Intelligent konjac glucomannan antibacterial composite film

Abstract

The invention provides an intelligent antibacterial konjac glucomannan composite film and a preparation method thereof. The intelligent antibacterial konjac glucomannan composite film is prepared by carrying out graft copolymerization on oxidized konjac glucomannan OKGM and amino-functionalized poly (N-isopropylacrylamide) PNIPAM-NH2 and then crosslinking with an antibacterial agent, the tail end of a molecular chain of the amino-functionalized poly (N-isopropylacrylamide) is provided with an amino group. The intelligent antibacterial konjac glucomannan composite film disclosed by the invention has temperature responsiveness, can control the release of an antibacterial agent according to environmental changes, ensures that the antibacterial agent is accurately released as required, and effectively prolongs the food preservation time and the food shelf life. The film is good in biocompatibility, has no obvious toxic reaction on human tissues and cells, and provides possibility for application in the field of biomedicine. Besides, the film has degradability, and the OKGM and the chitosan are natural high polymer materials and can be decomposed into harmless substances by microorganisms in the natural environment, so that the film meets the requirements of environmental protection and is beneficial to reducing environmental pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of fresh-keeping materials, and in particular relates to a preparation process of an intelligent antibacterial konjac glucomannan composite film. Background Art

[0002] Konjac flour film, a natural biodegradable material, is widely used in food packaging, pharmaceutical carriers, and agricultural coverings due to its excellent film-forming, barrier, biocompatibility, and biodegradability. However, existing konjac flour films have limited functionality, especially in terms of antibacterial controlled release. Currently, although some studies have attempted to directly add antimicrobial agents to film substrates through physical mixing, the antimicrobial components are often released prematurely or fail to respond to changes in ambient temperature, making it difficult to achieve a long-lasting, precise antibacterial effect.

[0003] Patent document CN104558653A discloses a method for preparing a high-strength konjac glucomannan antibacterial nanocomposite film: konjac glucomannan is added to an aqueous solution of silica-coated silver nanowires at a mass ratio of 1:1 to 200, resulting in a uniform mixed solution. The solution is poured into a mold and dried to form a composite film. The composite film is then immersed in an aqueous alkaline solution, washed with water until neutral, and dried to produce a high-strength konjac glucomannan antibacterial nanocomposite film. Patent document CN116874890A discloses a konjac glucomannan-based antibacterial fresh-keeping film and its preparation method: a konjac glucomannan aqueous solution is mixed with a nisin solution, chitosan, cinnamon essential oil, and ginger essential oil are sequentially added and stirred, the pH is adjusted, the liquid is coated on a heat-resistant glass plate, dried, and cooled to produce the konjac glucomannan-based antibacterial fresh-keeping film. Although the konjac glucomannan antibacterial composite film prepared by this method has added antibacterial agents and has certain antibacterial capabilities, the release of antibacterial agents is disordered and cannot be precisely controlled according to changes in food and environmental temperature. Summary of the Invention

[0004] The present invention is committed to overcoming the problem that the antibacterial agents in the current antibacterial composite film are released in a disorderly manner and cannot be precisely regulated according to changes in food and environmental temperature. It provides a preservative material that has temperature responsiveness, good antibacterial properties and biocompatibility, can achieve long-term and precise antibacterial inhibition, thereby extending the storage time of food and the shelf life of food, and is green and environmentally friendly.

[0005] The invention discloses an intelligent antibacterial konjac glucomannan composite film, which is prepared by graft copolymerization of oxidized konjac glucomannan (OKGM) and amino-functionalized poly (N-isopropylacrylamide) PNIPAM-NH2, followed by cross-linking with an antibacterial agent. The oxidized konjac glucomannan has carboxyl groups on its molecular chain, and the amino-functionalized poly (N-isopropylacrylamide) has amino groups at its molecular chain ends.

[0006] The antibacterial agent is at least one of chitosan, tea polyphenols, citric acid or lactic acid.

[0007] A method for preparing an intelligent antibacterial konjac glucomannan composite film comprises the following steps: S1. Preparation of amino-functionalized poly (N-isopropylacrylamide): adding an initiator and a modifier to an N-isopropylacrylamide solution to react to obtain PNIPAM-NH2; S2. Preparation of OKGM grafted PNIPAM-NH2 copolymer: OKGM and PNIPAM-NH2 were mixed in water, a condensation agent and a coupling agent were added, and graft copolymerization was performed to obtain OKGM-PNIPAM-NH2; S3. Preparation of smart antibacterial film: OKGM-PNIPAM-NH2, antibacterial agent and plasticizer were mixed uniformly, and dried after the reaction to obtain a smart antibacterial film; The modifier described in S1 is at least one of 1-amino-2-methylpropane-2-thiol, 3-aminopropanethiol hydrochloride or mercaptoethylamine.

[0008] The preparation method of the OKGM is: (1) Disperse KGM in ethanol solution and add 2,2,6,6-tetramethylpiperidinyl nitroxide free radical TEMPO with stirring; (2) After adding hydrochloric acid to adjust the solution pH to 9.9-10.1, add the oxidant to start the oxidation reaction; during the reaction, add sodium hydroxide to control the pH of the reaction system to maintain at 9.9-10.1; (3) Add a reducing agent to terminate the reaction. After 1-1.5 h, add concentrated hydrochloric acid to adjust the pH to 4.9-5.1 and maintain for 1-1.5 h. (4) Wash and dry to obtain OKGM.

[0009] The mass ratio of the modifier described in S1 to N-isopropylacrylamide is 1:(200~250).

[0010] The initiator in S1 is at least one of sodium persulfate, azobisisobutyronitrile or lauroyl peroxide.

[0011] The condensing agent in S2 is diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; the coupling agent is N-hydroxyphthalimide or 1-hydroxybenzotriazole; the condensing agent and coupling agent are preferably a combination of diisopropylcarbodiimide and N-hydroxyphthalimide; the mass ratio of OKGM and PNIPAM-NH2 in S2 is 1: (0.4~0.6), and the mass ratio of OKGM, condensing agent and coupling agent is 1: (0.01~0.02): (0.03-0.05).

[0012] The plasticizer in S3 is at least one of glycerol, sorbitol or propylene glycol; the mass ratio of OKGM-PNIPAM-NH2 to the antibacterial agent in S3 is 1: (0.1~0.3).

[0013] The reaction temperature of S1 is 25~33℃, and the reaction time is 2~4 h; the reaction temperature of S2 is 20~25℃, and the reaction time is 8~10 h; the reaction temperature of S3 is 60~70℃, and the reaction time is 1~3 h.

[0014] The oxidizing agent is at least one of sodium hypochlorite, sodium periodate or hydrogen peroxide; the reducing agent is sodium sulfite; the mass ratio of TEMPO to KGM is 1:(900-1100), preferably 1:1000.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. In the intelligent antibacterial konjac glucomannan composite film of this invention, by mixing OKGM-PNIPAM-NH2 with the antibacterial agent, the antibacterial agent is effectively dispersed throughout the molecular chains of oxidized konjac glucomannan (OKGM), improving the uniformity of the antibacterial agent's dispersion. Furthermore, hydrogen bonds are formed between the amino groups of the antibacterial agent and the hydroxyl groups of the konjac glucomannan, enhancing their interaction and improving antibacterial properties.

[0016] 2. In the intelligent antibacterial konjac glucomannan composite film of this invention, oxidative modification of KGM selectively converts the primary alcohol groups at the C-6 position of the polysaccharide monosaccharide units (glucose and mannose) into carboxyl groups. This TEMPO oxidation treatment improves the solubility of the KGM, reduces the viscosity of the aqueous sol, and enhances its stability. The introduction of a large number of carboxyl groups as functional groups can form amide groups with the amino groups on amino-functionalized poly (N-isopropylacrylamide) (PNIPAM-NH2), significantly improving the film's temperature-responsive controlled-release properties.

[0017] 3. In the intelligent antibacterial konjac glucomannan composite film of the present invention, amino-functionalized synthetic modification of N-isopropylacrylamide (NIPAM) can significantly improve the reaction efficiency with KGM and increase the stability of the composite film.

[0018] 4. Poly (N-isopropylacrylamide) (PNIPAM) exhibits unique temperature sensitivity: By functionalizing poly (N-isopropylacrylamide) with amino groups, the film's temperature-sensitive controlled-release properties are improved, enabling sustained release of the antimicrobial agent over an extended period, resulting in a more durable antimicrobial effect. As the temperature fluctuates, the film automatically adjusts the release rate of the antimicrobial agent accordingly, maintaining an effective antimicrobial concentration under varying temperature conditions and preventing the shortening of the antimicrobial duration caused by overly rapid release of the antimicrobial agent. Specifically, when the ambient temperature is below the liquid crystal structure (LCST), the film's hydrophilicity increases, resulting in slower antimicrobial release. When the ambient temperature is above the LCST, the film's hydrophilicity decreases, leading to faster and more orderly release of the antimicrobial agent. This intelligent response to ambient temperature changes ensures precise, on-demand release of the antimicrobial agent, effectively extending the shelf life and shelf life of food.

[0019] 5. Enhanced antibacterial effect: The film formed by oxidized konjac flour and PNIPAM-NH2 has good biocompatibility and mechanical properties, and can provide a stable carrier environment for the antibacterial agent, allowing it to maintain its activity during the release process.

[0020] 6. Strong biodegradability: Konjac flour is a natural polymer material with good biodegradability. The film after use can be quickly degraded in the natural environment, reducing environmental pollution.

[0021] 7. High flexibility: The addition of OKGM and plasticizer improves the flexibility and ductility of the film, making it less likely to break during the packaging process and having good packaging performance. DETAILED DESCRIPTION

[0022] The embodiments of the present invention will be described in detail below with reference to examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0023] Example 1 (1) Preparation of oxidized konjac glucomannan (OKGM): 10 g of KGM was weighed and dispersed in 50 mL of 30% alcohol. The mixture was stirred with a magnetic stirrer and 0.01 g of 2,2,6,6-tetramethylpiperidinyl nitroxide (TEMPO) was added.

[0024] The solution was adjusted to pH 10 using hydrochloric acid, and 20 mL of pH 10 sodium hypochlorite solution was added to the solution, and the oxidation reaction began to occur.

[0025] When the pH value of the solution decreases, 0.5 mol / L sodium hydroxide solution is added dropwise to the solution to keep the pH value of the solution constant at 10.

[0026] When the amount of sodium hydroxide solution added reached 40 mL, 10 mL of sodium sulfite solution was added to terminate the reaction.

[0027] After 1 h, the solution was adjusted to pH = 5 with concentrated hydrochloric acid and maintained at this pH value for 1 h before stopping stirring.

[0028] The pellet was washed with ethanol three times and freeze-dried in vacuum to obtain OKGM.

[0029] (2) Synthesis of amino-functionalized poly (N-isopropylacrylamide) (PNIPAM-NH2): 10 g of monomer N-isopropylacrylamide was dissolved in 50 mL of water, and the solution was degassed by purging with nitrogen for 20 minutes.

[0030] 0.3 g of sodium persulfate and 0.05 g of 1-amino-2-methylpropane-2-thiol were dissolved in 5 mL of water, degassed, and then added to the monomer solution in sequence.

[0031] The reaction mixture was placed in a water bath at 25 °C for 3 h.

[0032] The product was purified and finally freeze-dried to obtain amino-functionalized poly (N-isopropylacrylamide) PNIPAM-NH2 with a molecular weight of 35 kDa.

[0033] (3) Preparation of OKGM grafted PNIPAM-NH2 copolymer: The grafting of PNIPAM-NH2 chains onto OKGM was achieved by using diisopropylcarbodiimide (DIC) as a condensing agent in the presence of N-hydroxyphthalimide (HOP) as a coupling agent. The specific steps are as follows: 1 g OKGM and 0.5 g PNIPAM-NH2 were mixed in water.

[0034] Then 0.0133 g of diisopropylcarbodiimide (DIC) was added, followed by 0.0405 g of N-hydroxyphthalimide (HOP).

[0035] After the mixture was reacted at 20° C. for 10 hours, the reaction mixture was neutralized to pH=6 using 1 M sodium hydroxide.

[0036] The obtained graft copolymer (OKGM-PNIPAM-NH2) was precipitated in 200 mL of acetone and freeze-dried.

[0037] (4) Formation of intelligent antibacterial film: Mix 1 g OKGM-PNIPAM-NH2, 0.2 g antibacterial agent chitosan and 1 g glycerol.

[0038] The mixture was placed in a reaction temperature of 60°C for 2 h, and then placed in an oven for 4 h to make its moisture content no more than 10%, thereby obtaining an intelligent konjac glucomannan antibacterial composite film material.

[0039] Example 2 The difference from Example 1 is that the sodium hypochlorite solution in step (1) is replaced by sodium periodate solution, and the rest is the same as Example 1.

[0040] Example 3 The difference from Example 1 is that the sodium hypochlorite solution in step (1) is replaced by a hydrogen peroxide solution, and the rest is the same as Example 1.

[0041] Example 4 The difference from Example 1 is that in step (3), diisopropylcarbodiimide is replaced by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxyphthalimide is replaced by 1-hydroxybenzotriazole. The rest is the same as in Example 1.

[0042] Example 5 The difference from Example 1 is that in step (2), 1-amino-2-methylpropane-2-thiol replaces 3-aminopropanethiol hydrochloride, and the rest is the same as Example 1.

[0043] Example 6 The difference from Example 1 is that in step (2), 1-amino-2-methylpropane-2-thiol is replaced by mercaptoethylamine, and the rest is the same as Example 1.

[0044] Comparative Example 1 The difference from Example 1 is that step (1) is cancelled and the oxidized konjac glucomannan (OKGM) in step (3) of Example 1 is replaced by konjac glucomannan (KGM).

[0045] Comparative Example 2 Mix 1 g KGM, 0.2 g antibacterial chitosan, and 1 g glycerol.

[0046] The mixture was placed in a reaction chamber at 60°C for 2 h, and then dried in an oven for 4 h until its moisture content did not exceed 10%, thereby obtaining the konjac glucomannan antibacterial film material.

[0047] Comparative Example 3 Mix 1 g KGM with 1 g glycerol.

[0048] The mixture was placed in a reaction chamber at 60°C for 2 h, and then dried in an oven for 4 h until its moisture content did not exceed 10%, thereby obtaining the konjac glucomannan film material.

[0049] Performance testing and analysis: Temperature responsiveness test: Primary amino groups in chitosan molecules react with ninhydrin under acidic conditions, forming a blue-purple condensation product with a maximum absorption at 570 nm. The absorbance is linearly correlated with the chitosan content within a certain range. The film was dispersed in aqueous solutions at 4°C, 20°C, and 35°C for 24 hours. Then, 1 mL of the solution was diluted and reacted with 10 g / L ninhydrin solution. The absorbance of the reaction solution at 570 nm was measured on a spectrophotometer. The chitosan content was calculated based on the absorbance of the solution and the regression equation of the standard curve.

[0050] Table 1: Temperature responsiveness test results (accumulated percentage of chitosan released after 24 h)

[0051] Antibacterial Performance Testing: The film prepared in Example 1 was cut into small pieces and sterilized to prepare a Staphylococcus aureus suspension. The film sample was placed in a sterile container containing the bacterial suspension. The suspension was incubated with shaking at 4°C, 20°C, and 35°C for a period of time to simulate the release of the antibacterial agent. After incubation, the bacterial suspension that had contacted the antibacterial film was diluted in a gradient. The bacterial suspensions at different dilutions were spread on nutrient agar plates. The plates were incubated in a 37°C incubator for 48 hours. The colony-forming units (CFU) on each plate were counted, and the antibacterial rate was calculated based on the dilution factor. The calculated antibacterial rates were 93.2% at 4°C, 97.9% at 20°C, and 98.3% at 35°C.

[0052] Comparative Example 1, in which unmodified konjac glucomannan was used to replace the oxidized konjac flour in Example 1, exhibited significantly lower temperature response compared to Example 1. This suggests that the amide group link between the oxidized konjac flour and PNIPAM-NH2 facilitates the regulation of the film's temperature response.

[0053] In Comparative Example 2, konjac glucomannan and chitosan were used to prepare the antibacterial film. The release of the antibacterial agent chitosan did not change with temperature, and its release curve was disordered, which could not ensure the effective utilization of the antibacterial agent.

[0054] It can be seen from the above data that the fresh-keeping material prepared by the present invention has good temperature response performance and sustained-release performance, can fully achieve long-term and precise antibacterial effect, thereby extending the storage time of food, extending the shelf life of food, and is green and environmentally friendly.

[0055] By selecting different film preparation formulas, the release amount of antibacterial agents at different temperatures can be adjusted to meet the diverse usage scenarios of various antibacterial films.

Claims

1. An intelligent antibacterial konjac glucomannan composite film, characterized in that: The invention is prepared by graft copolymerization of oxidized konjac glucomannan (OKGM) and amino-functionalized poly (N-isopropylacrylamide) PNIPAM-NH2, followed by cross-linking with an antibacterial agent. The oxidized konjac glucomannan has carboxyl groups on its molecular chain, and the amino-functionalized poly (N-isopropylacrylamide) has amino groups at its molecular chain end.

2. The intelligent antibacterial konjac glucomannan composite film according to claim 1, wherein The antibacterial agent is at least one of chitosan, tea polyphenols, citric acid or lactic acid.

3. A method for preparing an intelligent antibacterial konjac glucomannan composite film, characterized in that: The following steps are involved: S1. Preparation of amino-functionalized poly (N-isopropylacrylamide): adding an initiator and a modifier to an N-isopropylacrylamide solution to react to obtain PNIPAM-NH2; S2. Preparation of OKGM grafted PNIPAM-NH2 copolymer: OKGM and PNIPAM-NH2 were mixed in water, a condensation agent and a coupling agent were added, and graft copolymerization was performed to obtain OKGM-PNIPAM-NH2; S3. Preparation of smart antibacterial film: OKGM-PNIPAM-NH2, antibacterial agent and plasticizer were mixed uniformly, and dried after the reaction to obtain a smart antibacterial film; The modifier described in S1 is at least one of 1-amino-2-methylpropane-2-thiol, 3-aminopropanethiol hydrochloride or mercaptoethylamine.

4. The preparation method according to claim 3, characterized in that The preparation method of the OKGM is: (1) Disperse KGM in ethanol solution and add 2,2,6,6-tetramethylpiperidinyl nitroxide free radical TEMPO with stirring; (2) After adding hydrochloric acid to adjust the solution pH to 9.9-10.1, add the oxidant to start the oxidation reaction; during the reaction, add sodium hydroxide to control the pH of the reaction system to maintain at 9.9-10.1; (3) Add a reducing agent to terminate the reaction. After 1-1.5 h, add concentrated hydrochloric acid to adjust the pH to 4.9-5.1 and maintain for 1-1.5 h. (4) Wash and dry to obtain OKGM.

5. The preparation method according to claim 3, characterized in that The mass ratio of the modifier described in S1 to N-isopropylacrylamide is 1:(200~250).

6. The preparation method according to claim 3, characterized in that The initiator in S1 is at least one of sodium persulfate, azobisisobutyronitrile or lauroyl peroxide.

7. The preparation method according to claim 3, characterized in that The condensing agent in S2 is diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; the coupling agent is N-hydroxyphthalimide or 1-hydroxybenzotriazole; the condensing agent and coupling agent are preferably a combination of diisopropylcarbodiimide and N-hydroxyphthalimide; the mass ratio of OKGM and PNIPAM-NH2 in S2 is 1:(0.4~0.6), and the mass ratio of OKGM, condensing agent and coupling agent is 1:(0.01~0.02):(0.03-0.05).

8. The preparation method according to claim 3, characterized in that The plasticizer in S3 is at least one of glycerol, sorbitol or propylene glycol; the mass ratio of OKGM-PNIPAM-NH2 to the antibacterial agent in S3 is 1: (0.1~0.3).

9. The preparation method according to claim 3, wherein The reaction temperature of S1 is 25~33℃, and the reaction time is 2~4h; the reaction temperature of S2 is 20~25℃, and the reaction time is 8~10h; the reaction temperature of S3 is 60~70℃, and the reaction time is 1~3h.

10. The preparation method according to claim 4, characterized in that The oxidizing agent is at least one of sodium hypochlorite, sodium periodate or hydrogen peroxide; the reducing agent is sodium sulfite; the mass ratio of TEMPO to KGM is 1:(900-1100), preferably 1:1000.

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