A protein-chitosan oligosaccharide stabilized Pickering emulsion and preparation method thereof

Abstract

The invention discloses a protein-chitosan oligosaccharide-stabilized Pickering emulsion and a preparation method thereof, and belongs to the technical field of Pickering emulsion preparation. The preparation method of a protein-chitosan oligosaccharide-stabilized Pickering emulsion described in the present invention specifically comprises the following steps: firstly constructing protein-chitosan oligosaccharide composite nanoparticles by a thermally induced self-assembly method, then loading a fat-soluble active substance to obtain an aqueous nanoparticle suspension, and finally adding an oil phase or plant essential oil containing a fat-soluble active substance to the aqueous nanoparticle suspension, and preparing the Pickering emulsion by a high-energy emulsification method. The present invention enables the Pickering emulsion to achieve the purpose of high load or multi-function by adding the same or different active substances to the aqueous phase and the oil phase respectively.

Description

Technical Field

[0001] The invention relates to the technical field of Pickering emulsion preparation, and in particular to a protein-chitosan oligosaccharide stabilized Pickering emulsion and a preparation method thereof. Background Art

[0002] Emulsion is a uniform system composed of dispersed phase in the form of droplets dispersed in the continuous phase. From a thermodynamic point of view, emulsion is an unstable system. Under the influence of various factors, emulsion will demulsify over time. In order to make the emulsion exist stably for a long time, emulsifiers are usually added during the preparation of the emulsion to improve the stability of the emulsion. Pickering emulsion is a new type of emulsion prepared by using ultrafine solid particles or solid colloidal particles instead of traditional surfactants as emulsifiers. It has the advantages of good stability, easy regulation, environmental friendliness, low cost, relative safety and non-toxicity, etc. It has been widely used in many fields such as medicine, food, petroleum, water treatment, cosmetics, and material preparation.

[0003] Chitosan oligosaccharide is the main degradation product of chitosan / chitin by chemical hydrolysis or enzymatic degradation (including deacetylation and depolymerization processes). Chitosan oligosaccharide plays a great role in industries such as biomedicine, pharmaceuticals and agriculture. Chitosan oligosaccharide has a wide range of applications in industries such as cosmetics, medicines and nutritional products and wastewater treatment. Compared with chitosan and other available polysaccharides, chitosan oligosaccharide has a lower molecular weight, a higher degree of deacetylation, a higher degree of polymerization, a lower viscosity and complete water solubility, which gives it significant biological properties such as antibacterial, antioxidant, anti-inflammatory and antihypertensive, as well as drug delivery capabilities. In addition, it also shows anti-diabetic, anti-obesity, anti-HIV-1, anti-Alzheimer's disease, low cholesterol, calcium absorption and hemostasis effects. Protein not only has a high nutritional value, but also has good emulsification, and can be compounded with polysaccharides to obtain nano-colloidal particles with better emulsification and stability for the preparation of Pickering emulsions.

[0004] At present, the technology of preparing nanoparticles using protein and chitosan oligosaccharide mostly adopts the biocrosslinking agent genipin for crosslinking, glycosylation reaction for grafting, and simple electrostatic self-assembly by adjusting the pH value. There is no report on the preparation of protein-chitosan oligosaccharide composite nanoparticles by thermal induced self-assembly method. In addition, the existing Pickering emulsions all directly use simple nanoparticles to stabilize the Pickering emulsions, and the obtained Pickering emulsions have limited loading rate / embedding rate and single functionality. Summary of the invention

[0005] Technical problems to be solved: The purpose of the present invention is to overcome the above-mentioned shortcomings, provide a protein-chitosan oligosaccharide stabilized Pickering emulsion and a preparation method thereof, construct protein-chitosan oligosaccharide composite nanoparticles with a core-shell three-dimensional network structure by a heat-induced self-assembly method, and present a certain hydrophobic cavity inside the particles, which can load fat-soluble active substances to obtain an aqueous nanoparticle suspension, and finally add an oil phase or plant essential oil containing fat-soluble active substances to the aqueous nanoparticle suspension, and prepare the Pickering emulsion by a high-energy emulsification method. The method of the present invention can not only load the fat-soluble active substances more stably in the internal hydrophobic cavity of the nanoparticles, but also can achieve the purpose of high load or multi-function of the Pickering emulsion by adding the same or different fat-soluble active substances to the aqueous phase and the oil phase respectively.

[0006] Technical solution: A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion, comprising the following steps:

[0007] S1: Preparation of protein-chitosan oligosaccharide composite nanoparticles: weigh protein powder and chitosan oligosaccharide in a certain mass ratio, mix them evenly, and fully dissolve them in deionized water to obtain a mixed solution of protein and chitosan oligosaccharide with a certain mass concentration, adjust the pH value of the mixed solution to above the isoelectric point of the protein, then heat it in a water bath, and cool it to room temperature to obtain a protein-chitosan oligosaccharide composite nanoparticle suspension;

[0008] S2: Preparation of aqueous phase: fully dissolve the fat-soluble active substance with a solvent to obtain a fat-soluble active substance solution, stir the protein-chitosan oligosaccharide composite nanoparticle suspension at room temperature and add the fat-soluble active substance solution drop by drop, fully stir, centrifuge at 10000g for 20min, and the obtained supernatant is the aqueous phase;

[0009] S3: Preparation of oil phase: dissolving the fat-soluble active substance in the vegetable oil and mixing evenly to obtain the oil phase, or directly using the plant essential oil as the oil phase;

[0010] S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at a certain oil-water mass ratio, use a high-speed shear dispersing emulsifier for high-speed shearing to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion to obtain a Pickering emulsion.

[0011] Preferably, the protein comprises whey protein, soy protein, peanut protein, rice bran protein and pea protein.

[0012] Preferably, in step S1, the mass ratio of protein powder to chitosan oligosaccharide is 1:5-5:1, the mass concentration of the mixed solution of protein and chitosan oligosaccharide is 0.5%-5%, the water bath heating temperature is 70-98° C., and the water bath heating time is 1-60 min.

[0013] Preferably, the fat-soluble active substances in steps S2 and S3 include vitamins, lycopene, carotenoids, nisin, melatonin and tea polyphenols.

[0014] Preferably, the fat-soluble active substances in steps S2 and S3 are the same or different.

[0015] Preferably, the vegetable oil in step S3 includes soybean oil, peanut oil, sunflower oil, rapeseed oil and corn oil.

[0016] Preferably, the plant essential oil in step S3 includes thyme essential oil, oregano essential oil, citronella essential oil, lemon essential oil, clove essential oil and eucalyptus essential oil.

[0017] Preferably, the oil-water mass ratio in step S4 is (1:70) to 100.

[0018] Preferably, in step S4, the speed of high-speed shearing is 5000-20000 rpm, the shearing time is 10-600 s, the ultrasonic power of ultrasonic emulsification is 10-1000 W, the ultrasonic time is 1-30 min, the ultrasonic interval time is 2 s, and the ultrasonic frequency is 20 kHz. The present invention also provides a Pickering emulsion, which is prepared by the preparation method of a protein-chitosan oligosaccharide stabilized Pickering emulsion as described above.

[0019] Beneficial effects:

[0020] 1. The protein-chitooligosaccharide composite nanoparticles prepared by the heat-induced self-assembly method of the present invention present a core-shell three-dimensional network structure, with chitooligosaccharide as the outer shell and protein aggregated into a hydrophobic inner core. The method of the present invention can not only maximize the functional value of chitooligosaccharide, but also can more stably load the fat-soluble active substances in the internal hydrophobic cavity of the nanoparticles.

[0021] 2. The present invention can make the Pickering emulsion achieve the purpose of high load or multi-function and flexible conversion by adding the same or different active substances to the water phase and the oil phase respectively.

[0022] 3. The Pickering emulsion provided by the present invention is green and environmentally friendly, simple to prepare, and low in cost. Its ultimate purpose can be adjusted at will according to the type of active substance, and has broad application prospects and a wide range of application fields. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments, which are explanations of the present invention and are not limited to the following embodiments:

[0024] Example 1

[0025] A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion comprises the following steps:

[0026] S1: Preparation of peanut protein-chitosan oligosaccharide composite nanoparticles: peanut protein and chitosan oligosaccharide were weighed at a mass ratio of 2:1, mixed evenly, and fully dissolved with deionized water to obtain a peanut protein and chitosan oligosaccharide mixed solution with a mass concentration of 1%, and the pH value of the mixed solution was adjusted to 5.0, and then heated in a water bath at 80°C for 20 minutes, and cooled to room temperature to obtain a peanut protein-chitosan oligosaccharide composite nanoparticle suspension;

[0027] S2: Preparation of aqueous phase: Stir the peanut protein-chitosan oligosaccharide composite nanoparticle suspension at room temperature and add thyme essential oil drop by drop, stir thoroughly, centrifuge at 10000g for 20min, and the resulting supernatant is the aqueous phase;

[0028] S3: Preparation of oil phase: Thyme essential oil was directly used as the oil phase.

[0029] S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at an oil-water mass ratio of 10:100, use a high-speed shear dispersing emulsifier to perform high-speed shearing for 30 seconds at 13000rpm to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion at a power of 300W for 5 minutes to obtain a Pickering emulsion.

[0030] Example 2

[0031] A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion comprises the following steps:

[0032] S1: Preparation of rice bran protein-chitosan oligosaccharide composite nanoparticles: Rice bran protein and chitosan oligosaccharide were weighed at a mass ratio of 1:1, mixed evenly, and fully dissolved with deionized water to obtain a mixed solution of rice bran protein and chitosan oligosaccharide with a mass concentration of 1.5%, and the pH value of the mixed solution was adjusted to 4.8, and then heated in a water bath at 80° C. for 30 min, and cooled to room temperature to obtain a rice bran protein-chitosan oligosaccharide composite nanoparticle suspension;

[0033] S2: Preparation of aqueous phase: Melatonin was fully dissolved in 75% ethanol to obtain a 500 μg / mL melatonin solution, the rice bran protein-chitosan oligosaccharide composite nanoparticle suspension was stirred at room temperature and the melatonin solution was added dropwise at a volume ratio of 1:1, stirred thoroughly, and centrifuged at 10000 g for 20 min. The resulting supernatant was the aqueous phase;

[0034] S3: Preparation of oil phase: Dissolve 1 mg of nisin in 100 mL of sunflower oil and mix well to obtain an oil phase.

[0035] S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at an oil-water mass ratio of 15:100, use a high-speed shear dispersing emulsifier to perform high-speed shearing for 90 seconds at 9000rpm to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion at a power of 500W for 1 minute to obtain a Pickering emulsion.

[0036] Example 3

[0037] A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion comprises the following steps:

[0038] S1: Preparation of whey protein isolate-chitosan oligosaccharide composite nanoparticles: whey protein isolate and chitosan oligosaccharide were weighed in a mass ratio of 1:2, mixed evenly, and fully dissolved with deionized water to obtain a whey protein isolate and chitosan oligosaccharide mixed solution with a mass concentration of 2%, and the pH value of the mixed solution was adjusted to 6.0, and then heated in a water bath at 85°C for 15 minutes, and cooled to room temperature to obtain a whey protein isolate-chitosan oligosaccharide composite nanoparticle suspension;

[0039] S2: Preparation of aqueous phase: Vitamin A was fully dissolved in anhydrous ethanol to obtain a 450 μg / mL vitamin A solution, the whey protein isolate-chitosan oligosaccharide composite nanoparticle suspension was stirred at room temperature and the vitamin A solution was added dropwise at a volume ratio of 1:1, stirred thoroughly, and centrifuged at 10000 g for 20 min. The resulting supernatant was the aqueous phase;

[0040] S3: Preparation of oil phase: Dissolve 1 mg of vitamin A in 100 mL of soybean oil and mix well to obtain an oil phase.

[0041] S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at an oil-water mass ratio of 30:100, use a high-speed shear dispersing emulsifier to perform high-speed shearing for 60 seconds at 10,000 rpm to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion for 2 minutes at a power of 200 W to obtain a Pickering emulsion.

[0042] Example 4

[0043] A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion comprises the following steps:

[0044] S1: Preparation of soy protein isolate-chitosan oligosaccharide composite nanoparticles: Soy protein isolate and chitosan oligosaccharide were weighed at a mass ratio of 1:3, mixed evenly, and fully dissolved with deionized water to obtain a mixed solution of soy protein isolate and chitosan oligosaccharide with a mass concentration of 3%. The pH value of the mixed solution was adjusted to 5.0, and then heated in a 90°C water bath for 5 minutes. After cooling to room temperature, a soy protein isolate-chitosan oligosaccharide composite nanoparticle suspension was obtained;

[0045] S2: Preparation of aqueous phase: fully dissolve lycopene in acetone to obtain a 100 μg / mL lycopene solution, stir the soy protein isolate-chitosan oligosaccharide composite nanoparticle suspension at room temperature and add the lycopene solution dropwise at a volume ratio of 1:1, stir well, centrifuge at 10000 g for 20 min, and the resulting supernatant is the aqueous phase;

[0046] S3: Preparation of oil phase: dissolve 1 mg of lycopene in 100 mL of peanut oil and mix well to obtain the oil phase.

[0047] S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at an oil-water mass ratio of 60:100, use a high-speed shear dispersing emulsifier to perform high-speed shearing for 40 seconds at 12000rpm to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion at a power of 400W for 1 minute to obtain a Pickering emulsion.

[0048] Comparative Example 1

[0049] The difference between this comparative example and Example 1 is that after the pH value of the mixed solution is adjusted to 5.0 in step S1, no water bath heating is performed to obtain a purely electrostatically bonded traditional peanut protein-chitosan oligosaccharide composite nanoparticle suspension without a hydrophobic cavity, which is used for the subsequent preparation of the Pickering emulsion.

[0050] Comparative Example 2

[0051] The difference between this comparative example and Example 2 is that the operation of step S3 is not performed, and the water phase in step S2 is directly used for high-speed shearing and ultrasonic emulsification.

[0052] Comparative Example 3

[0053] The difference between this comparative example and Example 2 is that the operation of step S2 is not performed, and the rice bran protein-chitosan oligosaccharide composite nanoparticle suspension is directly used as the aqueous phase.

[0054] Comparative Example 4

[0055] The difference between this comparative example and Example 3 is that after the pH value of the mixed solution is adjusted to 6.0 in step S1, no water bath heating is performed to obtain a purely electrostatically bonded traditional whey protein isolate-chitosan oligosaccharide composite nanoparticle suspension without hydrophobic cavities, which is used for the subsequent preparation of Pickering emulsion.

[0056] Comparative Example 5:

[0057] The difference between this comparative example and Example 4 is that after the pH value of the mixed solution is adjusted to 5.0 in step S1, no water bath heating is performed to obtain a purely electrostatically bonded traditional soy protein isolate-chitosan oligosaccharide composite nanoparticle suspension without a hydrophobic cavity, which is used for the subsequent preparation of the Pickering emulsion.

[0058] Performance Test 1: Pickering emulsion loading rate determination:

[0059] Take 1 mL of each Pickering emulsion in Example 1 and Comparative Example 1, add 5 mL of n-hexane, vortex oscillate for 20 s, centrifuge at 5000 r / min for 10 min, dilute the supernatant 100 times after centrifugation, measure the absorbance at 274 nm, and calculate the emulsion loading rate by the standard curve and the following formula.

[0060]

[0061] The results show that the loading rate of thyme essential oil by the Pickering emulsion in Example 1 is as high as 96%, while the loading rate of thyme essential oil by the Pickering emulsion in Comparative Example 1 is only 72%, indicating that the Pickering emulsion provided by the present invention can achieve the effect of high loading of fat-soluble active substances.

[0062] Performance Test 2: Determination of the Multifunctionality of Pickering Emulsions:

[0063] The functional properties of the Pickering emulsions in Example 2, Comparative Example 2 and Comparative Example 3 were measured. The results are shown in Table 1.

[0064] Table 1 Functional properties of Pickering emulsion

[0065] sample Antioxidant Antibacterial Example 2 + ++ Comparative Example 2 + + Comparative Example 3 — ++

[0066] Note: + represents the sample has antioxidant or antibacterial effect, where the more + the stronger the antioxidant or antibacterial property of the sample.

[0067] As can be seen from Table 1, the Pickering emulsion in Example 2 has the strongest antioxidant and antibacterial properties. Compared with Comparative Example 2, due to the loading of nisin in Example 2, the antibacterial property of the Pickering emulsion is significantly enhanced, and there is no negative impact on the antioxidant property of melatonin loaded in the nanoparticles; compared with Comparative Example 3, due to the loading of melatonin by nanoparticles in Example 2, the Pickering emulsion shows a significant antioxidant effect, and there is no negative impact on the antibacterial property of nisin in the oil phase. This shows that the present invention can achieve a multifunctional effect of the prepared Pickering emulsion by adding different active substances to the water phase and the oil phase, and there is no antagonism between the active substances in the water phase and the oil phase.

[0068] Performance Test 3: Determination of Antibacterial Activity of Pickering Emulsion:

[0069] The antibacterial effect of Pickering emulsion on Staphylococcus aureus in Example 3 and Comparative Example 4 was studied. The antibacterial experiment was divided into five groups, namely, a blank group without any material, a bacterial spore suspension with pure vitamin A (the amount added was consistent with the content of vitamin A in the Pickering emulsion), a bacterial spore suspension with pure chitosan oligosaccharide (the amount added was consistent with the content of chitosan oligosaccharide in the Pickering emulsion), and a bacterial spore suspension with the same volume of Pickering emulsion in Example 3 and Pickering emulsion in Comparative Example 4. The experimental results showed that the blank group gradually multiplied spores in 12-18h, and the bacterial diameter became larger and larger, but the bacterial diameter of the Pickering emulsion in Comparative Example 4 was always smaller than that of the blank group, and the bacterial diameter of the Pickering emulsion in Example 3 was always kept at the minimum, and there was no significant difference in the size of the bacterial diameter with the pure chitosan oligosaccharide group, indicating that the core-shell protein-chitosan oligosaccharide composite nanoparticles prepared by the method of heat-induced self-assembly can maximize the functional value of chitosan oligosaccharide.

[0070] Performance Test 4: Antioxidant Determination of Pickering Emulsion:

[0071] The antioxidant properties of the Pickering emulsions in Example 4 and Comparative Example 5 were determined by the DPPH free radical scavenging ability method. 2 mL of 0.1 mmol / L DPPH solution and the sample solution were added to a test tube, mixed, and allowed to stand for 0.5 h before measuring the absorbance A at 517 nm. 0 ; Add 2mL DPPH· solution and 2mL anhydrous ethanol to another test tube, let it stand for 0.5h, and measure the absorbance A 1 Take another test tube, add 2mL of sample solution and anhydrous ethanol, let stand for 0.5h, and measure the absorbance A 2, the clearance rate is calculated as shown in the following formula:

[0072]

[0073] The test results are shown in Table 2:

[0074] Table 2 DPPH radical scavenging rate of Pickering emulsion

[0075]

[0076] It can be seen from Table 2 that the stability of lycopene is poor. Compared with Comparative Example 5, the core-shell protein-chitosan oligosaccharide composite nanoparticles prepared by heat-induced self-assembly in Example 4 can better load and protect the active substances, and the lycopene Pickering emulsion stabilized therewith has stronger and longer-lasting antioxidant capacity.

[0077] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion, characterized in that: The following steps are involved: S1: Preparation of protein-chitosan oligosaccharide composite nanoparticles: weigh protein powder and chitosan oligosaccharide respectively according to a certain mass ratio, mix them evenly, and fully dissolve them in deionized water to obtain a mixed solution of protein and chitosan oligosaccharide with a certain mass concentration, adjust the pH value of the mixed solution to above the isoelectric point of the protein, then heat it in a water bath, and cool it to room temperature to obtain a protein-chitosan oligosaccharide composite nanoparticle suspension; the mass ratio of the protein powder and chitosan oligosaccharide is 1:5-5:1, the mass concentration of the protein and chitosan oligosaccharide mixed solution is 0.5%-5%, the water bath heating temperature is 70-98°C, and the water bath heating time is 1-60min; S2: Preparation of aqueous phase: fully dissolve the fat-soluble active substance with a solvent to obtain a fat-soluble active substance solution, stir the protein-chitosan oligosaccharide composite nanoparticle suspension at room temperature and add the fat-soluble active substance solution drop by drop, fully stir, centrifuge at 10000g for 20min, and the obtained supernatant is the aqueous phase; S3: Preparation of oil phase: dissolving the fat-soluble active substance in the vegetable oil and mixing evenly to obtain the oil phase, or directly using the plant essential oil as the oil phase; S4: Preparation of Pickering emulsion: Add the oil phase to the water phase at a certain oil-water mass ratio, use a high-speed shear dispersing emulsifier for high-speed shearing to obtain a crude emulsion, and finally use an ultrasonic cell crusher to ultrasonically emulsify the crude emulsion to obtain a Pickering emulsion.

2. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: The protein is selected from the group consisting of whey protein, soy protein, peanut protein, rice bran protein and pea protein.

3. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: In the steps S2 and S3, the fat-soluble active substances are selected from vitamins, lycopene, carotenoids, nisin, melatonin and tea polyphenols.

4. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: The fat-soluble active substances in steps S2 and S3 are the same or different.

5. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: In step S3, the vegetable oil is selected from soybean oil, peanut oil, sunflower oil, rapeseed oil and corn oil.

6. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: In step S3, the plant essential oil is selected from thyme essential oil, oregano essential oil, citronella essential oil, lemon essential oil, clove essential oil and eucalyptus essential oil.

7. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: The oil-water mass ratio in step S4 is (1:70)-100.

8. The method for preparing a protein-chitosan oligosaccharide stabilized Pickering emulsion according to claim 1, characterized in that: In step S4, the speed of high-speed shearing is 5000-20000 rpm, the shearing time is 10-600 s, the ultrasonic power of ultrasonic emulsification is 10-1000 W, the ultrasonic time is 1-30 min, the ultrasonic interval time is 2 s, and the ultrasonic frequency is 20 kHz.

9. A protein-chitosan oligosaccharide-stabilized Pickering emulsion prepared by the method for preparing a protein-chitosan oligosaccharide-stabilized Pickering emulsion according to any one of claims 1 to 8.

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