Double emulsion, double emulsion for encapsulating probiotics, and preparation method and application of double emulsion

Abstract

The invention belongs to the technical field of emulsions, and particularly relates to a double-emulsion, a probiotic-encapsulated double-emulsion and a preparation method and application of the double-emulsion. The double-emulsion provided by the invention is prepared by taking ultrasonic modified ovomucin as an outer layer to wrap water-in-oil type emulsion droplets and through two-step shearing and homogenizing, the preparation process flow is simple, the quality is easy to control, and the raw materials can be selected from food raw materials, so that the double-emulsion is safe and reliable. The double emulsion prepared by the preparation method can be used for encapsulating various substances such as water-soluble drugs and probiotics, has high encapsulation efficiency, can obviously improve the endurance capacity of the encapsulated substance to gastrointestinal digestive juice after oral administration, and improves the stability of the encapsulated substance in the gastrointestinal digestive juice. The probiotic-encapsulated double emulsion prepared on the basis of the double emulsion system can obviously improve the endurance capacity of the probiotics to various environments such as acidity, alkalinity, cholate, digestive enzymes and the like, and is helpful for the probiotics to exert effects in intestinal tracts.

Description

Technical Field

[0001] The invention belongs to the technical field of emulsions, and in particular relates to a double emulsion and a probiotic-encapsulated double emulsion as well as a preparation method and application thereof. Background Art

[0002] Drugs and probiotics play an important role in treating diseases and maintaining health, but many drugs and probiotics are only absorbed or work in the colon, and are intolerant to gastrointestinal digestive juices or irritate the stomach and small intestine. Direct administration will lead to decreased efficacy and even adverse reactions such as gastrointestinal discomfort. The current solution is to make them into colon-localized preparations to avoid contact with gastric and intestinal juices. However, on the one hand, the preparation process of such preparations is cumbersome, strict requirements are placed on process parameters, and product quality is difficult to control. On the other hand, most of the excipients of such preparations can only be chemically synthesized, and residual solvents, intermediate impurities, by-products, etc. will bring certain safety hazards to the human body.

[0003] Therefore, it is necessary to demand new delivery systems to deliver drugs and probiotics to the intestine without the use of chemical synthetic excipients so that they can exert better effects. Summary of the invention

[0004] In view of the above problems, the present invention provides a double emulsion and a double emulsion encapsulating probiotics, and a preparation method and application thereof. The double emulsion provided by the present invention can be prepared from raw materials of natural origin, is safe for the human body, and its preparation method is simple and the quality is easy to control. The double emulsion has a high encapsulation rate for substances such as probiotics, can significantly improve the tolerance of its encapsulated substances to gastrointestinal digestive juices, and can be used as a highly efficient embedding and delivery of various substances such as drugs and probiotics.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical scheme: The first aspect of the present invention provides a method for preparing a double emulsion, which specifically comprises the following steps: S1, shearing and mixing the water phase and the oil phase containing the water-in-oil emulsifier in a volume ratio of 1-4:6-9, and homogenizing them into an emulsion as the inner layer emulsion; S2, shearing and mixing the inner layer emulsion and the outer layer solution at a volume ratio of 1-4:6-9, and homogenizing into an emulsion to obtain a water-in-oil-in-water (W1 / O / W2) double emulsion (DE), wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

[0006] According to the above preparation method, ultrasonically modified ovomucin (OVM) is used as an outer layer stabilizer to wrap the oil-in-water type emulsion droplets, and a water-in-oil-in-water double emulsion is prepared by two-step shearing and homogenization. Compared with the existing enteric preparation process, the preparation method has a simple process flow and is easy to control the quality. In addition, the ovomucin in the preparation method is extracted from eggs, and other raw materials can also be selected from food raw materials, so the raw materials are safe and have certain probiotic effects.

[0007] The double emulsion prepared by the preparation method can be used to encapsulate various substances such as water-soluble drugs and probiotics, with a high encapsulation rate, and can significantly improve the tolerance of the encapsulated drugs, probiotics and other substances to gastrointestinal digestive fluid after oral administration, and improve the stability of the encapsulated substances in the gastrointestinal digestive fluid, thereby helping to improve the efficacy of the substances in the intestine. Therefore, the double emulsion prepared by the preparation method can be used as a highly efficient embedding and delivery of various substances such as drugs and probiotics. In addition, the double emulsion prepared by the preparation method can also be further made into other embedding products that are convenient for daily or clinical applications.

[0008] Preferably, the oil phase in S1 is edible oil, which can be selected from common edible oils that do not react with proteins, such as olive oil and medium-chain triglycerides.

[0009] Preferably, the volume ratio of the water phase to the oil phase in S1 is 2:8.

[0010] Preferably, the shearing speed in S1 is 10000-14000 rpm, and the homogenization time is not less than 2 min. More preferably, the shearing speed is 12000.

[0011] Preferably, the preparation method of the ultrasonically modified ovomucin described in S2 is: dissolving the ovomucin in distilled water at room temperature to a concentration of 2% to 3%wt, and subjecting the solution to pulsed ultrasonic treatment for 20 to 40 min at an ultrasonic power of 350 to 450 W at a temperature not exceeding 40°C, wherein the pulsed ultrasonic treatment is: on for 3 seconds and off for 5 seconds.

[0012] Exemplarily, ovomucin can be prepared by the following method: after separating the egg yolk from the egg white, the egg white is diluted with 5 volumes of 0.1 mol / L NaCl solution and stirred for 30 min, and then the pH of the mixture is adjusted to 6 with 1 mol / L HCl. Subsequently, the dispersion is placed at 4°C for 12 h and centrifuged at 8000g for 20 min at 4°C. The precipitate is added to a 0.5 mol / L NaCl solution and placed at 4°C for 6 h. Then, the precipitate is centrifuged at 8000g for 20 min at 4°C, and the precipitate is repeatedly washed with distilled water, and finally freeze-dried in a vacuum freeze dryer for 48 hours to obtain ovomucin.

[0013] Preferably, the volume ratio of the inner layer emulsion to the outer layer solution in S2 is 3-4:6-7, and more preferably 3:7.

[0014] Preferably, the concentration of the ultrasonically modified ovomucin aqueous solution in S2 is 2.5%wt.

[0015] Preferably, the pH of the ultrasonically modified ovomucin aqueous solution in S2 is 8.5-9.5, and more preferably the pH is 9.

[0016] Preferably, the shearing speed in S2 is 10000-14000 rpm, and the homogenization time is 2-4 min.

[0017] Preferably, the water-in-oil emulsifier is polyglycerol polyricinoleate (PGPR).

[0018] More preferably, the amount of the water-in-oil emulsifier added is 2-8% v / v of the oil phase, and more preferably 4% v / v.

[0019] The second aspect of the present invention provides a double emulsion, and the double emulsion delivery system is prepared according to the above preparation method.

[0020] In a third aspect, the present invention provides use of the double emulsion as a drug encapsulation and delivery system.

[0021] In a fourth aspect, the present invention provides use of the double emulsion as a probiotic encapsulation delivery system.

[0022] In a fifth aspect, the present invention provides a method for preparing a double emulsion encapsulating probiotics, which specifically comprises the following steps: Step a, dispersing probiotics in a NaCl aqueous solution as an aqueous phase; shearing and mixing the aqueous phase and an oil phase containing an oil-in-water emulsifier at a volume ratio of 1-4:6-9, and homogenizing into an emulsion as an inner layer emulsion; Step b, shearing and mixing the inner layer emulsion and the outer layer solution at a volume ratio of 1-4:6-9, and homogenizing into an emulsion to obtain a double emulsion encapsulating probiotics, wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

[0023] The preparation method is based on the double emulsion of ultrasound-modified ovomucin to achieve efficient encapsulation of probiotics, with a high encapsulation rate. The obtained double emulsion encapsulating probiotics can significantly improve the tolerance of probiotics to various environments such as acidity, alkalinity, bile salts, digestive enzymes, etc., and can be used for efficient encapsulation and delivery of probiotics to help probiotics exert their efficacy in the colon. The double emulsion encapsulating probiotics prepared by the preparation method can also be further made into other encapsulated products containing probiotics that are convenient for daily or clinical use.

[0024] Preferably, the aqueous phase in step a is a NaCl aqueous solution, and its concentration is selected based on the type of probiotics to maintain the osmotic pressure of the probiotics. For example, for Bacillus cereus, the concentration of the NaCl aqueous solution can be 0.15%wt.

[0025] Preferably, the oil phase in step a is edible oil, which can be selected from common edible oils that do not react with proteins, such as olive oil and medium-chain triglycerides.

[0026] Preferably, in step a, the volume ratio of the water phase to the oil phase is 2:8.

[0027] Preferably, the shearing speed in step a is 10000-14000 rpm, and the homogenization time is not less than 2 min. More preferably, the shearing speed is 12000.

[0028] Preferably, the preparation method of the ultrasonically modified ovomucin in step b is: dissolving the ovomucin in distilled water at room temperature to a concentration of 2% to 3%wt, and subjecting the mixture to pulsed ultrasonic treatment for 20 to 40 min at an ultrasonic power of 350 to 450 W at a temperature not exceeding 40°C, wherein the pulsed ultrasonic treatment is: on for 3 seconds and off for 5 seconds.

[0029] Exemplarily, ovomucin can be prepared by the following method: after separating the egg yolk from the egg white, the egg white is diluted with 5 volumes of 0.1 mol / L NaCl solution and stirred for 30 min, and then the pH of the mixture is adjusted to 6 with 1 mol / L HCl. Subsequently, the dispersion is placed at 4°C for 12 h and centrifuged at 8000g for 20 min at 4°C. The precipitate is added to a 0.5 mol / L NaCl solution and placed at 4°C for 6 h. Then, the precipitate is centrifuged at 8000g for 20 min at 4°C, and the precipitate is repeatedly washed with distilled water, and finally freeze-dried in a vacuum freeze dryer for 48 hours to obtain ovomucin.

[0030] Preferably, the volume ratio of the inner layer emulsion to the outer layer solution in step b is 3-4:6-7, and more preferably 3:7.

[0031] Preferably, the concentration of the ultrasonically modified ovomucin aqueous solution in step b is 2.5%wt.

[0032] Preferably, the pH of the ultrasonically modified ovomucin aqueous solution in step b is 8.5-9.5, and more preferably the pH is 9.

[0033] Preferably, the shearing speed in step b is 10000-14000 rpm, and the homogenization time is 2-4 min.

[0034] The water-in-oil emulsifier in the preparation method should be one that does not react with protein. Preferably, the water-in-oil emulsifier is polyglycerol ricinoleate.

[0035] In a sixth aspect, the present invention provides a double emulsion encapsulating probiotics, which is prepared by the above method.

[0036] In a seventh aspect, the present invention provides use of the double emulsion encapsulating probiotics in the preparation of a medicament for preventing and treating ulcerative colitis.

[0037] Preferably, the probiotic is bread-associated Lactobacillus ( Companilactobacillus crustorum ).

[0038] The present invention has found through experiments that the double emulsion encapsulating bread-associated lactobacillus can effectively alleviate the physiological indicators, inflammatory response and pathological symptoms caused by ulcerative colitis, maintain the integrity of the intestinal barrier, and can be used to prepare a drug for preventing and treating ulcerative colitis.

[0039] Preferably, the drug for treating ulcerative colitis further comprises pharmaceutically acceptable oral preparation excipients. The specific excipients and corresponding preparation methods can be conventionally selected according to the specific dosage form of the oral preparation, and they do not adversely affect the pharmacological activity of the probiotics and the physicochemical stability of the emulsion and meet the clinical medication requirements, and the present invention does not limit this.

[0040] In an eighth aspect, the present invention provides a drug for preventing and treating ulcerative colitis, wherein the active ingredient of the drug includes the above-mentioned double emulsion encapsulating probiotics.

[0041] The beneficial effects of the present invention are as follows: the present invention uses ultrasonically modified ovomucin as an outer stabilizer to encapsulate oil-in-water type emulsion droplets to form a water-in-oil-in-water double emulsion, and the raw materials can be selected from natural substances, which is highly safe for the human body. The double emulsion can be used to encapsulate various substances such as water-soluble drugs and probiotics, with a high encapsulation rate, and can better maintain the double emulsion structure in gastrointestinal fluid, and can significantly improve the tolerance of the encapsulated substances to gastrointestinal digestive fluid, and can be used as a high-efficiency embedding and delivery of various substances such as drugs and probiotics. The double emulsion encapsulating probiotics provided by the present invention is prepared based on the above-mentioned double emulsion system, which can significantly improve the tolerance of probiotics to various environments such as acidity, alkalinity, bile salts, digestive enzymes, etc., and can be used for the efficient embedding and delivery of probiotics to help probiotics exert their efficacy in the colon. Experiments have shown that the double emulsion encapsulating bread-associated lactobacillus can effectively alleviate the physiological indicators, inflammatory reactions and pathological symptoms caused by ulcerative colitis, and maintain the integrity of the intestinal barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the appearance of the emulsion at different emulsifier dosages in Test Example 1 of the present invention; Figure 2 The appearance of the double emulsions prepared by ultrasonically modified ovomucin at different concentrations in Test Example 2 of the present invention; Figure 3 The ζ-potential of the double emulsions prepared by ultrasonically modified ovomucin at different concentrations in Test Example 2 of the present invention; Figure 4 is the particle size of the double emulsion prepared by ultrasonically modified ovomucin of different concentrations in Test Example 2 of the present invention; Figure 5 The particle size distribution of the double emulsions prepared by ultrasonically modified ovomucin at different concentrations in Test Example 2 of the present invention; Figure 6 The droplet morphology of the double emulsion prepared by ultrasonically modified ovomucin at different concentrations in Test Example 2 of the present invention; Figure 7 The rheological properties of the double emulsions prepared by ultrasonically modified ovomucin at different concentrations in Test Example 2 of the present invention; Figure 8 The appearance of the double emulsions prepared by ultrasonically modifying ovomucin at different pH values ​​in Test Example 3 of the present invention; Fig. 9 The ζ-potential of the double emulsions prepared by ultrasonically modifying ovomucin at different pH values ​​in Test Example 3 of the present invention; Fig.10 is the particle size of the double emulsion prepared by ultrasonically modifying ovomucin at different pH in Test Example 3 of the present invention; Fig.11 The particle size distribution of the double emulsion prepared by ultrasonically modifying ovomucin at different pH in Test Example 3 of the present invention; Fig.12 The droplet morphology of the double emulsion prepared by ultrasonically modifying ovomucin at different pH in Test Example 3 of the present invention; Fig.13 The rheological properties of the double emulsions prepared by ultrasonically modifying ovomucin at different pH values ​​in Test Example 3 of the present invention; Fig.14 The microscopic morphology of the emulsion after staining the oil droplets and proteins in Test Example 3 of the present invention; Fig.15 The circular dichroism test results in Test Example 4 of the present invention (pH 9+ in the figure is a double emulsion encapsulating probiotics); Fig.16 The morphological observation of the fluorescently stained bacteria after encapsulation in Test Example 5 of the present invention; Fig.17 The morphological observation of the emulsion encapsulating probiotics in Test Example 5 of the present invention after in vitro digestion with simulated gastric fluid and the comparison results of the encapsulation of probiotics; Fig.18 The morphological observation and probiotic encapsulation comparison results of the emulsion in Test Example 5 of the present invention after being digested by simulated small intestinal fluid; Fig.19 The encapsulation efficiency and the colony count after gastric juice digestion calculated by the colony count in Test Example 5 of the present invention; Fig. 20 This is the modeling-intervention scheme for mice in Example 13 of the present invention; Fig.21 The weight change of mice during DSS modeling in Example 13 of the present invention; Fig. 22 is the inflammatory factor in the mouse serum in Example 13 of the present invention; Fig.23 is the disease activity index of the mice during the modeling period in Example 13 of the present invention; Fig.24 This is the H&E staining pathological observation of the upper end section of the mouse colon in Example 13 of the present invention; Fig.25 These are the peripheral immune organ index and colon length of the mice in Example 13 of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0044] Many drugs and probiotics are only absorbed or effective in the colon, and are intolerant to the gastrointestinal digestive fluid environment or irritate the stomach and small intestine. The current solution is to make them into colon-targeted preparations. However, the preparation process of colon-targeted preparations is cumbersome, the process parameters are strictly required, the product quality is difficult to control, and most of the excipients of colon-targeted preparations can only be chemically synthesized, which poses certain safety risks. Therefore, it is necessary to develop new solutions to solve the above problems.

[0045] The present invention has obtained a preparation method of a double emulsion through extensive research. The preparation method can use natural materials as raw materials, has high safety, and has a simple preparation process. The double emulsion prepared by the method can encapsulate probiotics, drugs and other substances in emulsion droplets, has a high encapsulation rate, has good tolerance to gastrointestinal digestive fluid, can deliver the encapsulated substances to the colon for release, and can be used as a delivery carrier for probiotics, drugs and other substances.

[0046] Based on the above research, an embodiment of the present invention provides a double emulsion, and the preparation method thereof is as follows: S1, shearing and mixing the water phase and the oil phase containing the water-in-oil emulsifier in a volume ratio of 1-4:6-9, and homogenizing them into an emulsion as the inner layer emulsion; S2, the inner layer emulsion and the outer layer solution are shear-mixed at a volume ratio of 1-4:6-9, and homogenized into an emulsion to obtain a water-in-oil-in-water double emulsion, wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

[0047] The embodiment of the present invention further provides the use of the double emulsion as a drug encapsulation delivery system or a probiotic encapsulation delivery system, and provides a double emulsion encapsulating probiotics, the preparation method of which is as follows: Step a, dispersing probiotics in a NaCl aqueous solution as an aqueous phase; shearing and mixing the aqueous phase and an oil phase containing an oil-in-water emulsifier at a volume ratio of 1-4:6-9, and homogenizing into an emulsion as an inner layer emulsion; Step b, shearing and mixing the inner layer emulsion and the outer layer solution at a volume ratio of 1-4:6-9, and homogenizing into an emulsion to obtain a double emulsion encapsulating probiotics, wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

[0048] The embodiment of the present invention also provides the use of the double emulsion encapsulating probiotics in the preparation of a medicine for preventing and treating ulcerative colitis.

[0049] The scheme of the present invention is described below through specific embodiments.

[0050] The preparation method of ovomucin in the following examples is as follows: after separating the egg yolk from the egg white, the egg white is diluted with 5 volumes of 0.1 mol / L NaCl solution and stirred for 30 min, and then the pH of the mixture is adjusted to 6 with 1 mol / L HCl. Subsequently, the dispersion is placed at 4°C for 12 h and centrifuged at 8000g for 20 min at 4°C. The precipitate is added to a 0.5 mol / L NaCl solution and placed at 4°C for 6 h. Then, the precipitate is centrifuged at 8000g for 20 min at 4°C, and the precipitate is repeatedly washed with distilled water, and finally freeze-dried in a vacuum freeze dryer for 48 hours to obtain ovomucin.

[0051] The bread-associated Lactobacillus MN047 in the following examples has been disclosed in the non-patent document “Purification and characterization of a novel bacteriocin produced by Lactobacillus crustorum MN047 isolated from koumiss from Xinjiang, China” (Lanhua Yi, Ying Dang, Jingli Wu, Lihui Zhang, Xiaojiao Liu, Bianfang Liu, Yuan Zhou, and Xin Lu. 2016. J. Dairy Sci. 99:7002–7015).

[0052] The simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) used in the following examples were purchased from Shanghai Yuanye Technology Co., Ltd., dextran sodium sulfate (DSS) was purchased from MP Biomedicals, with a molecular weight of 36-50 kDa; C57B / 6J mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd., the occult blood test kit was purchased from Nanjing Jiancheng; and the ELISA kit was purchased from Shanghai Fankewei Biotechnology Co., Ltd.

[0053] Unless otherwise specified, other raw materials, reagents, drugs or instruments used in the following examples are conventional commercial products obtained from commercial channels. The methods used in the following examples are conventional methods in the art unless otherwise specified.

[0054] Example 1 This embodiment provides a double emulsion, and the preparation method thereof is as follows: Preparation of ultrasound-modified ovomucin: Ovomucin was dissolved in distilled water at room temperature at a concentration of 2.5% wt and gently stirred. Ultrasonic treatment was performed in an ultrasonic processor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at a power of 400 W for 30 min (pulse duration: on 3 s, off 5 s) to obtain ultrasound-modified ovomucin. During the ultrasonic treatment, the sample beaker was immersed in an ice bath to keep the temperature below 40 min.

[0055] The water phase was added to the oil phase at a volume ratio of 2:8: water phase (0.15% NaCl solution): oil phase (extra virgin olive oil, containing 4% v / v PGPR), and sheared at 12000 rpm, and then homogenized with a homogenizer for 2 min to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of inner emulsion: outer solution (2.5%wt ultrasonically modified ovomucin aqueous solution) = 3:7, and sheared at a speed of 12000 rpm, and then homogenized with a homogenizer for 2 min to prepare a W1 / O / W2 double emulsion.

[0056] Embodiment 2~4 Examples 2 to 4 provide a double emulsion, respectively, and the preparation methods thereof are substantially the same as that of Example 1, except that the contents of PGPR in the oil phase are 2%, 6%, and 8%, respectively.

[0057] Example 5 This embodiment provides a double emulsion, and the preparation method thereof is as follows: Preparation of ultrasonically modified ovomucin: same as in Example 1.

[0058] The water phase was added to the oil phase at a volume ratio of 2:8: water phase (0.15% NaCl solution): oil phase (extra virgin olive oil, containing 4% v / v PGPR), and sheared at 12000 rpm, and then homogenized with a homogenizer for 2 min to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 3:7 (inner emulsion: outer solution (2.5%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9 with 1 mol / L NaOH)), and sheared at 12,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to prepare a W1 / O / W2 double emulsion.

[0059] Example 6 This embodiment provides a double emulsion, and the preparation method thereof is as follows: Preparation of ultrasound-modified ovomucin: Ovomucin was dissolved in distilled water at room temperature at a concentration of 2% wt and gently stirred. Ultrasonic treatment was performed in an ultrasonic processor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at a power of 350 W for 40 min (pulse duration: on 3 s, off 5 s) to obtain ultrasound-modified ovomucin. During the ultrasonic treatment, the sample beaker was immersed in an ice bath to keep the temperature below 40 min.

[0060] The water phase was added to the oil phase at a volume ratio of 3:7, and the mixture was sheared at 10,000 rpm. The mixture was then homogenized for 3 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 4:6 (inner emulsion: outer solution (2%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 8.5 with 1 mol / L NaOH)), and sheared at 10,000 rpm. The mixture was then homogenized for 3 min using a homogenizer to prepare a W1 / O / W2 double emulsion.

[0061] Example 7 This embodiment provides a double emulsion, and the preparation method thereof is as follows: Preparation of ultrasound-modified ovomucin: Ovomucin was dissolved in distilled water at room temperature at a concentration of 3% wt and gently stirred. Ultrasonic treatment was performed in an ultrasonic processor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at a power of 450 W for 20 min (pulse duration: on 3 s, off 5 s) to obtain ultrasound-modified ovomucin. During the ultrasonic treatment, the sample beaker was immersed in an ice bath to keep the temperature below 40 min.

[0062] The water phase was added to the oil phase at a volume ratio of 4:6, and the mixture was sheared at 10,000 rpm. The mixture was then homogenized for 4 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 3:7 (inner emulsion: outer solution (3%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9.5 with 1 mol / L NaOH)), and sheared at 10,000 rpm. The mixture was then homogenized for 4 min using a homogenizer to prepare a W1 / O / W2 double emulsion.

[0063] Example 8 This embodiment provides a double emulsion, and the preparation method thereof is as follows: Preparation of ultrasound-modified ovomucin: Ovomucin was dissolved in distilled water at room temperature at a concentration of 2.6% wt and gently stirred. Ultrasonic treatment was performed in an ultrasonic processor (JY92-2D, Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China) at a power of 450 W for 25 min (pulse duration: on 3 s, off 5 s) to obtain ultrasound-modified ovomucin. During the ultrasonic treatment, the sample beaker was immersed in an ice bath to keep the temperature below 40 min.

[0064] The water phase was added to the oil phase at a volume ratio of 1:9, and the mixture was sheared at 14,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of inner emulsion: outer solution (2.6%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9 with 1 mol / L NaOH) = 1:9, and sheared at 14000 rpm, and then homogenized with a homogenizer for 2 min to prepare a W1 / O / W2 double emulsion.

[0065] Comparative Examples 1 to 3 This comparative example provides a double emulsion, and its preparation method is basically the same as that of Example 1, except that the concentrations of the ultrasonically modified ovomucin aqueous solution are 1%, 2%, and 3% respectively (pH is 6.3-6.6).

[0066] Comparative Examples 4 to 6 This comparative example provides a double emulsion, and its preparation method is basically the same as that of Example 1, except that the pH values ​​of the ultrasonically modified ovomucin aqueous solution are 2, 4.5, and 6.3, respectively.

[0067] Test Example 1 This test example conducted a preliminary observation on the stability of the inner layer solutions in Examples 1 to 4. The inner layer solutions obtained in the preparation process of Examples 1 to 4 were placed in glass bottles with lids of the same specifications and placed at room temperature for 2 hours. The results are as follows: Figure 1 As shown, the preparation methods of each embodiment can obtain a uniform inner layer emulsion.

[0068] Test Example 2 In this test example, the double emulsions obtained in Example 1 and Comparative Examples 1 to 3 were tested for morphology, ζ-potential, particle size, particle size distribution, and rheological properties.

[0069] The double emulsions of each embodiment have the following appearance: Figure 2 As shown, the ζ-potential is Figure 3 As shown, the particle size Figure 4 The particle size distribution is shown in Figure 5 The droplet morphology is shown in Figure 6 The rheological properties are shown in Figure 7 shown.

[0070] The above results prove that the preparation methods of the double emulsions provided in Example 1 and Comparative Examples 1 to 3 can prepare effective double emulsions, but the stability of the double emulsions is significantly different. The double emulsion of Example 1 has the characteristics of uniform droplet distribution, non-disintegration of oil droplets, and non-aggregation of proteins, and rheology proves its shear thinning property.

[0071] Test Example 3 In this test example, the double emulsions obtained in Example 5 and Comparative Examples 4 to 6 were tested for morphology, ζ-potential, particle size, particle size distribution, and rheological properties, and the double emulsions of Example 5, Comparative Example 4, and Comparative Example 6 were observed for microscopic morphology after dyeing.

[0072] The appearance of double emulsions prepared by protein solutions at different pH values ​​is as follows Figure 8 As shown, the ζ-potential is Fig. 9 As shown, the particle size Fig.10 The particle size distribution is shown in Fig.11 The droplet morphology is shown in Fig.12 The rheological properties are shown in Fig.13 shown.

[0073] The results show that the double emulsion prepared with the ultrasonically modified ovomucin solution under isoelectric point (4.5) and acidic conditions has protein aggregation, which affects the stability and rheological properties of the emulsion. The double emulsion of Example 5 (the ultrasonically modified ovomucin aqueous solution is weakly alkaline) has better stability, fluidity, and uniform droplets. This may be due to the relaxation structure of the protein under alkaline conditions and the formation of a network structure caused by the stretching of protein filaments, which makes the double emulsion prepared with the ultrasonically modified ovomucin aqueous solution at pH 9 have better fluid properties and emulsion stability.

[0074] The microscopic morphology of the emulsion and its components was observed by staining the oil droplets (Nile Red) and protein (Fast Green) using a laser confocal microscope. Fig.14 As shown, the double emulsion droplets of Example 5 are uniform and non-aggregated, while the double emulsions of Comparative Examples 4 and 6 are aggregated, which further proves the previous observation results and the characteristics of the protein under acidic and alkaline conditions. It can be inferred that the double emulsion prepared under the condition that the ultrasound-modified ovomucin solution is pH 9 has good stability and fluid properties, and the coagulation of the ovomucin conformation under acidic conditions can effectively wrap the internal aqueous phase containing the probiotic droplets, thereby protecting the encapsulated probiotics to survive in gastric juice. Furthermore, the emulsion can still mainly maintain the double emulsion structure in the digestion of simulated small intestinal fluid, ensuring the survival rate of the double emulsion encapsulated probiotics in the upper digestive tract.

[0075] Example 9 This embodiment provides a method for preparing a double emulsion encapsulating probiotics.

[0076] Preparation of ultrasonically modified ovomucin: same as in Example 1.

[0077] Will contain about 10 9 The suspension of Lactobacillus MN047 with a CFU / mL was centrifuged, rinsed twice with PBS, and then resuspended in 0.15% NaCl solution as the aqueous phase; The water phase was added to the oil phase at a volume ratio of 2:8, and the mixture was sheared at 12,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 3:7 (inner emulsion: outer solution (2.5%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9 with 1 mol / L NaOH)), and sheared at 12,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to prepare a double emulsion encapsulating probiotics.

[0078] Example 10 This embodiment provides a method for preparing a double emulsion encapsulating probiotics.

[0079] Preparation of ultrasonically modified ovomucin: same as Example 6.

[0080] Will contain about 10 9 The suspension of Lactobacillus MN047 with a CFU / mL was centrifuged, rinsed twice with PBS, and then resuspended in 0.15% NaCl solution as the aqueous phase; The water phase was added to the oil phase at a volume ratio of 3:7, and the mixture was sheared at 10,000 rpm. The mixture was then homogenized for 3 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 4:6 (inner emulsion: outer solution (2%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 8.5 with 1mol / L NaOH)), and sheared at 10,000 rpm. The mixture was then homogenized for 3 min using a homogenizer to prepare a double emulsion encapsulating probiotics.

[0081] Embodiment 11 This embodiment provides a method for preparing a double emulsion encapsulating probiotics.

[0082] Preparation of ultrasonically modified ovomucin: same as Example 7.

[0083] Will contain about 10 9 The suspension of Lactobacillus MN047 with a CFU / mL was centrifuged, rinsed twice with PBS, and then resuspended in 0.15% NaCl solution as the aqueous phase; The water phase was added to the oil phase at a volume ratio of 4:6, and the mixture was sheared at 10,000 rpm. The mixture was then homogenized for 4 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 3:7 (inner emulsion: outer solution (3%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9.5 with 1mol / L NaOH)), and sheared at 10,000 rpm. The mixture was then homogenized for 4 min using a homogenizer to prepare a double emulsion encapsulating probiotics.

[0084] Example 12 This embodiment provides a method for preparing a double emulsion encapsulating probiotics.

[0085] Preparation of ultrasonically modified ovomucin: same as Example 8.

[0086] Will contain about 10 9 The suspension of Lactobacillus MN047 with a CFU / mL was centrifuged, rinsed twice with PBS, and then resuspended in 0.15% NaCl solution as the aqueous phase; The water phase was added to the oil phase at a volume ratio of 1:9, and the mixture was sheared at 14,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to obtain the inner emulsion: The inner emulsion was added to the outer solution at a volume ratio of 1:9 (inner emulsion: outer solution (2.6%wt ultrasonically modified ovomucin aqueous solution, pH adjusted to 9 with 1 mol / L NaOH)), and sheared at 14,000 rpm. The mixture was then homogenized for 2 min using a homogenizer to prepare a double emulsion encapsulating probiotics.

[0087] Test Example 4 This test example uses circular dichroism to test the double emulsions obtained in Examples 5, 9, Comparative Examples 4, and Comparative Examples 6, and further explores the changes in the proteins in the emulsions from the perspective of protein conformation. The results are as follows Fig.15 shown.

[0088] Fig.15 The circular dichroism results showed that as the alkalinity of the ultrasonic modified ovomucin solution increased before preparation, the α helix gradually decreased, which was most likely due to the relaxation of the protein structure, forming more hydrogen bonds, and forming a network structure in the β fold. After encapsulating probiotics, the β fold structure was relatively reduced, which may be because the encapsulation of probiotics caused a certain degree of damage to this network structure.

[0089] Test Example 5 In this test example, the morphology of the probiotics in the double emulsion encapsulating probiotics prepared in Example 9, the encapsulation rate after digestion, and the number of colonies after digestion were investigated by fluorescent staining.

[0090] 1. Experimental methods Encapsulation efficiency detection method: The double emulsion to be tested was centrifuged at 7000 rpm for 10 min at 4°C, the sediment at the bottom of the centrifuge tube was removed and resuspended to separate the unencapsulated bacteria from the emulsion system, and then the resuspended bacteria were graded diluted, plated, cultured (MRS agar, 30°C, 48 hours) and counted. The encapsulation efficiency (EE) was calculated according to the following formula:

[0091] Among them, N0 represents the initial bacterial concentration and N1 represents the number of unencapsulated bacteria.

[0092] Will C. crustorum MN047 was stained with FITC and washed twice with PBS, and then a double emulsion encapsulating probiotics was prepared according to the method of Example 9, observed using an inverted fluorescence microscope with a 10x objective lens in fluorescence mode, and an in vitro simulated digestion experiment was performed.

[0093] In vitro simulated digestion process: simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were preheated to 37°C. 10 mL of double emulsion encapsulating fluorescently stained MN047 was added to SGF, stirred at a constant temperature for 2 h at 37°C, and the pH value was continuously adjusted (maintained at about 2). Then, the system after SGF digestion was added to SIF and reacted at a constant temperature for 2 h at 37°C, with a pH value of 7. The morphology after digestion was observed, and the double emulsion system and SIF system after SGF digestion were gradiently diluted, plated, cultured and counted according to the encapsulation rate detection method to investigate the encapsulation situation. C. crustorum The relative survival rate (SR) of MN047 was calculated according to the following formula:

[0094] Where N2 is the number of viable bacteria counted after digestion, and N0 is the initial number of viable bacteria. When calculating the survival rate of SIF, N0 is the total number of viable bacteria after SGF digestion.

[0095] 2. Results The morphology of fluorescent dyed probiotics after encapsulation is as follows Fig.16 As shown, Figure A is an observation photo under bright field, Figure B is an observation photo under dark field, and Figure C is a comparison photo of naked bacteria under dark field.

[0096] The morphological observation of the emulsion encapsulating probiotics after in vitro digestion with simulated gastric fluid and the comparison of the encapsulation of probiotics are shown in the following figure. Fig.17 (The left picture is the observation photo under bright field, and the right picture is the observation photo under dark field).

[0097] The morphological observation of the emulsion after digestion by simulated small intestinal fluid and the comparison of the encapsulation of probiotics are shown in the following table. Fig.18 (The left picture is the observation photo under bright field, and the right picture is the observation photo under dark field).

[0098] The encapsulation efficiency calculated by colony count and the colony count after gastric juice digestion are shown in Fig.19 shown.

[0099] The above results confirm the speculation in Test Example 4. The aggregation behavior of protein in the presence of acid effectively protects the probiotics in simulated gastric fluid and maintains the structure of the double emulsion to a certain extent in intestinal fluid. The encapsulation rate of the double emulsion for probiotics reaches 93.55%, and the survival rates of probiotics in gastrointestinal fluid are 82.26% and 66.82%, respectively, which are much higher than the survival rate of naked bacteria.

[0100] Embodiment 13 This example provides a pharmacodynamic study on the double emulsion encapsulating probiotics prepared in Example 12 for preventing ulcerative colitis.

[0101] 1. Experimental Animals 48 male C57B / 6J mice, 8 weeks old (20 ± 2 g), were provided with sufficient water and food throughout the experiment, and the living conditions were 23 ± 2 °C and 12 h day-night cycle.

[0102] 2. Experimental methods: The mice were divided into 6 groups, 8 in each group, and the 6 groups were named CT, DSS, MB, M, B, and PC. All mice were first fed with adaptive feeding for one week before intervention and modeling experiments. Fig. 20 As shown: The control group mice (CT) were given normal saline by gavage at a dose of 200 µl / day for 21 days. Drinking water was provided for free drinking; The model group mice (DSS) were given normal saline by gavage at a dose of 200 µl / day for 21 days. They were provided with drinking water for free drinking in the first two weeks, and the drinking water was replaced with drinking water containing 2.5% DSS in the third week. Mice in the probiotics encapsulated double emulsion group (MB) were intragastrically administered with the probiotics encapsulated double emulsion prepared in Example 12 at a dose of 200 μl / day for 21 days. At the same time, drinking water was provided for free drinking in the first two weeks, and the drinking water was replaced with drinking water containing 2.5% DSS in the third week; Mice in the double emulsion group (M) were intragastrically administered with the double emulsion prepared in Example 1 at a dose of 200 μl / day for 21 days. In the first two weeks, drinking water was provided for free drinking, and in the third week, the drinking water was replaced with drinking water containing 2.5% DSS. Mice in the probiotic group (B) were given 200 µl / day by gavage. C. crustorumMN047 bacterial suspension for 21 days. Drinking water was provided for free drinking in the first two weeks, and the drinking water was replaced with drinking water containing 2.5% DSS in the third week. The mice in the aminosalicylic acid group (PC) were first gavaged with normal saline at a dose of 200 µl / day for 14 days, and then gavaged with aminosalicylic acid at a dose of 200 mg / kg for 7 days; at the same time, drinking water was provided for free drinking in the first two weeks, and the drinking water was replaced with drinking water containing 2.5% DSS in the third week.

[0103] Starting from the third week, the body weight and water intake of mice were recorded, and the fecal health and occult blood levels were measured.

[0104] On day 22, all mice were intraperitoneally injected with 100 mg / kg ketamine and 10 mg / kg xylazine, and samples were collected to record colon length, and calculate thymus weight index, spleen weight index and disease activity index.

[0105] The distal colon tissue was directly fixed in 4% paraformaldehyde and then embedded in paraffin when collected. 5 μm thick sections were made and stained with hematoxylin & eosin (H&E). The colon tissue sections were then observed under a microscope.

[0106] The venous blood was centrifuged at 7000 rpm for 15 min to separate the serum. The levels of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) in the serum were measured using ELISA kits.

[0107] 3. Results The weight changes of mice during DSS modeling Fig.21 shown.

[0108] Inflammatory factors in mouse serum such as Fig. 22 shown.

[0109] The disease activity index of mice during the modeling period was as follows Fig.23 shown.

[0110] The pathological observation results of H&E staining of the upper colon section of mice are as follows Fig.24 shown.

[0111] Peripheral immune organ index and colon length of mice Fig.25 shown.

[0112] The results showed that MB, M and B groups all effectively alleviated the physiological indicators, inflammatory reactions, and pathological symptoms caused by colitis, and maintained the integrity of the intestinal barrier, and the MB group had the best relief effect. The relief effects of the M group and the B group on different indicators were observed in the MB, proving the protective effect of the double emulsion. It can be seen that the double emulsion provided by the present invention combines the functional activity of the ultrasonically modified ovomucin itself, and plays a protective role and targeted release role for probiotics in vivo. And the results also prove that the double emulsion based on ultrasonically modified ovomucin provided by the present invention has good efficacy in drug delivery.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a double emulsion, characterized in that: The specific steps include: S1, shearing and mixing the water phase and the oil phase containing the water-in-oil emulsifier in a volume ratio of 1-4:6-9, and homogenizing them into an emulsion as the inner layer emulsion; S2, the inner layer emulsion and the outer layer solution are shear-mixed at a volume ratio of 1-4:6-9, and homogenized into an emulsion to obtain a water-in-oil-in-water double emulsion, wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

2. The preparation method according to claim 1, characterized in that: The oil phase in S1 is edible oil; and / or The volume ratio of the water phase to the oil phase in S1 is 2:8; and / or The shearing speed in S1 is 10000-14000 rpm, and the homogenization time is not less than 2 min; and / or The preparation method of ultrasonic modified ovomucin in S2 is: dissolving ovomucin in distilled water at room temperature to a concentration of 2% to 3%wt, and subjecting it to pulsed ultrasonic treatment for 20 to 40 min at an ultrasonic power of 350 to 450 W at a temperature not exceeding 40°C, wherein the pulsed ultrasonic treatment is: on for 3 seconds and off for 5 seconds; and / or The volume ratio of the inner layer emulsion to the outer layer solution in S2 is 3-4:6-7; and / or The concentration of the ultrasonically modified ovomucin aqueous solution in S2 is 2.5%wt; and / or The pH of the ultrasonically modified ovomucin aqueous solution in S2 is 8.5-9.5; and / or The shearing speed in S2 is 10000-14000 rpm, and the homogenization time is 2-4 min; and / or The water-in-oil emulsifier is polyglycerol ricinoleate.

3. A double emulsion, characterized in that: Prepared according to the preparation method of claim 1 or 2.

4. Use of the double emulsion according to claim 3 as a drug encapsulation delivery system.

5. Use of the double emulsion according to claim 3 as a probiotic encapsulation delivery system.

6. A method for preparing a double emulsion encapsulating probiotics, characterized in that: The specific steps include: Step a, dispersing probiotics in a NaCl aqueous solution as an aqueous phase; shearing and mixing the aqueous phase and an oil phase containing an oil-in-water emulsifier at a volume ratio of 1-4:6-9, and homogenizing into an emulsion as an inner layer emulsion; Step b, shearing and mixing the inner layer emulsion and the outer layer solution at a volume ratio of 1-4:6-9, and homogenizing into an emulsion to obtain a double emulsion encapsulating probiotics, wherein the outer layer solution is a 2%-3%wt ultrasonically modified ovomucin aqueous solution.

7. The method according to claim 6, characterized in that The aqueous phase in step a is a NaCl aqueous solution; and / or The oil phase in step a is edible oil; and / or The volume ratio of the water phase to the oil phase in step a is 2:8; and / or The shearing speed in step a is 10000-14000 rpm, and the homogenization time is not less than 2 min; and / or The preparation method of the ultrasonic modified ovomucin in step b is: dissolving the ovomucin in distilled water at room temperature to a concentration of 2% to 3%wt, and pulse ultrasonic treatment for 20 to 40 minutes at an ultrasonic power of 350 to 450 W at a temperature not exceeding 40°C, wherein the pulse ultrasonic treatment is: on for 3 seconds and off for 5 seconds; and / or The volume ratio of the inner layer emulsion to the outer layer solution in step b is 3-4:6-7; and / or The concentration of the ultrasonically modified ovomucin aqueous solution in step b is 2.5%wt; and / or The pH of the ultrasonically modified ovomucin aqueous solution in step b is 8.5-9.5; and / or The shearing speed in step b is 10000-14000 rpm, and the homogenization time is 2-4 min; and / or The water-in-oil emulsifier is polyglycerol ricinoleate.

8. A double emulsion encapsulating probiotics, prepared according to the preparation method of claim 6 or 7.

9. Use of the double emulsion encapsulating probiotics according to claim 8 in the preparation of a medicament for preventing and treating ulcerative colitis.

10. The use according to claim 9, characterized in that: The probiotics are bread-associated Lactobacillus ( Companilactobacillus crustorum ).

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