Method for preparing water-in-oil-in-water type multiple emulsion

Abstract

The present invention provides a method for preparing a water-in-oil-in-water type multiple emulsion, which comprises: (a) mixing an active ingredient with an internal water phase to form a homogenous mixture; (b) mixing the homogenous mixture with an oil phase to form a water-in-oil emulsion; (c) mixing the water-in-oil emulsion with an external water phase to form the water-in-oil-in-water type multiple emulsion, wherein the external water phase comprises water and an excipient, wherein the excipient comprises whey protein concentrate and modified starch.

Description

Technical Field

[0001] The invention provides a method for preparing an emulsion, and particularly relates to a method for preparing a water-in-oil-in-water type multiple emulsion. Background Art

[0002] According to the statistical data analysis of the Food Institute's Industry & Technology Intelligence Service (ITIS), the scale of the dietary supplement market in Taiwan in 2013 was NT$54.1 billion, and the market continued to grow. Lactic acid bacteria accounted for 5% of the dietary supplement market, reaching NT$2.6 billion, indicating that the probiotics product market has its development value. In order to respond to the trend of developing novel products with health value using probiotics, how to improve the survival rate of probiotics during the processing process or the external environment, and further enhance the stability of the bacteria and the survival rate of the gastrointestinal tract will be the key technology. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for preparing a water-in-oil-in-water type multiple emulsion, which can improve the stability of active ingredients.

[0004] Therefore, the present invention provides a method for preparing a water-in-oil-in-water type multiple emulsion, comprising:

[0005] (a) mixing the active ingredient with an internal aqueous phase to form a homogeneous mixture;

[0006] (b) mixing the homogenous mixture with an oil phase to form a water-in-oil emulsion;

[0007] (c) mixing the water-in-oil emulsion with an external aqueous phase to form the water-in-oil-in-water multiple emulsion, wherein the external aqueous phase comprises water and an excipient, wherein the excipient comprises whey protein concentrate and modified starch.

[0008] The present invention further provides a composition prepared by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shows the results of 4°C stability analysis of lactic acid bacteria multi-emulsified powder.

[0010] Figure 2A Gastric acid resistance analysis results showing the re-dissolution of multiple emulsion powders in water.

[0011] Figure 2B Results of bile salt resistance analysis showing the re-dissolution of multiple emulsion powders in water.

[0012] Figure 3A The change over time of the number of lactic acid bacteria in mouse feces after administration of the test substance (D3, D7, D14) is shown.

[0013] Figure 3B The effect of administration of the test substance on the number of lactic acid bacteria contained in the cecal contents at the end of the test (D21) is shown.

[0014] Figure 4A The effect of test substance administration on the number of bifidobacteria in mouse feces over time (D3, D7, D14) is shown.

[0015] Figure 4B The effect of administration of the test substance on the number of Bifidobacteria contained in the cecal contents of mice at the end of the experiment (D21) is shown.

[0016] Figure 5A The effect of test substance administration on the number of Clostridium perfringens in mouse feces over time (D3, D7, D14) is shown.

[0017] Figure 5B The effect of administration of the test substance on the number of Bifidobacteria contained in the cecal contents of mice at the end of the experiment (D21) is shown.

[0018] Fig. 6A The effect of administration of the test substance on the intestinal maltase of mice is shown.

[0019] Figure 6B The effect of administration of the test substance on intestinal sucrase in mice is shown.

[0020] Figure 6C The effect of administration of the test substance on intestinal leucine aminopeptidase in mice is shown.

[0021] Fig.6D The effect of administration of the test substance on intestinal lipase in mice is shown.

[0022] Fig. 7A The change over time (Pre-D1, D7, D14) in the number of lactic acid bacteria in mouse feces after administration of the test substance is shown.

[0023] Figure 7B The effect of administration of the test substance on the number of lactic acid bacteria contained in the cecal contents of mice at the end of the experiment (D21) is shown.

[0024] Fig. 8A The effect of test substance administration on the number of bifidobacteria in mouse feces over time (Pre-D1, D7, D14) is shown.

[0025] Figure 8B The effect of administration of the test substance on the number of Bifidobacteria contained in the cecal contents of mice at the end of the experiment (D21) is shown.

[0026] Fig. 9A The effect of test substance administration on the number of Clostridium perfringens in mouse feces over time (Pre-D1, D7, D14) is shown.

[0027] Fig. 9B The effect of administration of the test substance on the number of Clostridium perfringens contained in the cecal contents of mice at the end of the experiment (D21) is shown.

[0028] Fig. 10A The effect of administration of the test substance on the intestinal maltase of mice is shown.

[0029] Fig. 10B The effect of administration of the test substance on intestinal sucrase in mice is shown.

[0030] Fig. 10C The effect of administration of the test substance on intestinal leucine aminopeptidase in mice is shown.

[0031] Fig. 10D The effect of administration of the test substance on intestinal lipase in mice is shown.

[0032] Fig.11A The effect of administration of the test substance on the gene expression level of Lactobacillus casei (L. casei) in the cecal contents of normal model mice is shown.

[0033] Fig. 11B The effect of administration of the test substance on the gene expression level of Lactobacillus casei in the cecal contents of antibiotic model mice is shown. DETAILED DESCRIPTION

[0034] The present invention may be understood more readily by reference to the following detailed description of its various aspects, examples and the chemical drawings and tables that accompany the related description.

[0035] The terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the invention.

[0036] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0037] Ranges are often expressed herein as "about" one particular value and / or to "about" another particular value. When such ranges are expressed, one aspect is a range that includes one particular value and / or to another particular value. Similarly, when values ​​are expressed as approximate values ​​by using the word "about," it is understood that the particular value may form another aspect. It is also understood that each endpoint of each range has significance, and one endpoint is both related to the other endpoint and independent of each other.

[0038] The present invention introduces multiple emulsification technology into the development of oral administration products, and establishes oral administration product coating technology through formulation and process design. Multiple emulsification technology uses a multi-layer membrane structure to create spatial separation, encapsulating the oral administration active ingredients in the internal water phase, which can achieve the effect of isolation and protection, and is conducive to the development of diversified products.

[0039] Emulsification technology refers to two originally immiscible phases, one of which is dispersed in the other phase as fine particles, which can be divided into oil-in-water (O / W) and water-in-oil (W / O). Multiple emulsions are a complex system in which W / O and O / W coexist. According to the properties of the dispersed phase and the continuous phase, they can be divided into two systems: water-in-oil-in-water (W / O / W) and oil-in-water-in-oil (O / W / O). Because multiple emulsions have the characteristics of multi-layer membranes, they can simultaneously embed water-soluble and oil-soluble functional substances to achieve isolation, protection, odor masking and controlled release, and have the potential for industrial application.

[0040] That is, the present invention provides a method for preparing a water-in-oil-in-water type multiple emulsion, comprising:

[0041] (a) mixing the active ingredient with an internal aqueous phase to form a homogeneous mixture;

[0042] (b) mixing the homogenous mixture with an oil phase to form a water-in-oil emulsion;

[0043] (c) mixing the water-in-oil emulsion with an external aqueous phase to form the water-in-oil-in-water multiple emulsion, wherein the external aqueous phase comprises water and an excipient, wherein the excipient comprises whey protein concentrate and modified starch.

[0044] The water-in-oil-in-water multiple emulsion of the present invention may include an inner aqueous phase, an oleaginous phase, and an outer aqueous phase. The inner aqueous phase and the outer aqueous phase may be composed of water or an aqueous solution, respectively, while the oil phase may be composed of a hydrophobic liquid or solution. In the following content of this article, the water-in-oil-in-water multiple emulsion may be referred to as a "multiple emulsion".

[0045] In the present invention, the active ingredient may be a drug or a microorganism. The drug may include, but is not limited to, a small molecule drug, a biological agent, or a traditional Chinese medicine preparation, etc., preferably soluble in water and stable in water.

[0046] The present invention does not limit the type of the microorganism, for example, it can be bacteria, fungi, actinomycetes, protozoa or algae, etc. In certain aspects, the microorganism can be a probiotic, such as Lactobacillus acidophilus, Bifidobacterium species, Lactobacillus casei, Lactobacillus reuteri, Bifidobacterium lactis and Bifidobacterium longum, etc.

[0047] In certain embodiments, the inner aqueous phase comprises water and an excipient. The excipient may be any excipient that can be used in a water-in-oil formulation, such as hydroxypropylmethylcellulose, carboxymethyl cellulose, sodium alginate, gelatin, gum arabic, sodium caseinate, soy protein, etc., and may comprise two or more excipients.

[0048] In addition, the inner aqueous phase may optionally further comprise one or more salts. Preferably, the salts are physiologically acceptable salts, such as sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, potassium sulfate, calcium sulfate, sodium malate, sodium citrate, disodium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and dipotassium hydrogen phosphate.

[0049] In certain embodiments, the internal aqueous phase comprises about 3% gelatin and about 2% sodium chloride, based on the total weight of the internal aqueous phase.

[0050] The present invention does not limit the method of mixing the active ingredient with the internal aqueous phase. However, when the active ingredient is a microorganism (e.g., a probiotic), the microorganism may be a dry powder, or may include a culture matrix thereof. In certain embodiments, the bacterial solution after microbial culture is centrifuged, the supernatant fermentation solution is removed, and then the solution is re-dissolved with an equal volume of the internal aqueous phase to form a homogenous mixture.

[0051] The oil phase of the present invention is any hydrophobic liquid that can be used in water-in-oil formulations, such as plant or animal fats, etc. For example, the oil phase can be a plant oil, such as sunflower oil, soybean oil, olive oil, canola oil, linseed oil and palm oil, etc., and can contain two or more plant oils.

[0052] In addition, the oil phase may optionally contain one or more lipophilic surfactants, such as polyglycerol polyricinoleate (PGPR), lecithin, sugar ester, emulsifying waxes polyglycerol fatty acid ester, polysorbate, monoglyceride and diglyceride, etc.

[0053] In certain embodiments, the oil phase comprises sunflower oil and about 8% polyglycerol ricinoleate, based on the total weight of the oil phase.

[0054] When the homogenous mixture is mixed with the oil phase, the homogenous mixture is preferably added to the oil phase and the activity of the active ingredient is maintained. Preferably, the mixture can be mixed at a low temperature, for example, about 0°C to about 10°C, preferably about 4°C. For example, the oil phase can be stirred at a low speed using a homogenizer, and the oil phase can be placed in an ice bath, and then the homogenous mixture is added to the oil phase, and then the speed is increased, for example, to about 12000 rpm for homogenization for about 3 minutes to obtain a water-in-oil emulsion. In some aspects, the homogenous mixture and the oil phase are mixed at a volume ratio of about 1:1.5.

[0055] The whey protein in the excipient in the external aqueous phase refers to a protein mixture separated from whey, which is a liquid byproduct produced during cheese production. Whey protein can be composed of α-lactalbumin, β-lactoglobulin, serum albumin and immunoglobulin. Whey protein concentrate (WPC) contains less fat and cholesterol, but compared with other whey proteins, whey protein concentrate contains more carbohydrates in the form of lactose and contains about 29-89% protein by total weight. In some aspects, the whey protein concentrate contains more than about 80% protein by total weight.

[0056] The modified starch in the excipient in the external aqueous phase is any modified starch that can be used as a food additive. In certain aspects, the modified starch can be octenyl succinate modified starch (or starch sodium octenyl succinate, OSA starch) .

[0057] In certain aspects, the weight ratio of the whey protein concentrate to the modified starch is 4:1 to 1:4, such as 3:1 to 1:3, or 2:1 to 1:2. Preferably, the weight ratio of the whey protein concentrate to the modified starch is 2:1.

[0058] In certain aspects, the excipients in the external aqueous phase may optionally further comprise one or more additional excipients selected from the group consisting of hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium alginate, gelatin, gum arabic, sodium caseinate and soy protein. And the additional excipients in the external aqueous phase may be the same as or different from the excipients in the internal aqueous phase.

[0059] When the excipient in the external aqueous phase further comprises sodium alginate, gelation is preferably not performed, i.e., sodium alginate does not form a gel. Therefore, the multiple emulsifier preferably does not contain divalent ions, such as Ca, that can gel the sodium alginate. 2+ or Mg 2+ Ions, etc.

[0060] In some aspects, the additional excipient is hydroxypropyl methylcellulose. In some aspects, based on the total weight of the excipients in the external aqueous phase, the weight percentage of the whey protein concentrate is 55-70%, the weight percentage of the modified starch is 25-35%, and the weight percentage of the additional excipient is 1-10%. Preferably, the weight ratio of the whey protein concentrate, the modified starch and the additional excipient is 38:19:3. In some aspects, based on the total weight of the external aqueous phase, the excipient is present in the external aqueous phase at a weight ratio of about 25%.

[0061] When the water-in-oil emulsion is mixed with the external aqueous phase, similar to the above, a homogenizer can be used to stir the external aqueous phase at a low speed, and then the water-in-oil emulsion is added to the external aqueous phase, and the volume ratio of the water-in-oil emulsion to the external aqueous phase can be about 2:8, and then the speed is increased, for example, increased to about 8000 rpm and homogenized for about 2 minutes to obtain the water-in-oil-in-water multiple emulsion.

[0062] In the present invention, the active ingredient can be coated with multiple layers by the above method, thereby improving its stability and achieving the effects of isolation, protection, odor masking and controlled release. When the active ingredient is a microorganism, its survival rate can also be improved.

[0063] In certain embodiments, the water-in-oil-in-water multiple emulsion may be spray dried to obtain multiple emulsion powder particles. For example, the inlet and outlet temperatures of the spray drying may be about 130° C. and about 80° C., respectively. By spray drying, all or part of the water in the water-in-oil-in-water multiple emulsion may be removed, thereby making the formed multiple emulsion powder particles have high stability and are easy to store.

[0064] The present invention further provides a composition obtained by the above method, that is, the composition can be the above water-in-oil-in-water type multiple emulsion or a multiple emulsion powder obtained by spray drying thereof.

[0065] The following examples are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0066] Example

[0067] 1. Materials

[0068] Material source Sunflower Oil Standard Foods Co., Ltd. PGPR Cheng Ding Enterprise Co., Ltd. gelatin Sigma-Aldrich Whey Protein Concentrate Hao Long Food Enterprise Co., Ltd. Octenyl succinate starch San Jiang Industrial Co., Ltd. Hydroxypropyl methylcellulose Huimin Pharmaceutical Co., Ltd. Sodium alginate Sigma-Aldrich Gum Arabic Hanxin Technology Co., Ltd. MRS medium Difco Agar Pronadisa

[0069] 2. Experimental Methods

[0070] Preparation of water-in-oil-in-water multiple emulsions

[0071] The multiple emulsions were prepared by a two-step method, with an inner aqueous phase, an oil phase containing a lipophilic surfactant, and an outer aqueous phase containing an excipient (the formula of the excipient is described in the "Results and Discussion" below), respectively. The formula is shown in the following table.

[0072]

[0073] The Lactobacillus casei bacterial solution was centrifuged at 6000 rpm for 5 minutes, and after removing the supernatant fermentation liquid, the precipitate was dissolved back in an equal volume of water phase to form a homogenous mixture. 1 times the homogenous mixture and 1.5 times the corresponding oil phase were taken, and the homogenous mixture was added to the oil phase under the condition of low speed of the homogenizer, and the speed was increased to 12000 rpm, and homogenized for 3 minutes. In order to avoid the death of lactic acid bacteria during the process, homogenization was performed under an ice bath to form an oil-in-water emulsion.

[0074] Take an oil-in-water emulsion and an external aqueous phase in a volume ratio of 2:8, slowly add the oil-in-water emulsion to the external aqueous phase under low speed conditions of the homogenizer, increase the speed to 8000 rpm, and homogenize for 2 minutes to form an oil-in-water-in-oil multiple emulsion.

[0075] Multiple emulsification and granulation process

[0076] The aforementioned water-in-oil-in-water type multiple emulsion containing lactic acid bacteria is spray dried with an inlet / outlet temperature of 130°C / 80°C to form multiple emulsified powder particles. After the process is completed, the powder is collected and weighed, and the powder recovery rate is calculated (powder recovery rate = powder weight in the spray drying recovery barrel / solid weight before spray drying). The powder is sampled and coated on a plate for viable bacterial count analysis, and the bacterial survival rate of the spray drying process is calculated. The experiments described later in this article all use the aforementioned multiple emulsified powder particles containing lactic acid bacteria, or "multiple emulsified formulation probiotics / multiple emulsified formulation lactic acid bacteria".

[0077] Animal models

[0078] Pre-test screening

[0079] To help determine which formulation of multiple emulsions has the best protection effect on probiotics, a preliminary test was conducted using the changes in probiotics in feces as an indicator before the gastrointestinal efficacy evaluation test.

[0080] Animal experiments

[0081] Two-month-old healthy adult male ICR mice were selected and divided into a control group, a blank emulsion group, a multiple emulsion probiotic group with different formulations (the excipient formulation in the external aqueous phase is described in Table 3 in the "Results and Discussion" below), and an uncoated probiotic group. Each group had 10 mice, and distilled water (control group), a blank emulsion (its formulation was the same as that of the multiple emulsion probiotic group, but the probiotics were not coated), multiple emulsion probiotics, and uncoated probiotics were orally administered. The administration volume was 10 mL / kg.bw, and the live bacteria administration amount of the multiple emulsion probiotic group and the uncoated probiotic group was 1x10 8 cfu / kg.bw. Feces of mice were collected one day before administration of the test substance (Pre-D1) and on the 7th day (D7) and 14th day (D14) after administration for probiotic analysis.

[0082] Normal model

[0083] After preliminary tests, it was determined that a specific formula of emulsified dosage form was selected to evaluate the effect of multiple emulsified dosage forms of probiotics on improving the intestinal function of mice. This test also evaluated uncoated probiotics and microcapsule-coated probiotics at the same time.

[0084] Two-month-old healthy adult male ICR mice were selected and divided into a control group, a blank emulsion group, a multiple emulsion probiotic group (the excipient formula in the external aqueous phase is described in Table 3 in the "Results and Discussion" below), a microcapsule-coated probiotic (the external aqueous phase composition is soy protein: sodium alginate = 9:1, there is no oil phase, and it is a microcapsule prepared by a general emulsification system) and an uncoated probiotic group. Each group had 10 mice, and distilled water (control group), a blank emulsion (its formula is the same as that of the multiple emulsion probiotic group, but the probiotics are not coated), multiple emulsion probiotics, microcapsule probiotics and uncoated probiotics were orally administered. The administration volume was 10mL / kg.bw, and the live bacteria administration amount of the multiple emulsion probiotics, microcapsule probiotics and uncoated probiotic groups was 1x10 8 cfu / kg.bw. Distilled water and test materials were administered once a day for 3 consecutive weeks. The feces of mice were collected 1 day before administration of the test substance (Pre-D1) and on the 3rd day (D3), 7th day (D7) and 14th day (D14) after administration of the test substance for fecal bacterial analysis. In addition, the mice were sacrificed on the 21st day (D21) after administration of the test substance, and the small intestine and cecum were collected for analysis of intestinal digestive enzymes, cecal flora, cecal content pH, cecal weight and real-time polymerase chain reaction.

[0085] antibiotic model

[0086] The test substances were administered to mice that had been treated with broad-spectrum antibiotics to evaluate their effects on gastrointestinal function under the antibiotic model (Reference: Yang LC, Lu TJ, Lin WC. The prebiotic arabinogalactan of Anoectochilus formosanus prevents ovariectomy-induced osteoporosis in mice. J Func Food Oct 5(4); 1642-53).

[0087] Two-month-old healthy adult male ICR mice were selected and divided into a control group (no antibiotics were administered), a blank emulsion group, a multiple emulsion probiotic group (the excipient formula in the external aqueous phase is described in Table 3 in the "Results and Discussion" below), a microcapsule-coated probiotic group, and an uncoated probiotic group. There were 10 mice in each group. Except for the control group, 2 mg / mL of the broad-acting antibiotic streptomycin was added to the drinking water of the mice for 7 days before the administration of the test substance. Then, each group of mice was administered distilled water (control group), a blank emulsion, multiple emulsion probiotics, microcapsule probiotics, and uncoated probiotics, respectively. The administration volume was 10 mL / kg.bw, while the amount of live bacteria administered to the multiple emulsion probiotic group, the microcapsule probiotic group, and the uncoated probiotic group was 1x10 8 cfu / kg.bw. Distilled water and the test substance were administered once a day for 3 consecutive weeks. The feces of the mice were collected 1 day before the administration of the test substance (Pre-D1) and on the 7th day (D7) and 14th day (D14) after the administration of the test substance, and fecal microbial analysis was performed. In addition, the mice were sacrificed on the 21st day (D21) after the administration of the test substance, and the small intestine and cecum were collected for analysis of intestinal digestive enzymes, cecal flora, cecal content pH, cecal weight, and real-time polymerase chain reaction.

[0088] Intestinal digestive enzyme analysis

[0089] The methods for measuring the activity of digestive enzymes, such as lipase, leucine aminopeptidase and disaccharidase, are described in detail below.

[0090] Sample processing:

[0091] After sacrifice, the small intestine of the mouse was obtained, and the intestinal mucosa was scraped off (the intestinal mucosa does not need to be scraped off for lipase activity determination). 0.2 g of small intestine or intestinal mucosa was added to 2 mL of a solution containing protease inhibitors (1 M phenylmethylsulfonylfluoride (PMSF) and 2.2 mM iodoacetic acid) and 0.9% NaCl at 4°C, and homogenized with a homogenizer to form a small intestine homogenate or intestinal mucosa homogenate.

[0092] Disaccharidase activity assay

[0093] Objective: To determine the activities of disaccharide digestive enzymes (maltase and sucrase) in the small intestine.

[0094] Determination method: Basically according to the method disclosed by Dahlqvist, A. (Method for assay of intestinal disaccharidase). Anal. Biochem. 1964 January; 7: 18-25). Specifically, 30 μL of intestinal mucosal homogenate was added with 15 μL of 56 mM disaccharide solution (lactose, maltose or sucrose) at 37°C for 30 minutes, and then 100 μL of 0.6N sodium hydroxide solution was added to dissolve the cells, and then the intestinal mucosal homogenate was neutralized with 10 μL of 6N hydrochloric acid solution. 45 μL of treated intestinal mucosal homogenate or glucose standard solution (0-40 mg / mL) was taken and mixed with 625 μL of reaction buffer (containing 50 mM tris-maleate, 33 mM potassium sodium phosphate and 30 mM MgCl 2 , pH 6.8), 150 μL of 1 mM ATP, 150 μL of 1 mM NADP, 15 μL of 330 IU / mL hexokinase (EC 2.7.1.11) and 15 μL of 170 IU / mL glucose-6-phosphate dehydrogenase (EC 1.1.1.49) at 37° C. for 30 minutes. Finally, the sample was placed in an ice bath to terminate the reaction. At the beginning of the reaction, the absorbance value A1 is measured at a wavelength of 340nm using a spectrophotometer. The absorbance value A2 is measured after 30 minutes of reaction. ΔA=A2-A1 is the change (increase) of NADPH. The concentration of glucose is calculated based on the change of NADPH (refer to the instructions of the Randox GL 1611 kit). The protein content in the sample is determined using the improved Lowry method (Peterson, GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable.) Analytical Biochemistry (Anal. Biochem.) 83:346-356.), and the disaccharidase activity is expressed in IU / mg protein.

[0095] Lipase activity assay

[0096] Objective: To determine the activities of lipid-digesting enzymes in the small intestine.

[0097] Determination method: Basically according to the method disclosed by Verduin, PA et al. (Studies of the determination of lipase activity. Clin ChimActa. 1973 June 14; 46(1): 11-9). Specifically, a commercially available reagent kit (Sigma Lipase-PS TM ) determination. 900 μL of substrate solution (containing 1.1 mM 1,2-diacylglycerol, 2 mM sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-formate, 0.66 mM ATP, 860 U / L monoglyceride lipase, 1340 U / L glycerol kinase, 40,000 U / L 3-phosphoglycerol oxidase, 1340 U / L horseradish peroxidase, 40,000 U / L colipase and buffer) was added to 15 μL of small intestine homogenate or standard solution (attached to the reagent set) and incubated at 37°C for 3 to 5 minutes. Then, 300 μL of activator (containing 36 mM deoxycholic acid, 6 mM 4-aminoantipyrine, 0.05% sodium azide and buffer) was added and incubated at 37°C for 3 to 5 minutes. After 3 minutes at ℃, the sample was measured with a spectrophotometer at a wavelength of 550nm for 2 minutes, and the protein content in the sample was determined by the improved Lowry method. The lipase activity was expressed as KU / mg protein.

[0098] Leucine aminopeptidase (LAP) activity assay

[0099] Objective: To determine the activity of protein digestive enzymes on the brush border membrane of small intestine villi.

[0100] Determination method: Basically refer to the method disclosed by Martinek, PG et al. (Simplified estimation of leucine aminopeptidase (LAP) activity. Clin. Chem. 1964 Dec; 10: 1087-97.). Specifically, a commercially available reagent kit (Sigma LAP) determination. Take 0.5 mL of intestinal mucosal homogenate, mix with 0.5 mL of LAP substrate solution (20 mg / dL L-leucyl-β-naphthylamine dissolved in phosphate buffer, pH 7.1), and act at 37°C for 1 hour. Then add 0.5 mL of 2N HCl, 1.5 mL of treated intestinal mucosal fluid or 1.5 mL of standard solution (0-12 Sigma units / mL) and 0.5 mL of sodium nitrite solution. After acting at room temperature for 3 minutes, mix with 1.0 mL of 0.5% (w / v) ammonium sulfamate and act at room temperature for 3 minutes. Then add 2.0 mL of N-1-naphthylethylenediamine alcohol solution. After acting at room temperature for 45 minutes, measure the absorbance at a wavelength of 580 nm with a spectrophotometer, and determine the protein content in the sample with the modified Lowry method. The enzyme activity of leucine amino acid is expressed in Sigma units / mg protein. One Sigma unit is defined as the amount of enzyme that generates 1 mole of naphthylamine per hour at 37°C and pH 7.1.

[0101] Confirmation: Improvement in any of the above items means that the gastrointestinal digestive function can be improved.

[0102] Analysis of cecal flora

[0103] According to the experimental design time point, fresh feces of mice were collected to analyze the number of probiotics used in this experiment in the feces. When the mice were sacrificed, the contents of their cecum were collected to detect the intestinal flora, including the probiotics used in this experiment and 1 harmful (or unhelpful) bacteria: Clostridium perfringens.

[0104] Sampling and homogenization methods

[0105] Feces were collected by massaging in a sealed container (about 3 to 10 pieces). After weighing the feces, sterile anaerobic diluent (0.1% peptone solution) was added in an anaerobic operating box at a ratio of 1:9 (w / w), and then shaken and mixed into a homogeneous feces suspension. After the feeding experiment, the mice were anesthetized, dissected, and the cecal contents were removed. About 1 g was weighed and added to a test tube containing 9 mL of sterile anaerobic diluent, and the mixture was shaken to form a homogenous solution (Method for evaluating the improvement of gastrointestinal function of health foods. 880802 Health Bureau Food No. 88037803 Announcement; 920829 Health Bureau Food No. 0920401629 Amendment; and Yang LC (Yang, LC), Lu TJ (Lu, TJ), Lin WC (Lin, WC.) Prebiotic arabinogalactan from Taiwan silver line orchid prevents ovariectomy-induced osteoporosis in mice. Journal of Functional Foods, October 5 (4); 1642-53).

[0106] Take feces or cecal homogenate and dilute it tenfold in a series with anaerobic diluent in an anaerobic operating box. Take the appropriate dilution multiple and add it to the culture medium by the spread plate method, place it in an anaerobic operating box or culture it at 37°C for 24 to 72 hours, and count the number of colonies.

[0107] This test uses a culture medium of a probiotic strain, which will be cultured using a selective culture medium for the strain.

[0108] According to ISO 7937 (2004 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of Clostridium perfringens—Colony-count technique), Clostridium perfringens was cultured on sucrose sulfite cycloserine (TSC) agar medium.

[0109] Determination: If the probiotics in the cecum or feces are significantly increased, and Clostridium perfringens is reduced or has no significant change, it is said to have improved the intestinal bacterial function.

[0110] Statistical analysis

[0111] The data obtained from the experiment were analyzed by one-way analysis of variance and Dunnet or Duncan test, and a p value less than 0.05 was considered significant.

[0112] 3. Results and Discussion

[0113] Multiple emulsification system establishment

[0114] In this experiment, Lactobacillus casei was combined with an external water phase containing different excipients to prepare different oil-in-water-in-oil multiple emulsions by the aforementioned method, which were then spray-dried to prepare a powdered lactic acid bacteria product (i.e., multiple emulsified powder particles) to explore the effects of different excipients on the recovery rate of spray-dried powder and the number of viable bacteria in the powder. The formula of the excipients added to the external water phase is shown in Table 1 below. In Table 1 and the following text, whey protein concentrate is referred to as WPC, and octenyl succinate-modified starch is referred to as modified starch or OSA.

[0115] As shown in Table 1, the powder recovery rate of lactic acid bacteria in the powder of the group with only whey protein concentrate added to the external water phase is poor (less than 10%). If whey protein concentrate is combined with modified starch, the powder recovery rate can be improved. As shown in Table 1, the powder bacterial count of the pure whey protein concentrate group and the group with a ratio of whey protein concentrate to modified starch of 2:1 are higher, and the survival rate of lactic acid bacteria in the spray drying process (survival rate = powder bacterial count / bacterial count before spray drying) is higher, the bacterial count decreases by less than 2 logCFU / g, and the bacterial count is greater than 6×10 7 The comprehensive comparison of the powder recovery rate and powder bacterial count showed that the group with whey protein concentrate: modified starch at a ratio of 2:1 was the optimal formula, so this formula was selected for subsequent experiments.

[0116] Table 1: Effect of excipient formulation on powder bacterial count, lactic acid bacteria survival rate and recovery rate

[0117]

[0118] After selecting whey protein concentrate: modified starch as the main formula of 2:1, 5% of the total weight of the excipients in the external water phase was replaced with additional excipients (i.e. whey protein concentrate: modified starch: additional excipient = 38:19:3) to explore the effects of different types of additional excipients (as shown in Table 2 below) on the recovery rate of spray-dried powder and the number of viable bacteria in the powder.

[0119] As shown in Table 2, the powder recovery rate of the group using only whey protein concentrate and modified starch (and the ratio is 2:1) and the group replacing 5% of the total weight of the excipients with different types of additional excipients is 20% to 32%. As shown in Table 2, the analysis results of the viable count of the powder show that the group using hydroxypropyl methylcellulose and sodium alginate as additional excipients is preferred, and the powder count can reach 9×10 7 CFU / g and 8×10 7 CFU / g. As shown in Table 2, the survival rate of lactic acid bacteria in the spray drying process was analyzed. The group with hydroxypropyl methylcellulose as an additional excipient had a lactic acid bacteria survival rate of 7.5%, which was the best protection effect; the group with gelatin as an additional excipient had a lactic acid bacteria survival rate of 5.8%, which was the second best protection effect. Since many formula groups have a certain protection effect and recovery rate for lactic acid bacteria during spray drying, the powder viable bacteria count in the formula design is greater than 10 7 CFU / g groups were used for follow-up studies.

[0120] Table 2: Effect of excipient formulation on powder bacterial count, lactic acid bacteria survival rate and recovery rate

[0121]

[0122] In order to determine the stability of lactic acid bacteria powder embedded in the multiple emulsion system (i.e., lactic acid bacteria multiple emulsion powder particles), the multiple emulsion powder particle samples were placed at 4° C. for stability analysis. Figure 1 The results show that the initial bacterial count of each group was greater than 10 at 4°C. 7 CFU / g. After 14 days, the number of viable bacteria was measured. The decline in the number of viable bacteria in each group was less than 0.3log cfu / g. The group with the lowest survival rate was the group using only modified starch as an excipient, and its survival rate remained greater than 50%. After 28 days, the number of viable bacteria was measured. Compared with the initial number of bacteria, the survival rate of the group using only modified starch as an excipient was 64%, and the survival rates of the other groups were all higher than 64%. After 28 days at 4°C, the number of viable bacteria in all groups was higher than 2×10 7 CFU / g, indicating that the stability of lactic acid bacteria powder encapsulated by the multiple emulsification system is high.

[0123] The tolerance of the aforementioned multiple emulsified powder to water was analyzed. The multiple emulsified powder was dissolved in water, and then gastric acid and bile salt simulation liquid were added to analyze the survival rate of lactic acid bacteria after a certain period of time. Figure 2A As shown in the figure, the range of the decrease in the number of live bacteria in each group of multiple emulsified powder particles is from 0.7log cfu / g to 3.2log cfu / g; for the bile salt resistance test, the analysis results are as follows Figure 2B As shown in the figure, the range of the decrease in the number of live bacteria in each group of multiple emulsion powder particles is less than 1.0 log cfu / g. Compared with the unencapsulated group (the decrease in the number of bacteria is greater than 10 log cfu / g) and the microcapsule coated group, the multiple emulsion powder particles have a good protective effect, and the number of live bacteria in all formula groups after being placed at 4°C for 28 days is higher than 2×10 7 CFU / g, indicating that the multiple emulsion powder particles of the present invention have high powder stability.

[0124] Animal experiments

[0125] Preliminary experiment

[0126] The multiple emulsion powders (the excipient formula in the external aqueous phase is shown in Table 3 below) coated with lactic acid bacteria and blank multiple emulsion powders were orally administered to mice for 14 consecutive days. The feces of mice were collected on the day before the test (Pre-D1) and on the 7th day (D7) and 14th day (D14) after administration for plate coating and analysis of the number of lactic acid bacteria in the feces. As shown in Table 4 below, the number of lactic acid bacteria in the feces of Group 5 increased significantly on the 7th day. On the 14th day, the content of lactic acid bacteria in the feces of the unembedded group (Lactobacillus casei culture solution) and groups 2 to 7 increased significantly compared with their blank emulsions, among which the number of bacteria in formulas 5 to 7 increased the most.

[0127] Table 3: Excipient formulation in the external aqueous phase

[0128] 1 Whey Protein Concentrate 2 Whey protein concentrate: octenyl succinate modified starch = 2:1 3 Modified starch 4 Whey protein concentrate: modified starch: Hydroxypropyl methylcellulose = 38:19:3 5 Whey protein concentrate: modified starch: sodium alginate = 38:19:3 6 Whey protein concentrate: modified starch: gelatin = 38:19:3 7 Whey protein concentrate: modified starch: gum arabic = 38:19:3

[0129] Table 4: Effects of administration of test substances on the number of lactic acid bacteria in mouse feces

[0130]

[0131] * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001

[0132] Normal mouse model test

[0133] In the normal mouse model test, group 5 can quickly and significantly improve the mouse feces ( Figure 3A ) and the number of lactic acid bacteria in the cecal contents (Figure 3B), the feces ( Figure 4A ) and cecal contents ( Figure 4B ) and reduced the number of Bifidobacterium in mouse feces ( Figure 5A ) and cecal contents ( Figure 5B ) and increased intestinal maltase (maltolase) ( Fig. 6A )、sucrase (sucrase) Figure 6B ), Leucine aminopeptidase (Figure 6C) and Lipase ( Fig.6D ) digestive enzyme activity.

[0134] In the above-mentioned normal model, the mice were administered the test substances for up to 3 weeks, during which no toxic reactions were observed, and the various test substances had no significant effect on the weight of the mice. In terms of intestinal flora ecology, the administration of multiple emulsified powders and microcapsules coated with lactic acid bacteria can increase the number of lactic acid bacteria and bifidobacteria in the intestine, but the multiple emulsified powders coated with lactic acid bacteria alone can significantly reduce the number of bad bacteria in the intestine (the number of Clostridium perfringens). However, although the administration of test substances can improve the intestinal flora ecology, the main influencing factor should be lactic acid bacteria. Since the main location of lactic acid bacteria colonization is in the small intestine, not the cecum, it is speculated that this may be the reason why the cecum values ​​are not affected by the test substances.

[0135] The Lactobacillus casei used in the above test is a lactic acid bacteria with starch decomposition activity. Therefore, in the intestinal digestive enzyme activity test, it can be found that the administration of test substances such as bacterial liquid, multiple emulsified powder or microcapsules can increase the activity of disaccharide decomposing enzymes in the intestinal mucosa, and the expression of enzyme activity is positively correlated with the number of intestinal lactic acid bacteria. In addition, the group administered with the coated lactic acid bacteria multiple emulsified powder has the highest improvement in various types of digestive enzymes, indicating that the multiple emulsified powder of the present invention can significantly promote the number of lactic acid bacteria.

[0136] Antibiotic mouse model test

[0137] In the antibiotic mouse model test, after the pre-administration of broad-acting antibiotics, the number of lactic acid bacteria in the feces decreased significantly. Group 5 could quickly and significantly improve the feces of mice ( Fig. 7A ) and cecal contents ( Figure 7B ) and the number of lactic acid bacteria in mouse feces ( Fig. 8A ) and cecal contents ( Figure 8B ) and reduced the number of Bifidobacterium in mouse feces ( Fig. 9A ) and cecal contents ( Fig. 9B ) and increased the number of Clostridium perfringens in the intestine and increased intestinal maltase ( Fig. 10A )、Sucrase( Fig. 10B ), leucine aminopeptidase ( Fig. 10C ) and lipase ( Fig. 10D ) digestive enzyme activity.

[0138] Figures 7 to 9 show that antibiotic treatment significantly reduces the number of lactic acid bacteria, bifidobacteria, and Clostridium perfringens in the intestines of mice; however, after stopping the antibiotics and administering the test substance, the number of lactic acid bacteria, bifidobacteria, Clostridium perfringens, etc. gradually recovered in both the blank dosage form group and the lactic acid bacteria-containing group. The group administered with multiple emulsions of lactic acid bacteria (i.e., multiple emulsions) recovered faster in the growth of lactic acid bacteria and bifidobacteria in the intestines than the other groups; and probably because the probiotics recovered better, the results of Clostridium perfringens showed that the group administered with multiple emulsions of lactic acid bacteria had a slower increase rate.

[0139] In the antibiotic model, the lactic acid bacteria multi-emulsified powder particles affected the growth of Bifidobacterium, but this was not found in the normal model mouse test. It is speculated that this may be because after the administration of antibiotics, the growth of most bacteria is inhibited, while the administration of lactic acid bacteria multi-emulsified powder particles can improve the intestinal environment, making the environment beneficial to the growth of other probiotics such as Bifidobacterium.

[0140] The results of intestinal enzyme analysis showed that the group of probiotics coated with multiple emulsions can enhance the activity of disaccharide degrading enzymes, lipases, and proteases, and this result may correspond to the changes in the weight of mice. During the intestinal enzyme analysis, the weight of mice was recorded as shown in Table 5 below. After the administration of antibiotics, the weight of mice decreased, and then gradually recovered. However, 7 days after the administration of probiotics coated with multiple emulsions, the weight of mice recovered to the same level as that of mice not treated with antibiotics. It is speculated that the reason may be related to the improvement of intestinal nutrient digestion and absorption by the test substance.

[0141] Table 5:

[0142]

[0143]

[0144] Fecal or cecal content smear tests can only analyze the number of lactic acid bacteria, and it is impossible to know whether the lactic acid bacteria are proliferating in the mouse's own intestines or colonizing in the body after the test substance is administered. However, using quantitative real-time polymerase reaction and Lactobacillus casei specific primers, the colonization of Lactobacillus casei in the intestine can be tracked. In addition to the blank dosage form group and the control group, each group of mice was administered with a fixed number of Lactobacillus casei polymerase reaction results as shown in the figure. Fig.11A and 11B As shown, the results show that after administration of Lactobacillus casei, the strain can colonize in the intestine. Among them, the colonization of the strains in the groups coated with multiple emulsion formulations and microcapsule formulations is good, and there is a significant difference compared with the uncoated bacterial solution. It is speculated that it should be due to the effect of the coated formulation to protect the target substance from passing through gastric acid, bile salts, etc.

[0145] Referring to the above, through the gastrointestinal physiological activity related indicator test items such as mouse intestinal flora distribution, intestinal digestive enzyme activity, cecal physiological data, polymerase reaction, etc., it is confirmed that the multiple emulsified powder particles of the present invention can indeed exert protective properties against active ingredients such as probiotics (such as lactic acid bacteria), thereby enhancing the physiological activity of the gastrointestinal tract.

[0146] The above embodiments are only for explaining the principle and efficacy of the present invention, but not for limiting the present invention. The modifications and changes made by the technicians in the relevant field to the above embodiments still do not violate the spirit of the present invention. The scope of rights of the present invention shall be as listed in the claims described below.

Claims

1. A method for preparing multiple emulsion powder particles, comprising: (a) mixing an active ingredient with an internal aqueous phase to form a homogenous mixture, wherein the active ingredient is a probiotic; (b) mixing the homogenous mixture with an oil phase to form a water-in-oil emulsion; (c) mixing the water-in-oil emulsion with an external aqueous phase to form a water-in-oil-in-water multiple emulsion, wherein the external aqueous phase comprises water and an excipient, wherein the excipient in the external aqueous phase comprises whey protein concentrate and modified starch, the modified starch is octenyl succinate modified starch, and wherein the weight ratio of the whey protein concentrate to the modified starch is 2:1; and (d) spray drying the water-in-oil-in-water multiple emulsion to obtain multiple emulsion particles.

2. The method according to claim 1, wherein the probiotic is selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium, Lactobacillus casei, Lactobacillus lodneri, Bifidobacterium lactis, Bifidobacterium longum and any combination thereof.

3. The method according to claim 1 or 2, wherein the internal aqueous phase comprises water and excipients.

4. The method according to claim 3, wherein the excipient in the internal aqueous phase is selected from the group consisting of hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium alginate, gelatin, gum arabic, sodium caseinate, soy protein and any combination thereof.

5. The method according to claim 3, wherein the internal aqueous phase further comprises salts.

6. The method of claim 5, wherein the salt is selected from the group consisting of sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, potassium sulfate, calcium sulfate, sodium malate, sodium citrate, disodium citrate, sodium dihydrogen citrate, potassium dihydrogen citrate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, and any combination thereof.

7. The method according to claim 1 or 2, wherein the oil phase comprises a vegetable oil and a lipophilic surfactant.

8. The method of claim 7, wherein the vegetable oil is selected from the group consisting of sunflower oil, soybean oil, olive oil, canola oil, linseed oil and palm oil.

9. The method according to claim 7, wherein the lipophilic surfactant is selected from the group consisting of polyglycerol ricinoleate, lecithin, sugar esters, emulsifying wax, polyglycerol fatty acid esters, polysorbate, monoglycerides, diglycerides, and any combination thereof.

10. The method according to claim 1 or 2, wherein the excipient in the external aqueous phase further comprises an additional excipient selected from the group consisting of hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium alginate, gelatin, gum arabic, sodium caseinate, soy protein, and any combination thereof.

11. The method of claim 10, wherein the additional excipient is hydroxypropyl methylcellulose.

12. The method according to claim 10, wherein based on the total weight of the excipients in the external aqueous phase, the weight percentage of the whey protein concentrate is 55-70%, the weight percentage of the modified starch is 25-35%, and the weight percentage of the additional excipient is 1-10%.

13. The method according to claim 12, wherein the weight ratio of the whey protein concentrate, the modified starch and the additional excipient is 38:19:

3.

14. A composition obtained by the method according to any one of claims 1 to 13.

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