Acetylated chitin nanocrystal and preparation method of stable Pickering emulsion of acetylated chitin nanocrystal

Abstract

The invention discloses acetylated chitin nanocrystals and a preparation method of stable Pickering emulsion of the acetylated chitin nanocrystals, and belongs to the technical field of food colloid. According to the method, chitin is treated through an acidolysis and acetylation composite modification process, so that chitin nanocrystals with different acetylation substitution degrees are prepared. The obtained modified chitin nanocrystal has optimized surface properties and good particle size distribution, so that the modified chitin nanocrystal forms a stable physical barrier on an oil-water interface, and the stability and biocompatibility of an emulsion are remarkably improved. The Pickering emulsion prepared by using the modified nanocrystal solves the stability problem caused by hydrophobicity, high volatility and environmental sensitivity of origanum vulgare essential oil, and opens up a new path for application of natural biological materials in the field of Pickering emulsion.

Description

Technical Field

[0001] This invention relates to a method for preparing acetylated chitin nanocrystals and their stable Pickering emulsion. Background Technology

[0002] Oregano essential oil is a natural essential oil extracted from the oregano plant (Origanum vulgare) of the Lamiaceae family. It has a rich aromatic scent and various biological activities, such as antibacterial, antioxidant, and anti-inflammatory properties. However, the hydrophobicity, high volatility, and high sensitivity to environmental factors (air, light, temperature) of oregano essential oil limit its application in various industries.

[0003] In recent years, Pickering emulsions, based on solid particle stabilization, have attracted increasing attention due to their environmental friendliness and high stability. Compared with traditional emulsions, Pickering emulsions exhibit superior anti-agglomeration stability, are simpler to prepare, and have better biocompatibility. These advantages make Pickering emulsions show significant potential in enhancing the encapsulation effect of active substances and improving the stability of bioactive substances. Chitin is a natural high-molecular-weight polysaccharide, second only to the exoskeletons of arthropods and mollusks, as well as the cell walls of fungi and yeasts. As a green biomaterial, chitin has good biocompatibility and biodegradability, and therefore has attracted attention in the food, pharmaceutical, and environmental protection fields. Chitin nanocrystals (ChNCs) are nanoscale materials prepared based on natural high-molecular-weight chitin, and possess high aspect ratio, large specific surface area, and good surface reactivity, making them potential emulsifiers. However, ChNCs have strong surface hydrophilicity, and the strong hydrogen bonding between hydroxyl groups easily leads to aggregation between nanocrystals, hindering the formation of a stable interfacial structure at the oil-water interface. Therefore, how to stabilize Pickering emulsion with hydrophobic nanocrystals prepared from chitin to protect oregano essential oil and broaden the application scope of oregano essential oil in the food and pharmaceutical fields is a technical problem that this application urgently needs to solve. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing acetylated chitosan nanocrystals and their stable Pickering emulsion.

[0005] The technical solution for achieving the objective of this invention is as follows:

[0006] 1. A method for preparing acetylated chitosan nanocrystals and their stable Pickering emulsion, comprising the following steps:

[0007] Step (1) Preparation of chitin nanocrystals: Chitin was mixed with sulfuric acid solution, stirred and heated in a water bath for 12 hours, then centrifuged and washed, and then redispersed in deionized water to obtain chitin nanocrystals.

[0008] (2) The chitin nanocrystal suspension obtained in step (1) was centrifuged to remove water, then added to glacial acetic acid solution, stirred and treated, acetic anhydride reagent was added dropwise and then a catalyst was added dropwise, followed by centrifugation and washing, and redispersed in deionized water to obtain acetylated chitin nanocrystals.

[0009] (3) The acetylated chitin nanocrystals obtained in step (2) are mixed with oregano essential oil at a certain mass ratio and homogenized at high speed and then ultrasonically emulsified to obtain a milky white Pickering emulsion.

[0010] 2. The method for preparing chitin nanocrystals according to claim 1, wherein the mass ratio of chitin to sulfuric acid is 1:30.

[0011] 3. The water bath heating temperature for preparing chitin nanocrystals according to claim 1 is 90°C, and the stirring speed is 200 rpm / min.

[0012] 4. In the preparation of acetylated chitosan nanocrystals according to claim 1, the mass ratio of nanocrystals to glacial acetic acid is 1:20.

[0013] 5. The stirring speed in step (2) according to claim 1 is 200 rpm / min.

[0014] 6. In step (2) of claim 1, the mass ratio of chitin nanocrystals to acetic anhydride is 1:1 to 1:5.

[0015] 7. In step (3) of claim 1, the mass ratio of acetylated chitosan nanocrystals to oregano essential oil is 19:1 to 15:5; the high-speed dispersion speed is 12000 rpm / min and the time is 2 min; the emulsification ultrasonic power is 375 W and the ultrasonic time is 3 min.

[0016] The present invention discloses the following technical effects:

[0017] Acetylated chitin nanocrystals exhibit improved hydrophobicity and dispersibility, resulting in superior performance in stabilizing Pickering emulsions compared to unmodified chitin nanocrystals. Acetylation modification also imparts higher interfacial activity to the nanocrystals, enabling them to effectively adsorb at the oil-water interface and prevent droplet coalescence.

[0018] The hydrophobicity, high volatility, and high sensitivity to environmental factors (air, light, temperature) of oregano essential oil limit its further applications. This invention utilizes modified acetylated nanocrystals as a solid particle stabilizer to effectively stabilize oregano essential oil and improve its stability under environmental conditions. Furthermore, the oregano essential oil emulsion exhibits good stability during 21-day storage, with no stratification observed. This invention provides a new method for developing and encapsulating plant essential oils. Attached Figure Description

[0019] Figure 1 Infrared spectra of Examples 1-3.

[0020] Figure 2 Particle size distribution and Zeta potential diagrams of Examples 1-3.

[0021] Figure 3 Numerical diagrams of the water contact angle of acetylated nanocrystals in Examples 1-3.

[0022] Figure 4 Stability of the Pickering emulsion prepared in Example 1 and Comparative Example 3 under ultraviolet radiation

[0023] Figure 5 The thermal stability of the Pickering emulsion stabilized by acetylated chitin nanocrystals prepared in Example 1 compared with that of Comparative Example 3.

[0024] Figure 6 Storage stability of Pickering stabilized by acetylated chitin nanocrystals prepared in Example 1. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings.

[0026] Example 1

[0027] Preparation method of acetylated chitin nanocrystals and their stable Pickering emulsion:

[0028] 3g of chitin powder was mixed with 90g of 3mol / L sulfuric acid solution and stirred continuously at 90℃ for 12h. After the reaction was completed, ten times the volume of deionized water was added to the resulting pale yellow liquid to terminate the reaction. The mixture was then allowed to stand at 4℃ for 12h. The upper pale yellow clear liquid was discarded, and sulfuric acid was removed by centrifugation to obtain a chitin nanocrystal suspension. The prepared ChNC suspension was washed twice with anhydrous ethanol by centrifugation to remove water. The mixture was then mixed with glacial acetic acid and sonicated for 10min to disperse it in the solution. Acetic anhydride (nanocrystal to acetic anhydride mass ratio of 1:5) was then added dropwise. While stirring at 45℃, methanesulfonic acid was added dropwise as a catalyst, and the reaction continued for 4h. After the reaction was completed, the mixture was allowed to stand, the upper pale yellow clear liquid was removed, and the mixture was washed three times with anhydrous ethanol. The mixture was then re-dispersed in deionized water by sonication to obtain an acetylated modified chitin nanocrystal (aChNC) suspension. 8 mL of 2 wt.% acetylated chitin nanocrystal dispersion was mixed with 2 mL of oregano essential oil and homogenized for 2 min using a high-speed disperser at 12000 rpm / min. Then, the mixture was ultrasonicated for 5 min using an ultrasonic homogenizer at 375 W to obtain a stable Pickering emulsion with acetylated nanocrystals.

[0029] Example 2

[0030] 3g of chitin powder was mixed with 90g of 3mol / L sulfuric acid solution and stirred continuously at 90℃ for 12h. After the reaction was completed, ten times the volume of deionized water was added to the resulting pale yellow liquid to terminate the reaction. The mixture was then allowed to stand at 4℃ for 12h. The upper pale yellow clear liquid was discarded, and sulfuric acid was removed by centrifugation to obtain a chitin nanocrystal suspension. The prepared ChNC suspension was washed twice with anhydrous ethanol by centrifugation to remove water. The mixture was then mixed with glacial acetic acid and sonicated for 10min to disperse it in the solution. Acetic anhydride (nanocrystal to acetic anhydride mass ratio of 1:3) was then added dropwise. While stirring at 45℃, methanesulfonic acid was added dropwise as a catalyst, and the reaction continued for 4h. After the reaction was completed, the mixture was allowed to stand, the upper pale yellow clear liquid was removed, and the mixture was washed three times with anhydrous ethanol. The mixture was then re-dispersed in deionized water by sonication to obtain an acetylated modified chitin nanocrystal suspension.

[0031] Example 3

[0032] 3g of chitin powder was mixed with 90g of 3mol / L sulfuric acid solution and stirred continuously at 90℃ for 12h. After the reaction was completed, ten times the volume of deionized water was added to the resulting pale yellow liquid to terminate the reaction. The mixture was then allowed to stand at 4℃ for 12h. The upper pale yellow clear liquid was discarded, and sulfuric acid was removed by centrifugation to obtain a chitin nanocrystal suspension. The prepared ChNC suspension was washed twice with anhydrous ethanol by centrifugation to remove water. The mixture was then mixed with glacial acetic acid and sonicated for 10min to disperse it in the solution. Acetic anhydride (nanocrystal to acetic anhydride mass ratio of 1:1) was then added dropwise. While stirring at 45℃, methanesulfonic acid was added dropwise as a catalyst, and the reaction continued for 4h. After the reaction was completed, the mixture was allowed to stand, the upper pale yellow clear liquid was removed, and the mixture was washed three times with anhydrous ethanol. The mixture was then re-dispersed in deionized water by sonication to obtain an acetylated modified chitin nanocrystal suspension.

[0033] Comparative Example 1

[0034] 3g of chitosan powder was mixed with 90g of 3mol / L sulfuric acid solution and stirred continuously at 90℃ for 12h. After the reaction was completed, ten times the volume of deionized water was added to the resulting pale yellow liquid to terminate the reaction. The mixture was then allowed to stand at 4℃ for 12h. The upper pale yellow clear liquid was discarded, and the sulfuric acid was removed by centrifugation. The mixture was then re-dispersed in deionized water by ultrasonication to obtain a chitosan nanocrystal suspension.

[0035] Comparative Example 2

[0036] 3g of chitosan powder was mixed with 90g of 3mol / L sulfuric acid solution and stirred continuously at 90℃ for 12h. After the reaction was completed, ten times the volume of deionized water was added to the resulting pale yellow liquid to terminate the reaction. The mixture was then allowed to stand at 4℃ for 12h. The upper pale yellow clear liquid was discarded, and the sulfuric acid was removed by centrifugation. The mixture was then re-dispersed in deionized water by ultrasonication to obtain a chitosan nanocrystal suspension.

[0037] 8 mL of chitin nanocrystal dispersion was mixed with 2 mL of oregano essential oil and homogenized for 2 min at 12000 rpm using a high-speed disperser. Then, the mixture was ultrasonicated for 5 min at 375 W using an ultrasonic homogenizer to obtain a Pickering emulsion stabilized with acetylated nanocrystals.

[0038] Comparative Example 3

[0039] Take unsealed oregano essential oil into a sample bottle and do not treat it in any way.

[0040] Example 1

[0041] Preparation of Pickering emulsion of oregano essential oil stabilized by acetylated chitosan nanocrystals:

[0042] The acetylated chitin nanocrystal dispersion prepared in Example 1 was diluted to 2 wt.%, and 8 mL of the acetylated chitin nanocrystal dispersion was mixed with 2 mL of oregano essential oil. The mixture was homogenized for 2 min at 12000 rpm / min using a high-speed disperser, and then ultrasonicated for 5 min at 375 W using an ultrasonic disruptor to obtain a stable Pickering emulsion of acetylated nanocrystals.

[0043] Result characterization:

[0044] (1) Fourier transform infrared spectrum

[0045] The chemical structure of the modified chitin nanocrystals was characterized using a Bruker Tensor 27 Fourier transform infrared spectrometer (FT-IR). The test conditions were as follows: the infrared spectra of the nanocrystals were measured in attenuated total reflectance (ATR) mode, with a wavenumber range of 4000-600 cm⁻¹. -1 64 scans, 4cm resolution -1 All example samples were measured at 1741 cm. -1 and 1260cm -1 A new absorption band appears at 1741 cm⁻¹. -1 The prominent ester bands are attributed to the stretching vibration of the C=O group in the acetyl group, and the intensity of these bands gradually increases with increasing degree of substitution (DS) value. (1260 cm⁻¹) -1 The new absorption band is due to the stretching vibration of CO in the acetyl group. Furthermore, at 1070 cm⁻¹... -1 and 1012cm -1 The peak intensity observed at [value] is significantly increased compared to ChNC, which is due to the stretching vibrations of COC and CO induced by the acetylation reaction. All these results indicate that chitin nanocrystals have been successfully acetylated.

[0046] (2) Particle size distribution and zeta potential

[0047] Using a Zetasizer Nano ZS instrument manufactured by Malvern Instruments, UK, at an ambient temperature of 25℃, the hydrodynamic diameter, particle size distribution, and zeta potential of chitin nanocrystals were determined by dynamic light scattering (DLS) and laser Doppler velocimetry. The average particle size of all samples was between 130-160 nm, indicating that the hydrodynamic diameter of the nanoparticles did not change significantly after modification. This is because acetylation mainly alters the surface functional groups of the nanoparticles without affecting their overall structure or aggregation state, thus maintaining the original particle size. The polydispersity index (PDI) reflects the uniformity of particle dispersion in the sample. A higher PDI value indicates a wider particle size distribution range in the tested sample solution, and a greater likelihood of particle aggregation and sedimentation. The PDI of ChNC was 0.356, which decreased to 0.276 ± 0.02 with increasing acetylation degree, indicating that the modified nanocrystals had a narrow particle size distribution and good dispersibility.

[0048] (3) Static water contact angle

[0049] The water contact angle of the nanocrystalline sheets before and after modification was determined using the sitting drop method to evaluate the hydrophilicity of the nanocrystals. The test drop volume was 2 μL. The contact angle of the sample in Comparative Example 1 was 34.3°, indicating strong hydrophilicity. This is because there are a large number of hydroxyl groups on the surface of chitin molecules. These polar groups easily form hydrogen bonds with water molecules, enhancing the interaction force. After different degrees of acetylation modification, the contact angle values ​​of the sample in the examples gradually increased. When the degree of substitution increased from 0.08 to 0.25, the contact angle of the examples increased from 34.3° to 70.9°, indicating a significant enhancement in the hydrophobicity of the samples, and the nanocrystals exhibited better amphiphilicity. Since acetyl groups are less polar than hydroxyl groups, the hydroxyl groups on the surface are replaced by acetyl groups after acetylation modification, reducing the number of hydrophilic groups and decreasing the overall polarity of the nanocrystals. In addition, since acetyl groups cannot form hydrogen bonds with water molecules, the interaction between the modified chitin nanocrystals and water molecules is weakened, making it difficult for water molecules to wet the surface of the nanocrystals. The modified nanocrystals exhibit improved overall hydrophobicity and increased affinity for oil, which helps them adsorb at the oil-water interface and stabilize the emulsion.

[0050] (4) UV stability

[0051] 10g of sample was placed open in a glass bottle and exposed to UV light emitted by a 20W, 365nm UV lamp under a sterile operating table. At regular intervals, 100mg of sample was placed in a 10mL centrifuge tube, and 5mL of ethanol and n-hexane (2:3, v / v) were added as solvents to extract the encapsulated essential oil. The mixture was then centrifuged (8000rpm, 5min), and the absorbance of the supernatant was measured at 276nm using a microplate reader. With increasing UV irradiation time, the final retention rates of the Pickering emulsion system and pure oregano essential oil were 63.1% and 53.08%, respectively, indicating that the essential oil substances directly exposed to UV radiation underwent the most severe volatile degradation. This may be because acetylated chitosan nanocrystals provided a certain physical barrier to UV radiation, thereby reducing the essential oil's exposure to UV light to some extent.

[0052] (5) Thermal stability

[0053] 10g of sample was sealed in a glass bottle and placed in a 55℃ incubator away from light. At regular intervals, 100mg of sample was transferred to a 10mL centrifuge tube, and 5mL of ethanol and n-hexane (2:3, v / v) was added as solvent to extract the encapsulated essential oil. The mixture was then centrifuged (8000rpm, 5min), and the supernatant was collected and its absorbance was measured at 276nm. The retention rate of the essential oil loaded in Pickering increased from 45.8% to 58.6% compared to the unencapsulated essential oil, and the thermal stability of the essential oil was significantly improved. The nanocrystals tightly encapsulated the essential oil, reducing its loss due to thermal degradation to a certain extent. These results indicate that the Pickering emulsion encapsulating essential oil has good UV resistance and thermal protection capabilities, providing protection for the essential oil and thus reducing its activity loss.

Claims

1. A method for preparing acetylated chitosan nanocrystals and their stable Pickering emulsion, characterized in that, Includes the following steps: (1) Chitin was dissolved in sulfuric acid solution, heated and stirred in a water bath, centrifuged and washed, and then redispersed in deionized water to obtain chitin nanocrystals. (2) The chitin nanocrystal suspension obtained in step (1) was centrifuged to remove water, then added to glacial acetic acid solution, stirred and treated, acetic anhydride reagent was added dropwise and then a catalyst was added dropwise, followed by centrifugation and washing, and redispersed in deionized water to obtain acetylated chitin nanocrystals. (3) The acetylated chitin nanocrystals obtained in step (2) are mixed with oregano essential oil at a certain mass ratio and homogenized at high speed and then ultrasonically emulsified to obtain a milky white Pickering emulsion.

2. The method for preparing chitin nanocrystals according to claim 1, characterized in that, The mass ratio of chitin to sulfuric acid is 1:

30.

3. The water bath temperature for preparing chitin nanocrystals according to claim 1 is 90°C, and the stirring speed is 200 rpm / min.

4. In the preparation of acetylated chitosan nanocrystals according to claim 1, the mass ratio of nanocrystals to glacial acetic acid is 1:

20.

5. In step (2) according to claim 1, the stirring speed is 200 rpm / min, and the mass ratio of chitin nanocrystals to acetic anhydride is 1:1 to 1:

5.

6. In step (3) of claim 1, the mass ratio of acetylated chitosan nanocrystals to oregano essential oil is 19:1 to 15:5; the high-speed dispersion speed is 12000 rpm / min and the time is 2 min; the emulsification ultrasonic power is 375 W and the ultrasonic time is 3 min.

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