CN113336968B

CN113336968B - Preparation method and application of ginger essential oil nanoemulsion hydrogel

Info

Publication number: CN113336968B
Application number: CN202110599141.XA
Authority: CN
Inventors: 孙芝兰; 唐敏敏; 刘芳; 诸永志; 王道营; 张新笑; 李坤朋; 严敏
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2022-03-04
Anticipated expiration: 2041-05-31
Also published as: CN113336968A

Abstract

The invention discloses a preparation method of ginger essential oil nanoemulsion hydrogel, which comprises the steps of firstly preparing Ginger Essential Oil Nanoemulsion (GEON) consisting of Zein and NaCas, then further preparing the ginger essential oil nanoemulsion hydrogel into nanoemulsion hydrogel, and improving the gel time, antibacterial ability and action time of the ginger essential oil nanoemulsion hydrogel. Therefore, GEONH can obviously inhibitE.coliAndS.aureusand the growth of common spoilage bacteria in the meat products is expected to be applied to the field of corrosion prevention and preservation of low-temperature meat products, and the shelf life is prolonged.

Description

Preparation method and application of ginger essential oil nanoemulsion hydrogel

Technical Field

The invention belongs to the field of food processing materials, and particularly relates to a preparation method and application of a ginger essential oil nanoemulsion hydrogel.

Background

Emulsification gives the essential oil smaller size and larger surface area, enhancing its physicochemical properties. The colloid system of the nano emulsion can better protect nutrient substances, improve the stability, water solubility and bioavailability of the essential oil, and cover up the pungent smell of the essential oil, so that the influence on the sensory characteristics of food is reduced. In general, the emulsification properties (e.g., particle size, potential, entrapment rate, stability, etc.) of nanoemulsions are controlled by the type of emulsifier and the emulsification technique. On the one hand, high-energy emulsification techniques require high mechanical energy and low energy utilization, resulting in high equipment costs in production, while low-energy emulsification techniques do not require specialized or expensive production equipment. On the other hand, some high-performance small-molecule emulsifiers (such as Tweens and Spans) have been reported to have potential toxicity, which greatly limits the application of the emulsifiers in the food and pharmaceutical industries. Therefore, the preparation of vegetable essential oil nanoemulsions using biomacromolecular emulsifiers (e.g. vegetable or animal proteins) and by low energy emulsification methods may be a promising strategy for the food industry.

There are many food emulsifiers used to encapsulate hydrophobic compounds such as curcumin, thymol and fish oil, among which Zein (Zein) is one of the more widely used. Compared with animal-derived proteins, Zein has low price, high hydrophobicity and heat resistance, and has a series of advantages of good biocompatibility, slow digestibility, strong biodegradability and the like, so that Zein becomes an ideal carrier of hydrophobic materials. However, Zein is insoluble in water under neutral pH conditions and typically needs to be dissolved in 70-80% (v/v) alcohol solution to prepare nanoparticles. Although these alcohol solutions can later be removed by anti-solvent precipitation or spray/freeze drying, they still do not meet the consumer's concept of "green consumption". At the same time, the redispersibility and stability of Zein nanoparticles in aqueous systems remains a great challenge, limiting their application as delivery vehicles.

Sodium caseinate (NaCas), also known as sodium caseinate, is a food grade material rich in casein and can be used as an additive in the food industry. Recently, NaCas was found to improve the redispersibility and stability of Zein nanoparticles in aqueous systems. Chen et al reported a method for electrostatic binding of Zein to NaCas by adjusting the ph.

The nano-emulsion hydrogel is a homogeneous and stable gel network structure colloid formed by adding nano-emulsion into a hydrogel matrix composed of natural high molecular materials (such as collagen, gelatin and cellulose derivatives), and aims to improve the viscosity of the nano-emulsion and improve the spreadability of the emulsion on the surface of a meat product so as to prolong the action time of essential oil in the emulsion.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides the ginger essential oil nanoemulsion hydrogel, which is prepared by preparing a Ginger Essential Oil Nanoemulsion (GEON) composed of Zein and NaCas, and further preparing the ginger essential oil nanoemulsion hydrogel into the nanoemulsion hydrogel, so that the gel time, the antibacterial ability and the acting time of the ginger essential oil nanoemulsion are improved.

In order to realize the purpose, the invention provides a preparation method of ginger essential oil nanoemulsion hydrogel, which comprises the following steps: (1) respectively dissolving Zein and sodium caseinate in pure water, adjusting the pH value to 11-11.5, stirring to obtain a Zein solution with the mass fraction of 2-3% (w/v) and a sodium caseinate solution with the mass fraction of 2-3% (w/v), mixing the Zein solution and the sodium caseinate solution to obtain a mixed protein solution, preferably, increasing the pH value of the Zein solution and the mixed protein solution to 11.5 by using 3M NaOH, wherein the pH value is the lowest pH value for dissolving Zein; (2) deprotonating the ginger essential oil, adding the ginger essential oil into the mixed protein solution obtained in the step (1), and then acidifying and stirring until the pH is reduced to 7.0 to obtain the ginger essential oil nanoemulsion;

(3) sodium periodate is used as an oxidant, hydroxyl on the surface of the microfibrillated cellulose is partially oxidized into aldehyde group to prepare nano dialdehyde microfibrillated cellulose, then the sodium periodate is removed by dialysis, and freeze drying is carried out after dialysis to obtain freeze-dried powder of the nano dialdehyde microfibrillated cellulose;

(4) and (3) respectively adding the nanometer dialdehyde microfibrillated cellulose freeze-dried powder and the carboxymethyl chitosan freeze-dried powder into the ginger essential oil nanoemulsion prepared in the step (2), stirring and mixing uniformly, then mixing the two, stirring in a water bath, and standing at room temperature to obtain the ginger essential oil nanoemulsion hydrogel.

In the step (1), the mass ratio of the zein to the sodium caseinate in the mixed protein solution is (0-2):1, and preferably the mass ratio of the zein to the sodium caseinate is 1: 1.

In the step (2), the deprotonation of the ginger essential oil comprises the following steps: dissolving ginger essential oil in 3M sodium hydroxide, heating at 100-120 deg.C for 10-15min, taking out, and cooling to room temperature to obtain deprotonated ginger essential oil with volume concentration of 10% (v/v).

In the step (2), citric acid monohydrate is used for acidification treatment. Preferably, the acidification is performed using 3M citric acid monohydrate.

Preferably, the GEO concentration in the ginger essential oil nanoemulsion is 0.5-2% (v/v).

Dialdehyde microfibrillated cellulose: carboxymethyl chitosan: the ginger essential oil nanoemulsion comprises the following components in percentage by mass (5-25): (95-75): (1000 to 2000). Preferably, the dialdehyde microfibrillated cellulose: carboxymethyl chitosan: the mass ratio of the ginger essential oil nanoemulsion is 1: 9: 270, wherein the concentration of the ginger essential oil in the ginger essential oil nanoemulsion is 1%.

The mass ratio of the sodium periodate to the microfibrillated cellulose is 1:1-1: 3.

The invention further provides application of the ginger essential oil nanoemulsion hydrogel prepared by the method as an antiseptic preservative for meat products

Has the advantages that: compared with the prior art, the method has the following technical effects:

(1) according to the application, Ginger Essential Oil (GEO) is embedded and prepared into nanoemulsion by utilizing the interaction of Zein and NaCas, because the Zein is dissolved in water under an alkaline condition, NaCas micelles can be dissociated, GEO containing hydroxyl groups can be deprotonated, and because the time of alkaline treatment is short, neither of two proteins is hydrolyzed or has primary structure change, the three components can interact to form a good mixed solution, NaCas micelles can be recombined in the later acid-base neutralization process, so that the co-assembly between the Zein and the NaCas is realized, and the GEO is embedded in a Zein/NaCas compound through hydrophobic attraction and potential electrostatic attraction, in the experiment, the Ginger Essential Oil Nanoemulsion (GEON) consisting of the Zein and the NaCas is prepared under the condition of not using special equipment and organic solvents, the deprotonated GEO is added into the mixture containing the Zein and the NaCas under the alkaline condition, the pH was then neutralized with acid from 11.5 to 7.0. The performance of GEON such as particle size, potential, embedding rate and the like is regulated and controlled by regulating the mass ratio of Zein to NaCas;

(2) according to the invention, aldehyde groups on the molecular chain of the DAMFC can have Schiif base reaction with amino groups on the molecular chain of the CMCS to form hydrogel, then GEON is used as a solvent to prepare GEONH together with the DAMFC and the CMCS, and the structure and performance of the GEONH are regulated and controlled by taking the addition amount of the DAMFC as a variable.

Drawings

FIG. 1 is the appearance of 1% (v/v) GEO nanoemulsion prepared under the conditions of (A)0:1, (B)1:1, (C)1:2, (D)2:1, respectively, Zein/NaCas mass ratio at pH 7.0;

FIG. 2 shows the embedding rate of 1% (v/v) GEON prepared under the conditions of Zein/NaCas mass ratio of 0:1, 1:2 and 2: 1;

FIG. 3 is the embedding rate, particle size and zeta potential of GEON prepared from 0.5-2% oil concentration at a Zein/NaCas mass ratio of 1: 1;

FIG. 4 is S of Zein/NaCas dispersions with different mass ratios₀；

FIG. 5 is an SEM image of 1% (v/v) GEON prepared under conditions of a Zein/NaCas mass ratio of (A)0:1, (B)1:1, (C)1:2 and (D)2: 1;

FIG. 6 is a graph of the fluorescence spectra at λ ex-280 nm of (A) Zein, (B) NaCas and (C) Zein/NaCas (1:1) solutions containing different concentrations of GEO;

FIG. 7 is a UV spectrum of (a) Zein, (b) NaCas and (c) Zein/NaCas (1:1) solutions containing different concentrations of GEO;

FIG. 8 is a FT-IR spectrum of Zein/NaCas (1:1) and 1% (v/v) GEO nanoemulsion;

FIG. 9 is an XRD spectrum of lyophilized powders of Zein, NaCas, Zein/NaCas (1:1) and 1% (v/v) GEO nanoemulsion;

FIG. 10 is an SEM image of (A) Pseudomonas aeruginosa and (B) Staphylococcus aureus before and after GEON treatment;

FIG. 11 is a LSCM image of (A) Pseudomonas aeruginosa and (B) Staphylococcus aureus before and after GEON treatment;

FIG. 12 is a graph of the effect of GEON treatment on extracellular ATP concentrations of (A) Pseudomonas aeruginosa and (B) Staphylococcus aureus;

FIG. 13 shows the gel times of GEONH at DAMFC/CMCS mass ratios of 5/95, 10/90, 15/85, 20/80, 25/75;

FIG. 14 shows the gel strength of GEONH at DAMFC/CMCS mass ratios of 5/95, 10/90, 15/85, 20/80, 25/75;

FIG. 15 is a graph of the swelling curves (A)0-48h for GEONH at DAMFC/CMCS mass ratios of 5/95, 10/90, 15/85, 20/80, 25/75; (B)0-80 min;

fig. 16 is SEM images of nanofibers after lyophilization (a) MFC, (B) DAMFC;

FIG. 17 is an SEM image of GEONH at various DAMFC/CMCS mass ratios after lyophilization (A)5/95, (B)10/90, (C)15/85, (D)20/80, (E) 25/75;

FIG. 18 is a graph of the FT-IR spectra of MFC and DAMFC;

FIG. 19 is a schematic diagram of the preparation of DAMFC;

FIG. 20 is a FT-IR spectrum of DAMFC, CMCS, and DAMFC/CMCS (10/90) formulated in GEON;

FIG. 21 is a Schiff base reaction scheme;

FIG. 22 is an XRD spectrum of MFC and DAMFC;

FIG. 23 is an XRD spectrum of GEON formulated DAMFC, CMCS, and DAMFC/CMCS (10/90).

Detailed Description

The present invention is further illustrated in detail below with reference to specific examples, which are provided to facilitate the understanding of the present invention, and the detailed embodiments and the specific procedures are given, but the scope of the present invention is not limited to the following examples.

Example 1 preparation of Ginger Essential Oil Nanoemulsion (GEON).

Zein (not less than 92 percent) and ginger essential oil (not less than 90 percent) are purchased from winged snow reagent, Inc., and NaCas (not less than 90 percent) is purchased from Solaibao reagent, Inc. 2g of Zein and NaCas sample powders were weighed and dissolved in 100mL of pure water at 25 ℃. The pH of both was raised to 11.5, which is the lowest pH value for solubilizing Zein, using 3M NaOH (the same applies below). Both protein solutions were then magnetically stirred at 600rpm for 2 h. GEO deprotonation is referred to and slightly modified by the method of Wang et al (Wang L, Zhang Y. eugenol nanoemusion Stabilized with Zein and Sodium casemate by Self-Assembly [ J ]. Journal of aggregative and food chemistry,2017,65(14): 2990-2998.). 2mL of GEO and 18mL of NaOH were added to the flask in succession to obtain a GEO mixture, which was heated at 120 ℃ for 15min and then taken out and cooled to room temperature. Then, protein solutions of Zein and NaCas are mixed in different volume ratios to obtain mixed protein solutions with Zein/NaCas mass ratios of 0:1, 1:2 and 2: 1. After magnetic stirring at 600rpm for 30min, 2mL of deprotonated GEO was added to 18mL of mixed protein solution and mixed to obtain a mixture with a total GEO concentration of 1% (v/v). The mixture was then acidified with 3M citric acid monohydrate while magnetic stirring at 600rpm until the pH dropped to 7.0. GEON can form spontaneously during acidification.

The appearance of 1% (v/v) GEO nanoemulsion prepared by Zein and NaCas under different mass ratio conditions is shown in FIG. 1. As can be seen from the figure, the nanoemulsions are uniformly dispersed in water at the Zein/NaCas mass ratios of 0:1, 1:1 and 1:2, and show a good yellowish milky appearance; at a Zein/NaCas mass ratio of 2:1, a more pronounced precipitation occurred due to the lack of sufficient NaCas to stabilize Zein. The storage stability of the nanoemulsion can be assessed by measuring particle size and zeta potential of GEON (pH 7.0) using a Dynamic Light Scattering (DLS) NICOMP Z3000 nanometer particle size potentiostat. The results are shown in Table 1. It can be seen that the average particle size range for all the freshly prepared samples was 144-192nm, where the nanoemulsion with a Zein/NaCas mass ratio of 1:1 showed the smallest particle size (144nm) and the nanoemulsion with a mass ratio of 0:1 measured the largest particle size of 196.46 nm. The zeta potential value represents the stability of the emulsion, when the potential value is more than or equal to 30mV, the emulsion can be stabilized by electrostatic repulsion among colloid particles, and the larger the electrostatic repulsion, the smaller the possibility of particle aggregation, and the higher the stability. Table 1 shows the particle size and zeta potential of 1% (v/v) GEO nanoemulsions prepared with Zein and NaCas at different mass ratios for 4 weeks stored at 4 ℃, the zeta potential of freshly prepared samples ranged between-27.07 and-33.87 mV, with emulsions having a Zein/NaCas mass ratio of 1:1 bearing the largest negative charge (-33.87 mV). Under the condition of the same total protein content, the sample with the Zein/NaCas mass ratio of 1:2 has larger negative charge than the sample with the 2:1, which shows that the addition of NaCas can enable Zein colloidal particles to obtain larger electrostatic repulsion force, thereby showing better stability. The results of the storage experiments showed that no significant change in average particle size and zeta potential was observed for the samples with mass ratios of 0:1, 1:1 and 2:1 during storage (p >0.05), whereas the 1:2 samples showed significant changes in particle size and potential at 3 weeks of storage (p <0.05), indicating that the samples with mass ratio of 1:2 were less storage stable.

In conclusion, when the Zein/NaCas mass ratio is 1:1, the emulsion presents the smallest particle size and the largest negative charge, which indicates that the nano-emulsion prepared under the conditions has the strongest electrostatic repulsion and the best stability in the water phase.

TABLE 1

Note that the results are mean ± standard deviation; the different capitalization of the different treatment groups indicated significant differences (p < 0.05); different lower case letters at different times indicate significant differences (p < 0.05).

Based on the Zein/NaCas as the optimal mass ratio, different volumes of deprotonated GEO were added to 18mL of mixed protein solution to obtain various concentrations of GEON ranging from 0.5% to 2% (v/v). Part of the emulsion is freeze-dried, pulverized, and sealed at-20 deg.C for storage.

The embedding rate (EE value) is an important index for evaluating the quality of a delivery system. The encapsulation efficiency of the GEO was determined by subtracting the free oil content from the total oil content.

The embedding rate of 1% (v/v) GEON prepared at Zein/NaCas mass ratios of 0:1, 1:2 and 2:1, respectively, under neutral conditions is shown in FIG. 2. At a Zein/NaCas mass ratio of 1:2, the entrapment rate of the sample was higher than that of the sample at a mass ratio of 2:1, which is consistent with the observation of particle size and zeta potential. When the Zein/NaCas mass ratio is 0:1, the entrapment rate of the sample is lowest (76.31%) because too many NaCas particles aggregate and do not entrap the GEO. At a mass ratio of 1:1, the EE of the sample reached the highest value of 86.48%, which is higher than that of Zein stabilized emulsions prepared with aqueous ethanol (EE 16%) or high speed homogenization (EE 65-75%). Therefore, the GEON stability with the Zein/NaCas mass ratio of 1:1 is best, the EE value is highest,

the results of the embedding rate, the particle size and the zeta potential of the nano-emulsion prepared from ginger essential oil with different concentrations under the condition that the Zein/NaCas mass ratio is 1:1 are shown in FIG. 3. As the GEO concentration increased, EE decreased from 90.95% to 81.48%, the average particle size increased from 131.31nm to 195.15nm, and the zeta potential increased from-37.28 mV to-27.25 mV. This is due to the lack of protein, which cannot completely cover the surface of the oil molecules when the GEO concentration in the emulsion is too high, so that collisions between oil molecules and aggregation into oil droplets, which escape into the external phase through the Zein/NaCas matrix structure, resulting in a decrease in stability of the nanoemulsion. In conclusion, when the Zein/NaCas mass ratio is 1:1 and the GEO concentration is 0.5%, the prepared nano emulsion has the best stability and the highest EE value. Therefore, in practical application, the concentration of GEO in the emulsion is reduced as much as possible under the condition of meeting the required bacteriostatic requirement.

Zein: s of mixed protein solution with NaCas mass ratio of 0:1, 1:2 and 2:1₀The value is obtained. S₀Can reflect the distribution of hydrophobic amino acid on the surface of protein, is an evaluation index of protein adsorption to the interface oil side, and has great influence on the emulsifying capacity of protein. To better understand the potential synergy between Zein and NaCas, S for Zein/NaCas mixtures of different mass ratios was measured₀The value is obtained. As shown in FIG. 4, the sample containing only NaCas had the lowest S₀Value (1656.23) followingThe S content of the sample is increased along with the increase of the Zein content in the Zein/NaCas mixture₀The value also increased (5824.57 for Zein/NaCas ═ 1:2 and 7599.93 for 2: 1). This indicates that Zein helps to increase the hydrophobicity of the sample. The reason is that Zein can increase the surface area of Zein/NaCas nano-particles, so that more hydrophobic residues are exposed, thereby promoting the interaction of protein arrangement with better hydrophilic-lipophilic balance at a water-oil interface and enhancing the emulsifying property of the emulsion. However, when more hydrophobic surfaces are exposed, the nanoparticles aggregate, resulting in a decrease in emulsion stability, as the Zein/NaCas mass ratio of 2:1 samples have the highest S₀The values, however, are inferior to the 1:1 mass ratio samples in both the embedding rate (FIG. 2) and the stability (Table 1).

The morphology and distribution of the nanoparticles in the emulsion matrix was observed using a scanning electron microscope. As shown in fig. 5, the morphology of 1% GEO (v/v) nanoemulsion particles of different Zein/NaCas mass ratios are spherical and smooth surface, and no aggregation indicates that these emulsions are well dispersed in the aqueous phase, which also confirms that GEO, Zein and NaCas successfully self-emulsify during late acid-base neutralization. The images show that the size of the emulsion particles with Zein/NaCas mass ratios of 1:1 and 1:2 is small, while the size of the emulsion particles with mass ratios of 0:1 and 2:1 is larger, but some small size particles can be seen, which is consistent with the above analysis of particle size, zeta potential and EE.

The influence of GEO on the conformation of Zein and NaCas is researched by utilizing a fluorescence spectrum, an ultraviolet spectrum, an FT-IR spectrum and an XRD spectrum, and reference data is provided for the extended application range of Zein and NaCas and the reasonable use of GEO. FIG. 6 shows fluorescence spectra of Zein, NaCas and Zein/NaCas (1:1) complex protein solutions containing different concentrations of GEO at an excitation wavelength of 280nm (the trend of the fluorescence spectra is shown by the arrow direction as the concentration increases). Under the excitation wavelength of 280nm, GEO has fluorescence quenching effect on both Zein and NaCas and a Zein/NaCas (1:1) composite protein, which indicates that GEO can be combined with the two proteins at the same time and has obvious influence on the conformation.

FIG. 7 shows the UV absorption spectra of solutions of Zein, NaCas and Zein/NaCas (1:1) containing GEO at different concentrations (the trend of the fluorescence spectrum changes as the concentration increases, as indicated by the arrows). The ginger essential oil is proved to have pi-pi accumulation with Zein and NaCas, and then to be adsorbed on the surface of protein.

FT-IR spectra of Zein/NaCas (1:1) and GEON after lyophilization are shown in FIG. 8. After the compound protein and the ginger essential oil are prepared into the nano-emulsion, the compound protein is 1633.9cm^-1And 1446.3cm^-1The peak disappears, which shows that the nanoemulsion absorbs the trans-double bond, and indicates that the ginger essential oil and the compound protein have bonding effect.

Figure 9 shows XRD spectra of Zein, NaCas, Zein/NaCas (1:1) and GEON lyophilized powders (NaCas, Zein/NaCas ═ 1:1, GEON lyophilized powders in the order of the arrow). Upon addition of GEO to make a nanoemulsion, the characteristic peak absorption intensity associated with the protein crystalline structure becomes weaker and the relative crystallinity of the material decreases, probably because the presence of certain components in the composition hinders the progress of the crystallization process, thereby promoting the formation of an amorphous structure. It is well known that amorphous solids are generally more soluble and more hygroscopic. Therefore, the prepared GEON can be better applied to the field of food preservation and fresh-keeping.

Solid culture media with different concentrations of GEON (0.625-5 mg/mL) are prepared by a double dilution method, and the minimum inhibitory concentrations of the nanoemulsion to the P.aeruginosa strain and the S.aureus strain are determined to be 5mg/mL and 2.5mg/mL respectively by observing the growth conditions of the P.aeruginosa strain and the S.aureus strain on the surface of the solid culture media. It can be seen that GEON has a better inhibitory effect on S.aureus strains.

The appearance of p.aeruginosa and s.aureus was observed by SEM in sequence with or without geo treatment. As shown in fig. 10, untreated p. aeruginosa and s. aureus bacterial cells were intact and independent, the cell membranes were smooth, and the cell bodies were full. After GEON treatment with the concentration of 5mg/mL, the P.aeruginosa thalli are seriously damaged in surface, shriveled in shape and stacked mutually; after GEON treatment with a concentration of 2.5mg/mL, S.aureus lost smooth spherical shape, the cells were sunken, and the shape was shriveled and wrinkled. The above results show that GEON can change the external structure and morphology of the indicator strain cell, and presumably the decay-causing capability of GEON on the dominant putrefying bacteria commonly used in meat products is to destroy the cytoplasmic membrane of the bacteria, and the destruction effect on gram-positive bacteria is higher than that on gram-negative bacteria, but further basis is provided for guesswork.

The LSCM is used for observing the staining result of bacteria, so that the change condition of the permeability of the bacterial cell membrane after different bacteriostatic treatments can be visually seen. According to FIG. 11, 2 indicator cells showed green fluorescence in the control group, and only a few cells showed red fluorescence due to decay, indicating that the cell membrane of the bacteria in the control group was not destroyed and the structure was substantially intact; in the treatment group subjected to GEON treatment, most of 2 indicator bacterial cells show red fluorescence, only part of the cells show green, which indicates that the GEON treatment causes cell membranes of most of bacteria to be damaged and permeability to be enhanced, part of the bacteria even die, and PI enters the cells through the damaged cell membranes to be combined with nucleic acid, so that the cells show red fluorescence.

As shown in FIG. 12, the change of extracellular ATP concentration of 2 indicator bacteria in the control group is relatively small, and the concentration is always maintained within 25nmol/mL in the experimental process; after GEON treatment, the extracellular ATP concentrations of the 2 strains all increased to a large extent in a short time and gradually became stable after 60 min. Wherein, the extracellular ATP content of the P.aeruginosa is increased to 73.37nmol/mL after the nanoemulsion is treated for 90min, but is reduced at 120min, which may be caused by ATP degradation; for the s. aureus strain, the extracellular ATP concentration increased to 99.19nmol/mL after 120min nanoemulsion treatment. The above results show that the cell membrane permeability of s.aures and p.aeruginosa after geo treatment is increased, non-selective pores are formed, thereby inducing massive ATP leakage in cells, and the damage degree of s.aures is significantly higher than that of p.aeruginosa.

Experiments show that when the Zein/NaCas mass ratio is 1:1, the particle size of the emulsion is 144nm at the minimum, the EE is 86.48% at the maximum, and the storage stability is the strongest.

On the basis, fluorescence, ultraviolet and other spectrum experiments are carried out, and analysis results show that multiple interactions are generated between GEO and Zein and NaCas, and GEO not only changes the secondary structures of two proteins, but also has a bonding effect with the proteins, so that the formation of an amorphous structure of the proteins is promoted.

In an antibacterial experiment, researches show that the minimum inhibitory concentrations of GEON to P.aeruginosa and S.aureus are 5mg/mL and 2.5mg/mL respectively; SEM observation results show that after GEON treatment, the cell surfaces of the two indicator bacteria are seriously damaged and the shapes of the indicator bacteria are shriveled; the results of LSCM observation and extracellular ATP concentration determination show that, compared with gram-negative bacteria, GEO has good inhibition effect on gram-positive bacteria, and the action mechanism is mainly to destroy the cell membrane of the bacteria, increase the permeability of the bacteria, overflow intracellular substances, cause the bacteria to fail to repair the damage of the GEO to the cell membrane in time, and cause cell death.

Example 2 preparation of ginger essential oil nanoemulsion hydrogel.

Preparing nano-emulsion hydrogel.

(1) Preparation of MFC: 40g of NaOH was weighed out and dissolved in 340mL of pure water to prepare a NaOH solution. To this NaOH solution was added 20g of microcrystalline cellulose (MCC), placed in a 60 ℃ water bath, magnetically stirred at 300rpm for 2 hours, and cooled to room temperature. The sample was centrifuged at 8000rpm for 5min in a centrifuge, the precipitate was retained, and it was suction filtered repeatedly with pure water (filter pore size 10 μm) until the sample appeared neutral. Then, the sample is dispersed uniformly by 1000mL of pure water, and is homogenized under high pressure under the condition of 15000PSI, thus obtaining milky microfibrillated cellulose (MFC) suspension, and the milky microfibrillated cellulose (MFC) suspension is put into a refrigerator at 4 ℃ for standby.

(2) Preparation of DAMFC: 200g of MFC suspension are weighed into a conical flask according to NaIO₄Adding NaIO into a conical flask in a mass ratio of MFC (micro-fuel cell) of 1:2₄And then placing the mixture in a water bath kettle at 25 ℃ to be stirred for 48 hours in a dark place, so that the MFC is fully oxidized. Then the sample is put into a dialysis bag with the molecular weight cutoff of 8000-₄And after dialysis, carrying out freeze drying to obtain DAMFC freeze-dried powder, and putting the powder into a dryer for later use.

(3) Sample pretreatment: respectively weighing 5 groups of 1g CMCS, placing the CMCS into a 50mL beaker, adding 20g GEON (the Zein/NaCas mass ratio is 1:1 and the GEO concentration is 1%) respectively, and magnetically stirring the mixture under the condition of water bath at 45 ℃ until the mixture is uniformly mixed. Meanwhile, DAMFC freeze-dried powder with different mass is respectively weighed and put into a 50mL beaker filled with 10g GEON according to the mass ratio of 5/95, 10/90, 15/85, 20/80 and 25/75 of DAMFC/CMCS, and the serial numbers are a to e. Under the same water bath condition, stirring by magnetic force until the mixture is uniform.

(4) Preparing GEONH: under the condition of constant temperature water bath at 45 ℃, substances in the a-E beakers are correspondingly added into the A-E beakers, and the DAMFC and the CMCS react quickly, so that magnetic stirring is carried out immediately after the addition, and the DAMFC and the CMCS are uniformly mixed. Removing bubbles on the surface after stirring, then quickly pouring into a 24-hole plate, and standing at room temperature for 2h to obtain the nano GEONH.

And (II) sample characterization.

In order to obtain optimal production conditions for GEONH and to determine whether MFC was successfully oxidized to DAMFC, and to further observe the interaction between DAMFC and CMCS, a series of characterization assays were performed.

(1) Gel time.

The pure CMCS nanoemulsion samples exhibited a flowable viscous state until no DAMFC was added. After the DAMFC is added, the Schiff base reaction between the DAMFC and the CMCS is rapidly carried out, so that the mixed system of the DAMFC and the CMCS gradually loses fluidity and a nano-emulsion hydrogel is formed. The time from the moment the DAMFC was in contact with the CMCS until the solution formed a spherical irregular gel solid that was completely separated from the bottom of the beaker was recorded as the gel time.

FIG. 13 shows the gel times required to prepare GEONH from different DAMFC/CMCS mass ratios, and it can be seen that the amount of DAMFC has a significant effect on how fast the mixed system forms a hydrogel. When DAMFC/CMCS is 5/95, the mixed system formed a hydrogel for 9.4 min; the required gel time is also rapidly reduced with increasing specific gravity of the DAMFC, and when the DAMFC/CMCS is 25/75, the hydrogel can be formed in only about 1 min. This is because the amount of DAMFC directly determines the number of aldehyde groups in the mixed system, which in turn affects the number of Schiff base reactions that are critical for the formation of GEONH.

(2) Gel strength

As shown in fig. 14, the gel strength of the resulting hydrogel was gradually increased as the amount of the dacfc in the mixed system was increased. The gel strength was 3.31N minimum when dacfc/CMCS ═ 5/95; the gel strength was 12.17N when DAMFC/CMCS was 25/75, which is a 3-fold increase over when DAMFC/CMCS was 5/95. It can be seen that the gel strength results correspond to the gel time, i.e. within a certain range of ratios, the shorter the time required to form the gel, the stronger the gel strength formed. The reason is that the Schiff base reaction of the DAMFC with the CMCS enables the mixture to form an integral body, and the DAMFC simultaneously acts as a reinforcing agent and a chemical crosslinking agent in the integral body, so that the strength and the chemical reaction rate of the whole hydrogel system are improved when the content of the DAMFC is increased.

(3) Swelling Performance analysis

The prepared GEONH samples were freeze-dried, and the freeze-dried samples were weighed and recorded as initial masses. The samples were soaked in PBS buffer (pH 7.4,) and taken out at intervals, after absorbing residual moisture on the surface with filter paper, weighed on an analytical balance and recorded. Buffer solution is replaced every 12h, and each group of samples needs to be provided with 5 parallels so as to reduce experimental errors. The swelling degree calculation formula of the composite nano-emulsion hydrogel is as follows:

wherein SR represents the degree of swelling (%) of the sample, and W_tMass (g) at the time of swelling of the sample to time t, W₀The initial mass (g) of the sample after freeze-drying was weighed.

When the hydrogel is used as a preservative film, the hydrogel can absorb moisture in the air and on the surface of food products through water absorption swelling performance. The proper swelling performance can keep the surface of the product moist and prevent the loss of nutrient substances caused by water loss; meanwhile, the growth of microorganisms caused by excessive accumulation of water on the surface of the product can be avoided. As can be seen from FIG. 15, the swelling ratio of GEONH in PBS buffer (pH 7.0) increases rapidly between 0 and 10 min. Of these, the hydrogel with a DAMFC/CMCS mass ratio of 5/95 had a swelling ratio of up to 1087% at 10min (FIG. B), while the hydrogel with a mass ratio of 25/75 had a swelling ratio of only 526% (FIG. B). With time, the rate of water absorption by GEONH slowed, and the samples of the remaining components reached a state of swelling equilibrium after 48h, with the exception of the sample with a DAMFC/CMCS mass ratio of 5/95, and maintained good gel morphology throughout. In addition, it can be seen that the swelling ratio of GEONH is inversely related to the content of DAMFC in the mixed DAMFC/CMCS system. This is because the DAMFC acts as a cross-linking agent and a reinforcing agent in the whole gel system, and when the content of the DAMFC is increased, the number of reactions with the Schiff base of the CMCS is increased, and the formed hydrogel has a denser and stable three-dimensional network structure; in addition, the DAMFC itself occupies a part of the space structure in the hydrogel system, so the increase of the content thereof can reduce the moving space of free water molecules, and the swelling performance of the hydrogel is reduced. In the course of the progress of this experiment, it was found that GEONH with a mass ratio of 5/95 dissolved after 8h of water absorption and completely dissolved in PBS buffer after 12h, and the swelling ratio was 1767% at 8h (FIG. 15A). The reason for this is that the content of DAMFC in the mixed system of DAMFC/CMCS is too low, which results in insufficient crosslinking degree, and the three-dimensional network structure is too loose, so that when soaked in PBS buffer for a long time, the hydrogel system absorbs too many water molecules, which results in the whole system structure being crushed and further being dissolved.

(4) Composite hydrogel morphology analysis

FIG. 16 is a Scanning Electron Microscope (SEM) image of MFC and DAMFC. As shown in the figure (A), MFCs obtained after NaOH alkali treatment and micro-jet high-pressure homogenization are mutually wound and present a three-dimensional network shape, and the structure is relatively uniform. The morphology of DAMFC is shown in FIG. B and is compared with MFC by NaIO₄The oxidized DAMFC not only retains complete microfibers and three-dimensional network-like structures, but also has a more compact spatial structure. The main reasons are that MFC is insoluble in water and NaIO₄The amount of the oxidant used is low, so that the oxidation reaction can only stay on the surface of the MFC, and only part of the hydroxyl groups are converted into aldehyde groups.

FIG. 17 is an SEM image of GEONH prepared from different DAMFC/CMCS mass ratios after lyophilization. As can be seen, all GEONHs have a continuous three-dimensional network-like structure, and the pore size of the space network changes with the content of DAMFC in the mixed system. Table 2 reports the average pore size of the different GEONHs, which is larger at 80.2 μm when the DAMFC/CMCS is 5/95. As the amount of DAMFC increased, the average pore size gradually decreased and reached a minimum (44.7 μm) at a DAMFC/CMCS of 15/85, then gradually increased and reached a maximum (105.6 μm) at a DAMFC/CMCS of 25/75.

The reason is that the number of aldehyde groups is determined by the content of DAMFC, the content of aldehyde groups is small, the number of crosslinking points is small, and the average pore diameter is large; however, if the aldehyde group content is too high, the number of crosslinking points increases, and in this case, the DAMFC is likely to be unevenly dispersed in the CMCS matrix, resulting in an increase in pore size.

TABLE 2 mean pore diameters of different GEONH

(5) Interaction analysis

FT-IR spectra of MFC and DAMFC samples prepared with pure water are shown in FIG. 18. As can be seen from the figure, the product was obtained from NaIO in comparison with MFC₄The DAMFC obtained after oxidation was 1657cm^-1Characteristic absorption peaks of aldehydes appear. The result shows that the C2-C3 bond on the MFC glucose six-membered ring is destroyed, and part of the hydroxyl on the surface is indeed destroyed by NaIO₄Oxidized to form aldehyde groups. The oxidation reaction formula is shown in fig. 19.

FIG. 20 shows FT-IR spectra of DAMFC, CMCS, and DAMFC/CMCS (10/90) samples formulated using GEON after lyophilization. CMCS was found at 1579cm^-1And 1395cm^-1There is a characteristic peak respectively caused by COO-asymmetric stretching vibration and COO-symmetric stretching vibration. When the DAMFC is added to the CMCS, the aldehyde groups on the molecular chain of the DAMFC begin to Schiff base reactions with the amino groups on the molecular chain of the CMCS and form C ═ N bonds, as shown in FIG. 21. Thus, in the FT-IR spectrum of DAMFC/CMCS (10/90), 1572cm^-1The characteristic peak is caused by C ═ N stretching vibration in Schiff base reaction, and the characteristic peak group caused by COO-asymmetric stretching vibrationThis is the overlap. Compared with DAMFC, the length of the DAMFC/CMCS (10/90) was 1657cm^-1The characteristic peak of aldehyde groups disappears, which indicates that aldehyde groups on the molecular chain of DAMFC actually participate in the chemical crosslinking reaction and are consumed.

Fig. 22 is an XRD spectrum of MFC and dacmfc after freeze-drying, and it can be seen that MFC has a typical crystallization peak in the range of 10 to 60 ° 2 θ. The crystallization peaks at 14.9 °, 16.3 °, 21.9 ° and 34.1 ° each correspond to a different crystal plane of cellulose. Via NaIO₄The diffraction peak pattern of the obtained DAMFC after oxidation is similar to that of MFC, but the absorption intensities of the characteristic peaks of the related crystal structures are weakened, which indicates that NaIO₄In the process of oxidizing MFC, although the partial crystal structure of cellulose is damaged to a certain extent, the crystallinity of DAMFC is reduced, but the overall crystal structure is not changed. Fig. 23 is an XRD spectrum of samples of the DAMFC, CMCS and DAMFC/CMCS (10/90) solutions formulated with GEON after freeze-drying, and it can be seen that both the DAMFC and CMCS show diffraction peaks at 20.3 ° 2 θ, indicating that both samples contain the same crystal planes. While the DAMFC/CMCS (10/90) showed a broader diffraction peak at 21.5 ° 2 θ, indicating that the Schiff base reaction between DAMFC and CMCS altered the crystal structure of both.

GEONH is intended to be used as an antiseptic preservative for low-temperature meat products, and needs to have strong antibacterial ability, inhibit the growth of microorganisms and prolong the preservation period. Early experimental results show that GEON has a strong inhibition effect on both P.aeruginosa and S.aures, and CMCS itself also has a strong antibacterial property, so that the pure water hydrogel (PHW) also has a certain antibacterial effect. Therefore, the antibacterial rate of both PWH and GEONH was measured to investigate the antibacterial effect of GEONH prepared by GEON together with CMCS and DAMFC.

Table 3 shows the antibacterial ratio of the composite hydrogel and the composite nanoemulsion hydrogel to e.coli, p.aeruginosa and s.aures at a mass ratio of dacfc/CMCS of 10/90. As can be seen from the table, the antibacterial rate of PWH to three kinds of bacteria all reaches more than 90%, and the inhibition effect to e.coli is the best, which indicates that chitosan really gives a better antibacterial effect to PHW. After pure water is replaced by GEON, the antibacterial rate of the prepared GEONH to the three bacteria is improved to about 99 percent, which is probably because the GEON and the CMCS generate synergistic action after being mixed. Wherein the antibacterial rate of S.aureus is improved to 9.09% at most. This is probably because GEON had the best inhibitory effect on s.aureus, which is consistent with the minimum inhibitory concentration results for ginger essential oil nanoemulsion.

Therefore, the composite nano-emulsion hydrogel has obvious effect of inhibiting the growth of E.coli, P.aeruginosa and S.aureus, is expected to be applied to the corrosion prevention and the preservation of low-temperature meat products such as boiled salted ducks and the like, and can reduce the bacterial pollution on the surface of the products.

TABLE 3 antibacterial Rate of composite hydrogel and composite nanoemulsion hydrogel at a DAMFC/CMCS of 10/90

In conclusion, due to the existence of the CMCS, the PWH has a better bacteriostatic effect and has the best inhibitory effect on E.coli. After the hydrogel is combined with GEON, the antibacterial rate of the prepared hydrogel to three bacteria is improved to about 99%, wherein the antibacterial rate to S.aureus is improved most, which indicates that a synergistic effect is generated between GEON and CMCS. Therefore, GEONH can obviously inhibit the growth of common putrefying bacteria in meat products such as E.coli, S.aureus and the like, is expected to be applied to the field of corrosion prevention and preservation of low-temperature meat products, and prolongs the shelf life.

The invention provides a preparation idea and a preparation method of ginger essential oil nanoemulsion hydrogel, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and these improvements and decorations should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A preparation method of ginger essential oil nanoemulsion hydrogel is characterized by comprising the following steps:

(1) respectively dissolving zein and sodium caseinate in pure water, adjusting the pH to 11-11.5, stirring to obtain a 2-3% w/v zein solution and a 2-3% w/v sodium caseinate solution, and then mixing the zein and the sodium caseinate to obtain a mixed protein solution;

(2) deprotonating the ginger essential oil, adding the ginger essential oil into the mixed protein solution obtained in the step (1), and then acidifying and stirring until the pH is reduced to 7.0 to obtain the ginger essential oil nanoemulsion;

2. The method according to claim 1, wherein in the step (1), the mass ratio of the zein solution to the sodium caseinate solution in the mixed protein solution is not more than 2: 1.

3. The method according to claim 1, wherein in the step (2), the step of deprotonating the ginger essential oil is: dissolving ginger essential oil in 3M sodium hydroxide, heating at 100-120 deg.C for 10-15min, taking out, and cooling to room temperature to obtain deprotonated ginger essential oil with concentration of 10% v/v.

4. The method according to claim 1, wherein in the step (2), the acidification treatment is performed using citric acid monohydrate.

5. The preparation method of claim 1, wherein the concentration of the ginger essential oil in the ginger essential oil nanoemulsion is 0.5% -2% v/v.

6. The production method according to claim 1, characterized in that the dialdehyde microfibrillated cellulose: carboxymethyl chitosan: the ginger essential oil nanoemulsion comprises the following components in percentage by mass (5-25): (95-75): (1000 to 2000).

7. The method according to claim 1, wherein the mass ratio of the sodium periodate to the microfibrillated cellulose is 1:1 to 1: 3.

8. The application of the ginger essential oil nanoemulsion hydrogel prepared by the preparation method of any one of claims 1-7 as an antiseptic preservative for meat products.