阅读说明：本技术 一种基于丝素蛋白的纳米乳及其制备方法和应用 (Silk fibroin-based nanoemulsion and preparation method and application thereof ) 是由 王晓沁 郑兆柱 郭文君 王永峰 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种基于丝素蛋白的纳米乳及其制备方法和应用。本发明的基于丝素蛋白的纳米乳的制备方法,包括以下步骤：制备低分子量丝素蛋白,所述丝素蛋白溶液中丝素蛋白的分子量主要分布在40kDa以下。向处理后的丝素蛋白溶液中加入油相,将得到的混合液匀浆处理后得到初乳,所述混合液中,丝素蛋白的质量分数为0.05-2％,油相的质量分数为40％以下；向初乳中加入多元醇类,经高压均质或微射流处理后得到所述基于丝素蛋白的纳米乳。本发明的方法制备出纳米乳,且其具有高温、低温保存长期稳定性。(The invention relates to a silk fibroin-based nanoemulsion and a preparation method and application thereof. The preparation method of the silk fibroin-based nanoemulsion comprises the following steps: preparing low molecular weight silk fibroin, wherein the molecular weight of the silk fibroin in the silk fibroin solution is mainly distributed below 40 kDa. Adding an oil phase into the treated silk fibroin solution, and homogenizing the obtained mixed solution to obtain primary emulsion, wherein in the mixed solution, the mass fraction of silk fibroin is 0.05-2%, and the mass fraction of the oil phase is below 40%; and adding polyols into the colostrum, and carrying out high-pressure homogenization or micro-jet treatment to obtain the silk fibroin-based nanoemulsion. The nano-milk prepared by the method has long-term stability in high-temperature and low-temperature storage.)

1. A preparation method of a silk fibroin-based nanoemulsion is characterized by comprising the following steps:

(1) preparing low molecular weight silk fibroin; the molecular weight of silk fibroin in the silk fibroin solution is below 40 kDa;

(2) adding an oil phase into the silk fibroin solution treated in the step (1), wherein in the mixed solution, the mass fraction of the silk fibroin is 0.05-2%, and the mass fraction of the oil phase is below 40%;

(3) homogenizing the obtained mixed solution to obtain primary emulsion;

(4) and (3) homogenizing or carrying out micro-jet treatment on the colostrum solution to obtain the silk fibroin-based nanoemulsion.

2. The method of claim 1, wherein: in the step (1), the method further comprises the step of pretreating the low-molecular-weight silk fibroin to convert part of the silk fibroin into an aggregation state, wherein the granularity is 100-200 nm; the pretreatment method comprises the steps of mixing the low-molecular-weight silk fibroin with phosphate and carrying out high-temperature and high-pressure treatment; after mixing, the concentration of the silk fibroin is 0.01-8 wt%; the concentration of the phosphate is 0.05-20 mM; the pressure of the high-temperature high-pressure treatment is not higher than 0.4 MPa; the temperature is 121-140 ℃.

3. The method of claim 1, wherein: in step (2), the oil phase comprises one or any combination of essential oil, vegetable oil, animal oil, oil-soluble drug and wax.

4. The method of claim 1, wherein: in the step (3), the homogenizing treatment condition is that the rotating speed is not lower than 12000-25000 rpm, and the homogenizing time is not less than 1 minute.

5. The method of claim 1, wherein: in step (3), the homogenization treatment is divided into three steps: adding an oil phase into a fibroin solution, adding a polyol into oil phase-fibroin colostrum, and adding a preservative into the oil phase-fibroin-polyol colostrum.

6. The method of claim 5, wherein: the polyhydric alcohol comprises one or more of 1, 3-butanediol, ethanol, polyethylene glycol, 1, 2-propanediol and 1, 3-propanediol, and the preservative comprises one or more of phenoxyethanol, 2-phenethyl alcohol, methyl paraben, ethyl paraben, benzoic acid, p-hydroxyacetophenone, glyceryl caprylate and caprylyl hydroxamic acid; the addition ratio of the polyhydric alcohol is 5-30 wt%, and the addition ratio of the preservative is 0.5-1 wt%.

7. The method of claim 1, wherein: in the step (2), an auxiliary material is further added into the oil phase or the silk fibroin solution, wherein the auxiliary material comprises one or more of a skin feel modifier, a flavoring agent, essence, a trans-mucous membrane, a transdermal absorption enhancer, a pH regulator, a preservative, a thickening agent, a humectant, an antibacterial agent, an anti-inflammatory agent, a pigment, an aromatic agent, an antioxidant, an ultraviolet absorbent, vitamins, organic or inorganic powder, alcohol and sugar.

8. The method of claim 1, wherein: in step (4), the pressure of the homogenizing or microfluidizing treatment is not less than 400 bar.

9. A silk fibroin-based nanoemulsion prepared by the preparation method of any one of claims 1-8, characterized in that: the emulsion particle size of the nano-emulsion is less than 500 nm.

10. Use of the silk fibroin-based nanoemulsion of claim 9 in the preparation of cosmetics, skin care products, health products or foods.

Technical Field

The invention relates to the technical field of materials, in particular to a silk fibroin-based nanoemulsion and a preparation method and application thereof.

Background

The nano emulsion is a transparent or semitransparent homogeneous dispersion system which is spontaneously formed by aqueous substances, oily substances, interface substances and the like and has a thermodynamically stable particle size of 10-500 nm. In general, nanoemulsions are classified into three types, namely oil-in-water type nanoemulsions (O/W), water-in-oil type nanoemulsions (W/O) and bicontinuous type nanoemulsions (b.c), and this dispersion was first discovered and reported by hoor and Schulman in 1943. Until 1959, Schulman did not propose the concept of "microemulsions". Since then, theoretical and application studies of nanoemulsions have rapidly progressed. At present, the nano-emulsification technology has penetrated into the fields of daily chemical industry, fine chemical industry, petrochemical industry, material science, biotechnology, environmental science and the like, and becomes a research field with huge application potential in the world at present.

The nano-emulsion is generally prepared by taking an amphiphilic surfactant as an emulsifier, but the extraction process of the natural liposome amphiphilic surfactant is complex and high in cost, and chemical reagents used in the extraction process remain in the emulsion to damage skin surface tissues and form liver injury when entering a body. The synthetic liposome surfactant solves the problem of cost retention of the emulsifier, but cannot solve the problem of damage of residual substances to organisms. Along with the continuous improvement of living standard, the demands of people on green, environment-friendly, safe and high-quality and high-grade products are continuously enhanced, the traditional emulsifier can not meet the development demands of high and new technology industries, and the natural emulsifier with excellent biocompatibility, biodegradability and strong interface stability is bound to become the urgent demand and inevitable trend of the development of emulsion technology, especially the industries of cosmetics and biopharmaceuticals. Therefore, research and development of safer emulsifiers is a problem to be solved urgently in this field.

The silk fibroin has similar composition with human keratin, has strong affinity, safety and good biocompatibility with human skin, contains 18 amino acids, 11 of which are ingredients required by human body, and can enhance the activity of skin cells, prevent skin aging and promote metabolism by effective supplementation. Silk fibroin has been widely accepted in the industry as an ingredient of skin care products, for example, patent CN 107412023A, CN201710369732.1 discloses adding silk fibroin as an ingredient having skin cell repairing effect into essential oil emulsion containing sodium carboxymethylcellulose as an emulsifier. It is described in us patent (WO2014012105a1) that silk fibroin can be used as an emulsifier to stabilize emulsified phospholipid droplets, dispersed in silk fibroin-based materials "silk fibroin can be used as an emulsifier to stabilize lipid droplet emulsions dispersed in silk fibroin-based materials".

Silk fibroin consists of repeating units-Gly-Ser-Gly-Ala-and five units of non-hydrophilic groups-Gly-Ala Gly-for hydrophilicity-Ser-one unit, the hydrophilic-lipophilic balance (HLB) is in the medium range, and various emulsions ranging from O/W emulsions to W/O emulsions can be prepared. Thus, silk fibroin can be a solution to the "green" emulsifier. Many inventions have been developed to use silk proteins as surfactants. Fibroin protein based emulsions, microcapsules or drug loaded microspheres as disclosed in patents CN 107412023A, CN 1864833A, CN201110027281.6, CN 103417497B (1-10 microns), cn201710307628.x (100-200 microns), CN201810251754(3.4-5.3 microns), CN201810251755(2.5-4.7 microns), CN 101244277B (5.84-86.27 microns), CN 107157811A, CN201710369732.1, CN 108192731A. These inventions are useful as a surfactant containing sericin extracted from silk or raw silk, sericin or a decomposition product thereof as an active ingredient, and an emulsifier for cosmetics. However, compared with synthetic surfactants, silk fibroin emulsifiers have poor emulsifying capacity, and all formed micro-emulsions are formed. But the stability of the micro-emulsion is poor and can not reach the emulsion stability detection standard (freezing minus 18 ℃ for 1 month, high temperature 45 ℃ for 1 month, freezing and thawing cycle (minus 18 ℃ to thawing 45 ℃) for 5 times, centrifugation at 3000rpm and no demulsification within 30 minutes). Japanese patent (US7901668) uses silk to extract fibroin having an average molecular weight of 5,000 or more as an emulsifier to emulsify oil and fat, forming oil droplets in which protein is stabilized at the oil-water interface, has a large molecular weight and is mainly composed of a random coil structure. The ratio ranges of fibroin and oil phase capable of forming an emulsion are discussed in detail. The product is used as skin care material, and has low cell growth effect. But do not relate to the study of emulsion stability, and the stable layer of the emulsion is very important to the raw materials of the emulsion and the finished product processed by using the emulsion.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a silk fibroin-based nanoemulsion and a preparation method and application thereof. The method of the invention utilizes silk fibroin as an emulsifier to prepare the nano-emulsion, the diameter of the prepared nano-emulsion is less than 500nm, and the nano-emulsion has the long-term stability of centrifugation, high temperature, low temperature and freeze thawing.

The first purpose of the invention is to provide a preparation method of a silk fibroin-based nanoemulsion, which comprises the following steps:

(1) preparing the low molecular weight silk fibroin.

(2) And (2) adding an oil phase into the silk fibroin solution treated in the step (1), wherein in the mixed solution, the mass fraction of the silk fibroin is 0.05-2%, and the mass fraction of the oil phase is below 40%.

(3) Homogenizing the obtained mixed solution to obtain primary emulsion.

(4) And (3) homogenizing or carrying out micro-jet treatment on the colostrum solution to obtain the silk fibroin-based nanoemulsion.

Further, the molecular weight of silk fibroin in the silk fibroin solution is mainly distributed below 40 kDa.

Further, it is preferable to subject the low molecular weight silk fibroin to pretreatment. The pretreatment result is that part of silk fibroin is converted into an aggregation state, and the granularity is 100-200 nm. The pretreatment method comprises the steps of (1) mixing silk fibroin obtained in the step (1) with phosphate with a certain concentration, and carrying out high-temperature high-pressure treatment;

further, after mixing with phosphate, the concentration of the silk fibroin is 0.01-8 wt%;

further, after mixing with silk fibroin, the concentration of the phosphate is 0.05-20 mM; the pressure of the high-temperature high-pressure treatment is not higher than 0.4 MPa; the processing temperature is 121-140 ℃.

Further, in the step (2), the oil phase comprises one or any combination of essential oil, vegetable oil, animal oil, oil-soluble drug and wax.

Further, the essential oils include but are not limited to rose essential oil, tea tree essential oil, geranium essential oil, jasmine essential oil, sweet orange essential oil, chamomile essential oil, rosemary essential oil, orange flower essential oil, and the like, alone or in any combination; vegetable oils include, but are not limited to, avocado oil, camellia oil, grapefruit oil, argan, jojoba oil, grapeseed oil, perilla oil, coconut oil, corn oil, soybean oil, castor oil, peanut oil, walnut oil, sunflower oil, and the like, either alone or in any combination; animal oils include, but are not limited to, whale oil, fish oil, DHA, and the like, alone or in any combination.

Further, other auxiliary materials such as skin feel improving agents, flavoring agents, essences, trans-mucous membranes, transdermal absorption promoters, pH regulators, preservatives, thickeners, humectants, antibacterial agents, anti-inflammatory agents, pigments, fragrances, antioxidants, ultraviolet absorbers, vitamins, organic or inorganic powders, alcohols, sugars and the like may be added to the oil phase or the silk fibroin solution as long as the functions and effects of the present invention are not impaired. The silk fibroin-based nanoemulsion can be used for preparing cosmetics, skin care products, health products, foods or medical appliances.

Fig. 4 is a diagram of a proportional relationship between silk fibroin and oil phase when a nanoemulsion can be formed, in which the abscissa represents silk fibroin concentration, the ordinate represents oil phase concentration, solid circles indicate that a nanoemulsion can be formed, and open circles indicate that a nanoemulsion cannot be formed.

Further, in the steps (2) and (3), the homogenizing treatment is carried out at a rotation speed of not less than 12000-25000 rpm, preferably not less than 16000rpm, for a homogenizing time of not less than 1 minute. The effect after homogenization is that the oil phase in the mixed solution is uniformly dispersed in the water phase and no oil drop can be seen by naked eyes; oil-free water stratification occurred after 30 minutes of standing.

Further, the homogenization process is divided into three steps: adding the oil phase into the fibroin solution, adding the polyalcohol into the oil phase-fibroin colostrum, and adding preservatives such as phenoxyethanol and the like into the oil phase-fibroin-polyalcohol colostrum.

Further, the polyhydric alcohol includes, but is not limited to, 1,3 butylene glycol, ethanol and polyethylene glycol, 1,2 propylene glycol, 1,3 propylene glycol, and the like, as a single component or any combination thereof.

Further, the preservative includes, but is not limited to, phenoxyethanol, 2-phenylethyl alcohol, methylparaben, ethylparaben, benzoic acid, p-hydroxyacetophenone, caprylic glycerol, caprylic hydroxamic acid, and the like, either alone or in any combination thereof.

Furthermore, the adding proportion of the polyhydric alcohol is 5-30 wt%, and the adding proportion of the preservative is 0.5-1 wt%. The addition ratio of the polyhydric alcohol and the preservative is based on the total weight of the homogenate.

Further, in the step (4), the pressure of the homogenizing or microfluidizing treatment is not lower than 400 bar. The number of times of the circulating treatment is not less than 5.

The second purpose of the invention is to provide the silk fibroin-based nanoemulsion prepared by adopting the preparation method, wherein the emulsion particle size of the nanoemulsion is less than 500 nm.

The third purpose of the invention is to disclose the application of the silk fibroin-based nanoemulsion in preparing cosmetics, skin care products, health products and foods.

By the scheme, the invention at least has the following advantages:

the invention finds that the size of the emulsion is crucial to the stability of the emulsion, and the molecular weight of the silk fibroin and the proportion of the silk fibroin to the oil phase can regulate and control the particle size of the emulsion and obtain stable nano-scale emulsion. The invention takes silk fibroin with specific molecular weight as an emulsifier, and controls the structural change through a series of steps to realize the preparation of the nano emulsion. The diameter of the nano emulsion prepared by the method is less than 500nm, and the nano emulsion has long-term stability of centrifugation, high temperature, low temperature and freeze-thaw. Can be stored at-18 deg.C for more than 1 month, at 45 deg.C for more than 1 month, and can not be demulsified after being centrifuged at 3000rpm for 30 min. The silk fibroin-based nanoemulsion has good application prospect in preparing cosmetics, skin care products, health care products and foods.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of silk fibroin;

figure 2 is an optical photograph of a silk fibroin nanoemulsion;

FIG. 3 is a diagram of the state of avocado oil silk fibroin emulsion emulsified with silk fibroin solution at different treatment conditions after centrifugation;

FIG. 4 is a graph of silk fibroin versus oil phase ratio when capable of forming a nanoemulsion;

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.