W1 / O / W2 double-layer emulsion as well as preparation method and application thereof

Abstract

The invention provides a W1 / O / W2 double-layer emulsion and a preparation method and application thereof, and relates to the technical field of biological material encapsulation, the preparation method of the W1 / O / W2 double-layer emulsion comprises the following steps: mixing protein, polyphenol and a solvent, and heating to obtain a protein compound; mixing the protein complex with a polysaccharide solution to obtain a ternary complex; mixing an emulsifier, the first water phase and the oil phase, and dispersing to obtain a W1 / O emulsion; and mixing the ternary complex, the second water phase and the W1 / O emulsion, and dispersing to obtain a W1 / O / W2 double-layer emulsion. According to the technical scheme, the protein, the polyphenol and the polysaccharide solution are adopted to prepare the ternary complex, the ternary complex serves as the emulsifier between the second water phase and the W1 / O emulsion, the emulsion can be further emulsified to prepare the double-layer emulsion, and therefore the stability of the first water phase in the double-layer emulsion is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial encapsulation, and particularly relates to a W 1 / O / W 2 double emulsion and its preparation method and application. Background Art

[0002] Probiotics, as important microecological factors for regulating the intestinal flora homeostasis, enhancing the mucosal barrier function and alleviating inflammation, have broad application prospects in the fields of functional foods and intestinal disease intervention. Research shows that specific strains such as Escherichia coli Nissle 1917 (EcN) exhibit good anti-inflammatory and immunomodulatory effects in inflammatory bowel disease (IBD). However, after oral ingestion, probiotics need to cross complex gastrointestinal environments such as gastric acid, bile salts, and digestive enzymes, and are extremely vulnerable to damage, resulting in a sharp reduction in the number of viable bacteria and a decrease in bioavailability, which limits their effective colonization and function in the body. To improve the survival rate and targeted release effect of probiotics in the body, constructing a delivery system with protective and controlled release capabilities has become an important direction in current probiotic research.

[0003] In recent years, emulsion-based delivery systems, especially water-in-oil-in-water (W / O / W) multiple emulsions, have been widely used for delivering active substances such as small molecule drugs, proteins, polypeptides, and probiotics due to their "inner aqueous phase encapsulation - oil phase barrier - outer aqueous phase stabilization" structure. Multiple emulsions can not only construct an effective physical barrier through the oil phase to resist the destruction of the gastrointestinal digestion environment, but also precisely regulate the release behavior by controlling the type, ratio, and interfacial structure of emulsifiers. Research shows that the selection of food-grade natural emulsifiers (such as modified starch, protein, chitosan, etc.) can effectively improve the stability and biocompatibility of emulsions, making the emulsion system have good oral safety and structural stability.

[0004] Despite certain progress in the delivery research of multiple emulsions, there are still several key bottlenecks in the existing technologies. One is limited stability: the double emulsion structure is thermodynamically unstable and prone to demulsification and phase separation, affecting its protection and release functions. The second is the lack of targeting and functional synergy: traditional emulsion systems mostly focus on physical protection and lack the targeted release design for intestinal inflammation sites; at the same time, it is difficult to synergistically play a therapeutic role with anti-inflammatory factors or antioxidants. The third is the single structure: there is a lack of multi-level and biofunctionally complementary delivery strategies, making it difficult to meet the multi-objective requirements of "probiotic protection - colonization and release - intestinal repair". The fourth is the weak mechanism research: most studies focus on in vitro release or survival rate, lacking a systematic explanation of its comprehensive effects on regulating inflammation, repairing barriers, and intervening in the flora in the body.

[0005] Based on the above problems, there is an urgent need to develop a probiotic delivery system with protection, targeting, and multifunctional synergistic effects. This study aims to construct a functional delivery platform for intestinal inflammation intervention based on the structure of W / O / W multiple emulsions, combined with interfacial regulation of natural emulsifiers, bio-targeted functional modification, and co-loading of active ingredients. By introducing anti-inflammatory and antioxidant active substances such as natural polysaccharides and plant polyphenols into the outer aqueous phase, and utilizing the targeting of natural polysaccharides to intestinal cells and the intestinal adhesion of natural polyphenols, precise release and synergistic treatment of probiotics at the colon site can be achieved, providing new technical support and theoretical basis for the microecological intervention of inflammatory bowel disease. Summary of the Invention

[0006] The main object of the present invention is to propose a W 1 / O / W 2 double emulsion and its preparation method and application, aiming to improve the survival rate of probiotics encapsulated in the W 1 / O / W 2 double emulsion.

[0007] To achieve the above object, the present invention proposes a preparation method of a W 1 / O / W 2 double emulsion, and the preparation method of the W 1 / O / W 2 double emulsion comprises the following steps:

[0008] Mix and heat the protein, polyphenol, and solvent to obtain a protein complex;

[0009] Mix the protein complex with the polysaccharide solution to obtain a ternary complex;

[0010] Mix the emulsifier, the first aqueous phase, and the oil phase and disperse them to obtain a W 1 / O emulsion;

[0011] Mix the ternary complex, the second aqueous phase, and the W 1 / O emulsion and disperse them to obtain a W 1 / O / W 2 double emulsion.

[0012] In one embodiment, the protein includes zein; and / or,

[0013] the polyphenol includes tannic acid; and / or,

[0014] the polysaccharide solution includes sodium alginate solution; and / or,

[0015] the solvent includes an aqueous solution of ethanol.

[0016] In one embodiment, the step of mixing and heating the protein, polyphenol, and solvent to obtain a protein complex comprises the following steps:

[0017] After mixing the protein, polyphenol and solvent, adjust the pH value to 3 - 7, heat, disperse and dry to obtain the protein complex.

[0018] In one embodiment, in the step of mixing and heating the protein, polyphenol and solvent, the mass ratio of the protein to the polyphenol is 1:(0.1 - 0.5); and / or,

[0019] The heating duration is 30 - 60 min; and / or,

[0020] The heating temperature is 50 - 60 °C.

[0021] In one embodiment, in the step of mixing the protein complex with the polysaccharide solution, the mass concentration of the protein in the protein complex is 15 - 20 g / L; and / or,

[0022] The mass concentration of the polysaccharide in the polysaccharide solution is 15 - 20 g / L; and / or,

[0023] The volume ratio of the protein complex to the polysaccharide solution is (1 - 5):1.

[0024] In one embodiment, in the step of mixing and dispersing the emulsifier, the first aqueous phase and the oil phase, the emulsifier includes PGPR emulsifier; and / or,

[0025] The volume ratio of the first aqueous phase to the oil phase is (1 - 5):(5 - 9), and the mass ratio of the emulsifier to the oil phase is (1 - 5):100.

[0026] In one embodiment, in the step of mixing and dispersing the ternary complex, the second aqueous phase and the W 1 / O emulsion, the volume ratio of the second aqueous phase to the W 1 / O emulsion is (4 - 8):(2 - 6); and / or,

[0027] The mass ratio of the ternary complex to the second aqueous phase is (1 - 5):100.

[0028] In one embodiment, in the step of mixing and dispersing the emulsifier, the first aqueous phase and the oil phase, the dispersion speed is 12000 - 18000 rpm, and the separation duration is 2 - 5 min; and / or,

[0029] In the step of mixing and dispersing the ternary complex, the second aqueous phase and the W 1 / O emulsion, the dispersion speed is 12000 - 15000 rpm, and the separation duration is 1 - 3 min.

[0030] The present invention also provides a W 1 / O / W 2 double - layer emulsion, the W 1 / O / W 2The double emulsion is prepared according to the W as described above 1 / O / W 2 by the preparation method of the double emulsion.

[0031] The present invention also provides a W 1 / O / W 2 application of the double emulsion in encapsulating biological materials, wherein the W 1 / O / W 2 double emulsion includes the W 1 / O / W 2 double emulsion as described above;

[0032] The first aqueous phase includes the biological material to be encapsulated.

[0033] The technical solution of the present invention prepares a ternary complex by using protein, polyphenol and polysaccharide solutions, and uses it as an emulsifier between the second aqueous phase and the W 1 / O emulsion, and can further emulsify the emulsion to prepare a double emulsion, thereby improving the stability of the first aqueous phase in the double emulsion; by dispersing the biological material to be encapsulated in the first aqueous phase, it can be protected by the oil phase and the second aqueous phase, thereby improving the biological activity of the biological material to be encapsulated. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0035] Figure 1 It is a graph of the detection results of the EcN survival rate in Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention;

[0036] Figure 2 It is a graph of the results of the mouse experiment in the detection of the drug delivery efficiency in Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention;

[0037] Figure 3 It is a statistical graph of the survival rate of mice in the detection of the drug delivery efficiency in Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention;

[0038] Figure 4 It is a statistical graph of the disease activity index of mice in the detection of the drug delivery efficiency in Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention;

[0039] Figure 5 It is a statistical graph of the colon length of mice in the detection of the drug delivery efficiency in Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention;

[0040] Figure 6 Macroscopic images of the colon of mice in the drug administration efficiency detection experiments for Examples 1 to 2 and Comparative Examples 1 to 2 provided by the present invention.

[0041] The realization of the object, functional features and advantages of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0043] It should be noted that for those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, or solution B, or a solution where A and B are satisfied simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those skilled in the art can implement them. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0044] Currently, in the prior art, when using single-layer emulsions (such as W / O or O / W emulsions) to encapsulate probiotics, although the survival rate of probiotics can be improved to a certain extent, there are still problems of insufficient protection under extreme conditions.

[0045] In view of this, the present invention proposes a method for preparing a W 1 / O / W 2 double-layer emulsion. The method for preparing the W 1 / O / W 2 double-layer emulsion includes the following steps: mixing protein, polyphenol and a solvent and heating to obtain a protein complex; mixing the protein complex with a polysaccharide solution to obtain a ternary complex; mixing an emulsifier, a first aqueous phase and an oil phase and dispersing them to obtain a W 1 / O emulsion; mixing the ternary complex, a second aqueous phase and the W 1 / O emulsion and dispersing them to obtain a W 1 / O / W 2 double-layer emulsion.

[0046] It should be noted that the first aqueous phase and the second aqueous phase can be water or polar solvents such as acetonitrile, and are not limited herein.

[0047] The technical solution of the present invention prepares a ternary complex by using a protein, a polyphenol and a polysaccharide solution as an emulsifier between the second aqueous phase and the W 1 / O emulsion, and can further emulsify the single-layer emulsion to prepare a double-layer emulsion, thereby improving the stability of the first aqueous phase in the double-layer emulsion.

[0048] In one embodiment, the protein includes zein; and / or, the polyphenol includes tannic acid; and / or, the polysaccharide solution includes sodium alginate solution; and / or, the solvent includes an aqueous solution of ethanol.

[0049] The technical solution of the present invention improves the non-polarity of the ternary complex by using zein, so that the ternary complex can be dissolved in the oil phase; by using tannic acid, it can react with the protein to form a ternary complex and improve the polarity of the ternary complex, so that the ternary complex can be dissolved in the aqueous phase; by using sodium alginate, it can adsorb on the oil-water interface through its numerous hydroxyl groups, reduce the interfacial tension, promote the emulsification process, and improve the physical stability of the emulsion. At the same time, due to the good biocompatibility and low toxicity of sodium alginate, the stability of the bioactive materials to be encapsulated can be further improved. The types of the above-mentioned protein, polyphenol and polysaccharide can be defined separately or simultaneously; when defined simultaneously, the stability of the ternary complex is high.

[0050] In one embodiment, the step of mixing the protein, the polyphenol and the solvent and heating to obtain the protein complex includes the following steps: after mixing the protein, the polyphenol and the solvent, adjusting the pH value to 3-7, heating, dispersing and drying to obtain the protein complex.

[0051] The technical solution of the present invention maintains a weak acid environment by adjusting the pH value to 3-7, which can improve the stability of the protein complex; if the pH is less than 3, zein precipitates in a strong acid environment and the stability decreases; if the pH is greater than 7, the stability of the polyphenol decreases in an alkaline environment.

[0052] In one embodiment, in the step of mixing the protein, the polyphenol and the solvent and heating, the mass ratio of the protein to the polyphenol is 1:(0.1-0.5); and / or, the heating duration is 30-60 min; and / or, the heating temperature is 50-60 °C.

[0053] The technical solution of the present invention can adjust the polarity of the prepared protein complex by controlling the mass ratio of the protein to the polyphenol, so as to adjust the emulsifying ability of the prepared ternary complex to be suitable for different aqueous phases and oil phases, and further improve the W 1 / O / W 2 stability of the double-layer emulsion.

[0054] In one embodiment, in the step of mixing the protein complex with the polysaccharide solution, the mass concentration of the protein in the protein complex is 15-20 g / L; and / or, the mass concentration of the polysaccharide in the polysaccharide solution is 15-20 g / L; and / or, the volume ratio of the protein complex to the polysaccharide solution is (1-5):1.

[0055] The technical solution of the present invention adjusts the lipophilicity of the ternary complex by adjusting the mass concentration of the protein, so that the ternary complex can be dissolved in the oil phase; by adjusting the mass concentration of the polyphenol, a ternary complex can be formed by reacting with the protein, and the hydrophilicity of the ternary complex can be improved, so that the ternary complex can be dissolved in the aqueous phase; by adjusting the volume ratio of the protein complex to the polysaccharide solution to adjust the content of the polysaccharide, it can adsorb on the oil-water interface through its numerous hydroxyl groups, reduce the interfacial tension, promote the emulsification process, and improve the physical stability of the emulsion.

[0056] In one embodiment, in the step of mixing and dispersing the emulsifier, the first aqueous phase and the oil phase, the emulsifier includes PGPR emulsifier; and / or, the volume ratio of the first aqueous phase to the oil phase is (1-5):(5-9), and the mass ratio of the emulsifier to the oil phase is (1-5):100.

[0057] The technical solution of the present invention can stabilize the water-in-oil (W / O) system by using PGPR emulsifier, so as to maintain the stable mixing of the aqueous phase and the oil phase and prevent separation; by adjusting the volume ratio of the first aqueous phase to the oil phase, the proportion of the first aqueous phase in the W 1 / O emulsion can be adjusted, thereby adjusting the concentration of the biological material to be applicable to different fields; by adjusting the mass ratio of the emulsifier to the oil phase, the addition amount of the emulsifier can be adjusted, thereby adjusting the emulsification degree of the W 1 / O emulsion, and further adjusting the fluidity and stability of the W 1 / O emulsion.

[0058] In one embodiment, in the step of mixing and dispersing the ternary complex, the second aqueous phase and the W 1 / O emulsion, the volume ratio of the second aqueous phase to the W 1 / O emulsion is (4-8):(2-6); and / or, the mass ratio of the ternary complex to the second aqueous phase is (1-5):100.

[0059] The technical solution of the present invention can adjust the proportion of the W 1 / O emulsion in the W 1 / O / W 2 double-layer emulsion by adjusting the volume ratio of the second aqueous phase to the W 1 / O emulsion, thereby adjusting the concentration of the biological material to be applicable to different fields; by adjusting the mass ratio of the ternary complex to the second aqueous phase, the addition amount of the ternary complex can be adjusted, thereby adjusting the W1 / O / W 2 the emulsification degree of the double emulsion, thereby adjusting W 1 / O / W 2 the fluidity and stability of the double emulsion.

[0060] In one embodiment, in the step of mixing and dispersing the emulsifier, the first aqueous phase and the oil phase, the dispersion rotation speed is 12,000 to 18,000 rpm, and the separation duration is 2 to 5 minutes; and / or, in the step of mixing and dispersing the ternary complex, the second aqueous phase and the W 1 / O emulsion, the dispersion rotation speed is 12,000 to 15,000 rpm, and the separation duration is 1 to 3 minutes.

[0061] The technical solution of the present invention can fully disperse the first aqueous phase and the oil phase by adjusting the dispersion rotation speed and duration, improve the uniformity of the prepared W 1 / O emulsion, and further improve the uniformity of the W 1 / O / W 2 double emulsion, thereby improving the encapsulation effect of the W 1 / O / W 2 double emulsion on biomaterials and enhancing the stability of biomaterials.

[0062] The present invention also provides a W 1 / O / W 2 double emulsion, and the W 1 / O / W 2 double emulsion is prepared according to the preparation method of the W 1 / O / W 2 double emulsion as described above.

[0063] Since this W 1 / O / W 2 double emulsion adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be elaborated herein one by one.

[0064] The present invention also provides an application of a W 1 / O / W 2 double emulsion in encapsulating biomaterials, and the W 1 / O / W 2 double emulsion includes the W 1 / O / W 2 double emulsion as described above; the first aqueous phase includes the biomaterials to be encapsulated.

[0065] The technical solution of the present invention disperses the biomaterial to be encapsulated in the first aqueous phase, which can be protected by the oil phase and the second aqueous phase, thereby improving the biological activity of the biomaterial to be encapsulated. It should be noted that the biomaterial to be encapsulated can be probiotics or proteins, and the specific types are not limited, as long as it can maintain biological activity in water; specifically, in some embodiments of the present invention, the biomaterial to be encapsulated is Escherichia coli. In actual application, the biomaterial to be encapsulated can also be probiotics such as Bifidobacterium, Lactobacillus plantarum or Lactobacillus rhamnosus. The W of the present invention 1 / O / W 2 The specific components of the double emulsion have nothing to do with the biomaterial to be encapsulated. As long as it can maintain biological activity in the aqueous phase, it can be used as the biomaterial to be encapsulated, and thus be protected by the double emulsion.

[0066] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0067] Example 1

[0068] This example provides a W 1 / O / W 2 double emulsion, and its preparation method includes the following steps:

[0069] 1. Dissolve 2.5 g of zein powder in a 70% ethanol-aqueous solution, and stir until completely dissolved. Then add tannic acid (TA) powder, and the mass ratio of zein to tannic acid is 1:0.4. After mixing evenly, adjust the pH value of the mixture to 3, and heat it in a water bath at 55 °C for 50 minutes. Under the condition of rapid stirring, pour the mixture into 2.5 times the volume of deionized water in a thin stream, and continuously stir for 15 minutes. Use a rotary evaporator to remove ethanol and part of the water until the zein concentration in the solution is 3%. The prepared particle solution is stored in a 4 °C refrigerator or vacuum freeze-dried into a solid powder, named ZT-0.4;

[0070] 2. Mix the ZT-0.4 (1.8%) solution and the sodium alginate (SA, 1.8%) solution in a volume ratio of 5:1 to form a ZTS-5 ternary composite particle;

[0071] 3. Inoculate Escherichia coli Nissle 1917 (EcN) into a liquid medium and culture it overnight at 37 °C. Centrifuge at 3000 rpm for 5 minutes to collect the bacteria. Add EcN to 30 ml of 10 mM Tris-HCl buffer (pH 8.5) with a dopamine content of 0.5 mg / ml. During the 2-hour reaction process, add 10 μl of carboxymethyl chitosan solution (5 mg / ml) to the mixture every 30 minutes. After centrifuging at 3000 rpm for 5 minutes, resuspend the bacteria in 30 ml of Tris-HCl buffer, add another 10 μl of carboxymethyl chitosan solution (5 mg / ml), react for 30 minutes, and centrifuge to obtain the EcN@PCS bacterial solution;

[0072] 4. Uniformly disperse the EcN@PCS bacterial solution in the 1 inner aqueous phase. Use the lipophilic emulsifier PGPR (3%) dissolved in edible oil to obtain the oil phase. Mix the inner aqueous phase and the oil phase at a volume ratio of 1:9 and disperse them at high speed in a high-speed disperser at 15000 rpm for 3 minutes to obtain the 1 W 1 / O primary emulsion;

[0073] 5. Then add the 1 W 1 / O primary emulsion to the ZTS-5 solution at a volume ratio of 4:6 and disperse it at 13000 rpm for 2 minutes to obtain the 2 W 1 / O / W 2 double emulsion. The concentration of the EcN@PCS bacterial solution in the 9 W

[0074] / O / W double emulsion is 5×10

[0075] CFU / mL. Finally, prepare the W-EcN@PCS double emulsion. 1 / O / W 2 double emulsion. Its preparation method is similar to that in Example 1, except that step 3 is not performed. Step 4 includes the following steps:

[0076] Uniformly disperse the Escherichia coli Nissle 1917 (EcN) bacterial solution in the 1 inner aqueous phase. Use the lipophilic emulsifier PGPR (3%) dissolved in edible oil to obtain the oil phase. Mix the inner aqueous phase and the oil phase at a volume ratio of 1:9 and disperse them at high speed in a high-speed disperser at 15000 rpm for 3 minutes to obtain the 1 W 1 / O primary emulsion.

[0077] Finally, prepare the W-EcN double emulsion in this example.

[0078] Example 3

[0079] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that in step 1, the mass ratio of zein to tannic acid is 1:0.1, and the solid powder obtained in step 1 is named ZT-0.1.

[0080] Example 4

[0081] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that in step 1, the mass ratio of zein to tannic acid is 1:0.2, and the solid powder obtained in step 1 is named ZT-0.2.

[0082] Example 5

[0083] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that in step 1, the mass ratio of zein to tannic acid is 1:0.3, and the solid powder obtained in step 1 is named ZT-0.3.

[0084] Example 6

[0085] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that in step 1, the mass ratio of zein to tannic acid is 1:0.5, and the solid powder obtained in step 1 is named ZT-0.5.

[0086] Example 7

[0087] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that in step 4, the inner aqueous phase and the oil phase are mixed at a volume ratio of 5:5, and in step 5, the volume ratio of the W 1 / O primary emulsion to the ZTS-5 solution is 8:2. 1 / O primary emulsion and the ZTS-5 solution is 8:2.

[0088] Example 8

[0089] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1. The difference is that the probiotic is Bifidobacterium;

[0090] Example 9

[0091] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1, except that the probiotic is Lactobacillus plantarum;

[0092] Example 10

[0093] This example provides a W 1 / O / W 2 double emulsion, and its preparation method is similar to that in Example 1, except that the probiotic is Lactobacillus rhamnosus;

[0094] Comparative Example 1

[0095] This comparative example provides a modified Escherichia coli, and its preparation method includes the following steps:

[0096] Inoculate Escherichia coli Nissle 1917 (EcN) in a liquid medium and culture it overnight at 37 °C. Centrifuge at 3000 rpm for 5 minutes to collect the bacteria. Add EcN to 30 ml of 10 mM Tris-HCl buffer (pH 8.5), and the dopamine content is 0.5 mg / ml. During the 2-hour reaction process, add 10 μl of carboxymethyl chitosan solution (5 mg / ml) to the mixture every 30 minutes.

[0097] After centrifuging at 3000 rpm for 5 minutes, resuspend the bacteria in 30 ml of Tris-HCl buffer, add another 10 μl of carboxymethyl chitosan solution (5 mg / ml), react for 30 minutes, and centrifuge to obtain the EcN@PCS bacterial solution, which is stored at 4 °C for later use.

[0098] Comparative Example 2

[0099] This comparative example provides an Escherichia coli, specifically Escherichia coli Nissle 1917 (EcN).

[0100] EcN Survival Rate Detection Experiment

[0101] As Figure 1 shown in a, the survival rate of EcN continuously decreases over time in an environment with pH = 2. Among them, EcN encapsulated by double emulsion has a higher survival rate, and its survival rate after two hours is higher than that of EcN alone. This may be because when the pH is adjusted to strong acid, due to the flocculation effect of anionic alginate molecules bridging cationic protein-coated oil droplets, the double emulsion forms a gel and has the ability to resist strong acid.

[0102] In addition, as Figure 1As shown in Fig. b, double emulsion encapsulation and dopamine modification also increased the survival rate of EcN in strong base (pH 11). Among them, emulsion encapsulation had a better protective effect on EcN, and the simultaneous dopamine modification and double emulsion encapsulation had the best protective effect on EcN.

[0103] As Figure 1 shown in Fig. c, after half an hour of ultraviolet light irradiation, the activities of EcN (Comparative Example 2), EcN@PCS (Comparative Example 1), W-EcN (Example 2), and W-EcN@PCS (Example 1) were 4.69±0.53 logCFU / g, 5.08±0.32 logCFU / g, 8.54±0.12 logCFU / g, and 8.39±0.08 logCFU / g, respectively. Moreover, free EcN and dopamine-coated EcN had almost no survival after 2 h, while double emulsion and ECN surface modification significantly improved the survival rate of EcN, which may be because the double emulsion had poor transmission ability and could prevent EcN from being irradiated by ultraviolet light.

[0104] Drug delivery efficiency detection experiment

[0105] Establishment of colitis model

[0106] In this experiment, 6-8-week-old SPF-grade male BALB / C mice were purchased from Hubei Center for Disease Control and Prevention. After one week of adaptive feeding, they were randomly divided into 6 groups, namely normal group (Control), model group (DSS), EcN group, EcN@PCS group, W-EcN group, and W-EcN@PCS group, with 15 mice in each group. From the second week, different samples were intragastrically administered to the mice according to their body weight (0.1 mL / 10 g) every day. Among them, the normal group and the model group were intragastrically administered PBS, and the remaining groups were intragastrically administered EcN, EcN@PCS, W-EcN, and W-EcN@PCS, respectively, with the EcN content being 1×10 8 CFU / d. From the third week, DSS was used to induce colitis. Except for the normal group, the drinking water of the remaining groups was changed to 3% (w / v) DSS solution. From the start of DSS induction, the mice were weighed and recorded every day, and the fecal traits, occult blood or bloody stool conditions of the mice were observed daily.

[0107] During the entire DSS modeling period, the DAI score of the mice was mainly evaluated according to the weight loss, bloody stool, and fecal formation of the mice ( Figure 3 ).

[0108] During the experiment, the body weight of the mice in the normal group increased steadily, the fecal state was normal, and there was no occult blood condition, so the DAI index was close to 0.

[0109] Compared with the normal group, after administration of DSS, the mice in the model group showed phenomena such as dull hair color, listlessness, and reduced appetite. Moreover, fecal occult blood was detected on the second day and gradually became severe. Five days later, visible blood in the stool began to appear, the feces were loose, soft, unformed, and easy to stick to the anus. The mice began to visibly lose weight, so the DAI index was high. However, the W-EcN and W-EcN@PCS groups alleviated this phenomenon. Obvious blood in the stool gradually appeared only on the seventh day, the feces were more formed, and the degree of weight loss was smaller.

[0110] According to the macroscopic pictures of the mice 9 days after modeling, it can also be seen that the model group and the EcN group of mice were thinner in appearance and showed severe blood in the stool after modeling, and mouse deaths occurred on the seventh and eighth days respectively. However, administration of EcN@PCS and W-EcN alleviated both the weight loss and the blood in the stool in the mouse colitis phenomenon. The colitis phenomenon in the W-EcN@PCS group was less, and the weight loss and blood in the stool were greatly alleviated ( Figure 2 and Figure 3 ).

[0111] Therefore, during the entire DSS modeling process, the DAI score of the model group increased every day and was significantly higher than that of the normal group and the W-EcN@PCS group. However, W-EcN@PCS could significantly alleviate the physiological condition of colitis caused by DSS in mice, and the DAI index was smaller ( Figure 4 ).

[0112] In addition, the colon length is also one of the apparent phenomena reflecting the degree of mouse colitis. The more severe the colitis, the higher the degree of colon damage and the shorter the colon length. As Figure 5 and Figure 6 shown, the colon length of the mice in the model group (5.70 ± 1.10 cm) was extremely significantly shorter than that of the normal group (8.36 ± 0.50 cm), indicating that the colon of the mice in the model group was severely damaged. Among all the treatment groups, the W-EcN@PCS group showed the largest colon length (7.86 ± 1.14 cm), which was significantly increased compared with the model group. This may be because EcN protected by both dopamine and emulsion had a higher survival rate in the gastrointestinal tract and could better exert the effect of EcN in resisting enteritis, effectively alleviating mouse colitis.

[0113] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structural transformation made using the content of the specification of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A method for preparing a W1 / O / W2 double-layer emulsion, characterized in that: The preparation method of the W1 / O / W2 double-layer emulsion comprises the following steps: The protein, polyphenol and solvent are mixed and heated to obtain a protein complex; The protein complex is mixed with the polysaccharide solution to obtain a ternary complex; The emulsifier, the first water phase and the oil phase are mixed and dispersed to obtain a W1 / O emulsion; The ternary complex, the second aqueous phase and the W1 / O emulsion are mixed and dispersed to obtain a W1 / O / W2 double-layer emulsion.

2. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: The protein comprises zein; and / or, The polyphenols include tannic acid; and / or, The polysaccharide solution comprises a sodium alginate solution; and / or, The solvent includes an aqueous solution of ethanol.

3. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: The step of mixing and heating the protein, polyphenol and solvent to obtain the protein complex comprises the following steps: After the protein, polyphenol and solvent are mixed, the pH value is adjusted to 3-7, and the mixture is heated, dispersed and dried to obtain a protein complex.

4. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: In the step of mixing and heating the protein, polyphenols and solvent, the mass ratio of the protein to the polyphenols is 1:(0.1-0.5); and / or, The heating time is 30 to 60 minutes; and / or, The heating temperature is 50-60°C.

5. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: In the step of mixing the protein complex with the polysaccharide solution, the mass concentration of the protein in the protein complex is 15 to 20 g / L; and / or, The mass concentration of polysaccharide in the polysaccharide solution is 15-20 g / L; and / or, The volume ratio of the protein complex to the polysaccharide solution is (1-5):

1.

6. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: In the step of dispersing the emulsifier, the first aqueous phase and the oil phase after mixing, the emulsifier comprises a PGPR emulsifier; and / or, The volume ratio of the first water phase to the oil phase is (1-5):(5-9), and the mass ratio of the emulsifier to the oil phase is (1-5):

100.

7. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: In the step of mixing and dispersing the ternary complex, the second aqueous phase and the W1 / O emulsion, the volume ratio of the second aqueous phase to the W1 / O emulsion is (4-8): (2-6); and / or, The mass ratio of the ternary complex to the second aqueous phase is (1-5):

100.

8. The method for preparing the W1 / O / W2 double-layer emulsion according to claim 1, characterized in that: In the step of dispersing the emulsifier, the first aqueous phase and the oil phase after mixing, the dispersion speed is 12000-18000 rpm, and the separation time is 2-5 minutes; and / or, In the step of dispersing the ternary complex, the second aqueous phase and the W1 / O emulsion after mixing, the dispersion rotation speed is 12000-15000 rpm, and the separation time is 1-3 minutes.

9. A W1 / O / W2 double-layer emulsion, characterized in that: The W1 / O / W2 double-layer emulsion is prepared according to the preparation method of the W1 / O / W2 double-layer emulsion according to any one of claims 1 to 8.

10. Application of a W1 / O / W2 double-layer emulsion in encapsulating biomaterials, characterized in that: The W1 / O / W2 double-layer emulsion comprises the W1 / O / W2 double-layer emulsion as claimed in claim 9; The first aqueous phase includes the biological material to be encapsulated.

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