Special antibacterial and antioxidant biosensing nano-composite preservative film for fermented food

Abstract

The invention discloses a special antibacterial and antioxidant biosensing nano-composite preservative film for fermented food in the technical field of biochemistry, GOx and Lac are synergistically loaded on a nano-carrier to construct a multifunctional composite film, the loading capacity and catalytic efficiency of enzyme are remarkably improved by the high specific surface area and surface active sites of the nano-carrier, and the antibacterial and antioxidant biosensing nano-composite preservative film can be applied to the field of biochemistry. Meanwhile, the synergistic effect of the two enzymes endows the composite membrane with multiple functions: GOx catalyzes glucose to oxidize into hydrogen peroxide (H2O2), Lac catalyzes and oxidizes polyphenol substances, and the combination of GOx and Lac can realize antibacterial, antioxidant and biosensing functions at the same time. In addition, due to the introduction of the porous structure and the matrix material of the nano composite membrane, the mechanical property and the biocompatibility of the membrane are further enhanced, and the application potential of the membrane in food packaging, medical dressing and biosensors is widened.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and particularly to an antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods. Background Art

[0002] Glucose oxidase (GOx) and laccase are two important biocatalysts, which have wide applications in the fields of food, medicine, and environmental protection. GOx can catalyze the oxidation of glucose to produce gluconic acid and hydrogen peroxide, and has multiple functions such as antibacterial, antioxidant, and improving food texture. Laccase, on the other hand, can catalyze the oxidation of a variety of phenolic and non-phenolic substrates, showing good antioxidant and antibacterial activities. Combining these two enzymes can give full play to their synergistic effects and further improve the performance of the material.

[0003] However, there is currently a lack of a nanocomposite membrane material on the market that can simultaneously load GOx and laccase and has good stability and versatility. Traditional enzyme immobilization methods often have problems such as low immobilization efficiency, large loss of enzyme activity, and unstable performance of the membrane material. Therefore, the development of a new type of multifunctional nanocomposite membrane loaded with glucose oxidase and laccase is of great significance for expanding the application of enzymes in the fields of food preservation, packaging materials, etc. Summary of the Invention

[0004] The present invention aims to provide an antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods to solve

[0005] To solve the above technical problems, the present invention provides the following technical solution: An antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods, the nanocomposite fresh-keeping film comprising:

[0006] A nanocarrier material selected from silica nanoparticles, magnetic iron oxide nanoparticles, or graphene nanosheets;

[0007] Glucose oxidase and laccase are loaded on the surface of the nanocarrier by physical adsorption or covalent bonding;

[0008] The thickness of the composite film is 50 - 200 nanometers and has a porous structure.

[0009] Further, the surface of the nanocarrier material is modified with amino or carboxyl groups to enhance the enzyme immobilization efficiency.

[0010] Further, the composite film further comprises chitosan or sodium alginate as a film-forming matrix to improve the mechanical strength and biocompatibility.

[0011] Further, the mass ratio of the glucose oxidase to the laccase is 1:0.5 - 1:2.

[0012] Furthermore, a method for preparing an antibacterial and antioxidant biosensing nanocomposite preservative film for fermented foods as described above includes the following steps:

[0013] S1. Provide a nanocarrier material:

[0014] S2. Load glucose oxidase and laccase onto the nanocarrier material to form an enzyme-loaded nanocarrier;

[0015] S3. Mix the enzyme-loaded nanocarrier with a film-forming material to prepare a film-forming solution:

[0016] S4. Coat the film-forming solution on a substrate and dry it to form a multifunctional nanocomposite film loaded with glucose oxidase and laccase.

[0017] Furthermore, in the S3 step, the loading process is carried out in a buffer solution with a pH of 5.5 - 7.0, at a temperature of 25 - 37 °C, and for a time of 2 - 6 hours.

[0018] Furthermore, an application of the antibacterial and antioxidant biosensing nanocomposite preservative film for fermented foods as described above in food preservation, biosensing, or medical dressings.

[0019] Advantages of the present invention: 1. By simultaneously loading glucose oxidase (GOx) and laccase (Lac), the composite film realizes multiple functions of antibacterial, antioxidant, and biosensing by utilizing the synergistic catalytic effect of the two enzymes. GOx catalyzes glucose to generate hydrogen peroxide (H2O2), exerting antibacterial and antioxidant properties; Lac catalyzes the oxidation of polyphenolic substances, further enhancing the antioxidant ability. The combination of the two not only broadens the functional boundaries of a single enzyme but also improves the overall performance through a synergistic effect.

[0020] 2. By using nanocarrier materials such as silica, magnetic iron oxide, or graphene, their high specific surface area and surface active sites significantly improve the enzyme loading amount and catalytic efficiency. For example, amino-modified magnetic nanoparticles (Fe3O4-NH2) achieve the efficient immobilization of GOx and Lac through the dual actions of physical adsorption and covalent bonding.

[0021] 3. The porous structure (with a thickness of 50 - 200 nanometers) of the composite film combined with film-forming matrices such as chitosan and sodium alginate significantly improves the mechanical strength and biocompatibility of the film while maintaining the enzyme activity. The composite film prepared by electrospinning has good flexibility and is suitable for scenarios such as food packaging and medical dressings that require flexibility and durability. Its porous characteristics also promote the contact between the substrate and the enzyme, further optimizing the catalytic efficiency.

[0022] 4. This composite film can extend the shelf life of food in the field of food preservation through antibacterial and antioxidant effects, such as inhibiting the growth of microorganisms and delaying oxidative deterioration; it can be used for glucose detection in the field of biosensing, with a wide linear range and a low detection limit; in medical dressings, its antibacterial properties can be utilized to prevent infections, while the biocompatible matrix promotes wound healing. This versatility gives it commercial potential in multiple fields. Detailed implementation manners

[0023] The following is a further detailed description through specific implementation manners:

[0024] Example:

[0025] I. Preparation of nano-composite fresh-keeping film

[0026] 1. Synthesis of nano-carrier:

[0027] Disperse Fe3O4 nanoparticles in ethanol, add 3-aminopropyltriethoxysilane (APTES), and ultrasonically treat for 2 hours to obtain amino-modified magnetic nanoparticles (Fe3O4-NH2).

[0028] 2. Enzyme loading:

[0029] Mix the Fe3O4-NH2 suspension with the GOx solution (5 mg / mL, pH 6.0), and stir at room temperature for 4 hours. After centrifugation and washing, add the Lac solution (3 mg / mL, pH 5.5), and continue stirring for 2 hours to obtain magnetic nanoparticles loaded with dual enzymes (Fe3O4-NH2-GOx / Lac).

[0030] 3. Preparation of composite film:

[0031] Mix Fe3O4-NH2-GOx / Lac with the chitosan solution (2% w / v), and prepare the composite film by electrospinning. The spinning voltage is 15 kV, and the receiving distance is 15 cm to obtain a porous film with a thickness of about 100 nanometers.

[0032] II. Performance testing

[0033] 1. Antibacterial performance testing

[0034] Experimental method:

[0035] Bacterial strains: Escherichia coli (E.coli ATCC 25922), Staphylococcus aureus (S.aureus ATCC 25923)

[0036] Preparation of bacterial suspension: Inoculate the bacterial strains into LB medium, shake and culture at 37 °C for 18 hours, and adjust the bacterial suspension concentration to 1×106 CFU / mL.

[0037] Plate inoculation: Spread 100 μL of the bacterial suspension evenly on the LB agar plate.

[0038] Membrane material treatment: Place the composite membrane (diameter 10 mm) in the center of the plate. The blank control is the nano - membrane without loaded enzyme.

[0039] Cultivation conditions: Incubate upside - down at 37 °C for 24 hours.

[0040] Inhibition zone measurement: Use a vernier caliper to measure the diameter of the inhibition zone (including the membrane diameter).

[0041] Experimental data:

[0042]

[0043]

[0044] Conclusion: The composite membrane shows significant synergistic antibacterial effects against both Gram - negative bacteria (E. coli) and Gram - positive bacteria (S. aureus), and the inhibition zone is about 45% larger than that of the single - enzyme membrane.

[0045] 2. Antioxidant activity test

[0046] Experimental method:

[0047] DPPH free - radical scavenging experiment:

[0048] Prepare a 0.1 mM DPPH ethanol solution and store it in the dark. Add the composite membrane (0.1 g) to 2 mL of the DPPH solution and shake at room temperature for 30 minutes. After centrifugation, take the supernatant and measure the absorbance at 517 nm (A sample). The blank group is the DPPH solution (A blank), and the control group is the nano - membrane without loaded enzyme (A control).

[0049] Clearance rate calculation formula:

[0050]

[0051] Experimental data:

[0052] Sample type Absorbance (A) Clearance rate (%) Composite membrane 0.32 78.6 GOx single-enzyme membrane 0.45 54.3 Lac single-enzyme membrane 0.51 42.7 Blank membrane 0.68 0

[0053] Conclusion: The DPPH clearance rate of the composite membrane is significantly higher than that of the single - enzyme membrane, indicating that the dual - enzyme synergy enhances the antioxidant performance.

[0054] 3. Glucose detection performance test

[0055] Experimental method:

[0056] Sensor preparation: Fix the composite membrane on the surface of the screen - printed carbon electrode to form a working electrode.

[0057] Detection conditions:

[0058] Electrolyte: 0.1M PBS buffer (pH 7.0)

[0059] Detection potential: +0.6V (vs. Ag / AgCl)

[0060] Glucose concentration gradient: 0.1, 0.5, 1.0, 2.0, 5.0, 10.0 mM

[0061] Measurement of current response: Record the steady-state current each time glucose is added and plot a standard curve.

[0062] Experimental data:

[0063] Glucose concentration (mM) Current response (μA) Glucose concentration (mM) 0.1 1.2 0.1 0.5 5.8 0.5 1.0 11.5 1.0 2.0 22.3 2.0 5.0 55.6 5.0 10.0 108.4 10.0

[0064] Linear regression equation:

[0065] I (μA) = 10.6 [Glucose] (mM) + 0.8 (R 2 = 0.998)

[0066] Detection limit: 0.05 mM (S / N = 3)

[0067] Interference experiment:

[0068] Add 10 mM interfering substances (such as ascorbic acid, uric acid), and the change in current response is < 5%, indicating that the sensor has good selectivity.

[0069] 4. Stability test

[0070] Experimental method:

[0071] Store the composite membrane at 4°C and measure the glucose detection response value every 7 days. After continuous use 10 times, measure the retention rate of the detection current.

[0072] Experimental data:

[0073] Storage time (days) Response retention rate (%) 0 100 7 92 14 85 21 78

[0074] Conclusion: The composite membrane maintains 78% of its initial activity within 21 days and retains 89% of its response after repeated use 10 times, with better stability than the single enzyme membrane.

[0075] In this invention, a composite membrane with efficient antibacterial, antioxidant and glucose detection functions was successfully prepared by co-loading GOx and Lac with a nanocarrier. Experimental data show that the synergistic effect of the dual enzymes significantly improves the comprehensive performance of the membrane material, and its antibacterial effect, antioxidant activity and detection sensitivity are all better than those of the single enzyme membrane, and it has good stability, suitable for fields such as food preservation, biosensing and medical dressings.

[0076] The above are only embodiments of the present invention, and common knowledge such as specific structures and characteristics known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners and the like described in the specification can be used to interpret the content of the claims.

Claims

1. A special antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods, characterized in that, The nano-composite fresh-keeping film includes: A nano-carrier material selected from silica nanoparticles, magnetic iron oxide nanoparticles or graphene nanosheets; Glucose oxidase and laccase are loaded on the surface of the nano-carrier by physical adsorption or covalent bonding; The composite film has a thickness of 50-200 nanometers and has a porous structure.

2. The antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods according to claim 1, wherein: The surface of the nano-carrier material is modified with amino or carboxyl groups to enhance the enzyme immobilization efficiency.

3. The antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods according to claim 2, wherein: The composite film further comprises chitosan or sodium alginate as a film-forming matrix to improve mechanical strength and biocompatibility.

4. The antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods according to claim 3, characterized in that: The mass ratio of the glucose oxidase to the laccase is 1:0.5-1:

2.

5. The preparation method of an antibacterial and antioxidant biosensing nanocomposite fresh-keeping film special for fermented foods according to claim 1, wherein, It includes the following steps: S1. Provide a nano-carrier material: S2. Load glucose oxidase and laccase on the nano-carrier material to form an enzyme-loaded nano-carrier; S3. Mix the enzyme-loaded nano-carrier with a film-forming material to prepare a film-forming solution: S4. Coat the film-forming solution on a substrate and dry it to form a multifunctional nano-composite film loaded with glucose oxidase and laccase.

6. The preparation method of a special antibacterial and antioxidant biosensing nanocomposite fresh-keeping film for fermented foods according to claim 5, characterized in that: In the step S3, the loading process is carried out in a buffer solution with a pH of 5.5-7.0, at a temperature of 25-37 °C for 2-6 hours.

7. The application of the antibacterial and antioxidant biosensing nano-composite fresh-keeping film for fermented foods according to claim 1 in food preservation, biosensing or medical dressings.

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