CN115651276A - Chitosan composite membrane containing protein nanoparticles and preparation method and application thereof - Google Patents
Chitosan composite membrane containing protein nanoparticles and preparation method and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a chitosan composite membrane containing protein nanoparticles, a preparation method and application thereof, which are characterized in that zein and tannin form protein nanoparticles through a reverse solvent precipitation method, and the protein nanoparticles interact with chitosan to form a composite membrane, so as to inhibit at least one microorganism of Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa) and Listeria monocytogenes (Listeria monocytogenes). According to the invention, the zein-tannin nanoparticles are compounded in the chitosan film forming solution, so that the mechanical property of the chitosan film is enhanced, the antibacterial effect is obviously enhanced, the zein-tannin nanoparticles can be used as a food antibacterial film to be applied to the fields of fruits, meat products, aquatic products and the like, and the zein-tannin composite chitosan film is simple in preparation method, low in cost and wide in market prospect.
Description
Technical Field
The invention belongs to the food bacteriostasis technology, and particularly relates to a chitosan composite film containing protein nanoparticles, and a preparation method and application thereof.
Background
The food contains rich nutrient substances, and bacteria/mould are easy to grow and propagate on the outer surface/inside of the food, so that the food is easy to deteriorate, huge economic loss can be caused, and the health of a human body can be influenced. Food safety has been a major public health problem worldwide, with health hazards caused by food-borne pathogens being of great concern. Common food-borne pathogenic bacteria include Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), listeria monocytogenes (Listeria monocytogenes), salmonella (Salmonella), and the like. In the antibacterial film for food packaging, the antibacterial agent is used as a main functional component and plays a crucial role in the antibacterial activity of antibacterial materials and products. The edible antibacterial packaging film is increasingly applied to the fields of food packaging and preservation.
Chitosan is a linear polysaccharide mainly extracted from shrimp and crab shells, has good film forming property, antibacterial property and biodegradability, and is an excellent edible film forming raw material. However, chitosan itself has antibacterial property, but the antibacterial activity is not high after being made into a thin film, mainly because the chitosan molecules are slowly diffused in the film, so that only microorganisms directly contacting with the chitosan active site can be inhibited, and the antibacterial activity of the film is weaker. Therefore, the improvement of the antibacterial performance of the chitosan film has important significance for developing green packaging materials.
Tannic acid is mainly derived from vegetables and fruits, and has unique biological activity including antioxidant, antitumor and antibacterial activities. It is a potential component of stable protein colloidal particles with antibacterial activity. Zein is a class of alcohol soluble proteins that are the major storage proteins of corn. The zein microspheres formed by the anti-solvent precipitation technology can be used as a stabilizer, an emulsifier, a structure modifier and the like. The development of zein-based nanoparticles is emerging as a way to reduce loss of bioactivity during film manufacturing and/or packaging structure development. Therefore, the zein and the tannic acid are assembled to form the nano particles, and the chitosan membrane is modified and researched by using the nano particles, so that the mechanical property and the antibacterial property of the film are improved.
Due to different protonation, neutralization and ionization states of tannic acid and zein in solutions at different pH values, intermolecular interactions such as intermolecular hydrogen bonds, hydrophobic interactions, electrostatic interactions and the like, which are expressed by two molecules, are different, so that the properties of the formed nanoparticles and the modification effect of the nanoparticles on membranes are different. In addition, in the processing or fresh-keeping process, the food system is easy to be in different pH value environments, and the chitosan composite membrane containing the protein nanoparticles under the influence of different pH conditions is developed, so that the chitosan composite membrane has wide application value. However, the preparation of the chitosan composite membrane acted by plant-based protein nanoparticles obtained under the condition of different pH values and the application of the plant-based protein nanoparticles in inhibiting the growth of food-borne pathogenic bacteria are lacked at present.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems, the invention provides a chitosan composite membrane of protein nanoparticles as well as a preparation method and application thereof, aiming at utilizing the performance and bacteriostatic activity of the chitosan membrane modified by nano-scale particles.
The technical scheme is as follows: a chitosan composite membrane containing protein nano-particles is characterized in that the protein nano-particles are formed by a reverse solvent precipitation method through zein and tannic acid, and are interacted with chitosan to form a composite membrane, wherein the mass ratio of the zein to the tannic acid is 1: 0.1-1: 0.5, and the chitosan accounts for 1.2-2.5% of the total mass.
Preferably, the mass ratio of the zein to the tannic acid is 1: 0.5, and the chitosan accounts for 2.0% of the total mass. Further, the particle size of the protein nano-particles is less than 1 μm, and the thickness of the composite membrane is less than 0.06mm.
The preparation method of the chitosan composite membrane containing the protein nanoparticles is characterized by comprising the following steps of:
(1) Dissolving zein in 70% (v/v) ethanol solution, stirring at room temperature and 500rpm for 3h, standing for 12h to obtain 2% zein solution, adding tannin at a mass ratio of zein to tannin of 1: 0.1-1: 0.8, stirring the solution sufficiently, and adjusting pH to 2-7 with HCl or NaOH; preferably, the mass ratio of the zein to the tannic acid is 1: 0.5;
(2) Adding 2.5 times of water into the zein-tannin solution in the step (1) under the stirring condition of 1000rpm, stirring for 15min, performing rotary evaporation, removing ethanol and part of water in the solution to obtain zein-tannin nanoparticle suspension, and freeze-drying into powder with the particle size of less than 1 μm;
preferably, the particle size of the obtained zein-tannin nano particles is less than 200nm;
(3) Dissolving chitosan in 1% acetic acid solution, stirring in water bath at 65 ℃ for 30min, adding 50% (w/t) glycerol, adding zein-tannin nano particle freeze-dried powder with different pH values prepared in the step (2) into the solution according to the mass ratio of 5% -10% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film;
preferably, the mass ratio of the added nanoparticles is 5%.
The application of the chitosan composite membrane containing the protein nanoparticles in inhibiting the growth of microorganisms is also within the protection scope of the invention.
Wherein the microorganism is selected from Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa), listeria monocytogenes (Listeria monocytogenes).
The application comprises the following steps: the chitosan composite membrane containing the protein nanoparticles is cut into 2 x 2cm, added into a culture medium and inoculated with at least one microorganism of Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa) and Listeria monocytogenes (Listeria monocytogenes), and the bacteriostatic aging time is more than 24h.
Has the advantages that: compared with the prior art, the method has the following advantages: (1) According to the invention, the zein-tannin nanoparticles are used for modifying the chitosan composite membrane, so that the protein nanoparticles and chitosan under different pH conditions have a synergistic effect, and the chitosan composite membrane has a remarkable bacteriostatic effect on common food-borne pathogenic bacteria and has a broad spectrum. (2) The method is simple in operation method and low in cost, is suitable for preserving fruits, meat products, aquatic products and other food, and has wide market prospect.
Drawings
Fig. 1 is a graph of particle size and morphology of zein-tannin nanoparticles;
fig. 2 is a graph showing the inhibitory effect of the chitosan composite membrane containing zein-tannin nanoparticles on escherichia coli (e.coli), staphylococcus aureus (s.aureus), pseudomonas aeruginosa (p.aeruginosa) and l.monocytogenes (l.monocytogens) after culturing for 24 hours.
Detailed Description
Example 1 preparation of protein nanoparticles
2.0g zein was dispersed in 100mL 70% (v/v) ethanol solution, stirred at 500rpm for 3h at room temperature and left overnight. Adding tannic acid into zein solution at a mass ratio of 1: 0.5, stirring, and adjusting pH to 2 with 2M HCl. And removing ethanol and part of the aqueous solution by using a rotary evaporator to obtain a zein-tannin nanoparticle suspension, wherein the particle size of the nanoparticles is 131.9nm measured by adopting a Zeta potentiometer. And (3) freeze-drying the zein-tannin nano particle suspension into powder, and observing the microstructure of the powder by using a scanning electron microscope to obtain spherical particles.
Example 2 preparation of protein nanoparticles
2.0g zein was dispersed in 100mL 70% (v/v) ethanol solution, stirred at 500rpm for 3h at room temperature and left overnight. Adding tannic acid into zein solution at a mass ratio of 1: 0.5, stirring, and adjusting pH to 4 with 2M HCl. And removing ethanol and part of the aqueous solution by using a rotary evaporator to obtain a zein-tannin nanoparticle suspension, wherein the particle size of the nanoparticles is 106.3nm measured by adopting a Zeta potentiometer. Freeze drying zein-tannin nano particle suspension to powder, observing the microstructure of the zein-tannin nano particle suspension by adopting a scanning electron microscope to form spherical particles.
Example 3 preparation of protein nanoparticles
2.0g zein was dispersed in 100mL 70% (v/v) ethanol solution, stirred at 500rpm for 3h at room temperature and left overnight. Adding tannic acid into zein solution at a mass ratio of 1: 0.5, stirring, and adjusting pH to 7 with 2M NaOH. And removing ethanol and part of the aqueous solution by using a rotary evaporator to obtain zein-tannin nanoparticle suspension, wherein the particle size of the nanoparticles is 150.8nm measured by adopting a Zeta potentiometer. And (3) freeze-drying the zein-tannin nano particle suspension into powder, and observing the microstructure of the powder by using a scanning electron microscope to obtain spherical particles.
Example 4 preparation of chitosan composite membrane containing protein nanoparticles
Dissolving chitosan in 1% acetic acid solution, stirring in 65 ℃ water bath for 30min, wherein the content of chitosan is 2.0%, adding 50% (w/t) glycerol, adding the prepared zein-tannin nanoparticle freeze-dried powder with pH2 into the solution according to the mass ratio of 5% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film, wherein the thickness of the film is 0.059mm.
Example 5 preparation of chitosan composite membrane containing protein nanoparticles
Dissolving chitosan in 1% acetic acid solution, stirring in 65 ℃ water bath for 30min, wherein the content of chitosan is 2.0%, adding 50% (w/t) glycerol, adding the prepared zein-tannin nanoparticle freeze-dried powder with pH2 into the solution according to the mass ratio of 10% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film, wherein the thickness of the film is 0.05mm.
Example 6 preparation of chitosan composite membrane containing protein nanoparticles
Dissolving chitosan in 1% acetic acid solution, stirring in 65 deg.C water bath for 30min, adding chitosan 2.0%, adding 50% (w/t) glycerol, stirring at 750rpm, adding zein-tannin nanoparticle lyophilized powder with pH of 4 into the solution at 5% by mass ratio, stirring for 15min, ultrasonic degassing for 30min, pouring into a mold, drying at room temperature, and forming film with a thickness of 0.056mm.
Example 7 preparation of protein nanoparticle-containing Chitosan composite Membrane
Dissolving chitosan in 1% acetic acid solution, stirring in 65 deg.C water bath for 30min, adding chitosan 2.0%, adding 50% (w/t) glycerol, stirring at 750rpm, adding zein-tannin nanoparticle lyophilized powder with pH of 4 into the solution according to mass ratio of 10%, stirring for 15min, ultrasonic degassing for 30min, pouring into a mold, drying at room temperature, and forming film with thickness of 0.05mm.
Example 8 preparation of chitosan composite membrane containing protein nanoparticles
Dissolving chitosan in 1% acetic acid solution, stirring in 65 ℃ water bath for 30min, wherein the content of chitosan is 2.0%, adding 50% (w/t) glycerol, adding the prepared zein-tannin nano particle freeze-dried powder with pH7 into the solution according to the mass ratio of 5% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film, wherein the thickness of the film is 0.058mm.
Example 9 preparation of protein nanoparticle-containing Chitosan composite Membrane
Dissolving chitosan in 1% acetic acid solution, stirring in 65 ℃ water bath for 30min, wherein the content of chitosan is 2.0%, adding 50% (w/t) glycerol, adding the prepared zein-tannin nano particle freeze-dried powder with pH7 into the solution according to the mass ratio of 10% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film, wherein the thickness of the film is 0.059mm.
Example 10 bacteriostatic methods and assays
(1) Preparation of the bacterial suspension
Selecting 1-2 rings of the microorganism preserved on the inclined plane by using an inoculating ring, inoculating the microorganism into an LB culture medium, culturing for 12 hours in a full-temperature shaking table with the rotating speed of 37 ℃ and 180rpm, taking out 1mL of bacterial liquid into a centrifuge tube, centrifuging for 3-5 min at the conditions of 8000-15000 rpm and 4 ℃ to collect the thallus, washing and resuspending the thallus by using sterile physiological saline for at least 3 times, and finally preparing a bacterial suspension by using the sterile physiological saline, wherein the concentration is 1 multiplied by 107CFU/mL.
(2) Bacteriostatic effect of composite film
Cutting 2X 2cm composite membrane to process bacterial suspension, placing in an incubator at 37 deg.C for 24h, taking out, and measuring OD600 with enzyme labeling instrument. Six replicates of each sample were run against a blank supplemented with 1mL of broth and 1mL of LB broth. According to the OD600 results after 24h of culture of Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and Listeria monocytogenes shown in figure 2, compared with the control group (CK), the OD of the bacterial liquid co-cultured with the composite membrane is obviously reduced, which indicates that the prepared chitosan composite membrane containing the nano-protein particles has obvious inhibition effect on Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and Listeria monocytogenes. Wherein,
the chitosan composite membrane containing protein nanoparticles prepared in example 4 has an inhibition rate of 60.4% for escherichia coli, 37.8% for staphylococcus aureus, 9.5% for pseudomonas aeruginosa, and 27.8% for listeria monocytogenes.
The chitosan composite membrane containing protein nanoparticles prepared in example 5 has an inhibition rate of 76.8% for escherichia coli, 53.5% for staphylococcus aureus, 42.6% for pseudomonas aeruginosa and 53.4% for listeria monocytogenes.
The chitosan composite membrane containing protein nanoparticles prepared in example 6 has an inhibition rate of 84.6% on escherichia coli, 53.5% on staphylococcus aureus, 58.2% on pseudomonas aeruginosa, and 77.4% on listeria monocytogenes.
The chitosan composite membrane containing protein nanoparticles prepared in example 7 has an inhibition rate of 87.8% for escherichia coli, 72.3% for staphylococcus aureus, 57.5% for pseudomonas aeruginosa, and 73.0% for listeria monocytogenes.
The chitosan composite membrane containing protein nanoparticles prepared in example 8 has an inhibition rate of 18.0% on escherichia coli, 22.4% on staphylococcus aureus, 44.0% on pseudomonas aeruginosa, and 62.9% on listeria monocytogenes.
The chitosan composite membrane containing protein nanoparticles prepared in example 9 has an inhibition rate of 80.4% for escherichia coli, 73.9% for staphylococcus aureus, 72.1% for pseudomonas aeruginosa, and 65.6% for listeria monocytogenes.
Comparative example: bacteriostatic effect of chitosan membrane without protein nanoparticles
Dissolving chitosan in 1% acetic acid solution, stirring in 65 deg.C water bath for 30min to obtain chitosan content of 2.0%, adding 50% (w/t) glycerol, stirring at 750rpm for 15min, ultrasonic degassing for 30min, pouring into a mold, drying at room temperature, and forming film with thickness of 0.05mm.
According to the method of the embodiment 10, the chitosan composite membrane has an inhibition rate of 8.0% for escherichia coli, 14.9% for staphylococcus aureus, 9.5% for pseudomonas aeruginosa, and 27.8% for listeria monocytogenes.
As can be seen from the above, the chitosan composite film containing the protein nanoparticles has a remarkable bacteriostatic effect on common food-borne pathogens, and the protein nanoparticles prepared under different pH conditions have different bacteriostatic and synergistic effects on the chitosan film, so that the chitosan composite film is suitable for the fresh-keeping in the fields of fruits, meat products, aquatic products and the like and has a wide application prospect.
Claims (5)
1. The chitosan composite membrane containing protein nano-particles is characterized in that the protein nano-particles are formed by zein and tannic acid through an anti-solvent precipitation method and are interacted with chitosan to form a composite membrane, wherein the mass ratio of the zein to the tannic acid is 1: 0.1-1: 0.8, and the chitosan accounts for 1.2-2.5% of the total mass.
2. The method for preparing the chitosan composite membrane containing the protein nanoparticles as claimed in claim 1, comprising the steps of:
(1) Dissolving zein in 70% (v/v) ethanol solution, stirring at room temperature and 500rpm for 3h, standing for 12h to obtain 2% zein solution, adding tannin, wherein the mass ratio of zein to tannin is 1: 0.1-1: 0.8, stirring the solution sufficiently, and adjusting the pH range to 2-7 with HCl or NaOH;
(2) Adding 2.5 times of water into the zein-tannin solution in the step (1) under the stirring condition of 1000rpm, stirring for 15min, performing rotary evaporation, removing ethanol and part of water in the solution to obtain zein-tannin nanoparticle suspension, and freeze-drying into powder with particle size less than 1 μm;
(3) Dissolving chitosan in 1% acetic acid solution, stirring in 65 ℃ water bath for 30min, adding 50% (w/t) glycerol, adding zein-tannin nanoparticle freeze-dried powder with different pH values prepared in the step (2) into the solution according to the mass ratio of 5% -10% under the stirring condition of 750rpm, stirring for 15min, ultrasonically degassing for 30min, pouring into a mold, drying at room temperature, and forming a film.
3. Use of a chitosan composite membrane comprising protein nanoparticles as claimed in claim 1 for inhibiting the growth of microorganisms.
4. Use according to claim 3, wherein the microorganism is selected from the group consisting of Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa), listeria monocytogenes (Listeria monocytogenes).
5. The application according to claim 3, characterized in that it comprises the following steps: the chitosan composite membrane containing the protein nanoparticles is cut into 2 x 2cm, added into a culture medium and inoculated with at least one microorganism of Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), pseudomonas aeruginosa (Pseudomonas aeruginosa) and Listeria monocytogenes (Listeria monocytogenes), and the bacteriostatic aging time is more than 24h.
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