Method for producing lactic acid bacteria encapsulated in calcium alginate beads

Abstract

The present disclosure relates to a method of producing lactic acid bacteria encapsulated with calcium alginate beads; lactic acid bacteria encapsulated by calcium alginate beads produced by the method; the invention discloses a method for improving the production yield of calcium alginate beads encapsulating lactic acid bacteria. The invention further discloses a method for improving the survival rate of the freeze-dried lactic acid bacteria.

Description

Technical Field

[0001] The present disclosure relates to a method for producing lactic acid bacteria encapsulated with calcium alginate beads; lactic acid bacteria encapsulated with calcium alginate beads produced by the above method; a method for improving the production yield of calcium alginate beads encapsulating lactic acid bacteria; and a method for improving the survival rate of freeze-dried lactic acid bacteria. Background Art

[0002] Lactic acid bacteria, also known as lactic acid bacteria or lactobacilli, live in the intestines of mammals and prevent abnormal fermentation of saprophytes. Therefore, they are used as medicines for the treatment of intestinal disorders. For example, Lactobacillus bulgaricus (L.bulgaricus) is a long-known lactic acid bacteria, which is used in the production of yogurt and is also used as a starter for the production of cheese or cultured butter. In addition, aerobic lactobacilli found in the intestines of all mammals, including humans and other animals: Lactobacillus acidophilus (L.acidophilus) is used to produce butter or milk or for the treatment of autointoxication. In addition, Lactococcus lactis (L.lactis) produces DL-lactic acid and is used to produce butter or cheese because it is always found in milk. Lactococcus lactis is the most important bacterial species in dairy applications.

[0003] This beneficial lactic acid bacteria is present in the intestinal tract, and shows various physiological activities, such as active intestinal motility, inhibiting harmful bacteria, promoting the generation of vitamins and immunostimulants, improving atopic dermatitis, etc. However, this physiological activity only shows when the lactic acid bacteria quantity of intake is much more than the quantity of intake from foods such as yogurt. Therefore, the easy-to-eat form (such as powder and capsule) of lactic acid bacteria isolate is currently available on the market. However, when lactic acid bacteria are processed in the form of powder or capsule, they may not show inherent physiological activity, because most of them are likely to be killed during long-term storage, which is a shortcoming.

[0004] Usually, lactic acid bacteria are produced in the form of powder by freeze drying or spray drying. Among them, freeze drying is a method in which microorganisms are suspended in a freeze drying suspension solution, frozen, and then dried under reduced pressure. However, since lactic acid bacteria are anaerobic and very sensitive to the surrounding environment, the viability of powdered lactic acid bacteria is reduced at low temperatures and room temperature, thereby reducing the number of viable bacteria. Recently, in order to overcome these shortcomings, efforts have been made to develop methods for coating lactic acid bacteria with various coating materials. For example, methods using starch, gelatin, alginic acid, cellulose, hardened oils, various emulsifiers, etc. to maintain quality during storage are used as cryoprotectants, etc.

[0005] Temperature rise will kill live lactic acid bacteria; therefore, freeze drying is the most effective way to maintain cell activity during the preservation of lactic acid bacteria. Although freeze drying can greatly improve the storage and distribution of lactic acid bacteria, the survival rate of lactic acid bacteria is generally greatly reduced by the freeze drying process.

[0006] Representatively, studies have been conducted to confirm the effects of storage temperature and polymer on the stability of lactic acid bacteria by freeze-drying Lactobacillus rhamnosus coated with gelatin (Claude PC et al, Food. Res. Int., 1996, 29, 555-562). However, the above studies disclose that about 1% of lactic acid bacteria survive after 6 months of storage at 20°C, and only about 0.2% of lactic acid bacteria survive after 12 months; therefore, it is necessary to develop a method that can improve the survival rate and storage stability of lactic acid bacteria.

[0007] Meanwhile, the method of coating lactic acid bacteria with alginate capsules is known, but alginate is unstable to acid and heat. For this reason, according to the conventional method of producing capsules using alginate, they are produced by spraying a mixture of alginate and microorganisms into a coagulation solution such as a CaCl2 solution. Therefore, when alginate reacts with calcium ions of the coagulation solution on the particle surface of the spray mixture of alginate and microorganisms, hardening begins, and sufficient calcium ions cannot move into the particles in a short time. Therefore, it is difficult to mass-produce alginate capsules that are firm and densely hardened even inside alginate with high yield. Summary of the invention

[0008] [Technical issues]

[0009] The present inventors have developed a method for producing lactic acid bacteria encapsulated with calcium alginate beads, which increases the production yield of the beads and improves the survival rate and stability after freeze-drying, thereby completing the present disclosure.

[0010] [Technical solution]

[0011] An object of the present disclosure is to provide a method for producing lactic acid bacteria encapsulated with calcium alginate beads.

[0012] Another object of the present disclosure is to provide lactic acid bacteria encapsulated with calcium alginate beads produced by the above method.

[0013] Another object of the present disclosure is to provide a method for improving the production yield of calcium alginate beads encapsulating lactic acid bacteria.

[0014] Another object of the present disclosure is to provide a method for improving the survival rate of freeze-dried lactic acid bacteria.

[0015] [Beneficial Effects]

[0016] The method for producing lactic acid bacteria encapsulated with calcium alginate beads of the present disclosure increases the yield of beads and can provide lactic acid bacteria having improved survival rate and stability after freeze-drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram showing the production method of lactic acid bacteria encapsulated with calcium alginate beads of the present invention.

[0018] Figure 2 It is a graph showing the results of evaluating gastrointestinal stability according to the presence or absence of calcium alginate beads. DETAILED DESCRIPTION

[0019] The present disclosure will be described in detail below. Meanwhile, each description and embodiment disclosed herein can be applied to other descriptions and embodiments respectively. That is to say, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below.

[0020] In addition, those skilled in the art will be able to recognize or confirm many equivalents to the specific aspects of the disclosure described herein using only routine experimentation. In addition, it is intended that these equivalents be included in the present disclosure.

[0021] One aspect of the present disclosure provides a method for producing lactic acid bacteria encapsulated with calcium alginate beads, comprising:

[0022] (a) culturing lactic acid bacteria and recovering bacterial cells;

[0023] (b) mixing the lactic acid bacteria of step (a) with an alginate-containing solution; and

[0024] (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads,

[0025] The alginate solution comprises sucrose, sorbitol or soy peptone, and the calcium solution comprises trehalose or maltodextrin.

[0026] In one embodiment, the method may further comprise (d) freeze-drying the lactic acid bacteria encapsulated with calcium alginate beads of step (c), but is not limited thereto.

[0027] As used herein, the term "freeze drying" refers to a method in which the material to be dried is frozen by rapidly lowering the temperature of a container, the internal pressure of the container is then made into a vacuum, and the solidifying solvent contained in the material is immediately sublimated into steam for drying. Freeze drying is a method by which the damage to heat-sensitive materials can be minimized, and the material can be stored for a long time, and is useful in preventing pollution, storage, transportation, and economic benefits. However, in the freeze drying process of lactic acid bacteria, the activity and survival rate of lactic acid bacteria are rapidly reduced, and ice particles are formed during the freezing process, so there is a problem that the membrane structure of lactic acid bacteria cells is destroyed. In order to solve this problem, a material that is added together during the freeze drying process so that the function can be restored without destroying or killing the lactic acid bacteria during the rehydration process is called a "cryoprotectant", which is used to impart physicochemical stability to lactic acid bacteria to increase the survival rate.

[0028] In the present disclosure, the freezing temperature of the freeze-drying can be a sub-zero temperature, such as -10°C to -196°C (the boiling point of liquid nitrogen), -40°C to -196°C, -50°C to -196°C and -70°C to -196°C, and at such low temperatures, all biological activities of lactic acid bacteria including biochemical reactions leading to cell death are effectively stopped.

[0029] As used herein, the term "cryoprotection" means that when the lactic acid bacteria are freeze-dried and then stored, the tissue of the lactic acid bacteria is protected from damage by freezing to maintain its original activity.

[0030] For the purpose of the present disclosure, the lactic acid bacteria encapsulated with calcium alginate beads produced by the above method of producing lactic acid bacteria encapsulated with calcium alginate beads may have improved survival rate and stability after freeze-drying.

[0031] Hereinafter, a method for producing lactic acid bacteria encapsulated with calcium alginate beads of the present disclosure will be described in detail.

[0032] First, (a) lactic acid bacteria are cultured and bacterial cells are recovered.

[0033] As used herein, the term "lactic acid bacteria" is a generic term for bacteria that obtain energy by fermenting sugars and produce a large amount of lactic acid. Although not particularly limited thereto, the lactic acid bacteria may include at least one selected from the group consisting of Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., Leuconostoc sp., and Weissella sp., but is not limited thereto.

[0034] Specifically, the lactic acid bacteria may include at least one selected from the group consisting of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Streptococcus faecalis and Lactococcus lactis subsp. lactis, but is not limited thereto.

[0035] More specifically, the lactic acid bacteria may include at least one selected from the group consisting of Lactobacillus plantarum CJLP133, Lactobacillus plantarum CJLP243, Lactobacillus plantarum CJLP136, Lactobacillus plantarum CJLP55, and Lactobacillus plantarum CJLP56, but is not limited thereto.

[0036] These strains are deposited in the gene bank of the Korea Institute of Bioscience and Biotechnology and can be easily obtained therefrom.

[0037] As used herein, the term "culture" refers to the growth of lactic acid bacteria under suitable controlled environmental conditions. The culture process disclosed herein can be carried out under suitable culture media and culture conditions known in the art. Those skilled in the art can easily adjust this culture method for use according to the strain to be selected. Specifically, the culture can be batch culture, continuous culture and fed-batch culture, but is not limited thereto.

[0038] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for culturing the disclosed microorganism as the main component, which provides nutrients and growth factors and water necessary for survival and growth. Specifically, the culture medium and other culture conditions for culturing the disclosed microorganism can be any culture medium used for conventional culture of microorganisms, without any particular restrictions. However, the disclosed microorganism can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions, while adjusting the temperature, pH value, etc.

[0039] In the present disclosure, the carbon source may include carbohydrates such as glucose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; alcohols such as glycerol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source may include natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, cane molasses, and corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and in addition, various other carbon sources may be used in appropriate amounts without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0040] The nitrogen source may include an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0041] The phosphorus source may include monopotassium phosphate, dipotassium phosphate or corresponding sodium salts, etc. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and / or appropriate precursors, etc. may be included. These components or precursors may be added to the culture medium in a batch or continuous manner, but are not limited thereto.

[0042] In the present disclosure, the pH of the culture medium can be adjusted in an appropriate manner by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. during the culture of lactic acid bacteria. In addition, the use of a defoaming agent (such as fatty acid polyethylene glycol ester) during the culture process can prevent the formation of bubbles. In addition, in order to maintain the aerobic conditions of the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium; or in order to maintain anaerobic or microaerobic conditions, the culture can be carried out without injecting any gas into the culture medium, or nitrogen, hydrogen or carbon dioxide can be injected into the culture medium, but it is not limited thereto.

[0043] The temperature of the culture medium may be in the range of from 20° C. to 50° C., specifically from 30° C. to 37° C., but not limited thereto. Furthermore, the culture may be continued until a desired amount of useful substances is obtained, specifically for 10 to 100 hours, but not limited thereto.

[0044] In the step of recovering lactic acid bacteria cells, the desired bacterial cells can be collected using a suitable method known in the art according to the method for culturing lactic acid bacteria disclosed herein, such as batch culture, continuous culture or fed-batch culture method. For example, centrifugation, filtration, treatment with a protein crystallization precipitant (salting out), extraction, ultrasonic crushing, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or a combination of these methods can be used, but are not limited to these examples.

[0045] The recovery step may include an additional purification step, which may be performed using a suitable method known in the art and may purify the recovered lactic acid bacteria cells.

[0046] Subsequently, (b) the lactic acid bacteria of step (a) are collected by a method such as centrifugation and then mixed with the alginate-containing solution.

[0047] As used herein, the term "alginic acid or alginate" is a polymer of two uronic acids, having a degree of polymerization of 80 and a molecular weight of about 1,500, and having an unbranched structure in which hundreds of D-mannuronic acid (hereinafter referred to as M) and L-guluronic acid are linked by β-1,4 bonds.

[0048] In one embodiment of the present disclosure, in order to increase the cryoprotective effect and thus increase the survival rate of lactic acid bacteria encapsulated in calcium alginate beads after freeze drying, they are prepared by adding sugar and protein to alginate-containing solutions. Cryoprotectants commonly used for proteins usually include skim milk, but it has been confirmed that when skim milk is added, the calcium component of skim milk first reacts with alginate and hardens. Therefore, it was excluded from the experimental group because beads could not be produced normally.

[0049] Specifically, the alginate-containing solution may include sucrose, sorbitol or soy peptone, more specifically, soy peptone, but is not limited thereto.

[0050] As used herein, the term "soy peptone" refers to a protein derived from soybeans, which is mainly used as a nutrient source in microbial culture media. The protein content of isolated soy protein is about 90%, and globulin is the highest in soy peptone.

[0051] In one embodiment, the alginate-containing solution comprises sucrose, sorbitol or soy peptone, more specifically 5 to 10 wt %, more specifically 6 to 8 wt %, even more specifically about 7 wt % soy peptone, but is not limited thereto.

[0052] When sucrose, sorbitol or soy peptone is included in the above weight percentage, the production yield of the beads increases, and it can be used as a cryoprotectant to prevent lactic acid bacteria from being damaged or dying due to freeze drying and increase stability. When sucrose, sorbitol or soy peptone is included in an amount higher than the optimal range, the formation of alginate beads is hindered, and thus the beads are not densely formed, which reduces the protective effect against gastric acid.

[0053] In one embodiment, the alginate-containing solution is prepared by dissolving 1 wt % to 3 wt %, more specifically about 2 wt % of sodium alginate, and may additionally include trehalose or maltodextrin, but is not limited thereto.

[0054] When the weight percentage of sodium alginate is lower than the optimal range, the hardening of the beads may be weak, resulting in elution of lactic acid bacteria and cryoprotectant components, while when the weight percentage of sodium alginate is higher than the optimal range, the hardening of the beads may be too strong, making it difficult for lactic acid bacteria to come out during subsequent ingestion, or the bacteria may die, which is a problem.

[0055] As used herein, the term "trehalose" is a sugar widely present in nature, such as plants or microorganisms, etc. For the purpose of the present disclosure, trehalose contained in the alginate-containing solution can act as a cryoprotectant, which prevents damage or death of lactic acid bacteria caused by freeze-drying and helps to restore their functions during rehydration, thereby increasing the cryoprotection effect of lactic acid bacteria.

[0056] As used herein, the term "maltodextrin" is a white powder based on porous particles and is a food additive commonly used in common foods such as yogurt, sauce, salad dressing, etc. For the purpose of the present invention, the maltodextrin contained in the alginate solution can be used as a porous carrier in the freeze-drying process of lactic acid bacteria and as a cryoprotectant to prevent damage or death of lactic acid bacteria caused by freeze-drying. The porous carrier is based on the form of porous particles and is used to block the inflow of external moisture and air.

[0057] In one embodiment, the trehalose may be present in an amount of 5 to 10 wt %, more specifically 6 to 8 wt %, even more specifically about 7 wt %, and the maltodextrin may be present in an amount of 5 to 10 wt %, more specifically 6 to 8 wt %, even more specifically about 6 wt %, but are not limited thereto.

[0058] In one embodiment, the ratio of the weight of the alginate solution to the weight of the lactic acid bacteria in the mixture of step (b) may be 1:1 to 10:1, more specifically 1:1 to 3:1, and more specifically about 2:1, but is not limited thereto.

[0059] Since the solid weight of lactic acid bacteria is composed in an optimal ratio, when this method is used, the production cost can be kept at an appropriate level while maintaining the cryoprotection effect and the yield of dry lactic acid bacteria powder. When the ratio of the weight of the alginate-containing solution to the weight of the lactic acid bacteria deviates from the optimal ratio, there is a problem that the production yield per unit time and / or per kg of lactic acid bacteria is reduced, thereby reducing the process efficiency.

[0060] Furthermore, fluidity (viscosity) can be maintained by making the total solid content of the alginate-containing solution 20 wt % to 30 wt %.

[0061] In one embodiment, in the mixing process of step (b), a prebiotic may be optionally further added. Specifically, when the lactic acid bacteria are mixed with the coating agent, a prebiotic may be added. The prebiotic serves as food for the lactic acid bacteria. The prebiotic may be any one or more selected from oligofructose, oligogalactose, maltitol, lactitol and inulin, in particular oligofructose or inulin. The prebiotic may be added in an amount of 0.1% to 5% by weight based on the weight of the lactic acid bacteria.

[0062] Subsequently, (c) the mixture of step (b) is added to a calcium-containing solution to form calcium alginate beads.

[0063] Specifically, step (c) is an extrusion step. When the mixture of step (b) is added to a calcium-containing solution in a droplet form or by spraying, so that it reacts with the calcium-containing solution, a matrix is ​​formed by alginate-calcium cross-linking. As a result, the lactic acid bacteria are encapsulated in the beads in the calcium-containing solution and form calcium alginate beads, but are not limited thereto.

[0064] The formation of beads by the alginate-calcium reaction is carried out at a temperature below room temperature (e.g., 25°C). Since this reaction involves less physical stress, it has little effect on the survival rate of live bacteria. Calcium alginate beads formed by extrusion show pH-dependent release; therefore, the calcium alginate bead particles do not decompose under acidic conditions such as gastric acid, and decompose slowly in a neutral environment such as the intestinal environment, which greatly helps to improve the survival rate of lactic acid bacteria in the intestine.

[0065] Carbohydrate, protein, lipid etc. can be used as cryoprotectant.When protein is added in calcium-containing solution, the amphipathicity of amino acid prevents alginate from combining with calcium ions, thereby preventing the formation of alginate beads with compact structure.Lipid can not mix well with water, and is not suitable for use in calcium-containing solution.Therefore, in one embodiment of the present disclosure, it is confirmed that adding sugar to calcium-containing solution improves the production yield and survival rate of alginate beads.

[0066] Specifically, the calcium-containing solution may include trehalose or maltodextrin, more specifically, trehalose, but is not limited thereto.

[0067] When trehalose or maltodextrin is included, the production yield of beads increases, and it can be used as a cryoprotectant to prevent lactic acid bacteria from being damaged or dying due to freeze-drying and to increase stability.

[0068] In one embodiment, in order to make the concentration of the calcium-containing solution similar to the solid content of the alginate-containing solution mixed with the lactic acid bacteria precipitate, the calcium-containing solution may contain trehalose or maltodextrin, more specifically, the amount of trehalose is 15 wt % to 35 wt %, even more specifically, 20 wt % to 30 wt %, further more specifically, about 25 wt %, but not limited thereto.

[0069] By making the concentrations of the mixture in step (b) and the calcium-containing solution similar, it is possible to prevent as much as possible the cryoprotectant component in the solution containing lactic acid bacteria and alginic acid from dissolving into the calcium-containing solution after being added in the form of droplets.

[0070] In one embodiment, the calcium-containing solution may be a solution in which calcium chloride is dissolved in water at 0.5 to 1.5 wt %, more specifically about 1 wt %, but is not limited thereto.

[0071] When the weight percentage of calcium chloride is lower than the optimal range, the bead structure may not be densely produced and the cryoprotection effect may be insufficient, and when the weight percentage of calcium chloride is higher than the optimal range, the hardening of the beads may be too strong, thus causing problems in commercialization due to insufficient elution of lactic acid bacteria in the final product.

[0072] Specifically, after step (c), the method may further include storing the solution containing calcium alginate beads at a temperature of 4° C. to 20° C. for 30 to 60 minutes.

[0073] This step is the ripening process. When the sodium ions are replaced by calcium ions, the alginate forms a network structure and encapsulates. Through this ripening process, the density of the particle structure can be increased.

[0074] The method of the present disclosure may further comprise adding one or more types, specifically, one, two or three types, selected from the group consisting of a cryoprotectant, a porous carrier, and a nitrogen source.

[0075] Specifically, the method of the present disclosure may further include adding a component known as a conventional cryoprotectant, which does not include alginate solutions and calcium-containing solutions. That is, as used herein, the term "cryoprotectant" refers to a substance with cryoprotective efficacy commonly used in the technical field to which the present disclosure belongs, which is further added to produce lactic acid bacteria encapsulated with calcium alginate beads. As a cryoprotectant, commercially available products can be purchased and used, and their types are not particularly limited. Specifically, the cryoprotectant can be trehalose, sugars, amino acids, peptides, gelatin, glycerol, sugar alcohols, whey, alginic acid, ascorbic acid, yeast extract, skim milk, etc., but is not limited thereto.

[0076] As used herein, a "porous carrier" is used to block the inflow of external moisture and air, and to impart porosity to the freeze-dried lactic acid bacteria to promote rehydration. The porous carrier is a porous carrier commonly used in freeze-drying processes in the technical field to which the present invention belongs. As a porous carrier, commercially available products can be purchased and used, and their types are not particularly limited. The porous carrier can specifically be maltodextrin, alginate, chitosan, starch, polyethylene glycol, propylene glycol, triacetin, acetyl triethyl citrate, triethyl citrate, glycerol or a combination thereof, but is not limited thereto.

[0077] As used herein, the term "nitrogen source (N-source)" refers to a substance used as a nitrogen energy source for lactic acid bacteria, and is used to prevent bacterial cell damage caused by post-fermentation. When lactic acid bacteria are mixed with a composition for cryoprotection, lactic acid bacteria that survive in the absence of energy produce organic acids that cause a decrease in pH, thereby inducing the death of lactic acid bacteria. Accordingly, the nitrogen source prevents the generation of organic acids and the resulting pH reduction, and therefore, the death of lactic acid bacteria can be prevented.

[0078] The nitrogen source is a nitrogen source commonly used in freeze drying processes in the technical field to which the present invention belongs. As a nitrogen source, commercially available products can be purchased and used, and their types are not particularly limited. The nitrogen source can be skimmed milk powder, whey protein, yeast extract, malt extract, beef extract, casein hydrolyzate, malt extract, tryptone, cysteine, peptone, etc., in particular, the peptone can be soy peptone, fish peptone, peptone, casein peptone, peptone No. 3, etc.

[0079] Subsequently, (d) the lactic acid bacteria encapsulated with calcium alginate beads of the freeze drying step (c) may be further added. The freeze drying step may be performed by transferring the calcium alginate beads containing lactic acid bacteria produced in step (c) to a freeze drying tray while raising the temperature from -20°C to 30°C for 50 hours.

[0080] Another aspect of the present disclosure provides lactic acid bacteria encapsulated with calcium alginate beads produced by the above method.

[0081] The method and lactic acid bacteria encapsulated in calcium alginate beads are as described above.

[0082] Specifically, the lactic acid bacteria may have an improved survival rate after freeze-drying compared to lactic acid bacteria not encapsulated with calcium alginate beads, but is not limited thereto.

[0083] Specifically, the lactic acid bacteria may have improved stability compared to lactic acid bacteria not encapsulated with calcium alginate beads, but is not limited thereto.

[0084] For the purpose of the present disclosure, the term "stability" may refer to storage (distribution) stability or stability in the gastrointestinal tract, but is not limited thereto.

[0085] Specifically, it may refer to high temperature stability, acid resistance or bile resistance, and the like.

[0086] Another aspect of the present disclosure provides a method for improving the production yield of calcium alginate beads encapsulating lactic acid bacteria, comprising:

[0087] (a) culturing lactic acid bacteria and recovering bacterial cells;

[0088] (b) mixing the lactic acid bacteria of step (a) with a solution containing alginate; and

[0089] (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads,

[0090] The alginate solution comprises sucrose, sorbitol or soy peptone, and the calcium solution comprises trehalose or maltodextrin.

[0091] The method and lactic acid bacteria encapsulated in calcium alginate beads are as described above.

[0092] Another aspect of the present disclosure provides a method for improving the survival rate of freeze-dried lactic acid bacteria, comprising:

[0093] (a) culturing lactic acid bacteria and recovering bacterial cells;

[0094] (b) mixing the lactic acid bacteria of step (a) with a solution containing alginate;

[0095] (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads; and

[0096] (d) freeze-drying the lactic acid bacteria encapsulated by calcium alginate beads in step (c),

[0097] The alginate solution comprises sucrose, sorbitol or soy peptone, and the calcium solution comprises trehalose or maltodextrin.

[0098] The method and lactic acid bacteria encapsulated in calcium alginate beads are as described above.

[0099] Another aspect of the present disclosure provides lactic acid bacteria encapsulated with calcium alginate beads and freeze-dried, which are produced by the above method.

[0100] The method and lactic acid bacteria encapsulated in calcium alginate beads are as described above.

[0101] Specifically, the lactic acid bacteria may have an increased survival rate after freeze-drying compared to lactic acid bacteria not encapsulated with calcium alginate beads, but is not limited thereto.

[0102] Specifically, the lactic acid bacteria may have improved stability compared to lactic acid bacteria not encapsulated with calcium alginate beads, but is not limited thereto.

[0103] [Modes for Carrying Out the Invention]

[0104] Hereinafter, the present disclosure will be described in detail by way of examples. However, these examples are preferred examples given only for illustrative purposes, and therefore, the scope of the present disclosure is not intended to be limited by these examples.

[0105] Comparative Example 1. Preparation of freeze-dried powder containing lactic acid bacteria

[0106] Comparative Example 1-1. Cultivation of lactic acid bacteria

[0107] Lactobacillus plantarum CJLP133 was cultured in MRS liquid medium (Difco, USA) at 37°C for 10 hours. The number of lactic acid bacteria in the medium was confirmed to be approximately 8.68 x 10^9 CFU / mL. MRS medium components were prepared as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of dextrose, 1 g of polysorbate 80, 2 g of ammonium citrate, 5 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, and 2 g of potassium dihydrogen phosphate were mixed in 1 L of distilled water, dissolved, and sterilized.

[0108] Comparative Example 1-2. Preparation of Lactic Acid Solution Containing Cryoprotectant

[0109] The culture medium in which the lactic acid bacteria of Comparative Example 1-1 were cultured was centrifuged for 10 minutes, and the supernatant was discarded to obtain a precipitate. The bacterial cells thus obtained were stabilized by stirring with a sterilized cryoprotectant at a ratio of 1:4 (w / w) for 30 minutes. A cryoprotectant solution was prepared by mixing 140 g of maltodextrin, 140 g of trehalose, 30 g of skim milk, and 690 g of distilled water, followed by sterilization.

[0110] Comparative Example 1-3. Freeze-drying of lactic acid bacteria containing cryoprotectant

[0111] The lactic acid bacteria of Comparative Example 1-2 containing a cryoprotectant were rapidly frozen using a freeze dryer and then dried. Freeze drying was performed by raising the temperature from -20°C to 30°C and maintaining it for 50 hours.

[0112] Example 1. Preparation of lactic acid bacteria encapsulated with calcium alginate beads using a calcium-containing solution containing calcium chloride and freeze-drying thereof

[0113] Example 1-1. Cultivation of lactic acid bacteria

[0114] Lactobacillus plantarum CJLP133 was cultured in MRS liquid medium (Difco, USA) at 37°C for 10 hours. The number of lactic acid bacteria in the medium was confirmed to be approximately 8.68 x 10^9 CFU / mL. MRS medium components were prepared as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of dextrose, 1 g of polysorbate 80, 2 g of ammonium citrate, 5 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, and 2 g of potassium dihydrogen phosphate were mixed in 1 L of distilled water, dissolved, and sterilized.

[0115] Example 1-2. Preparation of sodium alginate solution containing lactic acid bacteria

[0116] The culture medium in which the lactic acid bacteria of Example 1-1 were cultured was centrifuged for 10 minutes, and the supernatant was discarded to obtain a precipitate. An alginate-containing solution (alginate solution) was prepared by mixing the composition shown in Table 1 below and then sterilizing. The bacterial cells thus obtained were stabilized by stirring the sterilized sodium alginate solution at a ratio of 1:2 (w / w) for 30 minutes.

[0117] [Table 1]

[0118] Sodium alginate 20g Trehalose 140g dextrin 60g Distilled water 780g

[0119] Example 1-3. Preparation of lactic acid bacteria encapsulated with calcium alginate beads

[0120] The calcium-containing solution was prepared by mixing the composition shown in Table 2 below, followed by sterilization.

[0121] Calcium alginate beads were prepared by adding the mixture of Example 1-2 to a calcium-containing solution in the form of drops using a syringe. Thereafter, the resultant was soaked for 1 hour, and as sodium ions were replaced by calcium ions, alginate formed a network structure, thereby achieving encapsulation.

[0122] [Table 2]

[0123] <![CDATA[CaCl2]]> 10g Distilled water 990g

[0124] Example 1-4. Freeze Drying

[0125] The lactic acid bacteria encapsulated with calcium alginate beads of Example 1-3 were rapidly frozen using a freeze dryer and then dried. Freeze drying was performed by raising the temperature from -20°C to 30°C and maintaining it for 50 hours.

[0126] Example 2. Preparation of lactic acid bacteria encapsulated in calcium alginate beads using a calcium-containing solution comprising calcium chloride and maltodextrin and freeze-drying thereof

[0127] Lactic acid bacteria were prepared in the same manner as in Example 1, except that a calcium-containing solution having a composition shown in Table 3 below was used in Examples 1-3.

[0128] [Table 3]

[0129] <![CDATA[CaCl2]]> 10g Maltodextrin 250g Distilled water 740g

[0130] Example 3. Preparation of lactic acid bacteria encapsulated with calcium alginate beads using a calcium-containing solution containing calcium chloride and trehalose and freeze-drying thereof

[0131] Lactic acid bacteria were prepared in the same manner as in Example 1, except that a calcium-containing solution having a composition shown in Table 4 below was used in Examples 1-3.

[0132] [Table 4]

[0133] <![CDATA[CaCl2]]> 10g Trehalose 250g Distilled water 740g

[0134] Example 4. Preparation of lactic acid bacteria encapsulated in calcium alginate beads using an alginate solution containing sucrose and freeze drying thereof

[0135] Lactic acid bacteria were prepared in the same manner as in Example 1, except that an alginate-containing solution having a composition shown in Table 5 below was used in Example 1-2, and a calcium-containing solution having a composition shown in Table 6 below was used in Example 1-3.

[0136] [Table 5]

[0137]

[0138]

[0139] [Table 6]

[0140] <![CDATA[CaCl2]]> 10g Trehalose 250g Distilled water 740g

[0141] Example 5. Preparation of lactic acid bacteria encapsulated in calcium alginate beads using an alginate solution containing sorbitol and freeze-drying thereof

[0142] Lactic acid bacteria were prepared in the same manner as in Example 1, except that an alginate-containing solution having a composition shown in Table 7 below was used in Example 1-2, and a calcium-containing solution having a composition shown in Table 6 above was used in Example 1-3.

[0143] [Table 7]

[0144] Sodium alginate 20g Trehalose 70g Sorbitol 70g Maltodextrin 60g Distilled water 780g

[0145] Example 6. Preparation of lactic acid bacteria encapsulated in calcium alginate beads using an alginate solution containing soy peptone and freeze-drying thereof

[0146] Lactic acid bacteria were prepared in the same manner as in Example 1, except that an alginate-containing solution having a composition shown in Table 8 below was used in Example 1-2, and a calcium-containing solution having a composition shown in Table 6 above was used in Example 1-3.

[0147] [Table 8]

[0148]

[0149]

[0150] Experimental Example 1. Measurement of the production yield of calcium alginate beads and the survival rate of lactic acid bacteria after freeze-drying according to the composition of calcium-containing solution

[0151] An attempt was made to measure the production yield of the calcium alginate beads of Examples 1 to 3 and the survival rate of lactic acid bacteria after freeze-drying.

[0152] The method for calculating the yield of calcium alginate bead production is as follows.

[0153] Specifically, the alginate solution mixed with the lactobacillus precipitation of embodiment 1 to 3 is diluted to a suitable concentration, and is applied on MRS agar medium to measure the number of bacteria (cfu / g). In addition, the pearl prepared is also diluted to a suitable concentration, and is applied on MRS agar medium with a suitable concentration to measure the number of bacteria (cfu / g). In order to calculate the production yield, the total bacteria count is calculated by multiplying the weight of the alginate solution containing the weight added and the pearl prepared by the bacteria count measured. The production yield (before drying) of calcium alginate beads is measured by comparing the total bacteria count measured.

[0154] In order to calculate the survival rate of the lactic acid bacteria of Examples 1 to 3 after freeze-drying, the total number of bacteria was calculated.

[0155] Specifically, the weight of the beads of Example 1-3 after freeze drying was measured, and the beads were diluted to a suitable concentration in MRS agar medium, and smeared to measure the number of bacteria (cfu / g). The total number of bacteria calculated was compared with the total number of bacteria in the alginate solution calculated above to measure the survival rate of lactic acid bacteria encapsulated with calcium alginate beads after freeze drying. The results are shown in Tables 9 to 11 below.

[0156] [Table 9]

[0157]

[0158]

[0159] [Table 10]

[0160]

[0161] [Table 10]

[0162]

[0163] Referring to Table 9 above, when the CaCl 2 solution consisting of only CaCl 2 of Example 1 was used, the production yield of beads was 58.21%, and the final survival rate after freeze-drying was very low at 7.38%.

[0164] Referring to Table 10 above, when the CaCl2 solution containing 25% maltodextrin of Example 2 was used, the production yield of beads was 89.29%, and the final survival rate after freeze-drying was 19.33%, confirming that the results were improved compared to Example 1. This shows that 25% maltodextrin (which has a concentration similar to that of the alginate-containing solution) greatly improved the production yield of beads, and the survival rate after freeze-drying was also improved accordingly.

[0165] Referring to Table 11 above, when the CaCl2 solution containing 25% trehalose of Example 3 was used, the production yield of beads was 97.07% and the final survival rate after freeze-drying was 45.73%, demonstrating that the results were further improved.

[0166] Experimental Example 2. Measurement of the production yield of calcium alginate beads and the survival rate of lactic acid bacteria after freeze-drying according to the composition of alginate-containing solution

[0167] In the same manner as in Experimental Example 1, the production yields of the calcium alginate beads of Examples 4 to 6 and the survival rates of lactic acid bacteria after freeze-drying were measured, and the results are shown in Tables 12 to 14 below.

[0168] [Table 12]

[0169]

[0170] [Table 13]

[0171]

[0172] [Table 14]

[0173]

[0174] Referring to Table 12 above, when the alginate-containing solution including sucrose of Example 4 was used, the production yield of beads was 94.3%, and the final survival rate after freeze-drying was 50.44%, which was similar to Example 3.

[0175] Referring to Table 13 above, when the alginate-containing solution including sorbitol of Example 5 was used, the production yield of beads was 91.49% and the final survival rate after freeze-drying was 68.36%, demonstrating that the results were improved compared to Example 4.

[0176] Referring to Table 14 above, when the alginate solution containing soy peptone of Example 6 was used, the production yield of the beads was 98.77%, and the final survival rate after freeze drying was 86.35%, confirming that the results were greatly improved. Therefore, the optimal composition capable of maximizing the production yield of the beads and the cryoprotection effect of the lactic acid bacteria was confirmed.

[0177] Experimental Example 3. Evaluation of high temperature stability based on the presence or absence of calcium alginate beads

[0178] An attempt was made to compare the high temperature stability of Example 6 and the comparative example. Specifically, freeze-dried and powdered samples of the comparative example and Example 6 were individually packaged and sealed in aluminum bag packages in fixed amounts. Each sample was stored in an incubator at 40°C for 12 weeks. Figure 2 The reduction of lactic acid bacteria during each storage period is shown.

[0179] The results are as follows Figure 2 As shown, the amount of lactic acid bacteria in the comparative example decreased by 1.674 log, while the amount of lactic acid bacteria in Example 6 decreased by 1.027 log, confirming that the high temperature stability of the lactic acid bacteria prepared by the method of the present disclosure is improved.

[0180] Experimental Example 4. Evaluation of Stability in the Gastrointestinal Tract Based on the Presence or Absence of Calcium Alginate Beads

[0181] An attempt was made to compare the gastrointestinal stability of Example 6 and the comparative example.

[0182] Specifically, the survival rates were compared by acid resistance and bile resistance tests according to a simulated gastroduodenal passage (SSDP).

[0183] For the acid resistance test, 5N hydrochloric acid (HCl) was added to MRS broth to prepare acidic MRS broth at a pH of 3. For the bile resistance test, 10% ox bile solution and artificial bile solution buffer were prepared.

[0184] In order to compare the survival rate of lactic acid bacteria, the freeze-dried powder prepared in the comparative example and the freeze-dried powder prepared in Example 6 were added to acidic MRS and reacted at 37°C for 1 hour. Thereafter, 10% ox bile solution and the buffer were added thereto to adjust the pH to 7, reacted for 2 hours, and then sampled to confirm the survival rate of lactic acid bacteria. The results are shown in Table 15 below.

[0185] [Table 15]

[0186]

[0187] Referring to Table 15 above, the lactic acid bacteria of the comparative example showed a survival rate of 0.37%, while the lactic acid bacteria encapsulated with calcium alginate beads of Example 6 showed a survival rate of 34.94%, demonstrating that the acid resistance and bile resistance thereof were greatly improved.

[0188] Based on the foregoing, those skilled in the art to which the present disclosure belongs will be able to understand that the present disclosure can be implemented in other specific forms without modifying the technical concepts or essential features of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments, but also various substitutions, modifications, equivalent substitutions and other embodiments that may be included in the spirit and scope of the present disclosure defined by the appended claims.

Claims

1. A method for producing lactic acid bacteria encapsulated with calcium alginate beads, comprising: (a) culturing lactic acid bacteria and recovering bacterial cells; (b) mixing the lactic acid bacteria of step (a) with a solution containing alginate; and (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads, The alginate-containing solution comprises sucrose, sorbitol or soy peptone, and the calcium-containing solution comprises trehalose or maltodextrin.

2. The method according to claim 1, further comprising (d) freeze-drying the lactic acid bacteria encapsulated with calcium alginate beads of step (c). 3 . The method according to claim 1 , wherein the lactic acid bacteria comprises at least one selected from the group consisting of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Enterococcus, Pediococcus, Leuconostoc and Weissella.

4. The method according to claim 1, wherein the lactic acid bacteria comprises at least one selected from the group consisting of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Streptococcus faecalis, and Lactococcus lactis subsp. lactis. 5 . The method according to claim 1 , wherein the lactic acid bacteria comprises at least one selected from the group consisting of Lactobacillus plantarum CJLP133, Lactobacillus plantarum CJLP243, Lactobacillus plantarum CJLP136, Lactobacillus plantarum CJLP55, and Lactobacillus plantarum CJLP56.

6. Lactic acid bacteria encapsulated with calcium alginate beads produced by the method according to any one of claims 1 to 5.

7. The lactic acid bacteria according to claim 6, wherein the lactic acid bacteria has an increased survival rate after freeze-drying compared to lactic acid bacteria not encapsulated with calcium alginate beads.

8. The lactic acid bacteria according to claim 6, wherein the lactic acid bacteria has improved stability compared to lactic acid bacteria not encapsulated with calcium alginate beads.

9. A method for improving the production yield of calcium alginate beads for encapsulating lactic acid bacteria, comprising: (a) culturing lactic acid bacteria and recovering bacterial cells; (b) mixing the lactic acid bacteria of step (a) with a solution containing alginate; and (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads, The alginate-containing solution comprises sucrose, sorbitol or soy peptone, and the calcium-containing solution comprises trehalose or maltodextrin.

10. A method for improving the survival rate of freeze-dried lactic acid bacteria, comprising: (a) culturing lactic acid bacteria and recovering bacterial cells; (b) mixing the lactic acid bacteria of step (a) with a solution containing alginate; (c) adding the mixture of step (b) to a calcium-containing solution to form calcium alginate beads; and (d) freeze-drying the lactic acid bacteria encapsulated by calcium alginate beads in step (c), The alginate-containing solution comprises sucrose, sorbitol or soy peptone, and the calcium-containing solution comprises trehalose or maltodextrin.

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