Oil-in-water emulsion synergistically stabilized by silk fibroin interfacial film and modified chitosan and preparation method of oil-in-water emulsion

Abstract

The invention belongs to the technical field of emulsion stabilization, and discloses a silk fibroin interfacial film and modified chitosan synergistically stabilized oil-in-water emulsion and a preparation method thereof.The preparation method comprises the steps that rhamnolipid modified chitosan is dissolved in deionized water according to the concentration of 5-10 mg / mL; dissolving silk fibroin in deionized water according to a concentration of 2-5 mg / mL, adding an oil phase with a volume fraction of 5-40%, and homogenizing to form a primary emulsion; and standing and aging the primary emulsion, adding a modified chitosan solution, and homogenizing to obtain the oil-in-water emulsion. According to the invention, a step-by-step strategy of homogenizing, aging and re-homogenizing is provided, the emulsion stabilizing effect for more than 9 months at room temperature is realized through electrostatic anchoring of the interfacial film constructed by the silk fibroin and the modified chitosan, the dense interfacial film can be beneficial to packaging and protecting active substances and controlling fixed-point release of the active substances, and commercialized application is expected to be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of emulsion stabilization, and specifically relates to an oil-in-water emulsion stabilized by silk fibroin interfacial film and modified chitosan and a preparation method thereof, and application of the oil-in-water emulsion as a natural biopolymer (silk fibroin and chitosan) in the fields of food, cosmetics, medicine, personal care products and the like. BACKGROUND

[0002] The oil-in-water emulsion system has unique structural advantages. The water continuous phase makes the emulsion have good spreading property and easy cleaning characteristics. The oil dispersed phase can uniformly disperse oil-soluble active ingredients in the water-based system to achieve antibacterial, antioxidant, encapsulation of nutrients and response-controlled release, and construction of biomedical materials, and has been widely studied in the fields of food, medicine, agriculture, cosmetics and medical engineering. Of course, the key to constructing and stabilizing the oil-in-water emulsion lies in the emulsifier. For a long time, traditional synthetic molecular emulsifiers mainly include Span, Tween, sodium dodecyl sulfate SLS and the like, which have stable chemical properties. However, this type of emulsifier has poor biodegradability and is highly "chemically sensitive", which may cause health and safety concerns. Therefore, with the increasing urgency of sustainable development and health and safety needs, bio-based macromolecular emulsifiers are leading a green revolution.

[0003] Chitosan (CS) is the only polysaccharide with positive charge in the system, and is an important component in the study of emulsion stability, with a production second only to cellulose. Considering its low interfacial activity and harsh dissolution conditions, pure chitosan is not a good emulsifier. Modified chitosan (such as carboxymethylation, quaternization, hydroxyalkylation) effectively solves the solubility and emulsification problems. Rhamnolipid (RL) can be used to modify chitosan (CS-RL). The introduction of rhamnolipid not only improves the solubility of chitosan, but also significantly improves its interfacial activity, making chitosan have amphiphilic properties.

[0004] Silk fibroin (SF) as an emulsifier has unique advantages that cannot be replaced by other proteins (such as whey protein, soy protein, casein, etc.). Silk fibroin has a high proportion of natural beta-fold (Silk II structure), and its conformation can be precisely controlled by temperature / pH / ionic strength, etc., which endows silk fibroin with the rigid hydrophobic properties of ceramic-like. In addition, the interaction between silk fibroin and chitosan can be driven by electrostatic or hydrophobic interaction, without the need for additional cross-linking agents such as glutaraldehyde or genipin. This system is simple to operate, mild in reaction, low in cost, green and healthy, and easy to industrialize, and can have a wider application field.

[0005] In the prior art, the use of natural biomacromolecules (such as silk fibroin and chitosan) to stabilize emulsion systems usually relies on a one-step strategy of direct blending. The selected two components are pre-dissolved in a phase, and the two components are co-adsorbed at the oil-water interface spontaneously through homogenization and other strategies. Chinese Patent Application CN 119655425 A discloses a chitosan-casein peptide nanoparticle preparation and emulsion application. Casein peptides and chitosan are physically blended, combined into a nanocomposite with a particle size of 10-1000 nm through electrostatic interaction, and a relatively stable oil-in-water emulsion is prepared. Although this method can improve the stability of the emulsion compared to a single component, it usually causes the blended components to arrange disorderly and loosely at the interface, and the entanglement of different molecules can lead to the embedding of charged groups, affecting the effective combination rate. Moreover, different molecules can interfere with each other, affecting their structure and adsorption properties, resulting in insufficient strength of the oil-water interface formed, making it difficult to resist Ostwald ripening and droplet coalescence, and thus leading to oil-water phase separation. Therefore, in order to further improve the stability, cross-linking agents such as glutaraldehyde and genipin are often introduced in the study, which may cause biological compatibility risks and increase the purification cost.

[0006] Chinese Patent Application CN 115364054 A proposes a colon-targeted oil-in-water Pickering emulsion based on shellac nanoparticles and chitosan. First, the strong hydrophobicity and high negative surface potential of shellac nanoparticles are used to adsorb to the oil-water interface, and then positively charged chitosan is introduced to impart partial hydrophilicity to the shellac nanoparticles, so that the shellac nanoparticles can stabilize the oil-water interface to form an emulsion. The introduction of chitosan through a distribution strategy can effectively improve the adsorption arrangement of shellac nanoparticles at the oil-water interface. Considering that the interface stability of this patent relies only on the electrostatic interaction between shellac nanoparticles and chitosan, the influence of chitosan on the stability of the interface is limited due to the low interfacial activity of chitosan itself. Moreover, electrostatic interaction is related to the pH and salt concentration of the water environment, and in some cases the electrostatic interaction may be weakened, leading to the disintegration of the complex and thus affecting the stability of the emulsion. The present invention modifies chitosan to improve its interfacial activity, making chitosan itself more easily diffuse to the oil-water interface, and in addition to the electrostatic interaction with silk fibroin, the modified chitosan can better complex with silk fibroin.

[0007] Silk fibroin itself has special structural characteristics, and the high repetitive sequence gives it predictable self-assembly. Fresh silk fibroin is mainly in the random coil (Silk I structure) conformation, and the interfacial adsorption of the oil-water interface has a low elastic modulus. It cannot effectively inhibit the coagulation of oil droplets, nor can it provide effective support sites for modified chitosan. However, through physical factors, silk fibroin will change from random coil to rigid beta-pleated conformation. A small amount of beta-pleated formation provides an ordered "template", and the surrounding random coil or alpha-helix conformation will gradually arrange around these templates through hydrogen bonding and hydrophobic interaction to form new beta-pleated structures, thereby adsorbing at the oil-water interface to form anti-parallel ordered stacks and increase the mechanical strength of the interface. Subsequently, the distribution strategy is used to precisely anchor the modified chitosan on the template charged sites through electrostatic and hydrophobic forces, realizing the dense coating with minimal defects.

[0008] Therefore, based on the characteristics of silk fibroin, a high-efficiency and template-based method is designed to further improve the emulsification effect of proteins and polysaccharides, which has considerable research value for industrial application. SUMMARY

[0009] The purpose of the present application is to provide a silk fibroin rigid interface film template induced by aging strategy, which can provide stable interface support sites for the adsorption of subsequent functional molecules (such as rhamnolipid modified chitosan) to form a bio-based oil-in-water emulsion with super strong stability.

[0010] To achieve the above purpose, the present application provides a preparation method of a silk fibroin interface film and modified chitosan synergistically stabilized oil-in-water emulsion, which comprises the following steps: S1, dissolving rhamnolipid modified chitosan in deionized water at a concentration of 5-10 mg / mL to prepare a modified chitosan solution; S2, dissolving silk fibroin in deionized water at a concentration of 2-5 mg / mL, and adding an oil phase with a volume fraction of 5-40% to form a primary emulsion; S3, aging the primary emulsion for 10-20 hours, then adding 1 / 5-1 / 6 volume ratio of the modified chitosan solution to the primary emulsion, and homogenizing to obtain an oil-in-water emulsion stabilized by silk fibroin forming a rigid interface film base at the oil-water interface and rhamnolipid modified chitosan.

[0011] As a further preferred technical solution of the present application, the preparation method of the rhamnolipid-modified chitosan provided by the present application specifically comprises: adjusting the pH of deionized water to 4.5-5.0 with an acetic acid solution, and then adding chitosan to be dissolved to prepare a chitosan acid solution; dissolving rhamnolipid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a mixed solution of ethanol and deionized water to prepare a light yellow rhamnolipid-modified solution; mixing the chitosan acid solution and the rhamnolipid-modified solution to react, and then performing dialysis through a dialysis bag after the reaction is completed, and then freeze-drying to obtain the rhamnolipid-modified chitosan.

[0012] It should be noted that the rhamnolipid-modified chitosan used in the present application is a known material, and is not limited to being prepared by the above method.

[0013] As a further preferred technical solution of the present application, the grafting degree of the rhamnolipid-modified chitosan is 10-30%; and / or, the pH of the modified chitosan solution is 2.0-11.0.

[0014] As a further preferred technical solution of the present application, the preparation temperature of the initial emulsion is 10-30 °C.

[0015] As a further preferred technical solution of the present application, the initial emulsion is aged by standing in an atmospheric environment, and the aging time is 12-48 h.

[0016] As a further preferred technical solution of the present application, the oil phase comprises at least one of medium-chain triglyceride, coconut oil, soybean oil, corn oil, peanut oil, olive oil, palm oil, and rapeseed oil.

[0017] As a further preferred technical solution of the present application, the rotation speed of the homogenizer is 8000-15000 rpm.

[0018] According to a second aspect of the present application, the present application further provides an oil-in-water emulsion prepared by the above preparation method.

[0019] According to a third aspect of the present application, the present application further provides an application of the oil-in-water emulsion in emulsion stabilization, i.e., the oil-in-water emulsion is used as a natural biological macromolecule in food, cosmetics, medicines, personal care products, and the like to achieve emulsion stabilization.

[0020] Compared with the prior art, the present application can achieve the following beneficial effects: The present application has excellent structural characteristics and adjustable interfacial activity from the material selection of naturally extracted degradable biological macromolecules, avoiding the potential risks of chemical synthesis emulsifiers. In the first aspect, the property of silk fibroin that can induce the transformation of beta-sheet conformation under shear force is utilized. The formed beta-sheet provides an ordered "template". During the aging process, the surrounding random coil or alpha-helix conformation will gradually arrange around these templates through hydrogen bonding and hydrophobic interaction to form more beta-sheet structures, thereby adsorbing at the oil-water interface to form an anti-parallel ordered stack, and constructing a dominant rigid interface film, which provides an ideal substrate for the subsequent embedding of modified chitosan. In the second aspect, the silk fibroin primary emulsion forms a rigid interface film during the aging process, which is simple and easy to operate and has universality for different types of positive polymers or biological macromolecules. The functional design can be carried out according to the demand. In the third aspect, after the rhamnolipid modified chitosan, the chitosan has amphiphilicity, and the interfacial activity is significantly improved. It can be anchored to the silk fibroin interface film through electrostatic or hydrophobic interaction, making the film more dense, continuous and rigid. This strong steric hindrance effect can effectively resist oil droplet coagulation, and significantly improve the macroscopic stability of the emulsion.

[0021] The present application proposes to use a step-by-step strategy of homogenization-aging-rehomogenization, to realize the stable emulsion effect for more than 9 months at room temperature by the electrostatic anchoring of the interface film constructed by silk fibroin and the modified chitosan. The dense interface film may help to encapsulate and protect active substances and control their site-specific release, and is expected to realize commercial application. BRIEF DESCRIPTION OF DRAWINGS

[0022] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0023] Figure 1 Figure 1 is the fluorescence micrograph of the dyeing of the beta-sheet content of SF in the one-step strategy of Comparative Example 1 and the step-by-step strategy of Comparative Example 2, wherein a is the fluorescence micrograph of SF+CS-RL before and after homogenization and after being placed at room temperature for 7 days in the one-step strategy; b is the fluorescence micrograph of SF before and after homogenization and after being placed at room temperature for 12 hours in the step-by-step strategy; c is the fluorescence micrograph of SF before and after homogenization, before and after the compounding of SF and CS-RL after being placed for 12 hours, and after being placed for 7 days after the compounding.

[0024] Figure 2 Figure 2 is the schematic diagram of the interface film extraction and release in the step-by-step aging strategy of silk fibroin and rhamnolipid modified chitosan in Example 1, wherein a is the schematic diagram of the oil droplet release in the SF aging aqueous phase and the interface film morphology after 30 minutes of extraction; b is the schematic diagram of the oil droplet release after adding CS-RL solution in the SF aging aqueous phase and the interface film morphology after 20 minutes of repeated extraction.

[0025] Figure 3is the oil-water interfacial tension change diagram of emulsifiers in the one-step blending strategy of Comparative Example 1, the step-by-step strategy of Comparative Example 2 and the step-by-step aging strategy of Example 1, wherein: a is the interfacial tension change curve of CS-RL; b is the interfacial tension change curve of SF+CS-RL; c is the interfacial tension change curve of SF after homogenization and then compounding with CS-RL; d is the interfacial tension change curve of SF after homogenization and then compounding with CS-RL after being placed at room temperature for 12 hours.

[0026] Figure 4 is the optical microscope diagram of the emulsion of the distribution aging strategy under the distribution strategy of silk fibroin and rhamnolipid modified chitosan in the embodiment of the application, wherein: a is the microscope diagram of the initial emulsion of SF before aging; b is the microscope diagram of the initial emulsion of SF after aging for 12 hours; c is the microscope diagram of the initial emulsion of SF after aging for 12 hours and then compounding with CS-RL.

[0027] Figure 5 is the emulsion macroscopic static stability diagram of the one-step blending strategy of Comparative Example 1 and the step-by-step strategy of Comparative Example 2 and the step-by-step aging strategy of Example 1; wherein: a is the emulsion stability diagram of SF+CS-RL, SF(t=0 h) / CS-RL and SF(t=12 h) / CS-RL respectively; b is the emulsion stability diagram of the initial emulsion of SF after aging and then compounding with CS-RL, lysozyme (LZM) and polyethyleneimine (PEI) respectively.

[0028] Figure 6 is the macroscopic static stability diagram of the oil-in-water emulsion of SF(t=12 h) / CS-RL, wherein: a is a medium-chain triglyceride oil phase; b is a coconut oil oil phase; c is a corn oil oil phase; d is an olive oil oil phase.

[0029] The purposes, functional features and advantages of the application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0030] The specific embodiments of the application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely intended to illustrate and explain the application, and are not intended to limit the application.

[0031] Unless defined, the technical terms used in the following examples have the same meaning as generally understood by those skilled in the art to which the present application belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods. All raw materials and / or reagents in the embodiments of the application are purchased on the market or prepared according to conventional methods well known to those skilled in the art, for example, chitosan, silkworm cocoons, rhamnolipid, etc. are all obtained by market purchase. The pH of deionized water is 6.8.

[0032] The rhamnolipid-modified chitosan and silk fibroin in the above embodiments and comparative examples of the present application are prepared according to the following methods, respectively. (1) Preparation of rhamnolipid-modified chitosan Accurately weigh 1 g of chitosan (3000 MW, degree of deacetylation 80-90%), adjust the pH of deionized water to 4.8 with 1 mol / L acetic acid solution, then add the chitosan to the solution and stir at room temperature at 300 rpm for 6 h until completely dissolved to form a chitosan acid solution; accurately weigh 0.5 g of rhamnolipid and 0.54 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, dissolve them in a solution of ethanol: deionized water = 1:1, stir at room temperature at 300 rpm for 30 min to obtain a light yellow solution; add the light yellow solution to the chitosan acid solution, stir at room temperature at 300 rpm overnight, then put it into a dialysis bag (3000 molecular weight cut-off), dialyze for 3 days, change deionized water every 4 h, after the end, freeze-dry to obtain rhamnolipid-modified chitosan (CS-RL).

[0033] (2) Preparation of silk fibroin Accurately weigh 10 g of commercially available cocoon, boil in 1 L of 0.02 M sodium carbonate water for 40 min to remove sericin, then wash with deionized water for 3 times, repeat the whole process twice, after the end, dry at room temperature overnight to obtain silk fibroin fibers. Dissolve the silk fibroin fibers in 9.5 M lithium bromide solution, stir at 60 °C at 300 rpm for 3 h until completely dissolved, defoam. Put it into a dialysis bag for 3 days, change deionized water every 4 h, after the end, collect the solution and put it into a centrifuge, centrifuge at a speed of 10600 rpm for 20 min, repeat twice, freeze-dry to obtain silk fibroin (SF).

[0034] Comparative Example 1 Step S1, accurately weigh 0.5 g of rhamnolipid-modified chitosan, dissolve it in 100 ml of deionized water, stir at room temperature at 200 rpm for 3 h until completely dissolved, prepare a modified chitosan (CS-RL) solution.

[0035] Step S2, weigh 1 g of silk fibroin, dissolve it in 200 ml of deionized water, stir at room temperature at low speed until completely dissolved, prepare a silk fibroin (SF) solution.

[0036] Step S3, 160 mL of the above dissolved silk fibroin solution and 40 mL of the modified chitosan solution were mixed and stirred to obtain a simple blended SF+CS-RL aqueous phase. Then, 200 mL of the SF+CS-RL aqueous phase was placed in a high-neck beaker, and 50 mL of the oil phase medium-chain triglyceride was added dropwise under stirring with a homogenizer, and stirred at 12000 rpm for 6 min to obtain the final blended one-step SF+CS-RL oil-in-water emulsion system.

[0037] Comparative Example 2 Step S1, 0.5 g of rhamnolipid-modified chitosan was accurately weighed and dissolved in 100 mL of deionized water, and stirred at room temperature at 200 rpm for 3 h until completely dissolved to prepare a modified chitosan solution.

[0038] Step S2, 1 g of silk fibroin was weighed and dissolved in 200 mL of deionized water, and stirred at room temperature until completely dissolved to prepare a silk fibroin solution. Then, 160 mL of the silk fibroin solution was placed in a high-neck beaker, and 50 mL of the oil phase medium-chain triglyceride was added dropwise under stirring with a homogenizer, and homogenized at 12000 rpm for 3 min to form a preliminary emulsion.

[0039] Step S3, 40 mL of the modified chitosan solution was added dropwise to the above preliminary emulsion, and homogenized at 12000 rpm for another 3 min to allow the modified chitosan to adsorb to the oil-water interface, and the SF (t=0 h) / CS-RL oil-in-water emulsion under the step-by-step strategy was obtained after completion.

[0040] Comparing the above Comparative Example 1 with Comparative Example 2, the results show that the homogenizing shear of the distribution strategy in Comparative Example 2 increases the content of the β-sheet fragments of the silk fibroin, enhances the hydrophobic properties, and also enhances the adsorption capacity at the oil-water interface. The specific test method is as follows: first, the silk fibroin aqueous solution is dyed with thionin, and it is known that thionin only dyes the β-sheet fragments of the silk fibroin. Figure 1 The a (I) and b (I) in FIG. 1 show that there is less fluorescence signal before and after the silk fibroin is compounded with the rhamnolipid-modified chitosan, indicating that the initial content of the β-sheet fragments of the silk fibroin is less. After homogenizing the silk fibroin aqueous solution, the content of the β-sheet fragments increases, as shown in b (II) in FIG. 1, indicating that mechanical means such as homogenizing shear can effectively induce the conformational transition of the β-sheet fragments of the silk fibroin and enhance its interfacial adsorption properties. At the same time, Figure 1 Figure 1 ​a (II), a (III) in FIG. 1 prove that once the silk fibroin is compounded with the modified chitosan by electrostatic force, the conformation of the silk fibroin will not be transformed to β-sheet again, and the interaction of the two will affect the conformation-induced transformation between the silk fibroin molecules. The particle size and potential of the silk fibroin, the modified chitosan and the blend of the two are shown in Table 1, which proves that the two are compounded by electrostatic force. At the same time, the stability of the emulsion under the distribution strategy of Comparative Example 2 and the direct blend one-step strategy of Comparative Example 1 is tested at room temperature, Figure 5 The results of a in FIG. 1 show that the stability of the distribution strategy is enhanced.

[0041] Example 1 Step S1, accurately weigh 0.5 g of rhamnolipid-modified chitosan, dissolve it in 100 mL of deionized water, stir at 200 rpm at room temperature for 3 h until completely dissolved, and prepare a modified chitosan solution.

[0042] Step S2, weigh 1 g of silk fibroin, dissolve it in 200 mL of deionized water, stir at low speed at room temperature until completely dissolved, and prepare a silk fibroin solution. Then, take 160 mL of the silk fibroin solution and place it in a beaker, and add 50 mL of the oil phase medium-chain triglyceride dropwise under stirring with a homogenizer at 12000 rpm for 3 min to form a primary emulsion.

[0043] Step S3, the primary emulsion is aged at room temperature for 12 h, and its conformation transformation characteristics are used to promote more conformation transformation to β-sheet, so as to ensure that the aged silk fibroin forms a membrane at the interface. After the end, 40 mL of the modified chitosan solution is added dropwise to the above-mentioned aged primary emulsion, and homogenized again at 12000 rpm for 3 min to promote the adsorption of the modified chitosan to the existing silk fibroin interface membrane. At this time, the SF(t=12 h) / CS-RL oil-in-water emulsion under the step-by-step aging strategy is obtained, which is marked as sample A.

[0044] Comparing the above-mentioned Example 1 with Comparative Example 2, the silk fibroin of Example 1 is aged for 12 h after the content of β-sheet is increased by homogenization. At this time, the β-sheet segment is dyed with fluorescence, Figure 1 b (II), b (III) in FIG. 1 show that the aging process can significantly increase the content of β-sheet. It is known that the β-sheet of silk fibroin has hydrophobic characteristics, which can promote its adsorption and stacking at the interface to form Figure 2 a shows the rigid primary interface membrane. Subsequently, after the introduction of the rhamnolipid-modified chitosan solution, Figure 1 c (II) in FIG. 1 shows the interpenetrating network formed by the synergistic effect of electrostatic force and hydrophobic force, which is embodied in Figure 2The high viscoelasticity of the composite interface film in b is significantly different from that of Comparative Example 2. Moreover, the primary interface film formed by the silk fibroin has universality, Figure 5 The replacement of the positively charged modified chitosan by lysozyme or polyethyleneimine in b shows that the replacement can effectively improve the standing stability of the emulsion, which indicates that the primary interface film structure of the silk fibroin provides a good composite substrate. Meanwhile, the distributed aging strategy of the present application can significantly affect the oil-water interfacial tension, Figure 3 The curves of the oil droplet surface tension measured by the pendant drop method in b and c show that the composite film of the silk fibroin and the rhamnolipid-modified chitosan in the distributed aging strategy can more greatly reduce the oil-water interfacial tension and stabilize the emulsion. Moreover Figure 4 b and c also show that there is an obvious interface film structure between the oil droplets, which has high mechanical strength and strong steric hindrance, and inhibits the coalescence of the oil droplets, thereby achieving the standing stability of the emulsion far exceeding that of Comparative Example 2.

[0045] To further prove the beneficial technical effects of the present application, other oil-in-water emulsions with different aging times were prepared under the premise of completely consistent with the above steps in Example 1, only adjusting the aging time to 6 h and 24 h respectively, namely SF(t=6 h) / CS-RL and SF(t=24 h) / CS-RL, so as to reveal the law of the evolution of the interface structure with time by exploring the emulsion state of different aging times (t=6 h, 12 h, 24 h). It is found that the interface film gradually tends to be complete and dense during the aging process of the emulsion system, and the main structural strengthening is usually completed within 6 to 12 h, at which time the composite modified chitosan can maintain excellent interface structure and stability. Specifically, during the aging to 6 hours, the interface film formed by the silk fibroin is insufficient, and the continuity and density of the interface film layer are low. At this time, the composite rhamnolipid-modified chitosan has weak adsorption degree of the interface, which makes the whole interface composite structure loose, and significantly affects the emulsion stability. When the aging time reaches 12 h, the storage modulus of the emulsion will significantly exceed the loss modulus, the system realizes the transition of the gel network, the interface film structure is dense and complete, and the emulsion stability reaches the best. In comparison, the sample aged to 24 h still maintains the gel state, but due to the long-time standing of the internal network or the excessive rearrangement of the interface molecules, local relaxation or weakening begins to appear, and the emulsion stability is obviously insufficient. Therefore, the research shows that the difference in the aging time can significantly affect the rapid and stable assembly characteristics of the oil-water interface and the longer-term stability.

[0046] The particle size, potential and dispersity of each component and the directly blended component in Example 1 are shown in Table 1.

[0047] Table 1

[0048] The data in Table 1 demonstrates that the CS-RL and SF components in Example 1 are tightly combined under electrostatic driving and are uniformly dispersed after combination.

[0049] Example 2 The only difference from Example 1 is that the medium-chain triglycerides in the oil phase were replaced with one of coconut oil, corn oil, or olive oil, and a series of oil-in-water emulsions were prepared in sequence, namely: Sample B (coconut oil), Sample C (corn oil), and Sample D (olive oil).

[0050] Sample A (medium-chain triglycerides) from Example 1, and samples B (coconut oil), C (corn oil), and D (olive oil) emulsions from Example 2 were stored upright at room temperature. Immediately after preparation, all four emulsions (A, B, C, D) exhibited a uniform milky white color, good fluidity, and no oil droplets floating, layering, or sedimentation observed macroscopically. Short-term storage observations were as follows... Figure 6 As shown, after 7 days of storage, all four emulsions remained highly homogeneous, with no flocculation or oil-water separation, and their color and texture showed no significant change compared to the initial preparation stage. Even after 30 days of storage at room temperature, all emulsions remained macroscopically stable, with only very slight emulsification observed even after 9 months. Therefore, the synergistic stabilizing system of this invention is not only applicable to medium-chain triglycerides, but also effective for coconut oil with high saturated fatty acid content, corn oil with predominantly unsaturated fatty acids, and olive oil, demonstrating broad applicability. By changing the oil phase type, the SF(t=12 h) / CS-RL synergistic stabilizing system was systematically demonstrated to have broad applicability and excellent stabilizing effect on various oil phases, which was verified by macroscopic stability testing.

[0051] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A method for preparing oil-in-water emulsion stabilized by silk fibroin interface film and modified chitosan in coordination, characterized in that, The method comprises the following steps: S1, dissolving rhamnolipid modified chitosan in deionized water to prepare a modified chitosan solution with a concentration of 5-10 mg / mL; S2, dissolving silk fibroin in deionized water to prepare a primary emulsion with a concentration of 2-5 mg / mL and adding an oil phase with a volume fraction of 5-40%; S3, aging the primary emulsion for 10-20 hours, then adding 1 / 5-1 / 6 volume of the modified chitosan solution to the primary emulsion and homogenizing to obtain an oil-in-water emulsion with a rigid interface film base formed by silk fibroin at the oil-water interface and stabilized by rhamnolipid modified chitosan.

2. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interface film and modified chitosan according to claim 1, characterized in that, The rhamnolipid modified chitosan is prepared by the following method: (1) adjusting the pH of deionized water to 4.5-5.0 with acetic acid solution, then dissolving chitosan to prepare a chitosan acid solution; (2) dissolving rhamnolipid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a mixture of ethanol and deionized water to prepare a light yellow rhamnolipid modified solution; (3) mixing the chitosan acid solution with the rhamnolipid modified solution for reaction, then dialyzing through a dialysis bag, freeze-drying to obtain the rhamnolipid modified chitosan.

3. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interface film and modified chitosan according to claim 1, characterized in that, The grafting degree of the rhamnolipid modified chitosan is 10-30%.

4. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interface film and modified chitosan according to claim 1, characterized in that, The preparation temperature of the primary emulsion is 10-30 °C.

5. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interfacial film and modified chitosan according to claim 1, characterized in that, The primary emulsion is aged under atmospheric environment for 12-48 h.

6. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interfacial film and modified chitosan according to claim 1, characterized in that, The oil phase comprises at least one of medium-chain triglyceride, coconut oil, soybean oil, corn oil, peanut oil, olive oil, palm oil, and rapeseed oil.

7. The method for preparing the oil-in-water emulsion stabilized by the silk fibroin interfacial film and modified chitosan according to claim 1, characterized in that, The rotation speed of the homogenization is 8000-15000 rpm.

8. An oil-in-water emulsion characterized in that, The preparation method is prepared by any one of claims 1-7.

9. The oil-in-water emulsion of claim 8 for emulsion stabilization in food, cosmetics, pharmaceuticals, and personal care products.

Images