阅读说明：本技术 一种埃洛石纳米管负载香芹酚缓释复合物及其制备方法 (Halloysite nanotube-loaded carvacrol sustained-release compound and preparation method thereof ) 是由 卢立新 苏英杰 潘嘹 蔡莹 林自东 于 2021-02-08 设计创作，主要内容包括：本发明提供了一种埃洛石纳米管负载香芹酚缓释复合物及其制备方法,旨在提高埃洛石纳米管中香芹酚的负载量以及减缓香芹酚的释放速率。一种埃洛石纳米管负载香芹酚缓释复合物的制备方法,该制备方法包括顺序进行的以下步骤,(1)埃洛石纳米管改性、(2)香芹酚负载,其特征在于：步骤(1)包括将埃洛石纳米管与H～+浓度为4M的强酸混合后刻蚀改性。本发明刻蚀改性后的埃洛石纳米管,其比表面积由29.506m～2/g增加到95.577m～2/g,不破坏埃洛石纳米管的管状结构以及管壁保持完整,并且香芹酚的负载量由6.7%提高到24.2%。(The invention provides a halloysite nanotube loaded carvacrol sustained-release compound and a preparation method thereof, and aims to improve the load capacity of carvacrol in halloysite nanotubes and slow the release rate of carvacrol. Halloysite nanotube loadThe preparation method of the carvacrol sustained-release compound comprises the following steps of (1) halloysite nanotube modification and (2) carvacrol loading, and is characterized in that: the step (1) comprises the steps of mixing halloysite nanotubes with H + And etching and modifying after mixing strong acid with the concentration of 4M. The specific surface area of the etched and modified halloysite nanotube is 29.506m 2 The/g is increased to 95.577m 2 The/g does not destroy the tubular structure of the halloysite nanotube and the tube wall is kept intact, and the carvacrol loading is improved from 6.7 percent to 24.2 percent.)

1. A preparation method of a halloysite nanotube-loaded carvacrol sustained-release compound comprises the following steps of (1) halloysite nanotube modification and (2) carvacrol loading in sequence, and is characterized in that: the step (1) comprises the steps of mixing halloysite nanotubes with H⁺And etching and modifying after mixing strong acid with the concentration of 4M.

2. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 1, wherein the preparation method comprises the following steps: in the step (1), the strong acid is hydrochloric acid, and the halloysite nanotubes and 4M hydrochloric acid are mixed according to the proportion of 0.0625g/mL, reacted, centrifuged, washed, dried and ground to obtain white and powdery modified halloysite nanotubes.

3. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 2, wherein the preparation method comprises the following steps: in the step (1), the reaction is carried out under the conditions of heating and magnetic stirring, the temperature is 80 ℃, the rotating speed is 600rpm, and the time is 8 hours.

4. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 2, wherein the preparation method comprises the following steps: in the step (2), the modified halloysite nanotube and carvacrol are mixed in a ratio of 0.1g/mL, and the halloysite nanotube-carvacrol compound is obtained after ultrasonic treatment, vacuum loading, centrifugation and drying.

5. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 4, wherein the preparation method comprises the following steps: in the step (2), the ultrasound was performed under ice bath conditions for 30 minutes at a power of 200W with the pulse turned on for 2 seconds and off for 3 seconds.

6. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 4, wherein the preparation method comprises the following steps: in the step (2), the vacuum loading step is to reduce the pressure to 0.1MPa for 30 minutes and then return to the normal pressure for 10 minutes.

7. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 1, wherein the preparation method comprises the following steps: the preparation method also comprises the following steps of (3) polyelectrolyte packaging, mixing the halloysite nanotube-carvacrol compound with the polyelectrolyte solution, stirring, centrifuging, washing, finishing layer packaging, drying the obtained solid, and grinding to obtain the finished product.

8. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 7, wherein the preparation method comprises the following steps: in the step (3), the polyelectrolyte comprises polyethyleneimine and sodium polystyrene sulfonate, and the weight ratio of the halloysite nanotube-carvacrol compound to the polyethyleneimine solution is 2:8, mixing, stirring, centrifuging, washing to finish the first layer of packaging, adding sodium polystyrene sulfonate with the same volume as the polyethyleneimine, stirring, centrifuging, washing to finish the second layer of packaging.

9. The preparation method of the halloysite nanotube-loaded carvacrol sustained-release compound according to claim 1, wherein the preparation method comprises the following steps: the concentration of the polyethyleneimine solution and the concentration of the sodium polystyrene sulfonate solution are both 2 mg/ml.

10. A halloysite nanotube-loaded carvacrol sustained-release compound prepared by the method of any one of claims 1-9.

Technical Field

The invention belongs to the field of packaging slow-release materials, and particularly relates to a halloysite nanotube-loaded carvacrol slow-release compound and a preparation method thereof.

Background

The shortening of the shelf life of food products by oxidation and microbial spoilage by pathogenic microorganisms has become a major concern worldwide, and food packaging materials intended to control food oxidation and microbial growth provide an effective solution to extending shelf life. Most of the currently used active packaging materials work after direct contact with food, and limited efficacy and safety for solid food are major factors limiting their widespread use.

Essential Oils (EOs) are extremely attractive compounds for incorporation into food packaging materials because they are natural and highly effective antimicrobial and antioxidant agents that can be released as vapors into the headspace of the package, sterilizing both the food surface and the headspace environment; and has been approved by the U.S. Food and Drug Administration (FDA) for food use as a GRAS (generally recognized as safe). Much research has been devoted to incorporating essential oils and sensitive actives in essential oils into packaging materials while minimizing their loss during processing and maintaining their antimicrobial and antioxidant functions.

Halloysite Nanotubes (HNT) are natural, inexpensive nanoparticles with good dispersion in polymeric materials. The loading of essential oils or their sensitive active substances on halloysite nanotubes and their subsequent incorporation into packaging materials is one of the possible directions for the development of active packaging films. How to improve the load capacity of carvacrol in the halloysite nanotube and slow down the release rate of carvacrol is one of the technical problems to be solved urgently in the industry.

Disclosure of Invention

The invention provides a halloysite nanotube loaded carvacrol sustained-release compound and a preparation method thereof, and aims to improve the load capacity of carvacrol in halloysite nanotubes and slow the release rate of carvacrol.

The technical scheme is that the preparation method of the halloysite nanotube-loaded Carvacrol sustained-release compound comprises the following steps of (1) Halloysite Nanotube (HNT) modification, (2) Carvacrol (Carvacrol, crv) loading and is characterized in that: the step (1) comprises the steps of mixing halloysite nanotubes with H⁺And etching and modifying after mixing strong acid with the concentration of 4M.

Further, in the step (1), the strong acid is hydrochloric acid, and the halloysite nanotubes and 4M hydrochloric acid are mixed according to the proportion of 0.0625g/mL, reacted, centrifuged, washed, dried and ground to obtain white and powdery modified halloysite nanotubes.

Further, in the step (1), the reaction is carried out under the conditions of heating and magnetic stirring, the temperature is 80 ℃, the rotating speed is 600rpm, and the time is 8 hours.

Further, in the step (2), the modified halloysite nanotubes and carvacrol are mixed in a ratio of 0.1g/mL, and the halloysite nanotube-carvacrol compound is obtained after ultrasonic treatment, vacuum loading, centrifugation and drying.

Further, in the step (2), the sonication was performed under ice bath conditions for 30 minutes at a power of 200W, with the pulse turned on for 2 seconds and the pulse turned off for 3 seconds.

Further, in the step (2), the vacuum loading step is to reduce the pressure to 0.1MPa for 30 minutes and then return to normal pressure for 10 minutes.

Further, the preparation method comprises the following steps of (3) performing polyelectrolyte encapsulation, mixing the halloysite nanotube-carvacrol compound with the polyelectrolyte solution, stirring, centrifuging, washing, completing layer encapsulation, drying the obtained solid, and grinding to obtain a finished product.

Furthermore, in the step (3), the polyelectrolyte comprises Polyethyleneimine (PEI) and sodium polystyrene sulfonate (PSS), and the weight ratio of the halloysite nanotube-carvacrol compound to the polyethyleneimine solution is 2:8, mixing, stirring, centrifuging, washing to finish the first layer of packaging, adding sodium polystyrene sulfonate with the same volume as the polyethyleneimine, stirring, centrifuging, washing to finish the second layer of packaging.

Furthermore, the concentration of the polyethyleneimine solution and the concentration of the sodium polystyrene sulfonate solution are both 2 mg/ml.

The invention also provides a halloysite nanotube-loaded carvacrol sustained-release compound which is prepared by the method.

The preparation method and the halloysite nanotube-loaded carvacrol sustained-release compound prepared by the method have the following beneficial effects:

(1) by means of H⁺The strong acid solution with the concentration of 4M is used for carrying out selective etching modification on the halloysite nanotube, and the specific surface area of the halloysite nanotube is 29.506M²The/g is increased to 95.577m²The,/g, the tubular structure of the halloysite is not damaged, the integrity of the tube wall is ensured, and compared with the unmodified halloysite nanotube, the load capacity of carvacrol is improved to 24.2% from 6.7%;

(2) carvacrol loading adopts a vacuum loading method, and air in the halloysite nanotube is removed through decompression, so that high-volume carvacrol loading is realized;

(3) two polyelectrolytes are selected to carry out two-layer autonomous encapsulation on the HNT-crv compound to form a controlled release system, so that the release rate of carvacrol in the halloysite nanotube is remarkably slowed down, and the halloysite nanotube-loaded carvacrol slow release compound prepared by the method has the potential of being used as a controlled release carrier to be applied to an antibacterial and antioxidant gas-phase controlled release packaging film.

Drawings

FIG. 1a is a transmission electron micrograph of an unetched modified HNT, scale 200 nm.

FIG. 1b is a transmission electron micrograph of HNT-4M, scale 200 nm.

FIG. 1c is a transmission electron micrograph of an unetched modified HNT (tube orifice) at 50nm scale.

FIG. 1d is a transmission electron micrograph of HNT-4M (orifice) at 100 nm.

FIG. 2 is a nitrogen adsorption-desorption isotherm plot of halloysite nanotubes before and after acid etching.

FIG. 3 is a thermogravimetric analysis of halloysite loaded carvacrol before and after acid etching.

FIG. 4 shows the Zeta potential of HNT-crv-4M composite at different numbers of package layers.

FIG. 5 is a thermogravimetric analysis of HNT-crv-4M complex after encapsulation.

FIG. 6 shows the release curves of HNT-crv-4M complexes with different numbers of encapsulating layers.

Detailed Description

The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention, and are not intended to limit the scope of the present invention. In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to the following embodiments.

Description of raw materials:

conventional Halloysite Nanotubes (HNT) having a length of 0.2-1.5 μm, an outer diameter of 50-75nm and an inner diameter of lumen of 10-30nm, are commercially available from Yunxin nanotechnology Co., Ltd, and a specific surface area of 29.506m²Per g, pore volume of 0.196 cm³/g。

Polyethyleneimine (PEI), Mw & lt & gt 10000, sodium polystyrene sulfonate (PSS), Mw & lt & gt 70000; purchased from Aladdin, Shanghai, Aladdin Biotechnology Ltd.

Example 1

A halloysite nanotube-loaded carvacrol sustained-release compound is prepared by the following steps:

(1) modifying halloysite nanotubes, weighing 5g of HNT, placing the HNT in a 100mL beaker, adding 80mL of 4M HCl solution prepared in advance, uniformly stirring, setting the heating temperature to 80 ℃, magnetically stirring at 600rpm, and reacting for 8 hours; and after the acid etching reaction is finished, cooling the halloysite nanotube suspension, centrifuging, washing a solid phase to be neutral through deionized water, drying and grinding to obtain a white and powdery modified halloysite nanotube which is named as HNT-4M.

(2) Carvacrol loading, namely mixing HNT-4M and carvacrol in a ratio of 0.1g/mL to obtain a mixture of HNT-4M and carvacrol, which is named as HNT-crv; sonicate HNT-crv in an ice bath for 30min (power 200W, pulse on for 2 seconds, pulse off for 3 seconds); the HNT-crv was then transferred to a vacuum desiccator connected to a vacuum pump, depressurized to a pressure of 0.1MPa for 30 minutes to remove air inside the HNT, then returned to atmospheric pressure for 10 minutes to get carvacrol molecules into the evacuated lumen of the HNT, and the cycle was repeated twice to increase the loading efficiency, the halloysite nanotubes loaded with carvacrol, i.e. the halloysite nanotube-carvacrol complex, named HNT-crv-4M; and (3) obtaining a solid phase in the HNT-crv-4M suspension through centrifugal separation, then centrifuging the HNT-crv-4M for 2 times by using ethanol to remove carvacrol molecules adsorbed on the outer surface, and then drying the carvacrol molecules in an open container at room temperature overnight to obtain a finished product of the halloysite nanotube-loaded carvacrol sustained-release compound, which is named as HNT-crv-4M.

Characterization of morphology before and after HNT-4M modification:

the transmission electron microscope images are shown in FIGS. 1a to 1 d. It can be clearly seen from fig. 1a and 1c that the HNT before modification is in a hollow tubular shape, the inner and outer tube walls are flat and smooth, and the tube openings are not regular; from fig. 1b and fig. 1d, it can be seen that the morphology of the outer tube wall of the modified HNT-4M is not changed, etching traces are formed at the tube opening and the inner tube wall, the inner surface becomes uneven, and the residual nano SiO2 particles can be observed.

Characterization of pore structures before and after HNT-4M modification:

the adsorption and desorption curves are shown in figure 2. At a lower P/P₀Zone, amount of adsorbed gas is dependent on P/P₀Is increased slowly, at 0.4<P/P₀<Hysteresis loops are observed in the range of 1.0, which isThe mesoporous pores of the halloysite nanotubes are caused by capillary condensation during absorption and desorption, and the hysteresis loop of HNT-4M with increased pore diameter is increased; the specific surface area of HNT-4M is calculated by analysis of a BJH model and is 29.506M²The/g is increased to 95.577m²Per g, pore volume from 0.196 cm³The/g is increased to 0.329 cm³This is consistent with the microstructure changes revealed by TEM images, [ AlO6 ] due to acid etching of the inner tube wall of the halloysite nanotubes]Octahedron is dissolved, and the formed micropores enlarge the inner diameter of the tube cavity and increase the specific surface area and the pore volume.

HNT-crv-nM thermogravimetric analysis:

the thermogravimetric analysis thereof is shown in FIG. 3. And (3) HNT-crv-nM, wherein n is the molar concentration of the HCl solution in the step (1), and the values of n are 0, 1, 2, 3, 4 and 5. Compared with HNT-crv (which is not subjected to hydrochloric acid etching modification), the HNT-crv-nM also has a weight loss phenomenon within the range of 95-180 ℃, which is mainly the thermal decomposition loss of carvacrol loaded in a tube cavity, the load capacity of carvacrol in the halloysite nanotube can be indirectly calculated through the weight loss rate at the stage, and the carvacrol load capacities of HNT-crv, HNT-crv-1M, HNT-crv-2M, HNT-crv-3M, HNT-crv-4M and HNT-crv-5M are respectively 6.7%, 10.08%, 11.6%, 16.51%, 24.2% and 13.71% by calculation. Therefore, the load capacity of the halloysite nanotube is the largest after being etched by 4M hydrochloric acid, and the load capacity of carvacrol is improved by nearly 4 times. When the molar concentration of the HCl solution is 5M, the transmission electron microscope observation shows that the inner tube wall is perforated after being etched, and carvacrol overflows from the perforated position during loading, so that the loading amount of the halloysite nanotube is reduced.

HNT-crv-4M Release assay:

the carvacrol release curve is shown in figure 6. Placing the prepared HNT-crv-4M compound in an open culture dish, uniformly distributing the compound at the bottom, and storing the culture dish in a constant temperature and humidity box with the temperature of 23 ℃ and the relative humidity of 50%; taking out 0.05 g every 24 h, adding absolute ethyl alcohol, placing in a 10 mL centrifuge tube, ultrasonically vibrating for 25 min, standing for 24 h, placing in a centrifuge, and centrifuging for 15 min at 5000 r/min; extracting the supernatant, diluting by 20 times, placing into a quartz cuvette, measuring absorbance at 276 nm by using an ultraviolet spectrophotometer, calculating the concentration of carvacrol in the extracting solution according to a previously determined carvacrol concentration standard curve so as to estimate the carvacrol content in the HNT-crv-4M compound, calculating the release rate of the HNT-crv-4M compound according to an initial value, and referring to figure 6 for the release rate. At 23 ℃ and 50% relative humidity, the unencapsulated HNT-crv-4M compound is rapidly released in the early stage, the release rate of carvacrol is almost in direct proportion to days, and the release rate begins to slow after the 20-day release amount reaches 67.4%.

Example 2

A halloysite nanotube-loaded carvacrol sustained-release compound is prepared by the following steps:

(1) halloysite nanotube modification 5g of Halloysite Nanotubes (HNT) were weighed into a 100mL beaker followed by the addition of 80mL of a pre-formulated 4M hydrochloric acid solution. After stirring uniformly, the heating temperature is set to 80 ℃, and the reaction is carried out for 8 hours by magnetic stirring. And after the acid etching reaction is finished, centrifugally washing the cooled halloysite suspension to be neutral, drying and grinding to obtain white powder and naming the white powder as HNT-4M, wherein the halloysite nanotube after acid etching is shown in figure 1 and figure 2.

(2) Carvacrol loading, HNT-4M and carvacrol are mixed in a ratio of 0.1 g/mL. The HNT-crv mixture was sonicated in an ice bath for 30 minutes (pulse on for 2 seconds, pulse off for 3 seconds). The HNT-crv complex was then transferred to a vacuum desiccator connected to a vacuum pump. The pressure was reduced to 0.1MPa for 30 minutes to remove air from the interior of the HNT and then returned to atmospheric pressure for 10 minutes to allow carvacrol molecules to enter the evacuated HNT lumen. Carvacrol loading operations were repeated twice to increase loading efficiency. The solid phase in the suspension was separated by centrifugation and the HNT-crv complex was centrifuged 2 times with ethanol to remove the carvacrol molecules adsorbed on the outer surface and then dried overnight in an open container at room temperature.

(3) And (3) polyelectrolyte encapsulation, namely preparing a PEI polyelectrolyte solution and a PSS polyelectrolyte solution with the concentration of 2mg/ml by using deionized water, uniformly stirring, and performing ultrasonic treatment for 30 min. Adding the HNT-crv-4M compound into a PEI solution according to the mass ratio of 2:8, stirring and mixing for 10 min at room temperature to uniformly disperse the compound, centrifugally separating out a solid phase, and washing with deionized water to finish the first layer of packaging; and adding the mixture into the PSS solution with the same volume, stirring for 10 min, and then separating and washing to finish second-layer packaging. And (3) drying the obtained solid in vacuum for 24 hours, and grinding the solid into powder to obtain a finished product of the halloysite nanotube-loaded carvacrol sustained-release composite, which is named as HNT-crv-4M/PEI/PSS.

HNT-crv-4M/PEI/PSS encapsulation characterization:

the Zeta potential diagram is shown in FIG. 4. In fig. 4, the abscissa is the number of the packaging layers, 0 is not packaged, 1 layer is only packaged with 1 PEI, 2 layers are sequentially packaged with 1 PEI and 1 PSS, 3 layers are sequentially packaged with 1 PEI, 1 PSS and 1 PEI, 4 layers are sequentially packaged with 1 PEI, 1 PSS, 1 PEI and 1 PSS, wherein the PEI of each layer and the front and back packaging processes of each PSS are the same. The HNT-crv-4M complex is electronegative, and the potential value is-23.5 mV, because the HNT has abundant hydroxyl on the outer surface and is electronegative in water. By using the action of electrostatic force, PEI polyelectrolyte with positive electricity and PSS polyelectrolyte with negative electricity are sequentially adsorbed, so that the halloysite nanotube can be effectively packaged. After a layer of PEI polyelectrolyte is packaged, the potential value of the HNT-crv-4M compound is changed to +30.2 mV, and the potential value is changed to-35.6 mV after the PSS is continuously packaged. Along with the encapsulation of positive and negative polyelectrolytes, the potential value of the HNT-crv-4M compound has positive and negative alternate changes, which indicates that the polyelectrolytes are well encapsulated on the surface of the nanotube.

Thermogravimetric analysis of HNT-crv-4M/PEI/PSS:

the thermogravimetric analysis thereof is shown in FIG. 5. The HNT-crv-4M compound after being packaged has three weight loss stages, which are 250-450 ℃ more weight loss stages than the HNT-crv-4M compound without being packaged, and the larger the number of packaging layers is, the more weight loss is, which is mainly the heat decomposition loss of the packaged PEI and PSS polyelectrolytes. On the other hand, the carvacrol loaded in the halloysite nanotube is reduced along with the increase of the number of the packaging layers, which is supposed to be caused by the loss of the carvacrol caused by operations such as stirring and centrifuging of the HNT-crv-4M compound in the packaging process, so that the number of the packaging layers needs to be controlled to be 1-2 layers to ensure higher carvacrol loading.

HNT-crv-4M/PEI/PS release assay:

the carvacrol release curve is shown in figure 6. Placing the prepared HNT-crv-4M/PEI and HNT-crv-4M/PEI/PS compound in an open culture dish, uniformly distributing the compound at the bottom, and placing the culture dish in a constant temperature and humidity box with the temperature of 23 ℃ and the relative humidity of 50% for storage. Taking out 0.05 g every 24 h, adding absolute ethyl alcohol, placing in a 10 mL centrifuge tube, ultrasonically shaking for 25 min, standing for 24 h, placing in a centrifuge, and centrifuging at 5000 r/min for 15 min. Extracting the supernatant, diluting by 20 times, placing the supernatant into a quartz cuvette, measuring the absorbance at 276 nm by using an ultraviolet spectrophotometer, calculating the concentration of carvacrol in the extracting solution according to a previously determined carvacrol concentration standard curve so as to estimate the carvacrol content in the HNT-crv-4M/PEI/PSS complex, and calculating the release rate of the HNT-crv-4M/PEI/PSS complex according to an initial value. At 23 ℃ and 50% relative humidity, the early release rate of carvacrol in the HNT-crv-4M/PEI/PSS complex after encapsulation is obviously slowed down, and after 20 days, the release rates of carvacrol in the HNT-crv-4M complex for encapsulating 1 layer (only 1 PEI), 2 layers (1 PEI and 1 PSS), 3 layers (1 PEI, 1 PSS and one PEI) and 4 layers (1 PEI, 1 PSS, 1 PEI and 1 PSS) are respectively 48.8%, 38.7%, 8.43% and 4.83%. Although the slow release speed of HNT-crv-4M/PEI/PSS/PEI and HNT-crv-4M/PEI/PSS/PEI/PSS which are packaged by 3 layers and 4 layers is greatly reduced compared with that of HNT-crv-4M/PEI and HNT-crv-4M/PEI/PS which are packaged by 1 layer and 2 layers, the carvacrol loading amount is low and is not considered, so that corresponding curves are not shown in the figure. The release rate of carvacrol is reduced mainly because the positive and negative polyelectrolytes form good encapsulation layers outside the nanotube and at the pipe orifice, a diffusion barrier is provided, the release of carvacrol is slowed down, and the more the number of encapsulation layers is, the more obvious the slow release effect is.