Multi-coated probiotics and Method for preparing the same

Abstract

The present application relates to multi-coated probiotics and a method for manufacturing the same.

Description

Technology Field

[0001] The present application relates to multilayer coated probiotics and a method for manufacturing the same. Background Technology

[0002] Probiotics are known to exhibit beneficial health effects, such as maintaining the balance of intestinal microorganisms, improving immune function, and enhancing digestive function, and are utilized in various fields including health functional foods, pharmaceutical compositions, and feed additives. However, as live microorganisms, probiotics are highly sensitive to the external environment, leading to the problem of easy mortality during manufacturing and distribution processes. Specifically, live probiotic bacteria can easily die under high temperature and high humidity conditions, resulting in low storage stability. Furthermore, cell damage may occur during the manufacturing process of probiotic powder, such as concentration and freeze-drying, which can reduce the final yield; additionally, there is a problem where survival rates rapidly decrease upon oral intake due to exposure to gastric acid and bile acids in the intestinal tract.

[0003] Therefore, there is a need to develop formulation technology that ensures a high viable cell count of probiotics after pulverization, enhances resistance to gastric acid and bile acid, and exhibits excellent stability even under harsh conditions such as high temperature and high humidity. The problem to be solved

[0004] In accordance with these technical requirements, the present application aims to provide a probiotic composition with excellent stability and a method for manufacturing the same, which secures a high viable cell count and freeze-drying yield of the probiotic after freeze-drying through multi-coating of the probiotic. means of solving the problem

[0005] One example of the present application relates to a method for preparing a multi-coated probiotic composition.

[0006] Another example of the present application relates to a method for increasing the freeze-drying survival rate of probiotics.

[0007] Another example of the present application relates to a method for increasing the acid resistance and / or bile resistance of probiotics.

[0008] Another example of the present application relates to a method for increasing the surface hydrophobicity of probiotics.

[0009] Another example of the present application relates to a multi-coated probiotic composition sequentially coated with a natural adsorbent, sugars, plant lecithin, and dietary fiber.

[0010] Another example of the present application relates to a food comprising a probiotic composition according to one example of the present application.

[0012] The present application will be described in more detail below.

[0013] As probiotics are living microorganisms sensitive to changes in the external environment, they are generally distributed in powder form after freeze-drying to maintain stable viability and enhance industrial utility. In particular, freeze-drying halts the metabolic activity of heat-sensitive microorganisms at low temperatures and removes moisture through sublimation, thereby preserving the genetic and biological activity of the strains in their original state. Furthermore, the powdered strains obtained through freeze-drying significantly reduce volume and weight, which not only lowers transportation and storage costs but also facilitate processing into various formulations such as capsules, tablets, and powders, maximizing convenience for end consumers.

[0014] However, the freeze-drying process causes severe physical and chemical stress to microbial cells, which drastically reduces their survival rate, and ensuring the survival rate of strains after freeze-drying is a very important technical factor in the production of probiotic products.

[0015] A probiotic composition according to one example of the present application is manufactured through a multi-coating process, and through multi-coating after probiotic culture, the number of viable cells and yield of freeze-dried bacteria can be increased.

[0016] Specifically, the multi-coating process described above may include the step of adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; the step of secondarily coating the first-coated probiotics with sugars; the step of thirdly coating the second-coated probiotics with plant lecithin; and the step of fourthly coating the third-coated probiotics with dietary fiber.

[0017] The step of primary coating and separation of the above-mentioned probiotics is a process of primary coating and separation of probiotics by adding a natural adsorbent to the probiotic culture and performing centrifugation before separating the probiotic cells after probiotic culture. Lactic acid, acetic acid, etc. produced during the probiotic culture process inhibit the growth of probiotics, preventing the cell mass from increasing sufficiently, which has an adverse effect on the separation of probiotic cells through centrifugation. In addition, metabolic products generated during the fermentation process of probiotics pose a problem of significantly reducing the centrifugation yield. Accordingly, the method according to one example of the present application can improve the separation yield of probiotics while simultaneously primary coating of probiotics by adding a natural adsorbent to the probiotic culture and performing centrifugation.

[0018] The above natural adsorbent may be an organic material derived from natural raw materials such as plants or animals. Specifically, the above natural adsorbent may be one or more selected from the group consisting of starch, cellulose, guar gum, chitosan, and gelatin.

[0019] The above starch may be natural starch or modified starch.

[0020] The modified starch mentioned above may be oxidized starch, cross-linked starch, dextrin, gelatinized starch, or a starch derivative.

[0021] The above starch may be one or more selected from the group consisting of corn starch, potato starch, sweet potato starch, tapioca starch, rice starch, wheat starch, corn maltodextrin, tapioca maltodextrin, resistant maltodextrin, and α-cyclodextrin.

[0022] The above natural adsorbent may be added to the above probiotic culture at a concentration of 0.05% (w / v) or more, more than 0.05% (w / v), 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, or 0.1% (w / v) or more.

[0023] Accordingly, the first coating and separation step may include the step of adding the natural adsorbent to the probiotic culture at a concentration of 0.05% (w / v) or more, more than 0.05% (w / v), 0.06% (w / v) or more, 0.07% (w / v) or more, 0.08% (w / v) or more, 0.09% (w / v) or more, or 0.1% (w / v) or more; and the step of applying centrifugal force to the culture.

[0024] The step of secondarily coating the first-coated probiotics with sugars is a process of secondarily coating the probiotics by adding sugars to the first-coated probiotic sample, mixing, and homogenizing. Since the sugars provide a cryoprotective effect, the sugars may be a cryoprotective coating agent for probiotics.

[0025] During the freeze-drying process of probiotics, rapid pH changes occur, which causes damage to the cell membranes of the probiotics. Accordingly, the second coating step may be performed after adjusting the pH of the first-coated probiotic sample. Additionally, the step of adjusting the pH may be performed prior to homogenization in the second coating step.

[0026] Specifically, the step of secondary coating may further include a step of adjusting the pH of the primary coated probiotic sample to 6 to 8, or 6 to 7.5.

[0027] In addition, since the above sugars can perform the function of protecting probiotics during the freeze-drying process, they may be performed immediately after the first coating and separation of probiotics in the multi-coating process of probiotics.

[0028] The above sugars may be reducing sugars or non-reducing sugars.

[0029] The above reducing sugar may be one or more selected from the group consisting of glyceraldehyde, erythrose, ribose, xylose, arabianose, ribulose, glucose, fructose, galactose, mannose, maltose, lactose, lactulose, chitobiose, cellobiose, and dextrin.

[0030] The above non-reducing sugar may be one or more selected from the group consisting of trehalose, sucrose, raffinose, mellezitose, gentianose, stachyose, and verbascose.

[0031] The above sugars are present in the primary coated probiotic sample at concentrations of 0.1% (w / v) to less than 1.0% (w / v), 0.1% (w / v) to 0.9% (w / v), 0.1% (w / v) to 0.8% (w / v), 0.1% (w / v) to 0.7% (w / v), 0.1% (w / v) to 0.6% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.1% (w / v) to 0.4% (w / v), 0.1% (w / v) to 0.3% (w / v), 0.15% (w / v) to less than 1.0% (w / v), and 0.15% (w / v) to 0.9% (w / v). ≤, 0.15%(w / v) or more and ≤0.8%(w / v), ≤0.15%(w / v) or more and ≤0.7%(w / v), ≤0.15%(w / v) or more and ≤0.6%(w / v), ≤0.15%(w / v) or more and ≤0.5%(w / v), ≤0.15%(w / v) or more and ≤0.4%(w / v), ≤0.15%(w / v) or more and ≤0.3%(w / v), ≤0.2%(w / v) or more and less than 1.0%(w / v), ≤0.2%(w / v) or more and ≤0.9%(w / v), ≤0.2%(w / v) or more and ≤0.8%(w / v), ≤0.2%(w / v) or more and ≤0.7%(w / v), It may be added at a concentration of 0.2% (w / v) or more and 0.6% (w / v) or less, 0.2% (w / v) or more and 0.5% (w / v) or less, 0.2% (w / v) or more and 0.4% (w / v) or less, or 0.2% (w / v) or more and 0.3% (w / v) or less.

[0032] Accordingly, the second coating step comprises applying sugars to the first-coated probiotic sample in the range of 0.1% (w / v) to less than 1.0% (w / v), 0.1% (w / v) to 0.9% (w / v) or less, 0.1% (w / v) to 0.8% (w / v) or less, 0.1% (w / v) to 0.7% (w / v) or less, 0.1% (w / v) to 0.6% (w / v) or less, 0.1% (w / v) to 0.5% (w / v) or less, 0.1% (w / v) to 0.4% (w / v) or less, 0.1% (w / v) to 0.3% (w / v) or less, 0.15% (w / v) or more to less than 1.0% (w / v), or 0.15% (w / v) or more 0.9%(w / v) or less, 0.15%(w / v) or more and 0.8%(w / v) or less, 0.15%(w / v) or more and 0.7%(w / v) or less, 0.15%(w / v) or more and 0.6%(w / v) or less, 0.15%(w / v) or more and 0.5%(w / v) or less, 0.15%(w / v) or more and 0.4%(w / v) or less, 0.15%(w / v) or more and 0.3%(w / v) or less, 0.2%(w / v) or more and less than 1.0%(w / v), 0.2%(w / v) or more and 0.9%(w / v) or less, 0.2%(w / v) or more and 0.8%(w / v) or less, 0.2%(w / v) or more and 0.7%(w / v) It may be added at a concentration of 0.2% (w / v) or less, 0.6% (w / v) or less, 0.2% (w / v) or more, 0.5% (w / v) or less, 0.2% (w / v) or more, 0.4% (w / v) or less, or 0.2% (w / v) or more, 0.3% (w / v) or less, and mixed and homogenized.

[0033] The step of tertiarily coating the above-mentioned secondary-coated probiotics with plant lecithin is a process of adding plant lecithin to the secondary-coated probiotic sample, mixing and homogenizing it to tertiarily coat the probiotics. The acid resistance and bile resistance of the probiotics can be improved through the above-mentioned plant lecithin coating. Accordingly, the above-mentioned plant lecithin may be a coating agent for improving the acid resistance and / or bile resistance of the probiotics.

[0034] The above third coating step comprises applying plant lecithin to the second-coated probiotic sample in an amount greater than 0.001% (w / v) and less than 0.05% (w / v), greater than 0.001% (w / v) and less than or equal to 0.04% (w / v), greater than 0.001% (w / v) and less than or equal to 0.03% (w / v), greater than 0.001% (w / v) and less than or equal to 0.025% (w / v), greater than 0.001% (w / v) and less than or equal to 0.02% (w / v), greater than 0.002% (w / v) and less than or equal to 0.05% (w / v), greater than 0.002% (w / v) and less than or equal to 0.04% (w / v), greater than 0.002% (w / v) and less than or equal to 0.03% (w / v), or greater than or equal to 0.002% (w / v). 0.025%(w / v) or less, 0.002%(w / v) or more and 0.02%(w / v) or less, 0.003%(w / v) or more and less than 0.05%(w / v), 0.003%(w / v) or more and 0.04%(w / v) or less, 0.003%(w / v) or more and 0.03%(w / v) or less, 0.003%(w / v) or more and 0.025%(w / v) or less, 0.003%(w / v) or more and 0.02%(w / v) or less, 0.004%(w / v) or more and less than 0.05%(w / v), 0.004%(w / v) or more and 0.04%(w / v) or less, 0.004%(w / v) or more and 0.04%(w / v) or more 0.03%(w / v) or less, 0.004%(w / v) or more and 0.025%(w / v) or less, 0.004%(w / v) or more and 0.02%(w / v) or less, 0.005%(w / v) or more and less than 0.05%(w / v), 0.005%(w / v) or more and 0.04%(w / v) or less, 0.005%(w / v) or more and 0.03%(w / v) or less, 0.005%(w / v) or more and 0.025%(w / v) or less, 0.005%(w / v) or more and 0.02%(w / v) or less, 0.006%(w / v) or more and less than 0.05%(w / v), 0.006%(w / v) or more and 0.006%(w / v) or more 0.04%(w / v) or less, 0.006%(w / v) or more and 0.03%(w / v) or less, 0.006%(w / v) or more and 0.025%(w / v) or less, 0.006%(w / v) or more and 0.02%(w / v) or less, 0.007%(w / v) or more and less than 0.05%(w / v), 0.007%(w / v) or more and 0.0.4%(w / v) or less, 0.007%(w / v) or more and 0.03%(w / v) or less, 0.007%(w / v) or more and 0.025%(w / v) or less, 0.007%(w / v) or more and 0.02%(w / v) or less, 0.008%(w / v) or more and less than 0.05%(w / v), 0.008%(w / v) or more and 0.04%(w / v) or less, 0.008%(w / v) or more and 0.03%(w / v) or less, 0.008%(w / v) or more and 0.025%(w / v) or less, 0.008%(w / v) or more and 0.02%(w / v) or less, 0.009%(w / v) or more and 0.05%(w / v) It may be added at a concentration of less than, 0.009%(w / v) or more and 0.04%(w / v) or less, 0.009%(w / v) or more and 0.03%(w / v) or less, 0.009%(w / v) or more and 0.025%(w / v) or less, 0.009%(w / v) or more and 0.02%(w / v) or less, 0.01%(w / v) or more and less than 0.05%(w / v), 0.01%(w / v) or more and 0.04%(w / v) or less, 0.01%(w / v) or more and 0.03%(w / v) or less, 0.01%(w / v) or more and 0.025%(w / v) or less, or 0.01%(w / v) or more and 0.02%(w / v) or less, and mixed and homogenized. there is.

[0035] The above plant lecithin may be one or more selected from the group consisting of sunflower lecithin, rapeseed lecithin, and soybean lecithin.

[0036] The step of coating the above-mentioned tertiary-coated probiotics with dietary fiber for the fourth time is a process of adding dietary fiber to a tertiary-coated probiotic sample, mixing, and homogenizing to tertiarily coat the probiotics. Through the above-mentioned dietary fiber coating, the stability and surface hydrophobicity of the probiotics are improved, which can improve intestinal adhesion and bioavailability. Accordingly, the above-mentioned dietary fiber may be a coating agent for improving the stability and / or surface hydrophobicity of the probiotics.

[0037] The above fourth coating step may involve adding dietary fiber to the third-coated probiotic sample at a concentration of 0.005% (w / v) or more, 0.006% (w / v) or more, 0.007% (w / v) or more, 0.008% (w / v) or more, 0.009% (w / v) or more, or 0.01% (w / v) or more, and mixing and homogenizing. At this time, even if the upper limit of the dietary fiber concentration is not specified, a person skilled in the art can clearly perform the tertiary coating using dietary fiber for the purpose of coating probiotics, but for example, the upper limit of the dietary fiber concentration is 10% (w / v) or less, 9% (w / v) or less, 8% (w / v) or less, 7% (w / v) or less, 6% (w / v) or less, 5% (w / v) or less, 4% (w / v) or less, 3% (w / v) or less, 2% (w / v) or less, 1% (w / v) or less, 0.9% (w / v) or less, 0.8% (w / v) or less, 0.7% (w / v) or less, 0.6% (w / v) or less, 0.5% (w / v) or less, 0.4% (w / v) or less, 0.3% (w / v) or less, It may be 0.2% (w / v) or less, or 0.1% (w / v) or less, but is not limited thereto.

[0038] The above dietary fiber may be α-glycosidically bonded dietary fiber or β-glycosidically bonded dietary fiber.

[0039] The above dietary fiber may be one or more selected from the group consisting of pectin, acacia dietary fiber, alginic acid, guar gum, chicory dietary fiber (inulin), beta-glucan, polydextrose, indigestible maltodextrin, levan, seaweed-derived polysaccharides, seed-derived polysaccharides, cellulose, hemicellulose, crystalline cellulose, lignin, dietary fiber derived from grains (brown rice, barley, oats, wheat, etc.), dietary fiber derived from mushrooms (shiitake mushrooms, wood ear mushrooms, etc.), and processed dietary fiber (Schisandra chinensis, soybeans, psyllium husk, etc.).

[0040] A manufacturing method according to one example of the present application may further include a step of freeze-drying the quaternarily coated probiotic. Accordingly, the probiotic composition according to one example of the present application may be a powder. Specifically, the probiotic composition according to one example of the present application may be a freeze-dried powder.

[0041] Another example of the present application relates to a method for increasing the freeze-drying survival rate of probiotics, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; fourthly coating the third-coated probiotics with dietary fiber; and freeze-drying the fourth-coated probiotics.

[0042] Another example of the present application relates to a method for increasing the acid resistance and / or bile resistance of probiotics, or a method for increasing the surface hydrophobicity of probiotics, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; and fourthly coating the third-coated probiotics with dietary fiber.

[0043] The above probiotics are of the genus Lactobacillus ( Lactobacillus spp.), genus Bifidobacterium ( Bifidobacterium spp.), genus Lactococcus ( Lactococcus spp.), genus Akkermansia ( Akkermansia spp.), Streptococcus genus ( Streptococcus ssp.), genus Enterococcus ( Enterococcus spp.), Bacillus genus ( Bacillus ssp.), genus Pediococcus ( Pediococcus spp.), genus Weissella ( Weissella ssp.), and the genus Leuconostoc ( Leuconostoc It may be one or more selected from the group consisting of spp.).

[0044] Another example of the present application relates to a multi-coated probiotic composition sequentially coated with a natural adsorbent, sugars, plant lecithin, and dietary fiber.

[0045] The above probiotic composition may be in the form of a tablet, troches, lozenge, suspension, water-soluble suspension, oily suspension, powder, prepared powder, granule, emulsion, capsule, hard capsule, soft capsule, syrup, or elixir.

[0046] Another example of the present application relates to a food comprising a probiotic composition according to one example of the present application. The food may be a health functional food.

[0047] The above food may be in the form of, for example, meat, bread, chocolate, candies, jellies, snacks, confectionery, kimchi, sauces, cheese, dairy products, powders, beverages, and vitamin complexes.

[0048] The above food may include, as an active ingredient, ingredients that are typically added during food manufacturing. The above added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. The carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., conventional sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings, natural flavorings (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) may be used.

[0049] For example, when a food product according to one example of the present application is manufactured as a drink, it may additionally include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, or licorice extract.

[0050] Foods according to one example of the present application include processed forms of all natural materials, such as functional foods, nutritional supplements, health foods, and food additives. Foods of the above types may be manufactured in various forms according to conventional methods known in the art. For example, as health foods, probiotic compositions may be manufactured in the form of tea, juice, and drink for consumption, or may be consumed after granulation, encapsulation, and powdering. In addition, food products may be in the form of beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and its processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, malt syrup, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). Furthermore, in order to use the probiotic composition according to one example of the present application as a food additive, it may be prepared and used in the form of a powder or a concentrate. Effects of the invention

[0051] A method for preparing a multi-coated probiotic composition according to one example of the present application can increase the number and yield of viable probiotics in freeze-dried powder and improve acid resistance, bile resistance, and surface hydrophobicity through a multi-coating process of probiotics. Specific details for implementing the invention

[0052] The present application will be explained in more detail below by means of the following examples. However, these examples are merely illustrative of the present application, and the scope of the present application is not limited by these examples.

[0054] Example 1. Primary coating of probiotics

[0055] (1) Probiotic coating and separation

[0056] After culturing probiotics, a centrifugation process using a continuous high-speed centrifuge is generally performed to obtain the target product, the cell pellets, from the culture. Since the principle of centrifugation utilizes the difference in specific gravity between the substances to be separated, the specific gravity of the probiotic culture has a significant impact on the efficiency of cell separation. However, during the probiotic culture process, the secretion of lactic acid and acetic acid due to sugar consumption inhibits growth, leading to a problem where cell mass does not increase sufficiently, and consequently, the specific gravity of the culture tends to decrease. In such cases where the specific gravity of the culture is low, a relatively high gravitational acceleration (G-force) is required for the smooth separation of the cell pellets.

[0057] Meanwhile, the fermentation forms of lactic acid bacteria, which are representative probiotics, can be classified into homofermentation, heterofermentation, and bifidus fermentation, and depending on the fermentation form, the characteristics differ, such as the generation of gas or the secretion of metabolites including peptides and polysaccharides. In particular, metabolites generated during fermentation can impede the separation ability in the centrifugation process, thereby significantly reducing the centrifugation yield, and in severe cases, may result in a problem where the centrifugation process cannot be performed.

[0058] To minimize these problems and secure a high level of centrifugation yield, a natural adsorbent was added to the culture medium prior to centrifugation, and the natural adsorbent was coated onto the bacterial cells using a strong G-force during the centrifugation process to improve the separation yield of probiotics. Specifically, 0.10% (w / v) of corn starch was added to the probiotic culture as a natural adsorbent, and the bacterial cells were separated using a centrifuge (Hanil Science, Supira R12) at 7,000 RPM and 4°C for 10 minutes. The recovery rate of probiotics with and without the natural adsorbent coating was calculated using the following formula and is shown in Table 1.

[0059] Recovery Rate (%) = [Number of Viable Cells in Culture - Number of Viable Cells in Filter] / [Number of Viable Cells in Culture] x 100

[0060] Probiotics Natural adsorbents not used Use of natural adsorbents Female fluid OD filtrate viable bacteria recovery rate Female fluid OD filtrate viable bacteria recovery rate Lactobacillus acidophilus ( L. acidophilus ) 0.058 8.9E+08 88.3% 0.032 1.2E+08 98.4% Bifidobacterium longum ( B. longum ) 0.168 2.2E+09 56.0% 0.086 7.8E+08 84.4% Lactococcus lactis ( L. lactis ) 0.124 2.8E+09 84.4% 0.055 1.0E+09 94.4%

[0061] Table 1 shows the results of verifying the change in strain recovery rate following the addition of corn starch (0.10% (w / v)) as a natural adsorbent, and the OD value is OD 610nm As shown in Table 1, it was verified that when corn starch, a natural adsorbent, is added to a probiotic culture and high-speed centrifugation is performed to carry out primary coating and separation of probiotics, the separation yield of the cells is significantly improved.

[0063] (2) Coating using various types of natural adsorbents

[0064] 0.10% (w / v) of tapioca starch, corn maltodextrin, tapioca maltodextrin, resistant maltodextrin, or α-cyclodextrin as a natural adsorbent L. acidophilus After adding to the culture, the separation recovery rate after coating and separating the probiotics in the same way as in (1) of Example 1 is shown in Table 2.

[0065] Types of natural adsorbents Natural adsorbents not used Use of natural adsorbents Female fluid OD filtrate viable bacteria recovery rate Female fluid OD filtrate viable bacteria recovery rate corn starch 0.058 8.9E+08 88.3% 0.032 1.2E+08 98.4% tapioca starch 0.038 2.8E+08 96.3% Corn maltodextrin 0.056 8.2E+08 89.2% Tapioca maltodextrin 0.057 8.3E+08 89.1% Resistant maltodextrin 0.052 6.3E+08 91.7% α-Cyclodextrin 0.049 5.8E+08 92.4%

[0066] Table 2 shows the results of verifying the change in recovery rate of probiotics according to the type of natural adsorbent. As shown in Table 2, when a natural adsorbent is added to a probiotic culture and the probiotics are coated and separated by centrifugal force, it was verified that the separation yield of probiotic cells is improved, and in particular, the effect of improving the separation yield was best when corn starch was used as the natural adsorbent.

[0068] (3) Coating using natural adsorbents of various concentrations

[0069] Corn starch as a natural adsorbent at concentrations of 0.01% (w / v), 0.05% (w / v), 0.10% (w / v), 0.25% (w / v), or 0.50% (w / v). B. longum After adding to the culture, the recovery rate after centrifugation in substantially the same way as in (1) of Example 1 is shown in Table 3.

[0070] Natural adsorbent concentration Natural adsorbents not used Use of natural adsorbents Female fluid OD filtrate viable bacteria recovery rate Female fluid OD filtrate viable bacteria recovery rate 0.01%(w / v) 0.168 2.2E+09 56.0% 0.152 1.9E+09 62.0% 0.05%(w / v) 0.124 1.2E+09 76.0% 0.10%(w / v) 0.086 7.8E+08 84.4% 0.25%(w / v) 0.084 7.4E+08 85.2% 0.50%(w / v) 0.083 7.3E+08 85.4%

[0071] Table 3 shows the results of verifying the change in recovery rate of probiotics according to the concentration of natural adsorbent. As shown in Table 3, it was verified that the separation yield of probiotic cells is significantly improved at natural adsorbent concentrations of 0.05% (w / v) or higher, especially at concentrations exceeding 0.05% (w / v) or 0.10% (w / v) or higher.

[0073] Example 2. Secondary coating of probiotics

[0074] (1) Types of secondary coating solution

[0075] To minimize the problem of cell membrane damage in probiotics caused by rapid pH changes resulting from the formation of ice crystals between free water and bound water during the freeze-drying process of probiotics, a second coating was performed after adjusting the pH.

[0076] The pH of the probiotic sample, to which 0.10% (w / v) of corn starch, a natural adsorbent, was added was centrifuged to complete the first coating, and the pH was adjusted using monopotassium phosphate and disodium phosphate. Specifically, the probiotic pellet coated first in Example 1 was mixed with 0.10% (w / v) of sterilized potassium phosphate and homogenized to adjust the pH to 6.8 to 8.0. Subsequently, a sterilized aqueous solution of a reducing sugar (maltose, lactose, or lactulose) or a non-reducing sugar (trehalose, sucrose, raffinose, or stachylose) was mixed with the pH-adjusted first-coated sample at a ratio of 0.25% (w / v) and homogenized to complete the second coating. A certain amount of the probiotic sample with the second coating completed was dispensed into the chamber of a freeze dryer (MG Industry, MG-VFD5) and rapidly frozen at -40℃ or lower, and then freeze-dried for ~96 hours while maintaining a vacuum inside the chamber.

[0077] 1 g of freeze-dried and ground sample was added to 9 ml of BPW solution and vortexed for 20 minutes to sufficiently suspend and elute the bacteria. Then, 1 ml of the suspension was added to 9 ml of BPW solution and vortexed for approximately 1 minute to dilute by the decimal method. After repeated dilution, when the expected titration multiplier was reached, 1 ml was taken and dispensed onto a plate. Prepared, sterilized, and cooled MRS agar or BL agar was poured (15–18 ml) at approximately 45°C and allowed to solidify over approximately 20 minutes. After incubating the solidified plates in a 37°C incubator for 48–72 hours, the colonies that emerged on the plates were counted. The number of viable bacteria after freeze-drying was analyzed and is shown in Table 4. The freeze-drying yield was calculated using the following formula and is shown in Table 5.

[0078] Freeze-drying yield = (Weight of freeze-dried powder x Number of viable bacteria in powder) / (Weight of coated sample x Number of viable bacteria in coated sample) x 100%

[0079] Probiotics reducing sugar non-reducing sugars Maltose Lactose Lactulose Trehalose Sucrose Raffinose Stachyose L. acidophilus 4.4E+11 3.8E+11 3.9E+11 5.2E+11 4.2E+11 4.4E+11 4.5E+11 B. longum 2.6E+11 1.8E+11 2.1E+11 3.4E+11 2.3E+11 2.5E+11 2.7E+11 L. lactis 8.5E+11 7.6E+11 8.0E+11 9.8E+11 7.8E+11 8.2E+11 8.6E+11

[0080] Probiotics reducing sugar non-reducing sugars Maltose Lactose Lactulose Trehalose Sucrose Raffinose Stachyose L. acidophilus 57.6% 49.7% 51.0% 68.0% 54.9% 57.6% 58.9% B. longum 47.5% 32.9% 38.3% 62.1% 42.0% 45.6% 49.3% L. lactis 63.4% 56.7% 59.7% 73.1% 58.2% 61.2% 64.2%

[0081] Table 4 shows the results of verifying the viable cell count of probiotic powder freeze-dried after secondary coating, and Table 5 shows the results of verifying the freeze-drying yield of probiotic powder freeze-dried after secondary coating. As shown in Tables 4 and 5, Trehalose exhibited the most superior effect as a non-reducing sugar for the secondary coating of probiotics. Trehalose is a glycosidic disaccharide of Glucose - α-1,1-Glucose.

[0083] (2) Concentration of the secondary coating solution

[0084] The primary coated in Example 1 L. acidophilus Table 6 shows the viable cell count and freeze-drying yield of the freeze-dried powder compared to the case where only the first coating was performed, after the samples were secondarily coated with trehalose at various concentrations (w / v).

[0085] Trehalose concentration Only the first coating was applied. After the second coating is completed viable bacteria count freeze-drying yield viable bacteria count freeze-drying yield 0.10%(w / v) 3.8E+11 38.0% 4.8E+11 55.4% 0.25%(w / v) 5.2E+11 68.0% 0.50%(w / v) 3.9E+11 66.0% 1.0%(w / v) 2.7E+11 66.5%

[0086] Table 6 shows the results of verifying the changes in viable cell count and freeze-drying yield of probiotic powder according to changes in the concentration of the secondary coating solution. As shown in Table 6, the freeze-drying yield was significantly superior at a secondary coating solution concentration greater than 0.10% (w / v), and the viable cell count of the freeze-dried powder was significantly superior at a secondary coating solution concentration less than 1.0% (w / v). Therefore, considering the freeze-drying yield and the viable cell count of the freeze-dried powder comprehensively, the improvement effect on the freeze-drying yield and the viable cell count of the freeze-dried powder was significantly superior at a secondary coating solution concentration greater than 0.10% (w / v) and less than 1.0% (w / v).

[0088] (3) Verification of the optimal pH of the probiotic culture solution before secondary coating

[0089] In addition, to verify the effect of the pH adjustment process prior to the second coating, the pH of a probiotic sample first coated with 0.10% (w / v) corn starch was adjusted in various ways using aqueous solutions of monopotassium phosphate and disodium phosphate, then secondarily coated with 0.25% (w / v) trehalose, and freeze-dried using substantially the same method as in Example 2. The viable cell count and freeze-drying yield of the probiotic powder are shown in Table 7. At this time, the yield for the freeze-dried powder was calculated using the following formula.

[0090] Freeze-drying yield calculation: [Weight of secondary coated sample powder x Number of viable cells] / [Weight of primary coated sample x Number of viable cells] x 100%

[0091] Probiotics viable bacteria count freeze-drying yield 5.5 6.0 6.5 7.0 7.5 5.5 6.0 6.5 7.0 7.5 L. acidophilus 4.8E+11 5.4E+11 5.8E+11 5.3E+11 4.9E+11 62.8% 70.6% 75.9% 69.3% 64.1% B. longum 3.0E+11 3.4E+11 3.6E+11 4.0E+11 3.3E+11 54.8% 62.1% 65.7% 73.0% 60.2% L. lactis 8.8E+11 9.4E+11 1.0E+12 1.1E+12 1.0E+12 65.7% 70.1% 74.6% 82.1% 74.6%

[0092] Table 7 presents the results of verifying the viable cell count and freeze-drying yield of the secondary-coated probiotic powder according to the pH change of the primary-coated probiotic samples. As shown in Table 7, the pH of the secondary coating solution that yielded excellent viable cell count and freeze-drying yield varied depending on the probiotic strain. Specifically, L. acidophilus When freeze-drying was performed after adjusting the pH to 6.5, the viable cell count and freeze-drying yield of the freeze-dried powder were the best, and B. longum and L. lactis When freeze-drying was performed after adjusting the pH to 7.0, the viable cell count and freeze-drying yield of the freeze-dried powder were the best.

[0094] Example 3. Tertiary coating of lactic acid bacteria

[0095] (1) Oil-in-Water (O / W) type plant lecithin-based tertiary coating

[0096] As in Example 2, 0.0125% (w / v) of vegetable sunflower or soybean lecithin, hydrolyzed in an O / W type at 25°C or lower, was added to the sample that had undergone pH adjustment and secondary coating with non-reducing sugar, and the tertiary coating was completed by homogenizing by stirring at 60 RPM for 20 minutes. Subsequently, the viable cell count and freeze-drying yield of the probiotic powder obtained by freeze-drying as in Example 2 are shown in Table 8.

[0097] Probiotics viable bacteria count freeze-drying yield sunflower lecithin Soy lecithin sunflower lecithin Soy lecithin L. acidophilus 5.8E+11 5.3E+11 75.9% 39.1% B. longum 4.1E+11 3.8E+11 74.8% 69.4% L. lactis 1.1E+12 9.8E+11 82.0% 73.1%

[0098] Table 8 shows the results of testing the viable cell count and freeze-drying yield of probiotic powders freeze-dried after a third coating with plant lecithin. As shown in Table 8, when probiotics were third-coated with sunflower lecithin, the viable cell count and freeze-drying yield of the freeze-dried powder for all three strains were superior compared to soybean lecithin.

[0099] In addition, the concentration of sunflower lecithin was adjusted to various levels, and a third coating was performed and freeze-dried. The viable cell count and freeze-drying yield were measured and are shown in Table 9.

[0100] Probiotics viable bacteria count freeze-drying yield 0.001% 0.005% 0.0125% 0.025% 0.05% 0.001% 0.005% 0.0125% 0.025% 0.05% L. acidophilus 5.1E+11 5.6E+11 5.8E+11 5.4E+11 5.3E+11 66.7% 73.3% 75.9% 70.6% 69.3% B. longum 3.6E+11 4.0E+11 4.1E+11 3.9E+11 3.7E+11 65.7% 73.0% 74.8% 71.2% 67.5% L. lactis 9.5E+11 1.1E+12 1.1E+12 1.0E+12 9.6E+11 70.9% 82.0% 82.0% 74.6% 71.6%

[0101] Table 9 shows the results of verifying the viable cell count and freeze-drying yield of probiotic powders tertiarily coated with sunflower lecithin at various ratios and then freeze-dried. As shown in Table 9, it was confirmed that the viable cell count and freeze-drying yield were best when the concentration of sunflower lecithin was greater than 0.001% (w / v) and less than 0.05% (w / v) (specifically, 0.005% (w / v) or more and less than 0.025% (w / v), or 0.01% (w / v) or more and less than 0.02% (w / v), for example, 0.0125% (w / v)).

[0103] (2) Verification of acid and bile resistance

[0104] To verify the acid resistance of the tertiary-coated probiotic powder sample, artificial gastric juice was prepared by adding pepsin to a pH 2.1 MRS broth solution corrected with 1M HCl solution to achieve a final concentration of 1,000 units / mL. Subsequently, 0.1g of the tertiary-coated sample was dissolved in 9.9mL of artificial gastric juice and incubated at 37°C for 2 hours. After analyzing the number of viable cells at 0 and 2 hours, the survival rate relative to 0 hours was calculated and is shown in Table 10.

[0105] In addition, to verify the bile resistance of the tertiary-coated powder, 0.1g of each sample was dissolved in 9.9ml of MRS broth solution containing 0.5% oxgall and incubated at 37℃ for 2 hours. Subsequently, the number of viable cells at 0 and 2 hours was analyzed, and the survival rate relative to 0 hours was calculated and is also shown in Table 10.

[0106] Probiotics acid resistance bile 0% 0.005% 0.0125% 0.025% 0% 0.005% 0.0125% 0.025% L. acidophilus 40.1% 52.8% 71.5% 63.8% 37.2% 47.7% 70.7% 61.8% B. longum 33.6% 49.7% 65.2% 59.6% 34.1% 51.6% 68.3% 57.4% L. lactis 26.7% 41.6% 60.8% 45.7% 29.5% 43.2% 57.9% 48.3%

[0107] Table 10 shows the results of verifying the acid resistance and bile resistance of probiotic powder tertiarily coated with sunflower lecithin. As shown in Table 10, the acid resistance and bile resistance of the raw powder were best at a sunflower lecithin concentration of 0.01% (w / v) to 0.02% (w / v) (for example, 0.0125% (w / v)).

[0109] Example 4. Quaternary coating of lactic acid bacteria

[0110] (1) Fourth coating using plant-based dietary fiber

[0111] The tertiary coated samples obtained according to Example 3 were subjected to a quaternary coating using various types of plant dietary fiber and freeze-dried, and the viable cell count of the probiotic powder (Table 11) and freeze-drying yield (Table 12) were verified in substantially the same manner as in Example 3. In addition, the viable cell count of the raw powder after freeze-drying and the freeze-drying yield according to the plant pectin concentration are shown in Table 13.

[0112] Probiotics α-glycosidically bonded dietary fiber β-glycosidically bonded dietary fiber pectin Acacia dietary fiber alginate Guar gum Chicory dietary fiber (inulin) L. acidophilus 6.3E+11 6.1E+11 5.4E+11 5.5E+11 5.8E+11 B. longum 4.3E+11 4.1E+11 3.7E+11 3.9E+11 4.0E+11 L. lactis 1.2E+12 1.1E+12 9.8E+11 1.0E+12 1.1E+12

[0113] Probiotics α-glycosidically bonded dietary fiber β-glycosidically linked dietary fiber pectin Acacia dietary fiber alginate Guar gum Chicory dietary fiber (inulin) L. acidophilus 83.4% 80.7% 71.5% 72.8% 76.8% B. longum 79.6% 75.9% 68.5% 72.2% 74.1% L. lactis 90.3% 82.8% 73.8% 75.3% 82.8%

[0114] Probiotics viable bacteria count freeze-drying yield 0.001% 0.005% 0.01% 0.05% 0.10% 0.001% 0.005% 0.01% 0.05% 0.10% L. acidophilus 5.7E+11 5.8E+11 6.3E+11 5.8E+11 5.3E+11 74.6% 76.3% 83.4% 80.3% 77.5% B. longum 4.0E+11 4.1E+11 4.3E+11 3.8E+11 3.5E+11 73.0% 75.4% 79.6% 74.4% 74.1% L. lactis 9.6E+11 9.8E+11 1.2E+12 1.0E+12 9.6E+11 71.6% 73.5% 90.3% 78.0% 78.0%

[0115] Table 11 shows the results of verifying the viable cell count of probiotic powder freeze-dried after 4th coating with natural dietary fiber, and Table 12 shows the results of verifying the freeze-drying yield of probiotic powder freeze-dried after 4th coating with natural dietary fiber. As shown in Tables 11 and 12, when the 4th coating was performed with plant pectin having α-glycosidic bonds, specifically α-1,4 glycosidic bonds among natural dietary fibers, the viable cell count of the original powder and the freeze-drying yield of the lactic acid bacteria after freeze-drying were the best.

[0116] Table 13 shows the results of verifying the viable cell count and freeze-drying yield of freeze-dried probiotic powder according to the concentration of plant pectin in the 4th coating sample. As shown in Table 13, the viable cell count and freeze-drying yield of the lactic acid bacteria after freeze-drying were best when the concentration of plant pectin in the 4th coating was 0.005% (w / v) or higher, specifically 0.005% to 0.5% (w / v), and more specifically 0.005% to 0.05% (w / v).

[0118] (2) Verification of acid and bile resistance

[0119] After the fourth coating, the acid resistance and bile resistance of the raw powder, freeze-dried in substantially the same manner as in Example 3, were verified and are shown in Table 14.

[0120] Probiotics acid resistance bile 0.001% 0.005% 0.01% 0.05% 0.001% 0.005% 0.01% 0.05% L. acidophilus 72.8% 77.2% 85.9% 84.3% 71.5% 75.6% 84.4% 82.9% B. longum 66.6% 70.9% 77.4% 78.0% 69.2% 72.1% 80.9% 81.4% L. lactis 62.1% 67.7% 75.3% 76.2% 59.1% 63.8% 73.6% 74.7%

[0121] Table 14 shows the results of verifying the acid resistance and bile resistance of probiotic powder after the fourth coating. As shown in Table 14, the acid resistance and bile resistance of the freeze-dried powder were best when the fourth coating was performed with a concentration of vegetable pectin of 0.005% (w / v) or higher, for example, 0.005 to 0.1% (w / v).

[0123] (3) Verification of harsh stability and surface hydrophobicity

[0124] To verify the stability of the probiotic powder that underwent up to the fourth coating, the coated samples were freeze-dried and ground, and the ground powder samples were divided into five 5g portions under conditions of 25℃ or lower and RH 40% or lower. The divided samples were cultured for 4 weeks in a constant temperature and humidity chamber configured for harsh conditions (40℃, relative humidity (RH) 75%), and the number of viable cells at weeks 1, 2, 3, and 4 was analyzed. Weekly stability was determined by the survival rate relative to the initial viable cell count and is shown in Table 15.

[0125] In addition, cell surface hydrophobicity Lactobacillus and Bifidobacterium It is widely used as an indirect indicator of the intestinal adhesion ability of representative probiotics. Accordingly, after washing uncoated and quadruple-coated freeze-dried probiotic samples twice with 1xPBS (phosphate buffered saline, pH 7.2), the OD of the bacterial cells 600 It was suspended in 1x PBS to a concentration of 0.5. Subsequently, toluene was added and mixed, then reacted in a 37°C water bath for 20 minutes; afterward, the toluene was removed, and the OD of the aqueous layer was removed. 600nm The values ​​were measured. The hydrophobicity of the 4th-coated samples was calculated using the following formula and is shown in Table 15:

[0126] [Initial Suspension OD - Supernatant OD] / [Initial Suspension OD] x 100% = Hydrophobicity %

[0127] Probiotics 4-week harsh stability surface hydrophobicity non-coated 4th coating non-coated 4th coating L. acidophilus 21.7% 65.5% 35.8% 66.2% B. longum 13.2% 46.3% 36.6% 73.4% L. lactis 17.4% 50.3% 42.2% 71.6%

[0128] Table 15 shows the results of verifying the 4-week severe stability and cell surface hydrophobicity of probiotics that have undergone a 4th coating. As shown in Table 15, freeze-dried probiotic powder coated with plant pectin showed superior stability under severe conditions compared to uncoated probiotics, and cell surface hydrophobicity, which can indirectly confirm intestinal adhesion, was also significantly improved compared to uncoated probiotics.

[0130] (4) Summary of results for 4th-coated probiotic powder compared to uncoated powder

[0131] The verification results of the freeze-drying yield, acid resistance, bile resistance, 4-week harsh stability, and surface hydrophobicity of the 4th-coated probiotic powder were combined and are shown in Table 16.

[0132] Probiotics freeze-drying yield acid resistance bile 4-week harsh stability surface hydrophobicity non-coated 4th coating p 1 non-coated 4th coating p non-coated 4th coating p non-coated 4th coating p non-coated 4th coating p L. acidophilus 48.7% 83.4% 0.0060(**) 40.1% 85.9% 0.0003(***) 37.2% 84.4% 0.0003(***) 21.7% 65.5% 0.0043(**) 35.8% 66.2% 0.0004(***) B. longum 32.3% 79.6% 33.6% 77.4% 34.1% 80.9% 13.2% 46.3% 36.6% 73.4% L. lactis 54.6% 90.3% 36.7% 75.3% 29.5% 73.6% 17.4% 50.8% 42.2% 71.6%

[0133] 1 Two samples t -test

[0134] As shown in Table 16, after the fourth coating, all three representative probiotic strains showed significantly improved bile resistance, acid resistance, stability, and cell surface hydrophobicity.

Claims

Claim 1 A method for preparing a multi-coated probiotic composition, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; and fourthly coating the third-coated probiotics with dietary fiber. Claim 2 A method of manufacturing in which, in claim 1, the natural adsorbent is an organic material derived from plants or animals. Claim 3 A method of manufacturing, wherein the natural adsorbent in paragraph 2 is one or more selected from the group consisting of starch, cellulose, guar gum, chitosan, and gelatin. Claim 4 A method of manufacturing in paragraph 3, wherein the starch is natural starch or modified starch. Claim 5 A method of manufacturing, wherein the starch is one or more selected from the group consisting of corn starch, potato starch, sweet potato starch, tapioca starch, rice starch, wheat starch, corn maltodextrin, tapioca maltodextrin, resistant maltodextrin, and α-cyclodextrin. Claim 6 A method of manufacturing according to claim 1, wherein the sugars are reducing sugars or non-reducing sugars. Claim 7 A method of manufacturing in which, in paragraph 1, the sugars are non-reducing sugars. Claim 8 In claim 6, the reducing sugar is one or more selected from the group consisting of glyceraldehyde, erythrose, ribose, xylose, arabianose, ribulose, glucose, fructose, galactose, mannose, maltose, lactose, lactulose, chitobiose, cellobiose, and dextrin, and the non-reducing sugar is one or more selected from the group consisting of trehalose, sucrose, raffinose, mellezitose, gentianose, stachyose, and verbascose. Manufacturing method. Claim 9 A method of manufacturing, wherein the plant lecithin in claim 1 is one or more selected from the group consisting of sunflower lecithin, rapeseed lecithin, and soybean lecithin. Claim 10 A method of manufacturing in claim 1, wherein the plant lecithin is sunflower lecithin. Claim 11 A method of manufacturing according to claim 1, wherein the dietary fiber is α-glycosidically bonded dietary fiber or β-glycosidically bonded dietary fiber. Claim 12 A method of manufacturing, wherein the dietary fiber is one or more selected from the group consisting of pectin, acacia dietary fiber, alginic acid, guar gum, chicory dietary fiber (inulin), beta-glucan, polydextrose, indigestible maltodextrin, levan, seaweed-derived polysaccharides, seed-derived polysaccharides, cellulose, hemicellulose, crystalline cellulose, lignin, cereal-derived dietary fiber, mushroom-derived dietary fiber, and processed dietary fiber. Claim 13 A method for manufacturing according to claim 1, wherein the first coating and separation step comprises: adding the natural adsorbent to the probiotic culture at a concentration of 0.05% (w / v) or higher; and applying centrifugal force to the culture. Claim 14 A method for manufacturing according to claim 1, wherein the step of secondary coating further comprises the step of adjusting the pH of the primary coated probiotic sample to 6 to 8. Claim 15 A method of manufacturing according to claim 1, wherein the step of secondary coating involves adding sugars to a primary coated probiotic sample at a concentration greater than 0.1% (w / v) and less than 1.0% (w / v), and mixing and homogenizing. Claim 16 A method of manufacturing according to claim 1, wherein the third coating step involves adding plant lecithin to a second-coated probiotic sample at a concentration greater than 0.001% (w / v) and less than 0.05% (w / v), and mixing and homogenizing. Claim 17 A manufacturing method according to claim 1, wherein the step of the fourth coating is to add dietary fiber to the third-coated probiotic sample at a concentration of 0.005% (w / v) or higher, and to mix and homogenize. Claim 18 In paragraph 1, the probiotic is of the genus Lactobacillus ( Lactobacillus spp.), genus Bifidobacterium ( Bifidobacterium spp.), genus Lactococcus ( Lactococcus spp.), genus Akkermansia ( Akkermansia spp.), Streptococcus genus ( Streptococcus ssp.), genus Enterococcus ( Enterococcus spp.), Bacillus genus ( Bacillus ssp.), genus Pediococcus ( Pediococcus spp.), genus Weissella ( Weissella ssp.), and the genus Leuconostoc ( Leuconostoc A manufacturing method comprising one or more selected from the group consisting of spp.). Claim 19 A method of manufacturing according to claim 1, further comprising the step of freeze-drying the 4th coated probiotic. Claim 20 A method of manufacturing, wherein the composition is a powder, in accordance with claim 1. Claim 21 A method of manufacturing in claim 1, wherein the composition is a freeze-dried powder. Claim 22 A method for increasing the freeze-drying survival rate of probiotics, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; fourthly coating the third-coated probiotics with dietary fiber; and freeze-drying the fourth-coated probiotics. Claim 23 A method for increasing the acid resistance and / or bile resistance of probiotics, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; and fourthly coating the third-coated probiotics with dietary fiber. Claim 24 A method for increasing the surface hydrophobicity of probiotics, comprising the steps of: adding a natural adsorbent to a probiotic culture and applying centrifugal force to first coat and separate the probiotics; secondly coating the first-coated probiotics with a sugar; thirdly coating the second-coated probiotics with plant lecithin; and fourthly coating the third-coated probiotics with dietary fiber. Claim 25 A multi-coated probiotic composition sequentially coated with a natural adsorbent, sugars, plant lecithin, and dietary fiber. Claim 26 A food comprising a probiotic composition according to paragraph 25. Claim 27 A health functional food comprising a probiotic composition according to paragraph 25.

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