Method for producing lactic acid bacteria collected with calcium-alginate beads

Abstract

The present application relates to a method for producing lactic acid bacteria captured by calcium-alginate beads, lactic acid bacteria captured by calcium-alginate beads produced by said method, a method for improving the production yield of calcium-alginate beads for capturing lactic acid bacteria, and a method for improving the survival rate of freeze-dried lactic acid bacteria.

Description

[Technical field]

[0001] The present application relates to a method for producing lactic acid bacteria captured by calcium-alginate beads, lactic acid bacteria captured by calcium-alginate beads produced by said method, a method for improving the production yield of calcium-alginate beads for capturing lactic acid bacteria, and a method for improving the survival rate of freeze-dried lactic acid bacteria. [Background technology]

[0002] Lactic acid bacteria are important bacteria that live in the intestines of mammals, prevent abnormal fermentation caused by various bacteria, and are also used as intestinal regulators. For example, Lactobacillus bulgaricus is the oldest known lactic acid bacterium, and is used to make yogurt and as a starter for cheese and fermented butter production. Aerobic lactic acid bacteria (L. acidophilus) are found in the intestines of humans and all mammals and other animals, and are used to make butter or milk and treat intestinal autointoxication. Lactococcus lactis produces DL-lactic acid, which is always present in milk and used to make butter or cheese, making it the most important dairy lactic acid bacterium.

[0003] As mentioned above, useful lactic acid bacteria settle in the intestine and exert various physiologically active effects such as activating intestinal motility, inhibiting harmful bacteria, promoting immune-enhancing substances including vitamins, and alleviating atopic skin. However, in order to exert the physiological effects, a much larger amount of lactic acid bacteria must be ingested than that ingested from conventional foods such as yogurt. Therefore, the method of isolating only lactic acid bacteria and simply consuming them in the form of powder or capsules has become widespread. However, when lactic acid bacteria are made into powder or capsule form, many lactic acid bacteria die during the long-term distribution process, and there is a limit at which the original physiologically active functions of lactic acid bacteria cannot be exerted.

[0004] Generally, lactic acid bacteria are prepared in powder form by freeze-drying or spray-drying, among which freeze-drying is a method of suspending microorganisms in a freeze-drying suspension, freezing them, and then drying them under reduced pressure. However, since lactic acid bacteria are obligate anaerobes and very sensitive to the surrounding environment, the survival rate of powdered lactic acid bacteria decreases at low and normal temperatures, and the number of live bacteria decreases. Recently, in order to overcome this drawback, methods have been developed in which lactic acid bacteria are coated with various coating agents, such as starch, gelatin, alginic acid, cellulose, hardened oil, and various emulsifiers, which are used as cryoprotectants to maintain the quality during the distribution period.

[0005] Since live lactic acid bacteria die due to an increase in temperature, freeze-drying is the most effective method for maintaining the activity of cells during storage of lactic acid bacteria. Although freeze-drying can greatly improve the storage and distribution of lactic acid bacteria, the freeze-drying process generally significantly reduces the viability of lactic acid bacteria.

[0006] For example, a study has been conducted to confirm the effects of storage temperature and polymers on the stability of lactic acid bacteria by freeze-drying gelatin-coated Lactobacillus rhamnosus (Claude PC et al., Food. Res. Int., 1996, 29, 555-562). However, the study disclosed that after 6 months of storage at 20°C, about 1% of lactic acid bacteria survived, and after 12 months, only about 0.2% of lactic acid bacteria survived. This means that there is a need to develop a method to further increase the survival rate and storage stability of lactic acid bacteria.

[0007] Meanwhile, a method of coating lactic acid bacteria using alginate capsules is known, but alginate is unstable to acid and heat. In the conventional method of producing capsules using alginate, a mixture of alginate and microorganisms is mixed with CaCl 2Since the alginate is produced by spraying it into a coagulating liquid such as a solution, hardening begins when the alginate reacts with the calcium ions in the coagulating liquid from the particle surfaces of the sprayed mixture of alginate and microorganisms. However, sufficient calcium ions cannot migrate to the inside of the particles in a short period of time, making it difficult to mass-produce alginate capsules with a high yield, in which the alginate inside is hard and finely hardened. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Claude PCet al,Food.Res.Int., 1996,29,555~562 Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have developed a process for producing lactic acid bacteria entrapped in calcium-alginate beads, which increases the production yield of the beads and improves their survival rate and stability after freeze-drying, and have completed this application. [Means for solving the problem]

[0010] One object of the present application is to provide a method for producing lactic acid bacteria entrapped in calcium-alginate beads.

[0011] Another object of the present application is to provide calcium-alginate bead-entrapped lactic acid bacteria produced by said method.

[0012] Another object of the present application is to provide a method for improving the production yield of calcium-alginate beads for capturing lactic acid bacteria.

[0013] Another object of the present application is to provide a method for improving the viability of freeze-dried lactic acid bacteria. Effect of the Invention

[0014] The method for producing lactic acid bacteria entrapped in calcium-alginate beads according to the present invention can provide lactic acid bacteria with increased bead production yield and improved survival rate and stability after freeze-drying. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing a method for producing lactic acid bacteria captured with calcium-alginate beads according to the present invention. [Diagram 2] FIG. 1 shows the results of evaluating gastrointestinal stability in the presence or absence of calcium-alginate beads. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific description described below is not considered to limit the category of this application.

[0017] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed by this application.

[0018] One aspect of the present application provides a method for producing lactic acid bacteria captured 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 alginic acid; and (c) adding the mixture of step (b) to a calcium-containing solution to form alginic acid-calcium beads, wherein the alginic acid-containing solution contains sucrose, sorbitol or soy peptone, and the calcium-containing solution contains trehalose or maltodextrin.

[0019] As one specific example, the method may further include a step (d) of freeze-drying the lactic acid bacteria captured by the calcium-alginate beads in step (c), but is not limited thereto.

[0020] In the present application, the term "freeze drying" refers to a method of drying a material to be dried by rapidly lowering the temperature of a container, freezing the material, and then reducing the pressure inside the container to a vacuum, thereby immediately sublimating the solidified solvent contained in the material into water vapor. Freeze drying is a method that minimizes damage to heat-sensitive materials and allows for effective long-term storage, and is useful in terms of contamination prevention, storage, transportation, and economy. However, there are problems in that the activity and survival rate of lactic acid bacteria rapidly decreases during the freeze-drying process of lactic acid bacteria, and ice particles are formed during freezing, damaging the membrane structure of lactic acid bacteria cells. To improve this, a substance that is added during freeze-drying so that lactic acid bacteria can recover their functions during rehydration without being damaged or killed is called a cryoprotectant, which serves to impart physicochemical stability to lactic acid bacteria and increase their survival rate.

[0021] In the present application, the freezing temperature for lyophilization may be a subzero temperature, for example, −10° C. to −196° C. (the boiling point of liquid nitrogen), −40° C. to −196° C., −50° C. to −196° C., or −70° C. to −196° C., at such low temperatures all biological activities of the lactic acid bacteria, including biochemical reactions that lead to cell apoptosis, are effectively stopped.

[0022] In the present application, the term "cryoprotection" means protecting the tissue of lactic acid bacteria from freezing when they are freeze-dried and stored to preserve the activity of the lactic acid bacteria.

[0023] For the purposes of this application, the lactic acid bacteria captured in calcium-alginate beads produced by the method for producing lactic acid bacteria captured in calcium-alginate beads may have improved viability and stability after freeze-drying.

[0024] Specifically, the method for producing lactic acid bacteria captured with calcium-alginate beads according to the present invention will be described in detail below.

[0025] First, (a) lactic acid bacteria are cultured and the bacterial cells are collected.

[0026] In the present application, the term "lactic acid bacteria" is a general term for bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid, and is 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.

[0027] 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.

[0028] 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.

[0029] The strain has been deposited at the Institute for Bioscience and Biotechnology, and is readily available to those skilled in the art from the Institute for Bioscience and Biotechnology.

[0030] In the present application, "culturing" means growing the lactic acid bacteria under appropriately controlled environmental conditions. The culturing process of the present application can be carried out according to suitable media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous or fed-batch culture.

[0031] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients required for culturing the microorganism as the main components, and supplies nutrients such as water essential for survival and growth, and growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular restrictions, and the microorganism of the present application can be cultured under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while controlling the temperature, pH, etc.

[0032] In the present application, the carbon source may include carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, etc.; alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid, etc.; organic acids, amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0033] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0034] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may further include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0035] In the present application, during the cultivation of lactic acid bacteria, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. Also, during the cultivation, foam generation may be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Also, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain anaerobic and microaerobic states, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected, but is not limited thereto.

[0036] The temperature of the medium may be, but is not limited to, 20° C. to 50° C., specifically, 30° C. to 37° C. The culture period may continue until a desired amount of useful substance is produced, specifically, 10 hours to 100 hours, but is not limited to this.

[0037] In the step of recovering the lactic acid bacteria cells, the target cells can be recovered from the medium using an appropriate method known in the art such as the lactic acid bacteria culture method of the present application, for example, a batch, continuous or fed-batch culture method, etc. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations of these methods can be used, but are not limited to these examples.

[0038] The recovery step may include an additional purification step, which may purify the recovered lactic acid bacteria cells using any suitable method known in the art.

[0039] Next, (b) the lactic acid bacteria from step (a) is collected by a method such as centrifugation, and then mixed with a solution containing alginic acid.

[0040] In this application, the term "alginic acid" refers to a polymer of two types of uronic acid, a substance with a degree of polymerization of 80 and a molecular weight of approximately 1,500, and having an unbranched structure in which several hundred units of D-mannuronic acid (hereinafter also referred to as M) and L-guluronic acid are linked by β-1,4 bonds.

[0041] In one embodiment of the present application, sugars and proteins were added to a solution containing alginic acid in order to increase the cryoprotective effect and the survival rate of lactobacillus captured in calcium-alginic acid beads after freeze-drying. Skim milk is a representative and widely used cryoprotectant for proteins, but it was confirmed that the calcium component of skim milk reacts with alginic acid first and hardens when it is added. Therefore, it was confirmed that beads were not sufficiently produced and was removed from the experimental group.

[0042] Specifically, the alginic acid-containing solution may contain sucrose, sorbitol or soy peptone, more specifically, soy peptone, but is not limited thereto.

[0043] In this application, the term "soy peptone" refers to a protein derived from soybeans, which is primarily used as a nutrient source in microbial culture media; the protein content of isolated soy protein is about 90%, with globulin being the most abundant component.

[0044] As one specific example, the solution containing alginic acid may contain sucrose, sorbitol or soy peptone, more specifically soy peptone at 5% by weight to 10% by weight, more specifically 6% by weight to 8% by weight, and even more specifically about 7% by weight, but is not limited thereto.

[0045] When the sucrose, sorbitol or soy peptone is contained in the above weight percent, the production yield of the beads is increased, and the beads can act as a cryoprotectant to prevent damage or apoptosis of the lactic acid bacteria caused by freeze-drying and to enhance safety. When the sucrose, sorbitol or soy peptone is contained in a weight percent higher than the optimal range, it may interfere with the formation of alginic acid beads, resulting in a problem that the beads are not formed densely and the protective effect by gastric acid is reduced.

[0046] As one specific example, the solution containing alginic acid is one in which sodium alginate is solubilized in water at 1% by weight to 3% by weight, more specifically, approximately 2% by weight, and may further contain trehalose or maltodextrin, but is not limited thereto.

[0047] If the content of sodium alginate is lower than the optimum range by weight, the bead hardening may be weak and the lactic acid bacteria and cryoprotective components may be eluted, whereas if the content is higher than the optimum range by weight, the bead hardening may be too strong, resulting in poor production of lactic acid bacteria or the bacteria may die when ingested thereafter.

[0048] In this application, the term "trehalose" refers to a carbohydrate that is widely present in nature, such as plants and microorganisms. For the purposes of this application, trehalose contained in the alginic acid-containing solution acts as a cryoprotectant that prevents the lactic acid bacteria from being damaged or killed by freeze-drying and helps them recover their functions upon rehydration, thereby enhancing the cryoprotective effect of the lactic acid bacteria.

[0049] In this application, the term "maltodextrin" refers to a white powder of a porous particle base, and is a food additive that is often used in general foods such as yogurt, sauce, and salad dressing. For the purpose of this application, the maltodextrin contained in the alginic acid-containing solution is used as a porous support during freeze-drying of lactic acid bacteria, and can act as a cryoprotectant to prevent the lactic acid bacteria from being damaged or killed by freeze-drying. The porous support is a base having porous particle properties, and serves to block the inflow of moisture and air from the outside.

[0050] As a specific example, the trehalose may be contained in an amount of 5% by weight to 10% by weight, more specifically 6% by weight to 8% by weight, and even more specifically about 7% by weight, and the maltodextrin may be contained in an amount of 5% by weight to 10% by weight, more specifically 6% by weight to 8% by weight, and even more specifically about 6% by weight, but is not limited thereto.

[0051] As one specific example, the ratio by weight of the solution containing alginic acid to the weight of the lactic acid bacteria in the mixture in step (b) may be 1:1 to 10:1, more specifically 1:1 to 3:1, and even more specifically about 2:1, but is not limited thereto.

[0052] By containing the lactic acid bacteria solid powder in the optimal ratio, the cryoprotection effect and the yield of the lactic acid bacteria dry powder can be maintained during the process, and the manufacturing cost can be kept at an appropriate level. If the ratio of the weight of the alginic acid-containing solution to the weight of the lactic acid bacteria is outside the optimal ratio, there is a problem that the lactic acid bacteria production yield per unit time and / or kg of production is reduced, resulting in a decrease in process efficiency.

[0053] Furthermore, by containing the total solid content of the alginic acid-containing solution at 20% by weight to 30% by weight, the fluidity (viscosity) can be maintained.

[0054] As a specific example, prebiotics may be optionally added during mixing in step (b). Specifically, prebiotics may be added and mixed when the lactic acid bacteria and the coating agent are mixed. The prebiotics serve as food for the lactic acid bacteria. The prebiotics may be any one or more selected from, but are not limited to, fructooligosaccharides, galactooligosaccharides, maltitol, lactinol, and inulin, and may be fructooligosaccharides or inulin. The prebiotics may be 0.1% to 5% by weight relative to the weight of the lactic acid bacteria.

[0055] Then, (c) adding the mixture from step (b) to a calcium-containing solution to form alginate-calcium beads.

[0056] Specifically, step (c) is an extrusion step, and when the mixture of step (b) is dropped or sprayed into a calcium-containing solution to react with the calcium-containing solution, the lactic acid bacteria are captured inside the beads in the calcium-containing solution by forming a matrix through alginate-calcium cross-linking, forming alginate-calcium beads, but is not limited thereto.

[0057] The production of bead particles through the alginate-calcium reaction is carried out under room temperature conditions (e.g. 25°C), which causes little physical stress and has little adverse effect on the survival rate of live bacteria. In addition, the formation of alginate-calcium bead particles using the extrusion method has pH-dependent release properties, and is not decomposed in acidic conditions such as gastric acid, but is gradually decomposed in the neutral environment of the intestine, which is very useful for improving the survival rate of lactic acid bacteria in the intestine.

[0058] The cryoprotectant component may be sugars, proteins, lipids, etc. When proteins are added to a calcium-containing solution, the cationic nature of amino acids prevents alginic acid from binding with calcium ions, preventing the formation of dense alginic acid beads. Lipids do not mix well with water and are not suitable for use in calcium-containing solutions. Therefore, in one embodiment of the present application, it was confirmed that the production yield and survival rate of alginic acid beads can be improved by adding sugars to a calcium-containing solution.

[0059] Specifically, the calcium-containing solution may contain trehalose or maltodextrin, more specifically, but is not limited to, trehalose.

[0060] When the trehalose or maltodextrin is included, the production yield of the beads is increased, and the trehalose or maltodextrin can act as a cryoprotectant to prevent damage or apoptosis of the lactic acid bacteria caused by freeze-drying, thereby enhancing safety.

[0061] As a specific example, in order to set the calcium-containing solution to a concentration similar to the solid content of the solution containing alginic acid mixed with lactic acid bacteria pellets, the calcium-containing solution may contain trehalose or maltodextrin, more specifically trehalose, at 15% by weight to 35% by weight, more specifically 20% by weight to 30% by weight, and even more specifically about 25% by weight, but is not limited thereto.

[0062] By setting the concentrations of the mixture in step (b) and the calcium-containing solution to be similar, it is possible to minimize the dissolution of the cryoprotectant components in the calcium-containing solution containing lactic acid bacteria and alginic acid after the droplets are administered.

[0063] As a specific example, the calcium-containing solution may be one in which calcium chloride is dissolved in water at 0.5% by weight to 1.5% by weight, more specifically, about 1% by weight, but is not limited thereto.

[0064] If the calcium chloride is contained in a weight percent lower than the optimum range, the bead structure may not be dense, resulting in insufficient cryoprotection effect, whereas if the calcium chloride is contained in a weight percent higher than the optimum range, the bead hardening may be excessively strong, resulting in insufficient elution of lactic acid bacteria in the final product, making commercialization difficult.

[0065] Specifically, after the step (c), the method may further include a step of storing the alginate-calcium bead-containing solution at a temperature of 4° C. to 20° C. for 30 to 60 minutes.

[0066] This is an aging process, in which sodium ions are replaced with calcium ions, and alginic acid forms a network structure, thus encapsulation proceeds. Through this aging process, the density of the particle structure can be increased.

[0067] The method of the present application may further include the step of adding one or more selected from the group consisting of a cryoprotectant, a porous support, and a nitrogen source, specifically one, two, or three.

[0068] Specifically, the method of the present application may further include a step of adding a component that is conventionally known as a cryoprotectant, in addition to the alginic acid-containing solution and the calcium-containing solution. That is, the "cryoprotectant" further included in the present application for preparing lactobacillus trapped in calcium-alginic acid beads means a substance having cryoprotective effect that is commonly used in the art. The cryoprotectant may be purchased from a commercial source and is not particularly limited in type. Specifically, the cryoprotectant may be, but is not limited to, trehalose, sugars, amino acids, peptides, gelatin, glycerol, sugar alcohol, whey, alginic acid, ascorbic acid, yeast extract, skimmed milk, etc.

[0069] In the present application, the "porous support" serves to block the inflow of moisture and air from the outside and to provide porosity to the freeze-dried lactic acid bacteria to facilitate rehydration. The porous support is a commercially available porous support that is commonly used in the art for freeze-drying, and is not particularly limited in type. Specifically, the porous support may be, but is not particularly limited to, maltodextrin, alginate, chitosan, starch, polyethylene glycol, propylene glycol, triacetin, acetyl triethyl citrate, triethyl citrate, glycerin, or a combination thereof.

[0070] In this application, the term "nitrogen source (N-source)" refers to a substance used as a nitrogen energy source for lactic acid bacteria, which plays a role in preventing damage to the bacterial cells due to post-fermentation. When lactic acid bacteria are mixed with a cryoprotective composition, the lactic acid bacteria living in the absence of an energy source will produce organic acids, which will cause a decrease in pH and induce apoptosis of the lactic acid bacteria. Therefore, the nitrogen energy source can prevent the production of organic acids and the resulting decrease in pH, thereby preventing apoptosis of the lactic acid bacteria.

[0071] The nitrogen source is a commercially available nitrogen source that is commonly used in the art for freeze-drying, and is not particularly limited in type.Specifically, the nitrogen source is not particularly limited, but may be skim milk powder, whey protein, yeast extract, malt extract, beef extract, casein hydrolysate, malt extract, tryptone, cysteine, or peptone, and specifically, the peptone may include soy peptone, fish peptone, proteose peptone, casein peptone, or peptone No. 3.

[0072] Then, optionally, the method may further include a step (d) of freeze-drying the lactic acid bacteria captured in the calcium-alginate beads in step (c). The freeze-drying step may be carried out by transferring the alginate-calcium beads containing the lactic acid bacteria produced after step (c) into a freeze-drying tray and raising the temperature from -20°C to 30°C for 50 hours.

[0073] Another aspect of the present application provides lactic acid bacteria entrapped in calcium-alginate beads, produced by the above method.

[0074] The method and the lactic acid bacteria captured with calcium-alginate beads were as described above.

[0075] Specifically, the lactic acid bacteria may have an improved survival rate after freeze-drying compared to lactic acid bacteria not captured by calcium-alginate beads, but is not limited thereto.

[0076] Specifically, the lactic acid bacteria may have improved stability compared to lactic acid bacteria not captured by calcium-alginate beads, but is not limited thereto.

[0077] For purposes of this application, said stability may mean, but is not limited to, storage (distribution) stability or gastrointestinal stability.

[0078] Specifically, it may mean high temperature stability, acid resistance, bile resistance, or the like.

[0079] Another aspect of the present application provides a method for improving the production yield of calcium-alginate beads for capturing 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 alginic acid; and (c) adding the mixture of step (b) to a calcium-containing solution to form alginic acid-calcium beads, wherein the alginic acid-containing solution contains sucrose, sorbitol or soy peptone, and the calcium-containing solution contains trehalose or maltodextrin.

[0080] The method and the lactic acid bacteria captured with calcium-alginate beads were as described above.

[0081] Another aspect of the present application provides a method for improving the viability of freeze-dried lactic acid bacteria, comprising the steps of: (a) culturing lactic acid bacteria and recovering bacterial cells; (b) mixing the lactic acid bacteria of step (a) with a solution containing alginic acid; (c) adding the mixture of step (b) to a calcium-containing solution to form alginic acid-calcium beads; and (d) freeze-drying the lactic acid bacteria captured by the calcium-alginate beads of step (c), wherein the alginic acid-containing solution contains sucrose, sorbitol or soy peptone, and the calcium-containing solution contains trehalose or maltodextrin.

[0082] The method and the lactic acid bacteria captured with calcium-alginate beads were as described above.

[0083] Another aspect of the present application provides freeze-dried, calcium-alginate bead-entrapped lactic acid bacteria produced by the above method.

[0084] The method and the lactic acid bacteria captured with calcium-alginate beads were as described above.

[0085] Specifically, the lactic acid bacteria may have an improved survival rate after freeze-drying compared to lactic acid bacteria not captured by calcium-alginate beads, but is not limited thereto.

[0086] Specifically, the lactic acid bacteria may have improved stability compared to lactic acid bacteria not captured by calcium-alginate beads, but is not limited thereto. EXAMPLES

[0087] The present application will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present application.

[0088] Comparative Example 1. Production of freeze-dried powder containing lactic acid bacteria Comparative Example 1-1. Cultivation of lactic acid bacteria Lactobacillus plantarum CJLP133 strain was cultured in MRS liquid medium (Difco, USA) at 37°C for 10 hours. The lactic acid bacteria in the culture were approximately 8.68x10 9 The number of CFU / mL bacteria was confirmed. The components of the MRS medium were as follows: 10g of proteose peptone, 10g of beef extract, 5g of yeast extract, 20g of dextrose, 1g of polysorbate 80, 2g of ammonium citrate, 5g of sodium acetate, 0.1g of magnesium sulfate, 0.05g of manganese sulfate, and 2g of dipotassium phosphate. The mixture was sterilized by adding 1L of water and hydrating.

[0089] Comparative Example 1-2. Preparation of a lactic acid bacteria solution containing a cryoprotectant The culture medium in which the lactic acid bacteria of Comparative Example 1-1 was cultured was centrifuged for 10 minutes, and the supernatant was discarded to obtain a pellet. The obtained bacteria was stabilized by stirring with a sterilized cryoprotectant in a ratio of 1:4 (w / w) for 30 minutes. The cryoprotectant solution was prepared by mixing 140g of matodextrin, 140g of trehalose, 30g of skim milk, and 690g of distilled water and sterilizing the mixture.

[0090] Comparative Example 1-3. Freeze-drying of lactic acid bacteria containing cryoprotectants The cryoprotectant containing the lactic acid bacteria of Comparative Example 1-2 was rapidly frozen and then dried using a freeze dryer. The freeze drying was carried out by increasing the temperature from -20°C to 30°C for 50 hours.

[0091] Example 1. Preparation of lactic acid bacteria captured by calcium-alginate beads using a calcium-containing solution containing calcium chloride and freeze-drying Example 1-1. Cultivation of lactic acid bacteria Lactobacillus plantarum CJLP133 strain was cultured in MRS liquid medium (Difco, USA) at 37°C for 10 hours. The lactic acid bacteria in the culture were approximately 8.68x10 9 The number of CFU / mL bacteria was confirmed. The components of the MRS medium were as follows: 10 g of proteose 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 dipotassium phosphate. The mixture was sterilized by adding 1 L of water and dissolving in water.

[0092] Example 1-2. Preparation of sodium alginate solution containing lactic acid bacteria The culture medium in which the lactic acid bacteria of Example 1-1 was cultured was centrifuged for 10 minutes, and the supernatant was discarded to obtain pellets. The alginate solution was prepared by mixing the components shown in Table 1 below and sterilizing them. The obtained bacteria was stabilized by stirring with sterilized sodium alginate solution in a ratio of 1:2 (w / w) for 30 minutes.

[0093] [Table 1]

[0094] Example 1-3. Production of lactic acid bacteria captured with calcium-alginate beads The calcium-containing solution was prepared by mixing the components in the composition shown in Table 2 below and sterilizing them.

[0095] The mixture of Example 1-2 was dropped into a calcium-containing solution using a syringe to prepare calcium-alginate beads, which were then immersed for 1 hour to allow the sodium ions to be replaced with calcium ions, resulting in the formation of a network structure of alginate and encapsulation.

[0096] [Table 2] Example 1-4. Freeze-drying The lactic acid bacteria captured by the calcium alginate beads in Example 1-3 were rapidly frozen and then dried using a freeze-dryer. The freeze-drying was carried out for 50 hours while increasing the temperature from -20°C to 30°C.

[0097] Example 2. Preparation and freeze-drying of lactic acid bacteria captured in calcium-alginate beads using a calcium-containing solution containing calcium chloride and maltodextrin The preparation was carried out in the same manner as in Example 1, except that in Examples 1-3, a calcium-containing solution having the composition shown in Table 3 below was used.

[0098] [Table 3]

[0099] Example 3. Preparation and freeze-drying of lactic acid bacteria captured on calcium-alginate beads using a calcium-containing solution containing calcium chloride and trehalose The preparation was carried out in the same manner as in Example 1, except that in Examples 1-3, a calcium-containing solution having the composition shown in Table 4 below was used.

[0100] [Table 4] Example 4. Production of lactic acid bacteria captured in calcium-alginate beads using a solution containing sucrose and alginic acid and freeze-drying In Example 1-2, a solution containing alginic acid as shown in Table 5 below was used. The same method as in Example 1 was used to prepare Examples 1-3, except that a calcium-containing solution having the composition shown in Table 6 below was used.

[0101] [Table 5]

[0102] [Table 6] Example 5. Preparation of lactic acid bacteria captured on calcium-alginate beads using a solution containing sorbitol and alginic acid and freeze-drying In Example 1-2, a solution containing alginic acid as shown in Table 7 below was used. The preparation was carried out in the same manner as in Example 1, except that the calcium-containing solution having the composition shown in Table 6 was used in Examples 1-3.

[0103] [Table 7]

[0104] Example 6. Production of lactic acid bacteria captured on calcium-alginate beads using a solution containing soy peptone and alginic acid and freeze-drying In Example 1-2, a solution containing alginic acid as shown in Table 8 below was used. The preparation was carried out in the same manner as in Example 1, except that the calcium-containing solution having the composition shown in Table 6 was used in Examples 1-3.

[0105] [Table 8] Experimental Example 1. Measurement of the production yield of calcium-alginate beads and the survival rate of lactic acid bacteria after freeze-drying depending on the composition of the calcium-containing solution The production yield of the calcium-alginate beads of Examples 1 to 3 and the survival rate of lactic acid bacteria after freeze-drying were measured.

[0106] The method for determining the production yield of calcium-alginate beads is as follows.

[0107] Specifically, the solution containing alginic acid mixed with the lactic acid bacteria pellets of Examples 1 to 3 was diluted to an appropriate concentration, smeared on MRS agar medium, and the number of bacteria (cfu / g) was measured. The produced beads were also diluted to an appropriate concentration, smeared on MRS agar medium at an appropriate concentration, and the number of bacteria (cfu / g) was measured. To calculate the production yield, the measured number of bacteria was multiplied by the weight of the solution containing alginic acid added and the weight of the produced beads, respectively, to calculate the total number of bacteria. The measured total number of bacteria was compared to measure the production yield (before drying) of calcium-alginate beads.

[0108] 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.

[0109] Specifically, the weight of the beads after freeze-drying in Examples 1 to 3 was measured, and the beads were diluted to an appropriate concentration and smeared on MRS agar medium to measure the number of bacteria (cfu / g). The calculated total number of bacteria was compared with the total number of bacteria in the solution containing alginic acid calculated above to measure the survival rate after freeze-drying of the lactic acid bacteria captured by the calcium-alginate beads (after drying), and the results are shown in Tables 9 to 11 below, respectively.

[0110] [Table 9]

[0111] [Table 10]

[0112] [Table 11]

[0113] Referring to Table 9 above, the CaCl 2 CaCl 2 When the solution was used, the bead production yield was 58.21%, and the final viability after freeze-drying was 7.38%, both of which were very low.

[0114] Referring to Table 10 above, the maltodextrin of Example 2 was added at 25% CaCl 2 When the solution was used, the bead production yield was 89.29%, and the final survival rate after freeze-drying was 19.33%, which was improved from Example 1. This suggests that the bead production yield was significantly improved by using 25% maltodextrin, which has a similar concentration to the alginic acid-containing solution, and therefore the survival rate after freeze-drying was also improved.

[0115] Referring to Table 11 above, the CaCl 2When the solution was used, the bead production yield was 97.07%, and the final viability after freeze-drying was 45.73%, demonstrating further improvement in the results.

[0116] Experimental Example 2. Measurement of the production yield of calcium-alginate beads and the survival rate of lactic acid bacteria after freeze-drying depending on the composition of the solution containing alginic acid In the same manner as in Experimental Example 1, the production yields and survival rates of lactic acid bacteria after freeze-drying of the calcium-alginate beads in Examples 4 to 6 were measured and are shown in Tables 12 to 14 below.

[0117] [Table 12]

[0118] [Table 13]

[0119] [Table 14]

[0120] Referring to Table 12 above, when the solution containing alginic acid containing sucrose in Example 4 was used, the bead production yield was 94.3% and the final viability after freeze-drying was 50.44%, showing results at a similar level to Example 3.

[0121] Referring to Table 13, when the solution containing alginic acid containing sorbitol in Example 5 was used, the bead production yield was 91.49%, and the final viability after freeze-drying was 68.36%, which was improved from Example 4.

[0122] Referring to Table 14, it was confirmed that the bead production yield was 98.77% and the final survival rate after freeze-drying was 86.35%, which was a significant improvement, when the solution containing alginic acid and soy peptone of Example 6 was used. This confirmed the optimal composition that maximizes the bead production yield and the freeze-drying protection effect of lactic acid bacteria.

[0123] Experimental Example 3. Evaluation of high temperature stability with and without calcium-alginate beads The high temperature stability of Example 6 and Comparative Example was compared. Specifically, a certain amount of the freeze-dried powdered samples of Comparative Example and Example 6 was put into an aluminum pouch, which was individually wrapped and sealed. The samples were stored in an incubator at 40°C for 12 weeks. The amount of lactic acid bacteria reduced by each storage period is shown in Figure 2.

[0124] As a result, as shown in FIG. 2, the amount of lactic acid bacteria reduced was 1.674 logs in the comparative example, while it was 1.027 logs in the example 6, confirming that the high temperature stability of the lactic acid bacteria produced by the method of the present application was improved.

[0125] Experimental Example 4. Evaluation of gastrointestinal stability with and without calcium-alginate beads The stability in the gastrointestinal tract of Example 6 and the Comparative Example was compared.

[0126] Specifically, we compared survival rates through acid tolerance and bile tolerance experiments using simulated stomach duodenum passage (SSDP).

[0127] For the acid tolerance experiment, 5N hydrochloric acid (HCl) was added to MRS broth to prepare acidic MRS broth with a pH of 3. For the bile tolerance experiment, 10% Oxgall solution and artificial bile buffer were prepared.

[0128] 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 an Acidic MRS and reacted at 37°C for 1 hour. Then, 10% Oxgall solution and a buffer were added to adjust the pH to 7, and the mixture was reacted for 2 hours, after which a sample was taken to check the survival rate of the lactic acid bacteria. The results are shown in Table 15 below.

[0129] [Table 15]

[0130] 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 captured with calcium-alginate beads of Example 6 showed a survival rate of 34.94%, confirming that acid resistance and bile resistance were greatly improved.

[0131] From the above description, a person skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. (a) The stage of culturing lactic acid bacteria and collecting the bacterial cells; (b) The step of mixing the lactic acid bacteria from step (a) with a solution containing alginic acid; and (c) The step of adding the mixture from step (b) to a calcium-containing solution to form alginate-calcium beads, The aforementioned solution containing alginic acid contains 5% to 10% by weight of sucrose, trehalose, and maltodextrin. The calcium-containing solution contains trehalose, and the method for producing lactic acid bacteria collected with calcium-alginate beads.

2. (d) The method according to claim 1, further comprising the step of freeze-drying the lactic acid bacteria collected with the calcium-alginate beads in step (c).

3. The method according to claim 1, wherein the lactic acid bacteria 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.

4. The aforementioned lactic acid bacteria include Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium breve. The method according to claim 1, comprising at least one selected from the group consisting of *Lactococcus breve*, *Streptococcus faecalis*, and *Lactococcus lactis subsp. lactis*.

5. The lactic acid bacteria mentioned above are Lactobacillus plantarum CJLP133, Lactobacillus plantarum CJLP243, Lactobacillus plantarum CJLP136, Lactobacillus plantarum CJLP55, and Lactobacillus plantarum CJLP56 The method according to claim 1, comprising at least one selected from the group consisting of (Lactobacillus plantarum CJLP56).

6. Lactic acid bacteria collected by calcium-alginate beads, produced by the method described in any one of claims 1 to 5.

7. The lactic acid bacteria according to claim 6, wherein the lactic acid bacteria have an improved survival rate after freeze-drying compared to lactic acid bacteria that were not captured with calcium-alginate beads.

8. The lactic acid bacteria according to claim 6, wherein the lactic acid bacteria have improved stability compared to lactic acid bacteria that have not been captured with calcium-alginate beads.

9. (a) The stage of culturing lactic acid bacteria and collecting the bacterial cells; (b) The step of mixing the lactic acid bacteria from step (a) with a solution containing alginic acid; and (c) The step of adding the mixture from step (b) to a calcium-containing solution to form alginate-calcium beads, The aforementioned solution containing alginic acid contains 5% to 10% by weight of sucrose, trehalose, and maltodextrin. The calcium-containing solution contains trehalose. A method for improving the production yield of calcium-alginate beads for lactic acid bacteria capture compared to the case where lactic acid bacteria are not captured by calcium-alginate beads.

10. (a) The stage of culturing lactic acid bacteria and collecting the bacterial cells; (b) A step of mixing the lactic acid bacteria from step (a) with a solution containing alginic acid; (c) Adding the mixture from step (b) to a calcium-containing solution to form alginate-calcium beads; and (d) The step of freeze-drying the lactic acid bacteria collected with the calcium-alginate beads in step (c), The aforementioned solution containing alginic acid contains 5% to 10% by weight of sucrose, trehalose, and maltodextrin. The calcium-containing solution contains trehalose. A method for improving the survival rate of freeze-dried lactic acid bacteria compared to lactic acid bacteria that have not been captured with calcium-alginate beads.

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