KR20070104140A - The method of preparing multi-microcapsule of lactic acid bacteria, the microcapsule prepared by the method, and article comprising the microcapsule - Google Patents

Abstract

A multi-microcapsule of lactic acid bacteria, a method for preparing the same, and a product comprising the same are provided to improve the dispersibility and the stability of the multi-microcapsule, and maintain a large quantity of lactic acid bacterial even during a long storage period. A first coating material is prepared by sterilizing and gelatinizing low-fat skimmed milk, glycerin, and a media solution, and homogenizing a mixture of lactic acid bacteria and the resultant material. A second coating material is prepared by mixing refined process oil, polyglycerol fatty acid ester, and glycerin succinylated fatty acid into the first coating material, and homogenizing the mixture. A third coating material is prepared by mixing, gelatinizing, and homogenizing carbohydrate, protein, germ type binder, and emulsifier, and homogenizing a mixture of the second coating material and the resultant material. The third coating material is sprayed in sterilized cool water.

Description

The method of preparing multi-microcapsule of lactic acid bacteria, the microcapsule prepared by the method, and article comprising the microcapsule}

Figure 1 shows a schematic diagram of a method for producing lactic acid bacteria and unsaturated fatty acid covalent multiple microcapsules according to the present invention.

Figure 2 shows the number of viable cells per capsule of 1 mL during the storage period in the 4 ° C refrigerated state of the microcapsules prepared in Example 1.

Figure 3 shows the number of viable cells according to the storage temperature and storage period of the microcapsules prepared in Example 1.

Figure 4 is an analysis chart measuring the size of the microcapsules prepared in Example 1.

5 shows the number of viable cells according to the storage period at 4 ° C. when the microcapsules prepared in Example 1 and 0.3% of the microcapsules prepared in Example 1 were added to milk.

Figure 6 shows the degradation time of the capsule in the body in vitro test results for pepsin of the microcapsules prepared in Example 1.

Figure 7 shows the number of viable cells by heat treatment when the microcapsules prepared in Example 1 are directly sterilized and sterilized after addition to milk.

Figure 8 shows the experimental results showing how the capsules are broken under heat sterilization conditions at 90 ℃, 30 seconds after the addition of the microcapsules prepared in Example 1 to the beverage.

9 is a chart showing the change in viable cell number and pH (20 ° C.) when 2% L. Lactis microcapsules prepared in Example 4 were added to kimchi.

Figure 10 shows the number of lactic acid bacteria viable bacteria from the date of manufacture 15 days when the capsule of L. Lactis prepared in Example 4 is added to 0.5% milk.

Figure 11 shows the number of lactic acid bacteria viable bacteria from the date of manufacture 15 days when the capsule of L. Lactis prepared in Example 4 is added to 0.5% soy milk.

FIG. 12 is a table showing the viable cell count (per mL) for 5.0 days of 5.0% Lactobacillus esidophilus and Lactococcus thermophilus covalent microcapsules prepared in Example 5 in a cold state at 4 ° C.

13 is an experimental result showing the viable cell count of the microcapsules prepared in Example 6.

Figure 14 is GC data analyzing the CLA content in the lactic acid bacteria and CLA covalent microcapsules prepared in Example 9a.

FIG. 15 is a chart showing the viable cell count of lactic acid bacteria prepared in Example 9a and bifidus lactic acid bacteria in CLA-covalent microcapsules.

FIG. 16 is a table showing data of measuring viable cell number and survival time (12 months) of the powder prepared in Example 10.

FIG. 17 is a table showing data of measuring the number of viable bacteria (per g) of the mixed strain of the powder prepared in Example 11 during the storage period.

The present invention relates to a method for producing multiple microcapsules containing lactic acid bacteria, microcapsules produced by the method, and products containing the microcapsules. The present invention also relates to a method for producing lactic acid bacteria-unsaturated fatty acid multi-microcapsules containing both lactic acid bacteria and unsaturated fatty acids, microcapsules produced by the production method, and products containing the microcapsules.

Lactic acid bacteria, also known as lactic acid bacteria and lactic acid, is an important bacterium that lives in the intestines of mammals to prevent abnormal fermentation by various bacteria and is also used as a dressing agent. For example, Lactobacillus: L. Bulgarcus is a Bulgarian lactic acid bacterium that has long been known. It is used in the production of yogurt, and the species is used as a starter for the production of cheese or fermented butter, and L. acidophilus is an aerobic lactic acid bacterium that exists in the intestines of humans and all mammals and other animals. It is used to make milk or to treat intestinal self-intoxication. L. lactis also produces DL-lactic acid, which is always present in milk and is used in butter and cheese production and is the most important species for dairy lactic acid bacteria. Streptococcus: S. faecalis is used as a dressing agent and S. lactis is used as a starter for cheese making.

This useful lactic acid bacteria settles in the intestines and exerts various physiological effects such as activating intestinal motility, inhibiting harmful bacteria, promoting vitamins and immune enhancing substances, and due to the structure of the human body, humans pass through the stomach when ingesting lactic acid bacteria, The lactic acid bacteria are often killed, so that the lactic acid bacteria can not exhibit the original bioactive function. Therefore, in order to minimize the destruction of the lactic acid bacteria in the past, the method has been used to enable the anti-cancer and immune function by arbitrarily adjusting the concentration of lactic acid bacteria. However, determining the optimal concentration of lactic acid bacteria has been very difficult and its use has been extremely limited.

Therefore, in order to overcome these disadvantages in recent years by coating various lactic acid bacteria using starch, gelatin, alginic acid, cellulose, hardened oil, various emulsifiers as a coating material using a large capsule or 50 micrometer or more microcapsules There has been developed a method for producing or encapsulating functional unsaturated fatty acids in higher concentrations than necessary to maintain quality within the shelf life. In addition, a method of encapsulating using various coating agents has been developed to increase the stability of functional lactic acid bacteria or functional materials to continuously supply them to the human body. However, the conventional encapsulation method has a disadvantage in that the number of viable bacteria of lactic acid bacteria in the capsule is 10 ² or less per millimeter or the capsules collapse within 5 days at 20 ° C. without maintaining the storage capacity of the lactic acid bacteria for 5 days or more. Therefore, in the current trading market, it is required to develop microcapsules having both stability and dispersibility of lactic acid bacteria.

Recently, various physiological effects of polyunsaturated fatty acids have been reported along with lactic acid bacteria. In particular, omega-3 fatty acids, which are unsaturated fatty acids, are distributed in nerve cell membranes and retinas, and rapidly transfer electrical stimulation from cell membranes to the next cell, protect cells in the human body, maintain cell structure, and smoothly budding. It is reported to help with metabolism. In addition, it has been reported to have the effect of inhibiting the film formation of blood, promoting the formation of bone and at the same time strengthening. Omega 3 fatty acids include docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA), and alpha-linolenic acid (DPA). The recommended daily amount of omega-3 fatty acids is 0.6 to 1g and is found in fish oil, plankton, seafood, soybean oil, and breast milk. Newborns and adolescents in particular need more amounts to support normal tissue development. Deficiency may cause depression, schizophrenia, attention deficit hyperactivity disorder, decreased vision, heart disease, etc., and is known to increase the stress is a big concern recently. However, omega 3 fatty acids are unstable to oxygen or light and are easily oxidized during storage, resulting in a unique fishy. To solve this problem, a method of microencapsulation has been proposed, and as an example, Roh et al. (Korean Patent Laid-Open Patent Publication No. 2000-0038444) propose a method of encapsulating unsaturated fatty acids such as DHA by using a polysaccharide material such as glutinous rice starch and a twin series emulsifier. A method for improving sensory properties of fish oil is presented. However, it does not maintain dispersibility for a long time in the aqueous solution and shows a phenomenon of separation. Another example, Park et al. (Korea Patent Publication No. 10-2004-0042987) encapsulated fish oil containing polyunsaturated fatty acid using a protein-based coating material, such as isolated soy protein, suggesting the applicability to various fields including food, The stability of appeared to be a problem.

The present invention has improved dispersibility and stability compared to the conventional lactic acid bacteria capsule technology, a multi-microcapsules manufacturing method containing lactic acid bacteria that retain a large number of lactic acid bacteria even during long storage period, microcapsules produced by the method, It is an object to provide a product containing microcapsules. It is also an object of the present invention to provide a multi-microcapsules manufacturing method comprising a primary coated lactic acid bacteria and a primary coated unsaturated fatty acid together, and microcapsules produced by the production method, a product containing the microcapsules.

In order to achieve the above object, the present invention

(a) sterile gelatinization of one selected from whey powder, low fat skimmed milk powder, glucose or bactotrypton, glycerin and a medium solution, and then adding lactic acid bacteria and homogenizing to prepare a primary coating material;

(b) preparing a secondary coating material by mixing and homogenizing the refined processed oil, polyglycerin fatty acid ester, and glycerin zucchini fatty acid ester to the primary coating material prepared above;

(c) lactic acid bacteria micromicrocapsule comprising mixing carbohydrates, protein components, gum thickener and emulsifier to gelatinize and homogenize, and then adding a homogeneous secondary coating material to prepare a tertiary coating material and spraying it into sterile cooling water. It provides a manufacturing method 1.

In the preparation step, the carbohydrate is starch, modified starch, maltodextrin, dextrin, cyclodextrin and cellulose components CMC (carboxymethylcellulose), MC (methylcellulose), EC (ethylcellulose), NC (nitrocellulose) , AC (acetyl cellulose) may be used, and protein components may be gelatin, whey protein, whey powder, SMP8515 (Sungfeng), gluten, casein, albumin, hemoglobin, peptides and the like. The gum thickener may be acacia gum, gum arabic, xanthan gum, guar gum, etc., and the emulsifier is twin 80, twin 60, twin 40, twin 20, span 85, span 80, span 60, span 40, span 20, poly Glycerin fatty acid ester, monoglycerin fatty acid ester, etc. can be used.

The lactic acid bacteria in the production method 1 of the present invention BP dobacterium breve (CBG-C2, registration number 0516377, accession number KACC 91001), BP, which is a CLA (conjugated linoleic acid, hereinafter referred to as 'CLA') production patent strain Dobacterium pseudocartenulumum (CBG-C4, reg. No. 0514592, accession number KACC 91003), enterococcus passium (CBG-C5, reg. No. 0514593, KACC 91002), Lactobacillus ecidophilus, It is preferable that at least one selected from Lactococcus thermophilus, Lactobacillus casein, Lactobacillus luteri or bifidus lactic acid bacteria (CBG-C2, CBG-C4 and CBG-C5, respectively) Deposited with the same accession number in the microbial preservation center).

The lactic acid bacteria in step (a) of the production method 1 of the present invention is preferably 1.0 to 5.0% (w / w) in the total amount of microcapsules. In addition, the whey powder, low fat skimmed milk powder, glucose or bactotryptone of step (a) of the manufacturing method 1 of the present invention is 0.05 to 0.5% (w / w) based on the total amount of microcapsules, and glycerin is micro It is preferably 0.1 to 1.0% (w / w) based on the total amount of the capsule, and the medium solution is preferably 0.05 to 0.5% (w / w) based on the total amount of the microcapsules.

In the present invention, the method of the present invention (1) of the purified co-sharing step is 1.5 to 5.0% (w / w) based on the total amount of microcapsules, polyglycerol fatty acid ester is 0.3 to based on the total amount of microcapsules 0.5% (w / w), and glycerin zucchini fatty acid ester is preferably 0.2 to 0.4% (w / w) based on the total amount of microcapsules.

Carbohydrate in step (c) of the manufacturing method 1 of the present invention is 0.1 to 0.3% (w / w) based on the total amount of microcapsules, protein component is 1.5 to 3.0% based on the total amount of microcapsules, galvanic thickener 0.1 to 0.3% based on the total amount of the capsule, the emulsifier is preferably 0.1 to 0.5% based on the total amount of the microcapsules.

The homogenization temperature of the steps (a), (b) and (c) of the production method 1 of the present invention is preferably 30 ° C to 50 ° C, more preferably 35 ° C to 45 ° C, and more preferably 40 ℃.

In the production method 1 of the present invention, the carbohydrate in step (c) is preferably starch or modified starch, and the protein component is preferably gelatin or agar. In addition, the gum thickener of step (c) in the production method 1 of the present invention is preferably guar gum or xanthan gum, and the emulsifier is preferably polyglycerol fatty acid ester.

The present invention also provides a microcapsules prepared according to the inventors of the production method 1.

The present invention also provides a product comprising the microcapsules prepared according to the present invention manufacturing method 1. The products include fermented milk, processed milk, fermented foods, fermented foods, kimchi fermented foods, functional foods, functional drinks, general foods, pharmaceuticals, cosmetics.

The present invention also provides a method for preparing lactic acid bacteria-unsaturated fatty acid covalent microcapsules in which unsaturated fatty acid coated separately with polyglycerol fatty acid ester and phosphate buffer is added to step (b) together with the primary coating material produced in step (a). To provide.

In the manufacturing method 2 of the present invention, the unsaturated fatty acid is preferably DHA (docosahexaenoic acid), EPA (Eicosapentaenoic acid) or CLA.

In the preparation method 2 of the present invention, the coated unsaturated fatty acid is 0.3 to 0.5% (w / w) of polyglycerol fatty acid ester, 0.5 to 1.0% (w / w) of 0.1 N based on the total amount of microcapsules and It is preferred to encapsulate homogenized with 5.0-10.0% (w / w) unsaturated fatty acid.

The present invention also provides microcapsules prepared by the preparation method 2.

The present invention also provides a product comprising the microcapsules prepared by the preparation method 2. Here, the product includes fermented milk, processed oil, fermented food, kimchi fermented food, functional beverage, functional food, general food, medicine, cosmetics.

 The present invention provides a powder production method 3 for lyophilizing by mixing the microcapsule prepared by the production method 1 with skim milk powder, dextrin, chlorella.

In Preparation Method 3 of the present invention, the microcapsules prepared in Preparation Method 1 are 0.1 to 0.5 wt%, skim milk powder is 5 to 15 wt%, dextrin is 5 to 15 wt%, and chlorella is 70 to 85 wt% Is preferably.

The present invention also provides a powder prepared by the preparation method 3.

The present invention also provides a product comprising the powder produced by the production method 3. The products include fermented milk, processed oil, fermented foods, fermented foods, kimchi fermented foods, functional drinks, functional foods, general foods, pharmaceuticals, cosmetics.

The invention also

(a) sterilizing the low fat skimmed milk powder, the medium solution, glycerin and cysteine, adding lactic acid bacteria and homogenizing it to prepare a primary coating material;

(b) mixing and homogenizing polyglycerol fatty acid ester and monoglycerin fatty acid ester to the primary coating material prepared above to prepare a secondary coating material;

(c) Lactobacillus with improved dispersibility, comprising the steps of mixing the protein component gum thickener and emulsifier to gelatinize and homogenize, and then adding a homogeneous secondary coating material to prepare a tertiary coating material and spraying it into sterilized cooling water. Provided is a method for preparing multiple microcapsules.

In this preparation step, the protein components, gum thickeners and emulsifiers that can be used are the same as those mentioned above.

The lactic acid bacteria in the production method 4 of the present invention is CPI production patent strain Bifidobacterium breve (CBG-C2, Registration No. 0516377), Bifidobacterium pseudocartenulumtum (CBG-C4, Registration No. 0514592) ), Enterococcus fascium (CBG-C5, registration number 0514593), Lactobacillus escidophilus, Lactococcus thermophilus, Lactobacillus casein, Lactobacillus luteri or bifidus lactic acid bacteria is preferably one or more selected.

The lactic acid bacteria in the production method 4 of the present invention is preferably 1.0 to 5.0% (w / w) in the total amount of microcapsules.

The low fat skim milk powder in step (a) of the preparation method 4 of the present invention is 0.05 to 0.5% (w / w) based on the total amount of microcapsules, and glycerin is 0.1 to 1.0% (w / w) based on the total amount of microcapsules. The medium solution is 0.05 to 0.5% (w / w) based on the total amount of microcapsules, and cysteine is preferably 0.01% to 0.1% (w / w) based on the total amount of microcapsules.

The cysteine of step (a) of the preparation method 4 of the present invention is preferably 0.05% (w / w) based on the total amount of microcapsules.

Polyglycerol fatty acid ester of step (b) of the production method 4 of the present invention is 5.0 to 10.0% (w / w) based on the total amount of microcapsules, and monoglycerin fatty acid ester is 0.2 to 0.4% (based on the total amount of microcapsules) w / w).

The protein component of step (c) of the manufacturing method 4 of the present invention is 1.5 to 3.0% based on the total amount of microcapsules, gum thickener is 0.3 to 0.5% based on the total amount of microcapsules, and emulsifier is based on the total amount of microcapsules. It is preferable that it is 0.1 to 0.5%.

The homogenization temperature of the steps (a), (b) and (c) of the production method 4 of the present invention is preferably 30 ℃ to 50 ℃, more preferably 35 ℃ to 45 ℃, more preferably 40 ℃.

The protein component in step (c) of the production method 4 of the present invention is preferably gelatin or agar, the gum thickener is preferably guar gum or xanthan gum, and the emulsifier is preferably polyglycerol fatty acid ester.

The present invention also provides a microcapsules prepared according to the inventors' production method 4.

The present invention also provides a product comprising the microcapsules prepared according to the inventors' production method 4. The products include fermented milk, processed oil, fermented foods, fermented foods, kimchi fermented foods, functional drinks, functional foods, general foods, pharmaceuticals, cosmetics.

The present invention also provides a method for preparing lactic acid bacteria-unsaturated fatty acid covalent microcapsules 5, in which unsaturated fatty acid coated separately with polyglycerol fatty acid ester and phosphate buffer is added to step (b) together with the primary coating material produced in step (a). to provide.

In the production method 5 of the present invention, the unsaturated fatty acid is preferably DHA (docosahexaenoic acid), EPA (Eicosapentaenoic acid) or CLA.

In the preparation method 5 of the present invention, the coated unsaturated fatty acid is 0.3 to 0.5% (w / w) of polyglycerol fatty acid ester, 0.5 to 1.0% (w / w) of 0.1 N based on the total amount of microcapsules, and It is preferable to encapsulate homogenized with 5.0-10.0% (w / w) unsaturated fatty acid.

The present invention also provides microcapsules prepared by the preparation method 5.

The present invention also provides a product comprising the microcapsules prepared by the manufacturing method 5. Here, the product includes fermented milk, processed oil, fermented food, kimchi fermented food, functional beverage, functional food, general food, medicine, cosmetics.

 The present invention provides a powder production method 6 of mixing the microcapsules prepared by the production method 4 with skim milk powder, dextrin, chlorella and lyophilization.

In the preparation method 6 of the present invention, the microcapsules prepared in Preparation Method 4 are 0.1 to 0.5% by weight, skim milk powder is 5 to 15% by weight, dextrin is 5 to 15% by weight, and chlorella is 70 to 85% by weight. It is preferable.

The present invention also provides a powder prepared by the production method 6.

The present invention also provides a product comprising the powder produced by the production method 6. The products include fermented milk, processed oil, fermented foods, fermented foods, kimchi fermented foods, functional drinks, functional foods, general foods, pharmaceuticals, cosmetics.

Hereinafter, examples will be described to help understand the advantages and features of the present invention and how to achieve them. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are to make the disclosure of the present invention complete, and the general knowledge in the technical field to which the present invention belongs. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example  One]

CLA  production Patent strain Bifidobacterium Of Breve (CBG-C2)  Multiple Microcapsule Preparation

One-step coating, 1L high-temperature resistant glass container with 0.05% (w / w) low fat skim milk powder, 0.15% (w / w) glycerin and 0.05% (w / w) medium solution, based on the total amount of microcapsules Sterilization at 121 ° C. for 10 minutes using a sterilizer (Vision VS-1221-60) was followed by mixing. The mixture was cooled to 40 ° C. and then mixed with cultured recovered 5.0% (w / w) CLA-producing Bifidobacterium breve (CBG-C2) at 6,400 rpm using a precision homogenizer (IKA T-50). Homogenized for 1 minute. After the two-stage coating, 5.0% (w / w) refined covalently heated to 80 ℃ in the primary coating material and then cooled to 40 ℃ 0.5% (w / w) polyglycerin fatty acid ester (Almax 9025), 0.4% (w / w) glycerin succinic acid fatty acid ester (Almax 5100) was mixed and homogenized for 5 minutes at 6,400 rpm using a precision homogenizer (IKA T-50) to complete the secondary coating of the CLA production patent strain. Three stages of coating followed by sufficient sterilization of 0.3% (w / w) starch, 3.0% (w / w) gelatin, 0.5% (w / w) polyglycerin fatty acid ester (Almax 9080), 0.3% (w / w) guar gum The mixture was mixed with water, gelatinized at 120 ° C., cooled to 40 ° C., and homogenized at 6,400 rpm for 5 minutes using a precision homogenizer (IKA T-50). Next, the secondary coating material of the CLA production patent strain was After addition and homogenization at 6,400rpm again for 1 minute and then completed the triple coating, and sprayed directly into the sterilized cooling water to prepare a multi-lactic acid bacteria microcapsules. The resulting CLA-producing Bifidobacterium breve (CBG-C2) lactic acid bacteria multi-microcapsules were 5.0% (w / w) lactic acid bacteria cured into white spheres with an average size of 1.5-1.7 μm, and the yield was 99%.

Example  1, physical property test:

Hereinafter, the properties of these capsules or powders prepared above were tested. However, the identification of the number of lactic acid bacteria viable bacteria of the prepared multiple CLA lactic acid capsules used here was made through the following method. The plate coated with BL-agar medium was inoculated according to the dilution ratio, and then evenly distributed and put in an anaerobic bottle and incubated for 48 hours in a 37 ° C. incubator to calculate the number of lactic acid bacteria colonies. In addition, the multi-coated lactobacillus capsule was stored for 3 months (or in some cases 12 months) at 4 ℃ while confirming the shelf life while calculating the number of viable bacteria in the BL-agar medium plate as described above.

Experimental Example 1 Refrigeration Storage Experiment at 4 ° C

After the lactic acid bacteria (CBG-C2) multiple microcapsules prepared according to Example 1 were stored at 4 ° C. in a refrigerated state for 95 days, the number of viable cells per capsule of 1 mL was measured. The results are shown in Figure 2, it was observed that even after 95 days the number of lactic acid bacteria is present.

Experimental Example 2 Storage Experiment at 25 ° C and 37 ° C

The lactic acid bacteria (CBG-C2) prepared according to Example 1 microcapsules were stored at 25 ° C. and 37 ° C. for 50 hours, and then the number of viable cells was measured. The results are shown in Figure 3, in Figure 3 BB1202 and BB1208 represents the serial number of the CBG-C2 multiple microcapsules.

Experimental Example 3 Measurement of Capsule Size

Pretreatment by diluting the size of the lactic acid bacteria (CBG-C2) multiple microcapsules prepared according to Example 1 with an autoclaved 3rd DIW using an ELS-Electrophoretic Light Scattering System, manufacturer: Ostuka, model name: ELS-8000 Measured after the procedure. The results are shown in FIG. 4, and were measured to have an average size of 1,500 nm to 1,700 nm.

Experimental Example 4 After adding 0.3% of capsules to milk, refrigerated storage at 4 ° C

After adding lactic acid bacteria (CBG-C2) multiple microcapsules prepared according to Example 1 and 0.3% of the present capsules to milk at 10 ° C. for 15 days at 4 ° C., the change in viable cell count was measured accordingly. saw. The result is the same as Figure 5, even in the case of milk containing 0.3% microcapsules according to the present invention, it was found that even after about 10 days a large number of lactic acid bacteria.

Experimental Example 5 pH Stability and Dispersibility Experiment

Example 1 Preparation of lactic acid bacteria in the (C2-CBG) pH stability and dispersibility of the microcapsules in multiple 37 ℃ Trizma-base Buffer (20mM Triz / 100mM NaCl) and then to an appropriate pH using 0.1N HCl & 0.1N NaOH , Measured over time. The results are shown in Table 1 below.

Table 1

  Time pH 15 min 30 min 45 min One hr One hr  15 min One hr  30 min 2 hr 2 hr  30 min 1.04 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 2.10 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 3.00 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 4.11 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 5.52 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 7.10 Stability Stability Stability Stability Stability Stability Stability Slowly loosening 8.13 Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Cap Schull En Kim 9.50 Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Capsule Tangle Cap Schull En Kim

That is, the capsules according to the present invention have good stability and dispersibility for about 2 hours in acid and neutral, but have been shown to greatly decrease in stability at 2 hours or more. Alkaline, however, was found to be poor dispersibility of the capsule.

Experimental Example 6 In Vitro Experiment on Pepsin

Lactic acid bacteria (CBG-C2) multiple microcapsules prepared in Example 1 was tested in vitro for pepsin at 37 ℃. As an in vitro test method for capsules using pepsin, a method was used to measure the viable cell count of lactic acid bacteria at each time period by preparing artificial gastric juice. The measuring method was 4ml pepsin solution (pH 1.2, 1mg / ml: from porcine gastric mucosa; powder, 800 to 2,500 units / mg protein), and then adjusted to pH 2 with 1N HCl, the total solution to about 100ml And stored at 37 degrees incubator. Lactobacillus capsule, a reference sample, was also used while stored in an incubator at 37 degrees. Three samples shall be selected for each time zone (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 hours). The lactic acid bacteria capsules were added to 10% of the total solution, and the number of viable cells was measured by sampling and plating each time period. The results are shown in Figure 6, the capsule according to the invention was found to be stable for pepsin for about 3 hours.

Experimental Example 7 Measurement of Viable Cell Number Change by Sterilization

After adding the lactic acid bacteria (CBG-C2) multi-microcapsules prepared in Example 1 directly or in milk, the number of viable cells was decreased when sterilized for 20 seconds at 72 ° C. and for 2 seconds at 135 ° C., respectively. saw. The results are shown in Figure 7, when the capsule sterilization and sterilization after adding directly to the milk, the number of viable cells of the capsule was not heat treated at 135 ℃, 2 seconds treatment than 72 ℃, 20 seconds treatment Viable cell numbers similar to were identified.

<Example 8> Change in the number of viable cells with time at 90 ℃

The lactic acid bacteria (CBG-C2) multi-microcapsules prepared in Example 1 were added to soymilk ( Vegemil ) and then measured how much the capsules were broken under heat sterilization conditions at 90 ° C. for 30 seconds. The results are shown in Figure 8, it was confirmed that the CLA-producing bifidus lactic acid microcapsules about 50% collapse by 90 ℃, 30 seconds heat treatment (in Figure 14, the vertical axis shows the cell concentration (OD)).

[ Example  2]

CLA  production Patent strain Of pseudocartonulatum (CBG-C4)  Preparation of Multiple Microcapsules

5.0% (w / w) CLA Production Patented Strain Bifidobacterium Pseudocarthenulatum (CBG-C4) Instead of 5.0% (w / w) CLA Production Bifidobacterium breve (CBG-C2) Except for using the same method as in Example 1 to obtain the title capsule. The obtained 5.0% (w / w) CLA-producing patented strain Bifidobacterium pseudocartenulumatum (CBG-C4) lactic acid bacteria multiple microcapsules were cured into white spheres having an average size of 1.5-1.7 μm, and the yield was 99 Was%.

[ Example  3]

CLA  production Patent strain Enterococcus Passion (CBG-C5)  Preparation of Multiple Microcapsules

Except for using 5.0% (w / w) CLA production patent strain Enterococcus fasium (CBG-C5) instead of 5.0% (w / w) CLA production Bifidobacterium breve (CBG-C2) in Example 1 Then the same method as in Example 1 was carried out to obtain the title capsule. The obtained 5.0% (w / w) CLA production patent strain Enterococcus fasium (CBG-C5) Lactic acid bacteria multi-microcapsules were cured into white spheres with an average size of 1.5 ~ 1.7㎛, the yield was 99%.

[ Example 4 ]

L. Lactis  Preparation of Multi-Coated Microcapsules of Lactic Acid Bacteria

Example 1 except that 5.0% (w / w) L. Lactis Lactobacillus (Rhodia Inc.) was used in place of 5.0% (w / w) CLA production Bifidobacterium breve (CBG-C2) The same procedure as in 1 was carried out to obtain the title capsule. The obtained multi-microcapsules of 5.0% (w / w) L. Lactis lactic acid bacteria were cured into white spheres having an average size of 1.5-1.7 μm , and the yield was 99%.

Example  4, physical property testing:

Experimental Example 9 Measurement of Viable Cell Number Change When Added to Kimchi

After adding 2% L. Lactis multi-microcapsules prepared in Example 4 to kimchi, and stored for 10 days at 20 ℃, the resulting changes in viable bacteria and pH was measured. The result is shown in FIG. As fermentation progressed, kimchi became acidified, and viable cell number was observed to have the maximum number at pH around 3.5.

Example 10 Measurement of the Number of Viable Cells in Milk

After adding 0.5% of multiple microcapsules of L. Lactis prepared in Example 4 to milk, stored at 4 ° C, the number of lactic acid bacteria was measured until 15 days from the date of manufacture. The results are shown in FIG. 10, and the viable cell count was similar to that at the time of manufacture even after 15 days.

Example 11 Measurement of Change of Viable Cell Number in Soymilk

After adding 0.5% of multiple microcapsules of L. Lactis prepared in Example 4 to soymilk, stored at 4 ° C, the number of lactic acid bacteria was measured until 15 days from the date of manufacture. The result is shown in Figure 11, even after 15 days viable cell count was similar to the time of manufacture.

[ Example 5 ]

5.0% Lactobacillus With Ecidophilus Lactococcus Thermophilus  Shared microcapsule manufacturing

In Example 1, Lactobacillus Esidophilus (Rhodia Inc.) and Lactococcus Thermophilus (Rhodia Inc.) were replaced with 5.0% (w / w) CLA-producing Bifidobacterium breve (CBG-C2). The title capsule was obtained in the same manner as in Example 1 except that% (w / w, L. ecidophilus: S. thermophilus = 1: 1) was used. The obtained covalent multi-microcapsules of 5.0% (w / w) Lactobacillus Escidophilus and Lactococcus thermophilus were cured into white spheres having an average size of 1.5-1.7 μm, and the yield was 99%.

Example  5, physical property testing:

<Example 12>

Multiple microcapsules prepared according to Example 5 were stored for 90 days in a refrigerated state at 4 ℃ while measuring the number of viable cells (per mL) over time. The results are shown in FIG. 12, and after 90 days, viable cell counts were calculated using a BL-agar medium plate.

[ Example  6]

Improved dispersibility CLA  production Patent strain Bifidobacterium Of Breve (CBG-C2)  Preparation of Multiple Microcapsules

One-stage coating of 0.05% (w / w) low fat skimmed milk powder, medium solution, 0.17% (w / w) glycerin, and 0.05% cysteine, respectively, in 1L of luxury glass container with sterilizer After sterilization for minutes and then cooled to 40 ° C., the mixture was incubated with the recovered 5.0% (w / w) CLA producing patent strain Bifidobacterium breve (CBG-C2) and homogenized at 6,400 rpm for 1 minute. 10.0% (w / w) polyglycerin fatty acid ester (Almax 9025) and 0.4% (w / w) monoglycerin fatty acid ester (Almax 1800) were added to the primary coating material, followed by homogenization at 6,400 rpm for 5 minutes. To complete the secondary coating of the CLA production patent strain. 3.0% (w / w) gelatin, 0.5% (w / w) polyglycerin fatty acid ester (Almax 9080) and 0.5% (w / w) guar gum were mixed in sufficient sterile water, gelatinized at 120 ° C, and cooled to 40 ° C. And homogenized at 6,400 rpm for 5 minutes. Then, the second coating material of the CLA production patent strain was added, homogenized again at 6,400 rpm for 1 minute, and then completed the third coating, and then sprayed directly into the sterilized cooling water by 5.0% (w / w) CFI production Bifidobacte Multiple microcapsules of L. breve (CBG-C2) lactic acid bacteria were prepared. As a result, the title capsule obtained had a hardening effect by maintaining dispersibility even in a refrigerated state, and a yield was 99%.

Example  6, physical property testing:

Experimental Example 13 Refrigerated Storage of Experimental Example 6 Capsules

The CLA production patent strain Bifidobacterium breve (CBG-C2) prepared in Example 6 was stored at 4 ° C. for 15 days, and the number of viable cells was measured according to the storage period. The results are shown in Figure 13, it was found that even after 15 days of storage, the number of lactic acid bacteria was not significantly reduced.

[ Example  7]

Improved dispersibility CLA  production Patent strain Of pseudocartonulatum (CBG-C4)  Preparation of Multiple Microcapsules

5.0% (w / w) CLA Production Patented Strain Bifidobacterium Pseudocarthenulatum (CBG-C4) Instead of 5.0% (w / w) CLA Production Bifidobacterium breve (CBG-C2) Except for using the same method as in Example 4 to obtain the title capsule. As a result, the title capsule obtained had a hardening effect by maintaining dispersibility even in a refrigerated state, and a yield was 99%.

[ Example  8]

Improved dispersibility CLA  production Patent strain Enterococcus Passion (CBG-C5)  Preparation of Multiple Microcapsules

Except for using 5.0% (w / w) CLA production patent strain Enterococcus fasium (CBG-C5) instead of 5.0% (w / w) CLA production Bifidobacterium breve (CBG-C2) in Example 4. And the same method as in Example 4 to obtain the title capsule. As a result, the title capsule obtained had a hardening effect by maintaining dispersibility even in a refrigerated state, and a yield was 99%.

[ Example  9a]

5.0% CLA  production Bifidobacterium Breve (CBG-C2) and CLA  Covalent Multi-Microcapsule Preparation

One-step coating of lactic acid bacteria, 0.05% (w / w) whey powder (or SMP8515), 0.15% (w / w) glycerin and 0.05% (w / w) medium solution, respectively, based on the total amount of microcapsules with high temperature resistance. After gelatinization and mixing, the mixture was homogenized at 6,400 rpm using a homogenizer (IKA T-50) for 5 minutes, and then cooled to 40 ° C. to produce 5.0% (w / w) CLA Bifidobacterium breve (CBG-C2). It was added and homogenized again at 6,400rpm using a homogenizer for 1 minute. Mix the 0.5% (w / w) polyglycerin fatty acid ester (Almax 9025) with 10.0% (w / w) CLA, 1.0% (w / w) 0.1 N-phosphate buffer in a one-stage coating of CLA, and then use a homogenizer. By homogenization at 6,400 rpm for 5 minutes.

Next, the first stage coated lactobacillus, which is a core material, and the first stage coated CLA, were 5.0% (w / w) purified covalent and 0.5% (w / w) polyglycerin fatty acid ester (Almax 9025), 0.4% ( w / w) mixed with glycerin succinic acid fatty acid ester (Almax 5100), mixed in a mixing vessel and homogenized at 6,400rpm again using a homogenizer for 1 minute, 5.0% (w / w) CLA-producing bifidus lactic acid bacteria and 10.0% (w / w) ) CLA shared secondary coating was completed.

Subsequently, 0.3% (w / w) starch, 3.0% (w / w) gelatin, 0.5% (w / w) polyglycerin fatty acid ester (Almax 9080), 0.3% (w / w) The gum was mixed with sufficient sterile water, gelatinized at 120 ° C, cooled to 40 ° C, homogenized again at 6,400rpm for 5 minutes using a homogenizer, and then homogenized at 6,400rpm for 1 minute by adding a secondary coating material. After the coating was completed, 5.0% (w / w) CLA-producing bifidus lactic acid bacteria and 10.0% (w / w) CLA-covalent multi-microcapsules were prepared by spraying directly into sterilized cooling water. The yield was 99%.

[ Example  9b]

5.0% CLA  production Bifidobacterium Pseudocarthenulatum (CBG-C4) and CLA  Shared Multimicrocapsules Manufacture

In Example 9a, 5.0% CLA-producing Bifidobacterium sudocartenulumatum (CBG-C4) was added instead of 5.0% CLA-producing Bifidobacterium breve (CBG-C2) and the title micro Capsules were prepared. The yield was 99%.

[ Example  9c]

5.0% CLA  production Enterococcus Passion ( CBG - C5 ) And CLA  Covalent Multi-Microcapsule Preparation

In Example 9a, instead of 5.0% CLA-producing Bifidobacterium breve (CBG-C2), 5.0% CLA-producing Enterococcus Passium (CBG-C5) was added to prepare the title microcapsules in the same manner as in Example 9a. . The yield was 99% at this time.

Example  9, physical property testing:

Experimental Example 14 Analysis of CLA Content in Capsules

The CLA content of the lactic acid bacteria (CBG-C2) and the CLA covalent microcapsules of the capsule prepared in Example 9a was analyzed by GC. The CLA content detection was measured as follows using GC analysis. Lactobacillus and CLA-covalent multiple microcapsules were placed in an incubator for 5 hours and the capsules collapsed. After capsule disintegration, the mixture was stored in a refrigerated state and centrifuged at 9000 rpm for 7 minutes at 4 ° C. to obtain a supernatant. After mixing isopropyl alcohol in the obtained supernatant and shaking for 5 minutes, hexane was added again, shaken for 5 minutes, left for 10 minutes, only the supernatant was separated and distilled under reduced pressure at 45˜50 ° C., followed by addition of 1N NaOH / MeOH. Suspended. Then, the mixture was heated at 100 ° C. for 10 minutes, and then reacted for 5 minutes at 100 ° C. by adding BF ₃ / MeOH, and cooled by methylation at 100 ° C. for 1 minute by adding hexane. The obtained CLA-OMe was calculated using the GC (Varian CP-3800) to calculate the CLA content in the capsule. As a result, the CLA content in the CLA covalent microcapsules was 4.45% (at 13.77 min residence time) (see FIG. 14).

Experimental Example 15 Effect of CLA on the Growth of Bifidus Lactic Acid Bacteria

After storing the viable cell counts of the CLA-producing bifidus lactic acid bacteria (CBG-C2) and CLA-covalent multi-microcapsules bifidus lactic acid bacteria, which were prepared in Example 9a, at 4 ° C. for 20 days, the number of viable cells was measured. The results are shown in Figure 15, CLA was found to be a significant inhibition on the growth and maintenance of bifidus lactic acid bacteria.

[ Example  10]

 5.0% CLA  production Bifidobacterium Breve ( CBG - C2 ) Manufacturing of Multi-microcapsules Powder

5.0% CLA-produced Bifidobacterium breve (CBG-C2) prepared according to Example (1), multi-microcapsules, skim milk powder, dextrin first mixed in an equal proportion by the content ratio of the following table 79.6% After mixing again with Chlorella, mix it equally and freeze-dried for 5 days at the initial temperature of -43 ℃, final temperature of 30 ℃, and vacuum internal pressure of 7 mTorr using lyophilizer (Ilshin, Model No: PITFD100R). Prepared.

subject Content (g) Mixing ratio (%) Remarks 5.0% CLA Production Bifidus Lactobacillus Microcapsules 37.35 0.37 Initial Viable Count 2.3 × 10 ⁹ / g Skim milk powder 1,000 10.00 Dextrin 1,000 10.00 Chlorella 7,962.65 79.63 Total content 10,000 100.00

Example  10 property results:

Experimental Example 16 Measurement of Viable Cell Number and Survival Period

The microbial powder prepared according to Example 10 was stored for 12 months at 25 ° C. and the number of viable cells was measured over time. The results are shown in FIG. 16, and when the initial viable cell count was 2.3 × 10 ⁹ / g, the viable cell count after 12 months was 1.3 × 10 ⁸ / g.

[ Example  11]

5.0% Lactobacillus With Ecidophilus Lactococcus Thermophilus  Powder production of covalent multi-microcapsules

5.0% Lactobacillus Escidophilus and Lactococcus Thermophilus Covalent Multi-microcapsules prepared according to Example 5 and CMC, starch, dextrin and skim milk powder are first mixed in an equal proportion by the content ratio of the following table and then chlorella After mixing again and mixed evenly, use lyophilizer (Ilshin, Model No: PITFD100R) to freeze-dry for 5 days at the initial temperature of -43 ℃, final temperature of 30 ℃ and vacuum internal pressure of 7 mTorr and then in powder form. Prepared.

subject Content (g) Mixing ratio (%) Remarks 5.0% Lactobacillus Escidophilus and Lactococcus Thermophilus Covalent Multiple Microcapsules 8,100 81.00 Initial viable count 8.5 × 10 ⁹ / g CMC 35 0.35 Starch 85 0.85 Dextrin 1,400 14.00 Skim milk powder 380 3.80 Total content 10,000 100.00

Example  11 physical properties results:

Experimental Example 17

The powder prepared in Example 11 was stored at 25 ° C. (room temperature) for 12 months to determine the viable bacterial count (per g) of the mixed strain. The results are shown in FIG. 17, and the viable cell count was measured to be maintained for 10 months at 10 ⁸ CFU / mL.

Lactic acid bacteria multi-microcapsules of the present invention exhibits the effect of maintaining the storage period (4 ℃ state) more than 3 months at 10 ⁸ CFU / mL or more after the production. In addition, the lactic acid bacteria multi-microcapsules according to the present invention exhibits the effect of maintaining at least 6 months at 4 ℃, at least 1 month at 20 ℃, at least 1 month at 25 ℃.

And the yield of the active ingredient in the lactic acid bacteria multi-microcapsules of the present invention is more than 99%, even if stored for a long time shows the effect of dispersibility in the aqueous solution.

 Therefore, the lactic acid bacteria multi-microcapsules of the present invention can supply a variety of stable formulations according to the product type and use while maintaining the function of the lactic acid bacteria.

Claims (54)

(a) sterilizing the low fat skimmed milk powder, glycerin and the medium solution, and adding lactic acid bacteria and homogenizing to prepare a primary coating material; (b) mixing and homogenizing the refined processing oil, polyglycerin fatty acid ester and glycerin zucchini fatty acid ester to the primary coating material prepared above to prepare a secondary coating material; (c) lactic acid bacteria, comprising carbohydrates, protein components and gum thickeners, emulsifiers, gelatinization and homogenization, followed by addition of a secondary coating material, homogenization, preparation of a tertiary coating material, and then spraying into sterilized cooling water. Microcapsules Preparation Method. The method according to claim 1, wherein the lactic acid bacteria of step (a) are CLA (conjugated linoleic acid) production Bifidobacterium breve, CLA production Bifidobacterium pseudocartenulumum, CLA production enterococcus fasium, lactose A method for producing at least one selected from Bacillus ecidophilus, Lactococcus thermophilus, Lactobacillus casein, Lactobacillus ruteri or bifidus lactic acid bacteria. The method according to claim 1 or 2, wherein the lactic acid bacteria of step (a) is 1.0 to 5.0% (w / w) in the total amount of microcapsules. The method of claim 1, wherein the low fat skim milk powder in step (a) is 0.01 to 0.5% (w / w) based on the total amount of microcapsules, and glycerin is 0.1 to 1.0% (w / w) based on the total amount of microcapsules. , The medium solution is 0.05 to 0.5% (w / w) based on the total amount of microcapsules. The method according to claim 1, wherein the purified covalent acid of step (b) is 1.5 to 5.0% (w / w) based on the total amount of microcapsules, and the polyglycerol fatty acid ester is 0.3 to 0.5% (w / based on the total amount of microcapsules. w), wherein the glycerin succinic acid fatty acid ester is 0.2 to 0.4% (w / w) based on the total amount of microcapsules. The method of claim 1, wherein the carbohydrate of step (c) is 0.1 to 0.3% (w / w) based on the total amount of microcapsules, the protein component is 1.5 to 3.0% based on the total amount of microcapsules, the gum thickener is a microcapsules 0.1 to 0.3% based on the total amount, the emulsifier is 0.1 to 0.5% based on the total amount of the microcapsules. The method according to claim 1, wherein the homogenization temperature of steps (a), (b) and (c) is 30 ° C to 50 ° C. The method according to claim 1, wherein the homogenization temperature of steps (a), (b) and (c) is 35 ° C to 45 ° C. The process according to claim 1, wherein the homogenization temperature of steps (a), (b) and (c) is 40 ° C. The method according to claim 1, wherein the carbohydrate is starch or modified starch. The method of claim 1, wherein the protein component is gelatin or agar. The method according to claim 1, wherein the gum thickener is guar gum or xanthan gum. The process according to claim 1, wherein the emulsifier is polyglycerol fatty acid ester. Microcapsules prepared by the method of claim 1. An article comprising the microcapsules of claim 14. 16. The product of claim 15, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional beverage, functional food, general food, medicine and cosmetic. According to claim 1, Lactic acid bacteria-unsaturated fatty acid covalently multi-microcapsules manufacturing method of adding a polyglycerol fatty acid ester and unsaturated fatty acid coated separately with a phosphate buffer in step (b) together with the primary coating material produced in step (a) . 18. The method of claim 17, wherein the unsaturated fatty acid is docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA) or CLA. 18. The method according to claim 17, wherein the encapsulated unsaturated fatty acid is 0.3-0.5% (w / w) polyglycerin fatty acid ester, 0.5-1.0% (w / w) 0.1N phosphate buffer and 5.0-based on the total amount of microcapsules. 10.0% (w / w) is homogenized with unsaturated fatty acid is coated method. Microcapsules prepared by the method of claim 17. A product comprising the microcapsules of claim 20. 22. The product of claim 21, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional beverage, functional food, general food, pharmaceutical and cosmetic.  The method for preparing powder by lyophilizing the microcapsule prepared according to claim 1 with skim milk powder, dextrin and chlorella. 24. The method of claim 23, wherein the microcapsules prepared in claim 1 are 0.1 to 0.5% by weight, 5 to 15% by weight of skim milk powder, 5 to 15% by weight of dextrin, and 70 to 85% by weight of chlorella. Powder prepared by the method of claim 23. A product comprising a powder prepared by the method of claim 25. 27. The product of claim 26, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional beverage, functional food, general food, pharmaceutical, and cosmetic. (a) sterilizing the low fat skimmed milk powder, the medium solution, glycerin and cysteine, adding lactic acid bacteria and homogenizing it to prepare a primary coating material; (b) mixing and homogenizing polyglycerol fatty acid ester and monoglycerin fatty acid ester to the primary coating material prepared above to prepare a secondary coating material; (c) lactic acid bacteria micromicrocapsule comprising mixing homogeneous protein component, gum thickener and emulsifier, homogenizing by adding secondary coating material, preparing tertiary coating material, and then spraying into sterilized cooling water Manufacturing method. 29. The method according to claim 28, wherein the lactic acid bacteria of step (a) are CLA-produced Bifidobacterium breve, CLA-produced Bifidobacterium pseudocartenulatum, CLA-produced enterococcus, Lactobacillus ecidophilus , Lactococcus thermophilus, Lactobacillus casein, Lactobacillus ruteri or bifidus lactic acid bacteria is at least one selected from the production method. 30. The method according to claim 28 or 29, wherein the lactic acid bacteria of step (a) is 1.0 to 5.0% (w / w) in the total amount of microcapsules. 29. The method according to claim 28, wherein the low fat skimmed milk powder in step (a) is 0.05 to 0.5% (w / w) based on the total amount of microcapsules, and glycerin is 0.1 to 1.0% (w / w) based on the total amount of microcapsules. ), Wherein the medium solution is 0.05 to 0.5% (w / w) based on the total amount of microcapsules, and cysteine is 0.01% to 0.1% (w / w) based on the total amount of microcapsules. The method according to claim 28, wherein the cysteine of step (a) is 0.05% (w / w) based on the total amount of microcapsules. The method according to claim 28, wherein the polyglycerol fatty acid ester of step (b) is 5.0 to 10.0% (w / w) based on the total amount of microcapsules, and the monoglycerin fatty acid ester is 0.2 to 0.4% (w) based on the total amount of microcapsules / w) manufacturing method. 29. The method according to claim 28, wherein the protein component of step (c) is 1.5 to 3.0% based on the total amount of microcapsules, gum thickener is 0.3 to 0.5% based on the total amount of microcapsules, and emulsifier is 0.1 based on the total amount of microcapsules. To 0.5% of the preparation method. The method according to claim 28, wherein the homogenization temperature of steps (a), (b) and (c) is 30 ° C to 50 ° C. 29. The method of claim 28, wherein the homogenization temperatures of steps (a), (b) and (c) are from 35 ° C to 45 ° C. The method according to claim 28, wherein the homogenization temperature of steps (a), (b) and (c) is 40 ° C. 29. The method of claim 28, wherein the protein component is gelatin or agar. 29. The method of claim 28, wherein the gum thickener is guar gum or xanthan gum. 29. The method of claim 28, wherein the emulsifier is a polyglycerin fatty acid ester. A microcapsule prepared by the method of claim 28. An article comprising the microcapsules of claim 41. 43. The product of claim 42, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional beverage, functional food, general food, pharmaceutical and cosmetic. 29. The method of claim 28, wherein the unsaturated fatty acid coated separately with the polyglycerol fatty acid ester and the phosphate buffer solution is added to the step (b) together with the primary coating material produced in the step (a). . 45. The method of claim 44, wherein the unsaturated fatty acid is docosahexaenoic acid (DHA), Eicosapentaenoic acid (EPA) or CLA. 18. The method according to claim 17, wherein the encapsulated unsaturated fatty acid is 0.3-0.5% (w / w) polyglycerin fatty acid ester, 0.5-1.0% (w / w) 0.1N phosphate buffer and 5.0-based on the total amount of microcapsules. 10.0% (w / w) is homogenized with unsaturated fatty acid is coated method. Microcapsules made by the method of claim 44. 48. An article comprising the microcapsule of claim 47. 49. The product of claim 48, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional food, functional beverage, general food, medicine and cosmetic.  A powder manufacturing method for lyophilizing the microcapsule prepared according to claim 28 by mixing with skim milk powder, dextrin and chlorella. 51. The method according to claim 50, wherein the microcapsules prepared in claim 1 are from 0.1 to 0.5% by weight, from 5 to 15% by weight of skim milk powder, from 5 to 15% by weight of dextrin and from 70 to 85% by weight of chlorella. A powder prepared by the method of claim 50. An article comprising a powder prepared by the method of claim 52. 54. The product of claim 53, wherein the product is fermented milk, processed milk, fermented fermented food, kimchi fermented food, functional food, functional beverage, general food, medicine and cosmetic.

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