KR20200001543A - Composition for cryoprotecting lactic acid bacteria containing citrate - Google Patents

Abstract

The present application relates to a composition comprising citrate for cryoprotecting lactic acid bacteria; a method for preparing cryoprotected lactic acid bacteria using the composition; and a method for preserving lactic acid bacteria using the composition. When using the composition comprising citrate for cryoprotecting lactic acid bacteria, provided in the present application, the storage stability of lactic acid bacteria is improved, and in particular, even at high temperatures, the activity of the lactic acid bacteria can be maintained to the maximum before and after freeze-drying.

Description

Composition for cryoprotecting lactic acid bacteria containing citrate

The present application is lactic acid bacteria freeze protection composition comprising citrate; Method for producing a cryoprotected lactic acid bacteria using the composition; And it relates to a preservation method of lactic acid bacteria using the composition.

Lactic acid bacteria (lactic acid bacteria) is also known as lactic acid bacteria or lactic acid bacteria, and is an important bacteria used as a formal agent by preventing abnormal fermentation by various bacteria inhabiting in the intestines of mammals. For example, L. bulgaricus is the oldest known lactic acid bacterium, used in the manufacture of yogurt and as a starter in the manufacture of cheese or fermented butter. Aerobic Lactobacillus ( L. acidophilus ) is present in the intestines of humans and all mammals and other animals, and is used in the manufacture of butter or milk or in the treatment of intestinal self-intoxication. In addition, L. lactis produces DL-lactic acid, which is always present in milk and used to make butter or cheese and is the most important bacterium for dairy lactic acid bacteria.

Useful lactic acid bacteria as described above to settle in the intestine exerts a variety of physiological activities, such as activation of intestinal motility, inhibiting harmful bacteria, promoting vitamins, including vitamins, atopic dermatitis. However, in order to exert the physiological effect, it is necessary to take a much larger amount of lactic acid bacteria than conventionally ingested food such as yoghurt. Therefore, the method of separating only lactic acid bacteria and eating easily in the form of powder or capsule has been popularized. However, when lactic acid bacteria are made in powder or capsule form, there are many lactic acid bacteria that die during the long-term distribution process, so that the lactic acid bacteria cannot exhibit the original bioactive function.

In general, lactic acid bacteria are prepared in powder form by freeze drying or spray drying, wherein freeze drying is a method of suspending microorganisms in a freeze-drying suspension solution to freeze and drying under reduced pressure. However, since lactic acid bacteria are unilateral anaerobic and very sensitive to the surrounding environment, the powdered lactic acid bacteria have a low viability at low temperature and room temperature, thereby reducing the number of live bacteria. Recently, in order to overcome these disadvantages, a method of coating lactic acid bacteria using various coating agents has been developed. For example, starch, gelatin, alginic acid, cellulose, hardened oil, various emulsifiers, etc. are used as a cryoprotectant to maintain quality within the shelf life. A method of making it work is being developed.

Representatively, studies have been conducted to determine the effect of storage temperature and polymer 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 discloses that about 1% of lactic acid bacteria survived after 6 months of storage at 20 ° C., and only about 0.2% of lactic acid bacteria survived after 12 months, further improving the survival rate and storage stability of the lactic acid bacteria. The development of a method is required.

Under this background, the present inventors have found that when the lactic acid bacteria are coated with a composition containing citrate salts, the freezing protection of the lactic acid bacteria greatly improves the storage stability of the lactic acid bacteria, and in particular, can maintain the activity of the lactic acid bacteria at a high temperature. Was completed.

One object of the present application is to provide a composition for lactic acid bacteria freeze protection comprising citric acid salt.

Another object of the present application, (a) culturing the lactic acid bacteria in the medium to recover the cells; (b) preparing a composition comprising citric acid salt; (c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And (d) lyophilizing the mixture of step (c) to powder to produce a freeze-protected lactic acid bacterium.

Another object of the present application, (a) culturing the lactic acid bacteria in the medium to recover the cells; (b) preparing a composition comprising citric acid salt; (c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And (d) freezing or drying the mixture of the step (c) to powder; to provide a method for preserving the lactic acid bacteria.

Another object of the present application is to provide a lactic acid bacteria freeze protection use of citric acid salts.

This will be described in detail as follows. In addition, each description and embodiment disclosed in this application may apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of the present application. In addition, the scope of the present application is not to be limited by the specific description described below.

One embodiment of the present application provides a composition for lactic acid bacteria freeze protection comprising citric acid salt.

In the present application, "freeze protection" means protecting the lactic acid bacteria tissue from freezing when stored lyophilized to preserve the activity of the lactic acid bacteria as it is. In the present application, the "freeze drying" refers to freezing the material to be dried by drastically lowering the temperature of the container, and then vacuuming the solidified solvent contained in the material by vaporizing the pressure inside the container. It is a method of drying. Freeze-drying is a method of minimizing damage to heat-sensitive materials and effectively preserving them for a long time and is useful in terms of pollution prevention, storage, transportation and economics. However, in the freeze-drying process of lactic acid bacteria, the activity and survival rate of the lactic acid bacteria is rapidly reduced, there is a problem that damage the membrane structure of the lactic acid bacteria cells as ice particles are made during freezing. To improve this, a substance added together with freeze-drying to restore its function upon rehydration without damaging or killing lactic acid bacteria is referred to as a freeze protection agent, which gives physicochemical stability to the lactic acid bacteria and thus the survival rate. It serves to increase.

In the present application, the freezing temperature of the lyophilization is below freezing temperature, such as -10 ℃ to -196 ℃ (boiling point of liquid nitrogen), -40 ℃ to -196 ℃, -50 ℃ to -196 ℃, -70 ℃ to- It may be at 196 ° C. and all biological activities of the lactic acid bacteria, including biochemical reactions resulting in cell death at such low temperatures, are effectively stopped.

In the present application, "citrate" means any salt of citric acid, which salt may be formed with sodium, magnesium or potassium. "Citric acid" is an organic acid used to add acidic or sour taste to foods or beverages, and the chemical formula is C ₆ H ₈ O ₇ . The citric acid may be hydrous citric acid or anhydrous citric acid, but is not limited thereto. Citric acid, which may form citrate that may be used in the cryoprotective composition, may be included without limitation. The citrate may be present as citrate trichloride, dihydrogen citrate, dihydrogen citrate, and the like, but is not limited thereto. For example, citrate may mean, but is not limited to, a basic citrate of the formula C ₆ H ₅ O ₇ ^{3 -} as the 3-form of citrate.

For the purposes of the present application, the citrate serves as a freeze protection agent to prevent damage or death of lactic acid bacteria due to freeze drying and to increase storage stability, which was first identified in the present application.

In the present application, the citrate is not particularly limited, but may be sodium citrate, trisodium citrate, magnesium citrate or magnesium citrate, potassium citrate, for example, sodium citrate or trisodium citrate, but is not limited thereto. Do not.

The citrate that may be included in the composition may be one or more selected from the group consisting of sodium citrate, trisodium citrate, and potassium citrate, but is not limited thereto.

The "trisodium citrate" is a compound having a chemical formula of C ₆ H ₅ Na ₃ O _{7 It} is also called trisodium citrate as a material in the form of a white powder. In the food manufacturing field, acidic depressants that are added to soft drinks and ionic drinks to relieve the acidity of citric acid, processed foods (such as sliced cheese), pH adjusters such as ice cream, jams, jelly, dairy products, dairy rancids, cheeses and fish smoke It has been mainly used as a thickener in products, emulsifiers and stabilizers in ice cream.

In the present application, when the citrate salt is used with a conventional cryoprotectant, it may further strengthen the structure of the lactic acid bacteria cells. When lactic acid bacteria are mixed with the composition for cryoprotection, live lactic acid bacteria produce organic acids in the absence of an energy source, resulting in lowering of the lactic acid bacteria. In particular, the citrate has a weak alkalinity and thus serves to buffer the pH change, thereby preventing damage and death of lactic acid bacteria.

The citric acid salt may be included as 0.1 to 50% by weight relative to the total weight of the composition for lactic acid bacteria freeze protection, specifically 0.5 to 30% by weight, 1 to 20% by weight, 1 to 10% by weight, 1 It may be 5% by weight, or 5% by weight, but is not limited thereto. The weight of the citric acid salt may be appropriately adjusted and used by those skilled in the art according to the type, size, amount, freeze-drying condition, type and content of other components included in the composition, and the like.

In addition, in the present application, the citric acid salt may be used by being solubilized at 0.1% by weight to 50% by weight. Specifically, 0.1 to 30% by weight, 0.1 to 10% by weight, 0.5 to 10% by weight, 3 to 10% by weight, 3 to 8% by weight, 3 to 5% by weight, or 5% by weight can be solubilized. This is not restrictive.

The cryoprotective composition may have a solid content of lactic acid bacteria of 15% by weight to 50% by weight, or more than 15% by weight to less than 50% by weight, 16% by weight to 45% by weight, 20% by weight to 49% by weight, and 20% by weight to 45%. It may include, but is not limited to, by weight%, 25% by weight to 45% by weight, 25% by weight to 40% by weight, 25% by weight to 35% by weight.

In general, when the solid content of lactic acid bacteria is 15% by weight to 50% by weight, while maintaining the yield of dry lactic acid bacteria during the use of this process, the manufacturing cost can be maintained at an appropriate level in the process, based on this solid content of lactic acid bacteria You can choose appropriately.

The cryoprotective composition is 5 to 25 parts by weight, 5 to 10 parts by weight, 5 to 15 parts by weight, 5 to 20 parts by weight, 5 to 21 parts by weight, 5 to 22 parts by weight, and 5 to about 100 parts by weight of lactic acid bacteria cells. 23 parts by weight, 5 to 24 parts by weight, 6 to 10 parts by weight, 6 to 15 parts by weight, 6 to 20 parts by weight, 6 to 25 parts by weight, 8 to 10 parts by weight, 8 to 15 parts by weight, 8 to 20 parts by weight Part, 8 to 25 parts by weight, 10 to 15 parts by weight, 10 to 20 parts by weight, 10 to 25 parts by weight, 15 to 20 parts by weight, or 15 to 25 parts by weight may be included, but is not limited thereto. .

The composition for lactic acid bacteria freeze protection of the present application may further include one or more selected from the group consisting of a cryoprotectant, a porous support and a nitrogen source. Specifically, it may further include one kind, two kinds and three kinds.

Specifically, the composition for lactic acid bacteria cryoprotection of the present application may further include a component known as a cryoprotectant, in addition to citrate. That is, the "freeze protection agent" further included in the composition for lactic acid bacteria freeze protection in the present application means a material having a freeze protection effect commonly used in the art, except for citrate salts. The cryoprotectant may be purchased from a commercially available one, and there is no particular kind. Specifically, trehalose, sugars, amino acids, peptides, gelatin, glycerol, sugar alcohols, whey, alginic acid, ascorbic acid, yeast extract, skim milk, and the like may be used as cryoprotectants, but may not be limited thereto.

In the present application, the "trehalose" is a substance having a chemical formula of C ₁₂ H ₂₂ O ₁₁ as a saccharide widely present in nature such as plants and microorganisms. Trehalose is a substance known to act as a cryoprotectant to prevent lactic acid bacteria from being damaged or killed by freeze drying and to restore its function upon rehydration, especially when citrate and trehalose are used together in the present application. It can increase the freezing protection effect of lactic acid bacteria.

The trehalose may be 10 to 40% by weight, 10 to 30% by weight, 15 to 25% by weight, or 20% by weight based on the total weight of the composition for lactic acid bacteria freeze protection, but is not limited thereto. The weight of the cryoprotectant, including trehalose, may be appropriately adjusted and used by those skilled in the art according to the weight of the citric acid salt included in the composition of the present application, the type, size, amount, freeze-drying condition, type and content of other components, and the like.

In the present application, the "porous support" serves to block the inflow of external moisture and air and to impart porosity to the freeze-dried lactic acid bacteria to facilitate rehydration. The porous support may be purchased by using a commercially available porous support commonly used during freeze-drying in the art, there is no particular limitation on the kind. Specifically, the porous support is not particularly limited thereto, but may be maltodextrin, alginate, chitosan, starch, polyethylene glycol, propylene glycol, triacetin, acetyltriethyl citrate, triethyl citrate, glycerin, or a combination thereof. And more specifically maltodextrin.

In the present application, the "maltodextrin" is a white powder based on porous particles, and is a food additive often used in general foods such as yogurt, sauce, salad dressing, and in the present application, as a porous support during freeze drying of lactic acid bacteria. Used.

The maltodextrin may be 1% to 50% by weight relative to the total weight of the composition for lactic acid bacteria freeze protection, specifically 1% to 30% by weight, 1% to 10% by weight, 1% to 5% by weight, or 5 The weight percentage may be, but is not limited thereto.

In the present application, the "nitrogen source (N-source)" refers to a material used as a nitrogen energy source of lactic acid bacteria, which serves to prevent damage to the cells by post-fermentation. When lactic acid bacteria are mixed with a cryoprotectant composition, live lactic acid bacteria produce organic acids in the absence of an energy source, which causes a decrease in pH, leading to the death of lactic acid bacteria. Therefore, the nitrogen energy source prevents the generation of organic acids and the resulting pH drop, thereby preventing the death of lactic acid bacteria.

The nitrogen source can be purchased and used commercially available as a nitrogen source commonly used in the freeze-drying in the art, there is no particular limitation on the kind. Specifically, the nitrogen source is not particularly limited thereto, but may be skim milk powder, whey protein, yeast extract, malt extract, beef extract, casein hydrolyzate, malt extract, tryptone, cysteine, peptone, and specifically as the peptone Soy peptone, fish peptone, proteose peptone, casein peptone, peptone No. 3, may be more specifically soy peptone.

The soy peptone may be 1% by weight to 50% by weight relative to the total weight of the composition for lactic acid bacteria freeze protection, specifically 1% by weight to 30% by weight, 1% by weight to 10% by weight, 1% by weight to 5% by weight, or 5 The weight percentage may be, but is not limited thereto.

In the present application, "lactic acid bacteria" is a generic term for bacteria that ferment saccharides to obtain energy and produce a large amount of lactic acid.Lactobacillus sp .), Bifidobacterium genus (Bifidobacterium sp .), Genus StreptococcusStreptococcus sp .), Genus Lactococcus (Lactococcus sp .), Genus EnterococcusEnterococcus sp .), Genus PediocaucusPediococcus sp .) In leukonostockLeuconostoc sp .) And Vicella genus (Weissella sp .It may be one or more selected from the group consisting of. Specifically, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus rhamnoser (Lactobacillus rhamnosus), Lactobacillus asidophilus (Lactobacillus acidophilus), Bifidobacterium Bifidem (Bifidobacterium bifidum), Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium breb (Bifidobacterium breve), Streptococcus faecalis (Streptococcus faecalis) And Lactococcus lactis (Lactococcus lactis subsp . lactisIt may be one or more selected from the group consisting of. More specifically, Lactobacillus plantarum CJLP243 disclosed in the Republic of Korea Patent No. 1178217 (Lactobacillus plantarum CJLP243), Lactobacillus plantarum CJLP133 disclosed in Korean Patent No. 1486999 (Lactobacillus plantarum CJLP133), Lactobacillus plantarum CJLP136 disclosed in Korean Patent No. 1075558 (Lactobacillus plantarum CJLP136), Lactobacillus plantarum CJLP55 disclosed in Korean Patent No. 1255050 (Lactobacillus plantarum CJLP55) And Lactobacillus plantarum CJLP56 disclosed in Korean Patent No. 1075557 (Lactobacillus plantarum CJLP56It may be one or more selected from the group consisting of, but is not limited thereto. The strain has been deposited with the Korea Biotechnology Research Institute Gene Bank, it can be easily obtained by those skilled in the art.

The lactic acid bacteria freeze protection composition of the present application may be applied in the form of a coating, but is not limited thereto. That is, the lactic acid bacteria freeze protection composition of the present invention may be mixed with lactic acid bacteria to coat the surface of the lactic acid bacteria, thereby protecting the lactic acid bacteria from the external environment and may increase storage stability.

Another aspect of the present application, (a) culturing the lactic acid bacteria in the medium to recover the cells; (b) preparing a composition comprising citric acid salt; (c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And (d) lyophilizing the mixture of step (c) to powder to provide a method for producing a freeze-protected lactic acid bacterium.

The lactic acid bacteria of step (a) are as described above.

In the present application, "culture" means to grow the lactic acid bacteria in a moderately controlled environmental conditions. Cultivation process of the present application may be made according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the strain selected. Specifically, the culture may be batch, continuous and fed-batch, but is not limited thereto.

In the present application, "medium" refers to a material mixed with the nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which is indispensable for survival and development. Specifically, the medium and other culture conditions used for the culture of the microorganism of the present application can be used without any particular limitation as long as the medium is used for the culture of ordinary microorganisms, the microorganism of the present application is suitable carbon source, nitrogen source, personnel, inorganic It can be cultured in a conventional medium containing a compound, an amino acid and / or vitamins, etc. under aerobic conditions, adjusting the temperature, pH and the like.

In the present application, the carbon source may include carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol, and the like; Alcohols such as sugar alcohols, glycerol, pyruvic acid, lactic acid, citric acid and the like; Amino acids such as organic acids, glutamic acid, methionine, lysine and the like. In addition, natural organic nutrients such as starch hydrolyzate, molasses, blackstrap molasses, rice winters, cassava, sugarcane residue and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (ie, reducing sugars). Carbohydrates, such as molasses), and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more kinds, but are not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or their degradation products, skim soy cakes or their degradation products Can be used. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

The personnel may include a first potassium phosphate, a second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like may be used, and other amino acids, vitamins and / or suitable precursors may be included. These components or precursors may be added batchwise or continuously to the medium, but are not limited thereto.

In the present application, during the cultivation of lactic acid bacteria, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, in order to maintain the aerobic state of the medium, it is possible to inject oxygen or oxygen-containing gas into the medium or to inject nitrogen, hydrogen or carbon dioxide gas without the injection of gas to maintain the anaerobic and unaerobic state, but is not limited thereto. Do not.

The temperature of the medium may be 20 ° C to 50 ° C, specifically 30 ° C to 37 ° C, but is not limited thereto. The incubation period may continue until the desired amount of useful material is obtained, specifically, may be 10 hours to 100 hours, but is not limited thereto.

The step of recovering the lactic acid bacteria cells may recover the desired cells from the medium using a suitable method known in the art according to the culture method of the lactic acid bacteria, for example, batch, continuous or fed-batch culture method of the present application. have. For example, centrifugation, filtration, treatment with crystallized protein precipitation agent (salting method), extraction, ultrasonic crushing, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography Various chromatography such as HPLC, HPLC, and the like can be used in combination, but is not limited to these examples.

The recovery step may comprise an additional purification process. The purification process may be used to purify the recovered lactic acid bacteria cells using a suitable method known in the art.

The citrate of step (b) is as described above. The citrate may be one or more selected from the group consisting of sodium citrate, trisodium citrate and potassium citrate, but is not limited thereto.

The composition of step (b) may further comprise one or more selected from the group consisting of a cryoprotectant, a porous support and a nitrogen source. More specifically, it may further include one or more selected from the group consisting of trehalose, maltodextrin and soypeptone.

Cryoprotectants, porous supports, nitrogen sources, trehalose, maltodextrin, soypeptone are as described above.

The composition of step (b) may be 15% to 50% by weight of the solid content of the lactic acid bacteria in the composition, but is not limited thereto.

The mixing of the step (c) may be to mix the composition of the step (b) with 5 to 25 parts by weight of citrate added to 100 parts by weight of lactic acid bacteria cells, but is not limited thereto.

The solid content range and the addition content range of citrate are as described above.

In addition, the step (c) is a step of mixing the lactic acid bacteria of step (a) and the lactic acid bacteria freeze protection composition of step (b) before freeze-drying, the mixing ratio of the lactic acid bacteria and the composition is 1: 0.1 to 1: 5 days by weight It may be, specifically, 1: 0.1 to 1: 4, 1: 0.5 to 1: 3, 1: 0.5 to 1: 2, or 1: 2, but is not limited thereto.

Step (d) is a step of drying and powdering the mixture of step (c), carrying the drying process by transferring the mixture to a drying tray (Tray). The drying method is not particularly limited, but may be air drying, spray drying, vacuum drying or freeze drying, and specifically, may be freeze drying. More specifically, after freezing by keeping at a temperature of -10 ℃ to -196 ℃, -40 ℃ to -196 ℃, or -40 ℃ to -70 ℃ for 12 hours to 24 hours, thaw in the freeze dryer to freeze the moisture It may be a step of removing.

The lactic acid bacteria are granules, powders, powders, granules, acupuncture agents, emulsions, fluids, tablets, pills, capsules, granules, ointments, suppositories, injection solutions, inhalants, aerosols, suspensions, syrups, emulsions, soft and hard capsules, elixirs It may be in the form of elixirs, troches or lozenges, but is not limited thereto.

Another aspect of the present application, (a) culturing the lactic acid bacteria in the medium to recover the cells; (b) preparing a composition comprising citric acid salt; (c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And (d) freezing or drying the mixture of step (c) to powderize the mixture.

The term "preservation method" in the present application means a method for maintaining the desired function and / or biological activity of the lactic acid bacteria for a long time in the specific temperature range for the purpose of the present application, more specifically, a method that is present at room temperature for one year or more. It may mean, but is not limited thereto. Specifically, the preferred function and / or biological activity of the lactic acid bacteria can be maintained at 10% or more, but is not limited thereto.

(a) lactic acid bacteria, medium, culture, recovery, (b) the citrate, the mixing ratio of (c) is as described above.

The drying of step (d) may be air drying, spray drying, vacuum drying or freeze drying, and in particular, may be freeze drying, although the method is not particularly limited thereto.

Another aspect of the present application is to provide a lactic acid bacteria freeze protection use of citric acid salts.

Citrate, and lactic acid bacteria freeze protection applications are as described above.

When the composition for lactic acid bacteria freeze protection comprising the citric acid salt provided in the present application is used, the storage stability of the lactic acid bacteria is improved, and in particular, the activity of the lactic acid bacteria before and after freeze-drying can be kept to the maximum.

Figure 1 shows the production method of the lactic acid bacteria of the present invention.

Hereinafter, the present application will be described in more detail with reference to Examples. These examples are intended to describe the present application in more detail, and the scope of the present application is not limited by these examples.

Example  1: as a cryoprotectant Trisodium citrate  Preparation of lactic acid bacteria dry powder containing ingredient

Lactobacillus plantarum CJLP133 strain was incubated at 37 ° C. for 18-24 hours using MRS liquid medium (Difco, USA). The supernatant was discarded using a centrifuge to recover only the lactic acid bacteria.

Then, trehalose (20% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), maltodextrin (5% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), soy peptone (5% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), citrate Trisodium acid (5% by weight relative to the total weight of the lactic acid bacteria freeze protection composition) and water (residual amount) were mixed and sterilized.

The collected lactic acid bacteria (solid content of 30% by weight) and the cryoprotective composition were mixed and suspended in a 1: 2 weight ratio, and transferred to a freeze-drying tray and held at about 12 to 24 hours under rapid freezing conditions (-10 ° C or less). Then, the water was removed while thawing in a freeze dryer to obtain a lactic acid bacteria powder coated with the freeze protection composition.

Comparative example  One: Trehalose  And Maltodextrin  Preparation of lactic acid bacteria dry powder containing

Lactobacillus plantarum CJLP133 strain was incubated at 37 ° C. for 18-24 hours using MRS liquid medium (Difco, USA). The supernatant was discarded using a centrifuge to recover only the lactic acid bacteria.

Thereafter, trehalose (20 wt% based on the total weight of the composition for lactic acid bacteria freezing protection), maltodextrin (5 wt% based on the total weight of the composition for lactic acid bacteria freezing protection), and water (residual amount) were mixed and sterilized. The lactic acid bacteria (solid content of 30% by weight) and the cryoprotectant were mixed and suspended in a 1: 2 weight ratio. The mixed solution was transferred to a freeze drying tray, held at about 12 to 24 hours under rapid freezing conditions (-10 ° C. or lower), and then water was removed while thawing in a freeze dryer.

That is, Comparative Example 1 omits Soypeptone and trisodium citrate when preparing the coating solution in comparison with Example 1.

Comparative example  2: Trehalose , Maltodextrin  And Soypeptone  Preparation of lactic acid bacteria dry powder containing

Lactobacillus plantarum CJLP133 strain was incubated at 37 ° C. for 18-24 hours using MRS liquid medium (Difco, USA). The supernatant was discarded using a centrifuge to recover only the lactic acid bacteria.

Then, trehalose (20% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), maltodextrin (5% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), soy peptone (5% by weight relative to the total weight of the composition for lactic acid bacteria freezing protection), and water (residual amount) ) Were mixed and sterilized. The lactic acid bacteria and the cryoprotectant were mixed and suspended in a 1: 2 weight ratio. The mixed solution was transferred to a freeze drying tray, held at about 12 to 24 hours under rapid freezing conditions (-10 ° C. or lower), and then water was removed while thawing in a freeze dryer.

That is, Comparative Example 2 omits trisodium citrate when preparing the coating solution in comparison with Example 1.

Experimental Example  One: Trisodium citrate  Evaluation of high temperature stability with or without (save 6 weeks at 40 ℃)

Lyophilized lactic acid bacteria powder gradually decreases in activity with storage temperature and storage period. In general, factors affecting activity include temperature, oxygen, and moisture. Firstly, the lyophilized lactic acid bacteria powder is very hygroscopic and a large amount of content reduction occurs at the beginning of storage. In order to improve the shelf life of storage, there are various methods for applying an oxygen scavenger to the packaging material or dehumidifying, but ultimately, the storage period depends on the coating degree of the lactic acid bacteria powder itself. Therefore, in order to alleviate hygroscopicity due to raw material properties, excipients such as glucose and dextrin are mixed and stored within 1 to 10 times of the powder. In this experiment, an excipient in which maltodextrin and anhydrous crystalline glucose were mixed in a 1: 1 ratio was mixed and stored in a ratio of powder and mixed excipient 1: 9. In order to exclude air permeability during the storage period, individually packaged and stored in an aluminum pouch, and stored for 4 weeks at 40 ℃ to analyze the survival rate in harsh conditions.

Specifically, the samples of Comparative Example 1, Comparative Example 2 and Example 1, which were lyophilized and powdered, were individually packaged and sealed in an aluminum pouch package. Samples were each stored for 4 weeks in a 40 ° C. incubator. After a certain time, the experimental group sample was diluted to a ratio of 1: 100 in Saline Buffer and placed in a sterile bag and homogenized. Samples that were serially diluted with Saline Buffer were plated on MRS Agar Plates. The plates were harvested and counted after standing for 24 hours under aerobic conditions at 37 ° C., and the results are shown in Tables 1 and 2 below. The numbers listed in the table below indicate the percentage survival rate compared to the initial lactic acid bacteria.

Comparative Example 1 Comparative Example 2 Example 1 Early 100.0% 100.0% 100.0% 1 week 55.3% 66.5% 79.8% 2 weeks 41.0% 43.7% 74.6% 3 weeks 35.4% 45.4% 69.7% 4 Weeks 23.5% 45.7% 75.4%

By applying harsh conditions, the activity of the bacteria was compared before and after. In Example 1 containing trisodium citrate, lactic acid bacteria could be recovered at a high rate even after harsh conditions, and it was confirmed that the survival rate of lactic acid bacteria was significantly higher than that of Comparative Examples 1 and 2 containing no trisodium citrate. These results suggest that trisodium citrate, a representative example of citrate, can be an excellent cryoprotectant component in lactic acid bacteria.

Experimental Example 2: Evaluation of Storage Stability According to the Content of Trisodium Citrate

Experiment of the same method as Experimental Example 1 was performed while changing the amount of trisodium citrate of Example 1 from 5% by weight. The contents of trehalose, maltodextrin, soypeptone and trisodium citrate were mixed as follows.

Example 2 Example 3 Example 4 Trehalose 20 wt% 20 wt% 20 wt% Maltodextrin 5 wt% 5 wt% 5 wt% Soypeptone 5 wt% 5 wt% 5 wt% Trisodium citrate 0.1 wt% 1 wt% 2.5 wt%

After counting the lactic acid bacteria to calculate the survival rate (%) compared to the initial lactic acid bacteria are shown in Table 3 below.

Example 2
Trisodium Citrate
0.1%) Example 3
Trisodium Citrate
One%) Example 4
Trisodium Citrate
2.5%) Example 1
Trisodium Citrate
5%) Early 100.0% 100.0% 100.0% 100.0% One 75.5% 97.3% 80.6% 79.8% 2 61.8% 84.1% 72.8% 74.6% 3 50.7% 73.0% 60.9% 69.7% 4 44.9% 61.6% 58.9% 75.4%

As can be seen in Table 3, in the case of Example 1 containing the highest amount of trisodium citrate, the survival rate of the first week did not appear the highest, but the number of lactic acid bacteria that died over time was the lowest, the survival rate decreases the most It was confirmed to appear low. Therefore, after 4 weeks, the survival rate of lactic acid bacteria was finally highest, and it was confirmed that the storage stability of lactic acid bacteria was the highest in Example 1 having the highest content of trisodium citrate.

Experimental Example 3: Evaluation of freeze-drying yield according to trisodium citrate content

Live lactic acid bacteria undergo a drying process to improve storage stability, and a freeze-drying method having a high yield of drying with the least heat energy is preferred. However, even the freeze-drying process can not prevent the death of lactic acid bacteria 100%, depending on the composition of the freeze protection agent will affect the yield. Survival rate (%) of the total number of bacteria after drying compared to the total number of bacteria of lactic acid bacteria before drying, the results are shown in Table 4.

Example 2
(0.1% trisodium citrate) Example 3
(Tridium sodium citrate 1%) Example 4
(Tridium sodium citrate 2.5%) Example 1
(5% trisodium citrate) yield 66.6% 71.9% 88.0% 99.1%

As can be seen in Table 4, the higher the content of trisodium citrate, the survival rate of lactic acid bacteria compared before and after drying increased proportionally. That is, trisodium citrate played a role of preventing the death of lactic acid bacteria during freeze drying, suggesting that the effect of the increase in the content of trisodium citrate increases.

Experimental Example 4: Evaluation of Storage Stability with or without Trisodium Citrate (CJLP243)

Instead of Lactobacillus plantarum CJLP133 Except for using the Lactobacillus plantarum CJLP243 strain, the same composition as in Comparative Example 1 and Example 1 was prepared and Experimental Example 1 was carried out in the same manner. The survival rate (%) compared to the initial lactic acid bacteria is shown in Table 5 below.

Comparative Example 1 Example 1 Early 100.0% 100.0% One 46.0% 66.2% 2 36.2% 60.9% 3 26.2% 42.5% 4 16.1% 40.8%

As shown in Table 5, even if the experiment was carried out by changing the lactic acid bacteria strain to Lactobacillus plantarum CJLP243, the lactic acid bacteria survival rate was remarkably high in Example 1 containing trisodium citrate, and time stability was also excellent over time. It was confirmed. That is, the effect of the storage stability according to the content of the trisodium citrate suggests that it can be expressed regardless of the lactic acid bacteria strain.

These results suggest that citrate can be freeze-protected while maintaining safety regardless of the type of lactic acid bacteria strain, suggesting that it can be used as a cryoprotectant for excellent lactic acid bacteria that can maintain long-term high lactic acid bacteria survival rate. It is.

From the above description, those skilled in the art will appreciate that the present application can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present application should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present application.

Claims (16)

Lactic acid bacteria freeze protection composition containing citrate.
The composition of claim 1, wherein the citrate is at least one selected from the group consisting of sodium citrate, trisodium citrate, and potassium citrate.
The composition of claim 1, further comprising one or more selected from the group consisting of a cryoprotectant, a porous support and a nitrogen source.
The composition of claim 1, further comprising one or more selected from the group consisting of trehalose, maltodextrin, and soypeptone.
According to claim 1, wherein the lactic acid bacteria Lactobacillus sp ( Lactobacillus sp . ), Bifidobacterium genus ( Bifidobacterium sp . ), Streptococcus sp . , Lactococcus sp . ), Enterococcus sp . ), Pediococcus sp . ) Leuconostoc sp . ) And Bisella genus ( Weissella sp. ) At least one selected from the group consisting of, lactic acid bacteria freeze protection composition.
According to claim 1, wherein the citrate is lactic acid bacteria freeze protection composition containing 0.1% to 50% by weight relative to the total weight of the composition for cryoprotectant, lactic acid bacteria freeze protection.
According to claim 1, wherein the cryoprotective composition comprises lactic acid bacteria solid content of 15% by weight to 50% by weight, lactic acid bacteria freeze protection composition.
The composition of claim 1, wherein the citrate is included in an amount of 5 to 25 parts by weight based on 100 parts by weight of the lactic acid bacteria cells.
(a) culturing the lactic acid bacteria in the medium to recover the cells;
(b) preparing a composition comprising citrate;
(c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And
(d) lyophilizing and powdering the mixture of step (c);
Containing, method for producing a cryoprotected lactic acid bacteria.
The method of claim 9, wherein the citrate of step (b) is at least one selected from the group consisting of sodium citrate, trisodium citrate, and potassium citrate.
The method of claim 9, wherein the composition of step (b) further comprises one or more selected from the group consisting of a cryoprotectant, a porous support and a nitrogen source.
The method of claim 9, wherein the composition of step (b) further comprises one or more selected from the group consisting of trehalose, maltodextrin and soypeptone.
The method of claim 9, wherein the composition of step (b) has a solid content of lactic acid bacteria in the composition of 15% by weight to 50% by weight.
The method of claim 9, wherein the mixing of step (c) comprises mixing the composition of step (b) with 5 to 25 parts by weight of citrate added to 100 parts by weight of the lactic acid bacteria cells.
(a) culturing the lactic acid bacteria in the medium to recover the cells;
(b) preparing a composition comprising citric acid salt;
(c) mixing the lactic acid bacteria of step (a) and the composition of step (b); And
(d) freezing or drying the mixture of step (c) to powder;
Containing, preservation method of lactic acid bacteria.
The method of claim 15, wherein the drying is air drying, spray drying, vacuum drying or freeze drying.

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