsive edible antibacterial cling film, which does not rely on complex experimental equipment and is economical and efficient.

[0008] To achieve the above object, the present invention adopts the following scheme: The invention provides an edible antibacterial cling film with ROS response, which consists of sodium alginate (SA), carboxymethyl chitosan (CS), a ROS response group (TK-NH2) and an antibacterial small molecule (AB). The invention is characterized in that TK-NH2 and SA are subjected to an acylation reaction of EDC to obtain TK-SA with ROS response characteristics, the antibacterial small molecule (AB) is loaded into TK-SA through an acylation reaction of EDC to obtain AB-TK-SA with antibacterial activity; AB-TK-SA is mixed with CS, sprayed onto a clean glass plate, and dried at room temperature to obtain the edible antibacterial cling film with ROS response (AB-TK-SA / CS).

[0009] Furthermore, the antibacterial small molecule (AB) is a natural plant small molecule or a processed synthetic small molecule. The natural plants include but are not limited to fruits, vegetables, and Chinese medicinal materials, such as chlorogenic acid in honeysuckle, gallic acid in gallnut, rhubarb, bistort, sanguisorba officinalis, and tea, ellagic acid in raspberry, pomegranate, strawberry, walnut, and blueberry, glycyrrhizic acid in licorice, rosmarinic acid in rosemary, and cinnamic acid in cinnamon. The synthetic small molecule is obtained by total synthesis, structural modification, and the like.

[0010] The molar concentration ratio of TK-NH2 to EDC is 1:1-1:5, and the molar concentration ratio of TK-NH2 to SA is 2:1-1:2.

[0011] The molar concentration ratio of the antibacterial small molecule to EDC is 1:1-1:5, and the molar concentration ratio of TK-SA to the antibacterial small molecule is 2:1-1:2.

[0012] The mass fraction of the AB-TK-SA is 2%, and the mass fraction of the CS is 0-5%.

[0013] The present invention also provides a method for preparing an edible antibacterial fresh-keeping film responsive to ROS, comprising the following steps: 1) Preparation of sodium alginate with ROS responsive properties: Dissolve TK-NH2 (0.2-1 mol) in water, add an appropriate amount of EDC (0.2-5 mol), stir for 0.5-1 h, add the above solution to the sodium alginate aqueous solution, react under nitrogen protection for 12-24 h, dialyze and freeze-dry the product to obtain sodium alginate (TK-SA) with ROS responsive properties.

[0014] 2) Dissolve the antibacterial small molecule (0.2-1 mol) in water, add an appropriate amount of EDC (0.2-5 mol), stir for 0.5-1 h, add the above solution to the TK-SA aqueous solution, react under nitrogen protection for 12-24 h, dialyze and freeze-dry the product to obtain sodium alginate (AB-TK-SA) loaded with antibacterial small molecules and having ROS response characteristics.

[0015] 3) Weigh an appropriate amount of AB-TK-SA into the aqueous solution and stir it evenly to make the mass fraction 2%. Add 0-5% CS aqueous solution, heat and stir evenly to obtain AB-TK-SA / CS mixed solution. Spray the mixed solution onto a clean glass plate and dry it at room temperature to obtain an edible antibacterial cling film with ROS response.

[0016] Furthermore, the AB-TK-SA and CS are mixed with each other in an aqueous solution, and the mixing process is not particularly limited. For example, the two can be mixed after each is made into an aqueous solution, or the AB-TK-SA and CS can be added to water in sequence or simultaneously for mixing. For example, the above-mentioned mixture can be made into a 2% AB-TK-SA aqueous solution and a 0-5% CS aqueous solution and then mixed. Preferably, the AB-TK-SA solution is dripped into the CS solution for mixing.

[0017] Furthermore, the mixing can be performed under normal pressure or under pressure. The mixing temperature can be -4 to 90°C, or preferably 0 to 80°C, but is not limited to these conditions.

[0018] Compared with the prior art, the present invention has the following advantages: The ROS-responsive edible antibacterial fresh-keeping film provided by the present invention has good antibacterial performance. The abundant quaternary ammonium salt groups and protonable tertiary amine groups provided in the carboxymethyl chitosan skeleton can be adsorbed to the surface of the bacteria after contacting with the bacteria, penetrate the cell wall, and disrupt the composition of the cell membrane through the change of osmotic pressure and the decomposition of organic matter, thereby promoting the leakage of intracellular substances (DNA, RNA), and the antibacterial performance is effective for a long time.

[0019] The ROS-responsive edible antibacterial cling film provided by the present invention has good ROS-responsive performance. The cling film can release the antibacterial small molecules connected in SA on demand when the ROS level is high.

[0020] The present invention selects SA and CS as carriers. Compared with artificially synthesized carriers, they have better biological stability and safety, almost no cytotoxicity and immunogenicity, improved biological safety, and have the advantages of wide sources, simple preparation, low cost, good stability, etc., which are convenient for large-scale mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the molecular formula of AB-TK-SA / CS of the present invention.

[0022] Figure 2 This is the synthesis reaction formula of TK-SA in Example 1.

[0023] Figure 3 It is the synthetic reaction formula of AB-TK-SA in Example 1.

[0024] Figure 4 It is the inhibition rate of SA, AB-TK-SA and AB-TK-SA / CS against Staphylococcus aureus in Example 1. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further illustrated by specific implementation methods below, but it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims. Example

[0026] The present embodiment provides an edible antibacterial fresh-keeping film responsive to ROS, which is composed of sodium alginate (SA), carboxymethyl chitosan (CS), a ROS responsive group (TK-NH2) and an antibacterial small molecule (AB). The preparation method thereof comprises the following steps: 1) Dissolve TK-NH2 (0.2 mol) in deionized water, add EDC (0.4 mol) aqueous solution dropwise, stir for 0.5 h, add the above solution dropwise into sodium alginate aqueous solution, react under nitrogen protection for 12 h, dialyze and freeze-dry the product to obtain sodium alginate (TK-SA) with ROS response characteristics.

[0027] 2) Chlorogenic acid (0.2 mol) was dissolved in deionized water, and an aqueous solution of EDC (0.4 mol) was added dropwise. After stirring for 0.5 h, the above solution was added dropwise to the TK-SA aqueous solution. The reaction was protected by nitrogen for 12 h. The product was dialyzed and freeze-dried to obtain sodium alginate (AB-TK-SA) loaded with antibacterial small molecules and having ROS-responsive properties.

[0028] 3) Weigh an appropriate amount of AB-TK-SA in an aqueous solution and stir evenly to make the mass fraction 2%. Add a 1% CS aqueous solution and stir evenly with a magnetic stirrer at 60°C to obtain an AB-TK-SA / CS mixed solution. Spray the mixed solution onto a clean glass plate and dry it at room temperature to obtain an edible antibacterial cling film with ROS response.

[0029] 4) The Staphylococcus aureus suspension was inoculated into a 96-well microplate, and lysozyme, SA, AB-TK-SA, and AB-TK-SA / CS (with the same lysozyme concentration) were added respectively. After mixing, the mixture was placed in a 37 ℃ incubator for 24 h, and the OD value was measured at 600 nm on a microplate reader. The results showed that AB-TK-SA / CS had the strongest antibacterial effect. Example

[0030] The present embodiment provides an edible antibacterial fresh-keeping film responsive to ROS, which is composed of sodium alginate (SA), carboxymethyl chitosan (CS), a ROS responsive group (TK-NH2) and an antibacterial small molecule (AB). The preparation method thereof comprises the following steps: 1) Dissolve TK-NH2 (0.2 mol) in deionized water, add EDC (0.4 mol) aqueous solution dropwise, stir for 0.5 h, add the above solution dropwise into sodium alginate aqueous solution, react under nitrogen protection for 12 h, dialyze and freeze-dry the product to obtain sodium alginate (TK-SA) with ROS response characteristics.

[0031] 2) Cinnamic acid (0.2 mol) was dissolved in deionized water, and an aqueous solution of EDC (0.4 mol) was added dropwise. After stirring for 0.5 h, the above solution was added dropwise to the TK-SA aqueous solution. The reaction was carried out under nitrogen protection for 12 h. The product was dialyzed and freeze-dried to obtain sodium alginate (AB-TK-SA) loaded with antibacterial small molecules and having ROS-responsive properties.

[0032] 3) Weigh an appropriate amount of AB-TK-SA in an aqueous solution and stir evenly to make the mass fraction 2%. Add a 1% CS aqueous solution and stir evenly with a magnetic stirrer at 60°C to obtain an AB-TK-SA / CS mixed solution. Spray the mixed solution onto a clean glass plate and dry it at room temperature to obtain an edible antibacterial cling film with ROS response.

[0033] 4) Four copies of the Staphylococcus aureus suspension were inoculated into a 96-well microplate, and lysozyme, SA, AB-TK-SA, and AB-TK-SA / CS (with the same lysozyme concentration) were added respectively. After mixing, the mixture was placed in a 37 ℃ incubator for 24 h, and the OD value was measured at 600 nm on a microplate reader. The results showed that AB-TK-SA / CS had the strongest antibacterial effect. Example

[0034] The present embodiment provides an edible antibacterial fresh-keeping film responsive to ROS, which is composed of sodium alginate (SA), carboxymethyl chitosan (CS), a ROS responsive group (TK-NH2) and an antibacterial small molecule (AB). The preparation method thereof comprises the following steps: 1) Dissolve TK-NH2 (0.2 mol) in deionized water, add EDC (0.4 mol) aqueous solution dropwise, stir for 0.5 h, add the above solution dropwise into sodium alginate aqueous solution, react under nitrogen protection for 12 h, dialyze and freeze-dry the product to obtain sodium alginate (TK-SA) with ROS response characteristics.

[0035] 2) Rosemarinic acid (0.2 mol) was dissolved in deionized water, and an aqueous solution of EDC (0.4 mol) was added dropwise. After stirring for 0.5 h, the above solution was added dropwise to the TK-SA aqueous solution. The reaction was carried out under nitrogen protection for 12 h. The product was dialyzed and freeze-dried to obtain sodium alginate (AB-TK-SA) loaded with antibacterial small molecules and having ROS-responsive properties.

[0036] 3) Weigh an appropriate amount of AB-TK-SA in an aqueous solution and stir evenly to make the mass fraction 2%. Add a 1% CS aqueous solution and stir evenly with a magnetic stirrer at 60°C to obtain an AB-TK-SA / CS mixed solution. Spray the mixed solution onto a clean glass plate and dry it at room temperature to obtain an edible antibacterial cling film with ROS response.

[0037] 4) Four portions of the Staphylococcus aureus suspension were inoculated into a 96-well microplate, and lysozyme, SA, AB-TK-SA, and AB-TK-SA / CS were added respectively. After mixing, the mixture was placed in a 37°C incubator for 24 h, and the OD value was measured at 600 nm on a microplate reader. The results showed that AB-TK-SA / CS had the strongest antibacterial effect.

[0038] The technical solutions of the present invention are not limited to the above-mentioned embodiments, and any technical solutions obtained by equivalent replacement methods fall within the scope of protection required by the present invention.

Claims

1. A ROS-responsive edible antibacterial cling film, comprising sodium alginate (SA), carboxymethyl chitosan (CS), a ROS-responsive group (TK-NH2) and an antibacterial small molecule (AB), characterized in that: TK-NH2 and SA were acylated with EDC to obtain TK-SA with ROS response characteristics, and the antibacterial small molecule (AB) was loaded into TK-SA through the acylation reaction of EDC to obtain AB-TK-SA with antibacterial activity; AB-TK-SA was mixed with CS, sprayed onto a clean glass plate, and dried at room temperature to obtain an edible antibacterial cling film (AB-TK-SA / CS) with ROS response.

2. The ROS-responsive edible antibacterial cling film according to claim 1, characterized in that: The antibacterial small molecule (AB) is a natural plant small molecule or a processed synthetic small molecule.

3. The ROS-responsive edible antibacterial cling film according to claim 2, characterized in that: The natural plant small molecule sources include but are not limited to fruits, vegetables, and Chinese medicinal materials, such as chlorogenic acid in honeysuckle, gallic acid in gallnut, scutellaria baicalensis, radix polygoni multiflori, and tea, ellagic acid in raspberry, pomegranate, strawberry, walnut, and blueberry, glycyrrhizic acid in licorice, rosmarinic acid in rosemary, and cinnamic acid in cinnamon.

4. The ROS-responsive edible antibacterial cling film according to claim 2, characterized in that: The synthetic small molecule is obtained through total synthesis and structural modification.

5. A ROS-responsive edible antibacterial cling film according to any one of claims 1 to 4, characterized in that: The molar concentration ratio of TK-NH2 to EDC is 1:1-1:5, and the molar concentration ratio of TK-NH2 to SA is 2:1-1:

2.

6. The method for preparing a fresh-keeping film according to any one of claims 1 to 4, characterized in that: The molar concentration ratio of the antibacterial small molecule to EDC is 1:1-1:5, and the molar concentration ratio of TK-SA to the antibacterial small molecule is 2:1-1:

2.

7. A method for preparing an edible antibacterial cling film responsive to ROS, characterized in that: The following steps are involved: Step 1, preparation of sodium alginate with ROS responsive properties: dissolve 2,2'-(propane-2,2-diylbis(sulfonamide))diethylamine (TK-NH2) in water, add an appropriate amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), stir for a period of time, add the above solution to the sodium alginate aqueous solution, react under nitrogen protection for 12-24 hours, dialyze and freeze-dry the product to obtain sodium alginate (TK-SA) with ROS responsive properties; Step 2: Dissolve the antibacterial small molecule in water, add an appropriate amount of EDC, stir for a period of time, add the above solution to the TK-SA aqueous solution, react under nitrogen protection for 12-24 hours, dialyze and freeze-dry the product to obtain sodium alginate (AB-TK-SA) loaded with antibacterial small molecules and having ROS response characteristics; Step three, weigh an appropriate amount of AB-TK-SA into an aqueous solution and stir evenly; add an appropriate amount of CS aqueous solution, heat and stir evenly to obtain an AB-TK-SA / CS mixed solution; spray the mixed solution onto a clean glass plate, dry at room temperature, and obtain an edible antibacterial cling film with ROS response.

8. The method for preparing a fresh-keeping film according to claim 5, characterized in that: In the above steps, the stirring time is 0.5-1 h.

9. The method for preparing a fresh-keeping film according to claim 5, characterized in that: In step 3, the mass fraction of AB-TK-SA is 2%, and the mass fraction of CS is 0-5%.

10. The method for preparing a fresh-keeping film according to claim 5, characterized in that: In the above steps, the mixing temperature is -4~90℃.

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