CN111808307B - Preparation method of spirulina antibacterial peptide composite membrane - Google Patents
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Abstract
The invention discloses a preparation method of a spirulina antibacterial peptide composite membrane, which is characterized in that spirulina powder is subjected to enzymolysis by neutral protease to prepare spirulina antibacterial peptide, and then the spirulina antibacterial peptide composite membrane is mixed with nano titanium dioxide, sodium alginate and the like by low vacuum frequency conversion ultrasonic and dried to prepare the spirulina antibacterial peptide composite membrane. The composite film prepared by the invention has uniform thickness, smooth surface, no obvious bubbles, good antibacterial property and greatly improved tensile property, and can be used as a packaging material.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a preparation method of a spirulina antibacterial peptide composite membrane.
Background
At present, the most common packaging film material is plastic, such as PE, PVC, PP and the like, which is not easy to degrade and has great influence on the environment. The nano titanium dioxide has strong stability, super-hydrophilicity and non-migration, and simultaneously has antibacterial, anti-aging and self-cleaning performances. However, the nano titanium dioxide and the film forming agent are directly used for forming the film, so that the tensile strength is poor, the film is easy to break, and the film is not suitable for being directly used as a packaging film.
Patent CN 110818956A discloses a chitosan-nano titanium dioxide composite membrane, but chitosan has poor water solubility, is generally dissolved in acid, and needs to be added with various chemical substances to promote the dissolution, and the process is complex.
Patent CN 104497336A discloses a yellow crucian protein antibacterial peptide-nano zinc oxide film, the yellow crucian used is high in price, inconvenient to store and transport, and not suitable for large-scale production outside the production area.
Disclosure of Invention
The invention aims to provide a preparation method of a spirulina antibacterial peptide composite membrane, and the obtained spirulina antibacterial peptide composite membrane has good antibacterial performance and tensile property and good application prospect.
In order to realize the purpose, the invention adopts the following technical scheme:
a preparation method of a spirulina antibacterial peptide composite membrane comprises the following steps:
1) Preparation of spirulina antibacterial peptide: adding spirulina powder into sodium phosphate buffer solution with the pH =7.0 according to the mass volume ratio of 1g of 10mL, repeatedly freezing and thawing for three times, freezing and centrifuging at low temperature for 30min, then taking supernate, adding neutral protease according to the amount of 0.01-0.03g/100mL, carrying out enzymolysis at 20-70 ℃ for 1-8 h, keeping the pH value at 7.0 in the period, heating the enzymolysis liquid in a water bath at 95 ℃ for 10 min for enzyme deactivation, freezing and centrifuging at low temperature for 30min again, and taking supernate for vacuum freeze drying to obtain the spirulina antibacterial peptide;
2) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol according to the mass volume ratio of 1g to 20mL, performing ball milling for 48 hours, and filtering to obtain nano titanium dioxide;
3) Preparing a composite membrane: adding the spirulina antibacterial peptide, the nano titanium dioxide, the film forming agent, the plasticizer and the dispersing agent into a small amount of a composite solvent (the volume ratio of the anhydrous ethanol to the deionized water is 1-20) formed by anhydrous ethanol and deionized water for dissolving, then adding the composite solvent (the volume ratio of the anhydrous ethanol to the deionized water is 1-20) formed by the anhydrous ethanol and the deionized water until the total volume of the solution is 100mL, stirring, uniformly mixing by low vacuum variable frequency ultrasound, pouring into a mold, drying at 60 ℃ for 3 h, cooling to room temperature, and then removing to obtain the spirulina antibacterial peptide composite film.
In the step 3), the content of the spirulina antibacterial peptide in the solution is 0.1-5 g/100mL, the content of the nano titanium dioxide is 1-5 g/100mL, the content of the film forming agent is 0.5-5 g/100mL, the content of the plasticizer is 0.1-3 g/100mL, and the content of the dispersing agent is 0.1-1.5 g/100mL.
Wherein the film forming agent is any one of sodium alginate, gelatin and hydroxypropyl methyl cellulose;
the plasticizer is glycerol or sorbitol;
the dispersant is polyethylene glycol 1000 or polyethylene glycol 2000.
In the step 3), the vacuum degree of the low-vacuum frequency conversion ultrasound is 100-300Pa, the ultrasound power is 250W, the ultrasound frequency variation range is 20kHz-60kHz, the variation frequency is 0.1-0.5s, the variation frequency is 100-200Hz each time, and the total ultrasound time is 30min.
The invention has the following remarkable advantages:
(1) The spirulina used in the invention has high yield and low cost, is easy to store and transport after being dried, and can overcome the problem of inconvenient storage and transport in the prior art. Meanwhile, the protein content in the spirulina is up to 70 percent, so the spirulina is a suitable raw material for preparing the antibacterial peptide.
(2) The pH value of the antibacterial peptide is maintained at neutral in the preparation process, the corrosion action is avoided, the process is simple, and the scale-up popularization is easy.
(3) The nano titanium dioxide is easy to aggregate together when being mixed with other raw materials in water. In order to solve the problem, the invention utilizes variable frequency ultrasound in a low vacuum state and adds a dispersing agent to promote the acceleration of the uniform mixing and dispersing process, so that the nano titanium dioxide is not easy to agglomerate in the subsequent process and is stably combined with the base material. Meanwhile, the low vacuum frequency conversion ultrasound also has a degassing function, so that the obtained composite membrane has no bubbles and is flat and smooth.
(4) The composite film obtained by the invention has uniform thickness, smooth surface and no obvious bubbles, and the antibacterial performance is obviously improved, and meanwhile, the tensile property is also greatly improved. Meanwhile, food-grade raw materials are used in the preparation process, so that the obtained composite membrane is safe, non-toxic, green, environment-friendly, biodegradable and slightly environment-friendly, and can be used as a packaging material.
Drawings
FIG. 1 is a process flow diagram of the preparation of the spirulina antibacterial peptide composite membrane of the present invention.
FIG. 2 is a graph showing a comparison of the morphology of the composite films obtained in example 1 (A) and comparative example 1 (B).
Detailed Description
In order to make the content of the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
1) Preparation of spirulina antibacterial peptide: adding 6g of spirulina powder into 120mL of sodium phosphate buffer solution with the pH =7.0, repeatedly freezing and thawing for three times in a freezing and room-temperature thawing mode at the temperature of minus 20 ℃, freezing and centrifuging for 30min at the low temperature of 4 ℃, then taking supernatant, adding neutral protease according to the amount of 0.0125g/100mL, carrying out enzymolysis for 4 h at the temperature of 45 ℃, keeping the pH value at 7.0 in the period, heating enzymolysis liquid in a water bath at the temperature of 95 ℃ for 10 min for enzyme deactivation, freezing and centrifuging for 30min at the low temperature of 4 ℃ again, and then taking the supernatant for vacuum freeze drying to obtain the spirulina antimicrobial peptide;
2) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol according to the mass volume ratio of 1g;
3) Preparing a composite membrane: adding 0.6g of spirulina antibacterial peptide, 1.2 g of nano titanium dioxide, 1g of sodium alginate, 0.5 g of glycerol and 0.1 g of polyethylene glycol 1000 into a composite solvent (1, 20, v/v) formed by 10 mL of absolute ethyl alcohol and deionized water for dissolving, then adding the composite solvent until the total volume of the solution is 100mL, stirring, carrying out ultrasonic treatment for 30min under the conditions of a vacuum degree of 100-300Pa and a power of 250W to uniformly mix the solution, wherein the ultrasonic frequency is circularly changed within the range of 40kHz to 55kHz, the change mode is changed once in 0.1s, each change is 100Hz, pouring the solution into a mold after ultrasonic treatment, drying for 3 h at 60 ℃, cooling to room temperature, and then taking off to obtain the spirulina antibacterial peptide composite membrane.
Example 2
1) Preparation of spirulina antibacterial peptide: adding 6g of spirulina powder into 120mL of sodium phosphate buffer solution with the pH =7.0, repeatedly freezing and thawing for three times in a freezing and thawing mode at the temperature of minus 20 ℃ and room temperature, freezing and centrifuging for 30min at the low temperature of 4 ℃, then taking supernate, adding neutral protease according to the amount of 0.0125g/100mL, carrying out enzymolysis for 8 h at the temperature of 30 ℃, keeping the pH value at 7.0 in the period, heating the enzymolysis solution in a water bath at the temperature of 95 ℃ for 10 min for enzyme deactivation, freezing and centrifuging for 30min at the low temperature of 4 ℃ again, and then taking supernate for vacuum freeze drying to obtain the spirulina antimicrobial peptide;
2) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol according to the mass volume ratio of 1g;
3) Preparing a composite membrane: adding 1g of spirulina antibacterial peptide, 2 g of nano titanium dioxide, 2 g of gelatin, 1g of glycerol and 1g of polyethylene glycol 2000 into a composite solvent (1.
Example 3
1) Preparation of spirulina antibacterial peptide: adding 6g of spirulina powder into 120mL of sodium phosphate buffer solution with the pH =7.0, repeatedly freezing and thawing for three times in a freezing and room-temperature thawing mode at the temperature of minus 20 ℃, freezing and centrifuging for 30min at the low temperature of 4 ℃, then taking supernatant, adding neutral protease according to the amount of 0.0125g/100mL, carrying out enzymolysis for 2 h at the temperature of 70 ℃, keeping the pH value at 7.0 in the period, heating enzymolysis liquid in a water bath at the temperature of 95 ℃ for 10 min for enzyme deactivation, freezing and centrifuging for 30min again at the low temperature, and then taking supernatant for vacuum freeze drying to obtain the spirulina antimicrobial peptide;
2) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol according to the mass volume ratio of 1g to 20mL, performing ball milling for 48 hours, and filtering to obtain nano titanium dioxide;
3) Preparing a composite membrane: adding 3g of spirulina antibacterial peptide, 3g of nano titanium dioxide, 3g of hydroxypropyl methyl cellulose, 3g of sorbitol and 1.5 g of polyethylene glycol 1000 into a composite solvent (1, 20 v/v) formed by 10 mL of absolute ethyl alcohol and deionized water for dissolving, then adding the composite solvent until the total volume of the solution is 100mL, stirring, carrying out ultrasonic mixing for 30min under the conditions of a vacuum degree of 100-300Pa and a power of 250W, wherein the ultrasonic frequency circularly changes within the range of 20kHz to 6060kHz, the change mode is once for 0.1s, the change is 200Hz every time, pouring the solution into a mold after ultrasonic treatment, drying for 3 h at 60 ℃, cooling to room temperature, and then removing to obtain the spirulina antibacterial peptide composite membrane.
Comparative example
1) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol according to the mass volume ratio of 1g to 20mL, performing ball milling for 48 hours, and filtering to obtain nano titanium dioxide;
2) Preparing a composite membrane: adding 1.2 g of nano titanium dioxide, 1g of sodium alginate, 0.5 g of glycerol and 0.1 g of polyethylene glycol 1000 into 10 mL of a composite solvent (1, 20 v/v) formed by absolute ethyl alcohol and deionized water for dissolving, then adding the composite solvent until the total volume of the solution is 100mL, stirring, and then carrying out ultrasonic mixing for 30min under the conditions of a vacuum degree of 100-300Pa and a power of 250W, wherein the ultrasonic frequency is cyclically changed within the range of 40kHz to 55kHz, the change mode is changed once for 0.1s, each change is 100Hz, pouring the solution into a mold after ultrasonic mixing, drying for 3 h at 60 ℃, cooling to room temperature, and then uncovering to obtain the composite membrane without the antibacterial peptide.
And (3) performance test results:
the composite membrane prepared in example 1 and the comparative example without the antibacterial peptide are subjected to morphology characterization and performance detection, and the results are shown in fig. 1 and table 1.
TABLE 1
As can be seen from Table 1, the composite membrane added with the spirulina antibacterial peptide has obviously improved mechanical property and tensile property compared with the composite membrane not added with the spirulina antibacterial peptide, the antibacterial rate of the composite membrane on escherichia coli is increased by 751%, and the elongation rate is increased by 126%.
As can be seen from the combination of FIG. 1, the composite membrane added with the spirulina antibacterial peptide has loose and porous surface, has larger adsorption area, and can improve the adsorption of bacteria, thereby effectively inhibiting the growth of the bacteria; and the crosslinking degree of the composite membrane added with the spirulina antibacterial peptide is higher, so that the mechanical property of the composite membrane is obviously improved.
The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.
Claims (7)
1. A preparation method of a spirulina antibacterial peptide composite membrane is characterized by comprising the following steps: the method comprises the following steps:
1) Preparation of spirulina antibacterial peptide: adding spirulina powder into a sodium phosphate buffer solution with pH =7.0, repeatedly freezing and thawing for three times, freezing and centrifuging at a low temperature of 4 ℃ for 30min, taking a supernatant, adding neutral protease, performing enzymolysis at a temperature of 20-70 ℃ for 1-8 h, maintaining the pH value at 7.0 in the period, heating the enzymolysis solution in a water bath at 95 ℃ for 10 min to inactivate enzyme, freezing and centrifuging at a low temperature of 4 ℃ for 30min again, and taking the supernatant for vacuum freeze drying to obtain the spirulina antimicrobial peptide;
2) Preparing nano titanium dioxide: washing titanium dioxide with deionized water, drying, adding absolute ethyl alcohol, ball-milling for 48 hours, and filtering to obtain nano titanium dioxide;
3) Preparing a composite membrane: adding the spirulina antibacterial peptide, the nano titanium dioxide, the film forming agent, the plasticizer and the dispersing agent into a small amount of a composite solvent formed by anhydrous ethanol and deionized water for dissolving, then adding the composite solvent formed by the anhydrous ethanol and the deionized water until the total volume of the solution is 100mL, stirring, ultrasonically mixing by low vacuum frequency conversion, pouring into a mold, drying at 60 ℃ for 3 h, cooling to room temperature, and then removing to obtain the spirulina antibacterial peptide composite film;
step 3) the solution contains 0.1-5 g/100mL of spirulina antibacterial peptide, 1-5 g/100mL of nano titanium dioxide, 0.5-5 g/100mL of film-forming agent, 0.1-3 g/100mL of plasticizer and 0.1-1.5 g/100mL of dispersing agent;
the vacuum degree of the low-vacuum frequency-variable ultrasound is 100-300Pa, the ultrasound power is 250W, the variation range of the ultrasound frequency is 20kHz-60kHz, the variation mode is changed once every 0.1-0.5s, each variation is 100-200Hz, and the total ultrasound time is 30min.
2. The method for preparing the spirulina platensis antibacterial peptide composite membrane according to claim 1, characterized in that: the mass-to-volume ratio of the spirulina powder to the sodium phosphate buffer solution in the step 1) is 1g.
3. The method for preparing the spirulina platensis antibacterial peptide composite membrane according to claim 1, which is characterized in that: the amount of neutral protease added in step 1) is 0.01-0.03g/100mL.
4. The method for preparing the spirulina platensis antibacterial peptide composite membrane according to claim 1, which is characterized in that: the mass volume ratio of the titanium dioxide to the absolute ethyl alcohol used in the step 2) is 1g.
5. The method for preparing the spirulina platensis antibacterial peptide composite membrane according to claim 1, which is characterized in that: and step 3), the volume ratio of absolute ethyl alcohol to deionized water in the composite solvent is 1-20.
6. The method for preparing the spirulina platensis antibacterial peptide composite membrane according to claim 1, characterized in that: the film forming agent is any one of sodium alginate, gelatin and hydroxypropyl methyl cellulose;
the plasticizer is glycerol or sorbitol;
the dispersant is polyethylene glycol 1000 or polyethylene glycol 2000.
7. A spirulina platensis antibacterial peptide composite membrane prepared by the method of claim 1.
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