KR102429539B1 - Composition for cryoprotecting lactic acid bacteria containing citrate - Google Patents

Abstract

The present application relates to a composition for cryoprotection of lactic acid bacteria containing citrate; A method for preparing a cryoprotected lactic acid bacterium using the composition; And it relates to a method of preserving lactic acid bacteria using the composition.
When using the composition for freeze protection of lactic acid bacteria containing citrate provided in the present application, the storage stability of the lactic acid bacteria is improved, and in particular, it is possible to maintain the activity of the lactic acid bacteria before and after freeze-drying at a high temperature to the maximum.

Description

Composition for cryoprotecting lactic acid bacteria containing citrate

The present application relates to a composition for cryoprotection of lactic acid bacteria containing citrate; Method for preparing a cryoprotected lactic acid bacterium using the composition; And it relates to a method of preserving lactic acid bacteria using the composition.

Lactic acid bacteria, also called lactic acid bacteria or lactic acid bacteria, are important bacteria that live in the intestines of mammals and are used as intestinal agents by preventing abnormal fermentation by various bacteria. For example, Bulgarian lactic acid bacteria ( L. bulgaricus ) is the oldest known lactic acid bacteria, used in the manufacture of yogurt, and also used as a starter in the manufacture of cheese or fermented butter. Aerobic lactic acid bacteria ( 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 autopoisoning. In addition, lactis lactate cocci ( L. lactis ) produces DL-lactic acid, which is always present in milk and used for butter or cheese production, and is the most important lactic acid bacteria for dairy.

As described 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 effect, it is necessary to ingest a much larger amount of lactic acid bacteria than the amount previously consumed as food such as yogurt. Therefore, the method of isolating only lactic acid bacteria and eating them in powder or capsule form has become popular. However, when the 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 there is a limitation in that the original physiological activity function of the lactic acid bacteria cannot be exhibited.

In general, lactic acid bacteria are prepared in powder form by a freeze-drying method or a spray-drying method. Among them, the freeze-drying method is a method of suspending microorganisms in a freeze-drying suspension solution, freezing them, and then drying them under reduced pressure. However, since lactic acid bacteria are anaerobic and very sensitive to the surrounding environment, the viability of powdered lactic acid bacteria is lowered at low temperature and room temperature, thereby reducing the number of viable bacteria. Recently, in order to overcome this disadvantage, a method of coating lactic acid bacteria using various coating materials has been developed. For example, starch, gelatin, alginic acid, cellulose, hydrogenated oil, various emulsifiers, etc. are used as cryoprotectants to maintain quality within the shelf life. Methods for doing so are being developed.

Typically, a study was conducted to confirm the effect of storage temperature and polymer on the stability of lactic acid bacteria by freeze-drying Lactobacillus rhamnosus coated with gelatin (Claude PC et al, Food. Res. Int. , 1996, 29, 555-562). However, the above 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, so the survival rate and storage stability of lactic acid bacteria can be further improved. There is a need to develop a method.

Under this background, the present inventors confirmed that, when the lactic acid bacteria are coated with a composition containing citrate and freeze-protected, the storage stability of the lactic acid bacteria is remarkably improved, and in particular, it is possible to maintain the activity of the lactic acid bacteria to the maximum even at a high temperature. was completed.

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

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

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

Another object of the present application is to provide a use of lactic acid bacteria cryoprotection of citrate.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

One aspect of the present application provides a composition for cryoprotection of lactic acid bacteria comprising a citric acid salt.

In the present application, "freeze protection" means protecting the lactic acid bacteria tissue from freezing when lyophilized and stored in order to preserve the activity of lactic acid bacteria as they are. In the present application, the "freeze drying" refers to freezing the material to be dried by rapidly lowering the temperature of the container, and then sublimating the solidified solvent contained in the material to water vapor by creating a vacuum inside the container. way to dry. Freeze-drying is useful in terms of pollution prevention, storage, transportation, and economics as a method for minimizing damage to heat-sensitive materials and effectively long-term preservation. However, in the freeze-drying process of the lactic acid bacteria, the activity and survival rate of the lactic acid bacteria are rapidly reduced, and there is a problem in that ice particles are formed during freezing, thereby damaging the membrane structure of the lactic acid bacteria cells. To improve this, substances added together during freeze-drying are called cryoprotectants so that lactic acid bacteria are not damaged or killed and their functions can be restored during rehydration. plays a role in increasing

In the present application, the freezing temperature of freeze-drying is a sub-zero temperature, such as -10 °C to -196 °C (the boiling point of liquid nitrogen), -40 °C to -196 °C, -50 °C to -196 °C, -70 °C to - It may be 196 °C, and at such a low temperature, all biological activities of lactic acid bacteria including biochemical reactions that cause cell death 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 acid or sour taste to food 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, and for the purpose of the present application, citric acid capable of forming a citrate that can be used in the composition for cryoprotection may be included without limitation. The citrate may be present in the form of tri-citrate, dihydrogen citrate or monodihydrogen citrate, but is not limited thereto. For example, the citrate may mean a basic citrate of the formula C ₆ H ₅ O ₇ ^{3 -} as the 3-form of the citrate, but is not limited thereto.

For the purpose of the present application, the citrate serves as a cryoprotectant 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 thereto, but may be sodium citrate, trisodium citrate, magnesium citrate or trimagnesium citrate, or potassium citrate, for example, sodium citrate or trisodium citrate, but is not limited thereto. does not

The citrate that may be included in the composition may be at least one 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 and is a material in the form of a white powder, also called trisodium citrate. In the field of food manufacturing, acidity emollient added to soft drinks and ion drinks to alleviate the sour taste of citric acid, pH adjuster for processed foods (such as sliced cheese), ice cream, jam, jelly, dairy products, etc., rancidity inhibitor for dairy products, cheese and fish tenderloin It has been mainly used as a thickener for products, as an emulsifier and stabilizer for ice cream.

In the present application, when the citrate salt is used with a conventional cryoprotectant, the structure of the lactic acid bacteria can be further strengthened. When lactic acid bacteria are mixed with the composition for cryoprotection, live lactic acid bacteria in the absence of an energy source produce organic acids, and the resulting pH drop induces the death of lactic acid bacteria. In particular, since the citrate has a weak alkalinity, it serves to buffer changes in pH, thereby preventing damage and death of lactic acid bacteria.

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

In addition, in the present application, the citric acid salt may be used by water-soluble in 0.1 wt% to 50 wt%. Specifically, it may be water-soluble in 0.1 wt% to 30 wt%, 0.1 to 10 wt%, 0.5 to 10 wt%, 3 to 10 wt%, 3 to 8 wt%, 3 to 5 wt%, or 5 wt% , but not limited thereto.

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

In general, when the solid content of lactic acid bacteria is 15% to 50% by weight, it is possible to maintain the production cost at an appropriate level in the process while maintaining the dry powder yield of lactic acid bacteria when using this process. can be appropriately selected.

The composition for cryoprotection is based on 100 parts by weight of lactic acid bacteria, 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, 5 to 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 Parts, 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 to be added, but is not limited thereto. .

The composition for cryoprotecting lactic acid bacteria of the present application may further include at least one selected from the group consisting of a cryoprotectant, a porous support, and a nitrogen source. Specifically, it may further include 1 type, 2 types, and 3 types.

Specifically, the composition for cryoprotecting lactic acid bacteria of the present application may further include a component known as a cryoprotectant in the prior art, except for citrate. That is, in the present application, the "cryoprotectant" additionally included in the composition for cryoprotection of lactic acid bacteria refers to a material having a cryoprotectant effect commonly used in the art, except for citrate. The cryoprotectant may be purchased and used commercially, and there is no particular type of the cryoprotectant. Specifically, trehalose, saccharides, amino acids, peptides, gelatin, glycerol, sugar alcohols, whey, alginic acid, ascorbic acid, yeast extract, skim milk, etc. may be used as the cryoprotectant, but not limited thereto, and more specifically, trehalose.

In the present application, the "trehalose (Trehalose)" is a saccharide widely present in nature, such as plants and microorganisms, and has a chemical formula of C ₁₂ H ₂₂ O ₁₁ . Trehalose is a substance known to act as a cryoprotectant that prevents damage or death of lactic acid bacteria due to freeze-drying and helps restore its function upon rehydration, especially when citrate and trehalose are used together in this application It is possible to increase the cryoprotection 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 freeze protection of lactic acid bacteria, but is not limited thereto. The weight of the cryoprotectant, including trehalose, may be appropriately adjusted 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, the 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 provide porosity to the freeze-dried lactic acid bacteria to facilitate rehydration. The porous support may be purchased and used commercially as a porous support commonly used in freeze-drying in the art, and there is no particular limitation on the type thereof. Specifically, the porous support is not particularly limited thereto, but maltodextrin, alginate, chitosan, starch, polyethylene glycol, propylene glycol, triacetin, acetyl triethyl 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, and salad dressing. used

The maltodextrin may be 1% to 50% by weight based on the total weight of the composition for freeze protection of lactic acid bacteria, specifically 1% to 30% by weight, 1% to 10% by weight, 1% to 5% by weight, or 5 % by weight, 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 due to post-fermentation. When lactic acid bacteria are mixed with the composition for cryoprotection, live lactic acid bacteria in the absence of an energy source produce organic acids, which causes a decrease in pH and induces death of lactic acid bacteria. Therefore, since the nitrogen energy source prevents the production of organic acids and the resulting pH drop, it is possible to prevent the death of lactic acid bacteria.

The nitrogen source may be purchased and used commercially as a nitrogen source commonly used during freeze-drying in the art, and there is no particular limitation on its type. 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, or peptone. Specifically, the peptone includes It may include soy peptone, fish peptone, proteose peptone, casein peptone, peptone No. 3, and more specifically, soy peptone.

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

In the present application, "lactic acid bacteria" is a generic term for bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid . ( Bifidobacterium sp . ), Streptococcus genus ( Streptococcus sp . ), Lactococcus genus ( Lactococcus ) sp . ), Enterococcus genus ( Enterococcus sp . ), Pediococcus genus ( Pediococcus ) sp . ) Leuconostoc genus ( Leuconostoc sp . ) and Weissella genus sp . ) may be at least one selected from the group consisting of. Specifically, Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus rhamnosus ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Bifidobacterium ( Bifidobacterium ) bifidum ), Bifidobacterium longum ( Bifidobacterium longum ), Bifidobacterium brev ( Bifidobacterium ) breve ), Streptococcus faecalis and Lactococcus lactis subsp . lactis ) may be at least one selected from the group consisting of. More specifically, Lactobacillus plantarum CJLP243 disclosed in Korean 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 may be at least one selected from the group consisting of, but is not limited thereto. The strain is deposited with the Korea Research Institute of Bioscience and Biotechnology Gene Bank, and can be easily obtained by those skilled in the art.

The composition for freeze protection of lactic acid bacteria of the present application may be applied in the form of a coating agent, but is not limited thereto. That is, the composition for freeze protection of lactic acid bacteria 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 increasing storage stability.

Another aspect of the present application, (a) culturing lactic acid bacteria in a medium to recover the cells; (b) preparing a composition comprising a citrate salt; (c) mixing the composition of step (b) with the lactic acid bacteria of step (a); And (d) freeze-drying the mixture of step (c) to powder;

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

In the present application, "cultivation" means growing the lactic acid bacteria in appropriately controlled environmental conditions. The culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. 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 nutrients required for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganism of the present application may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms, but a suitable carbon source, nitrogen source, personnel, inorganic It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids and/or vitamins.

As the carbon source in the present application, carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, sorbitol; 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 may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is 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; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include monopotassium phosphate, dipotassium 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, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium in a batch or continuous manner, but is not limited thereto.

In the present application, by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner during the culture of the lactic acid bacteria, the pH of the medium can be adjusted. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without injection of gas or without gas to maintain anaerobic and microaerobic conditions, but is not limited thereto. does 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 be continued until a desired production amount of the useful substance is obtained, and specifically, it may be 10 hours to 100 hours, but is not limited thereto.

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

The recovery step may include an additional purification process. The purification process may use a suitable method known in the art to purify the recovered lactic acid bacteria.

The citrate in step (b) is the same as described above. The citrate may be at least one 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 include at least one 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 soipeptone.

The cryoprotectant, the porous support, the nitrogen source, trehalose, maltodextrin, and soipeptone are as described above.

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

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

The solid content range and the added content range of citric lead are the same as described above.

In addition, the step (c) is a step of mixing the lactic acid bacteria of step (a) and the composition for freeze protection of step (b) before freeze-drying, and the mixing ratio of the lactic acid bacteria and the composition is 1:0.1 to 1:5 days by weight. 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.

The step (d) is a step of drying and powdering the mixture of step (c), and the mixture is transferred to a drying tray to perform a drying process. The drying method is not particularly limited thereto, but may be air drying, spray drying, vacuum drying or freeze drying, and specifically freeze drying. More specifically, after freezing by maintaining at a temperature of -10 °C to -196 °C, -40 °C to -196 °C, or -40 °C to -70 °C for 12 hours to 24 hours, thawing in a freeze dryer to remove moisture It may be a step to remove.

The lactic acid bacteria are granules, powders, powders, granules, needles, emulsions, fluids, tablets, pills, capsules, granules, ointments, suppositories, injections, inhalants, aerosols, suspensions, syrups, emulsions, soft/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 lactic acid bacteria in a medium to recover the cells; (b) preparing a composition comprising a citrate salt; (c) mixing the composition of step (b) with the lactic acid bacteria of step (a); And (d) freezing or drying the mixture of step (c) to powder;

In the present application, "preservation method" means a method for maintaining the desired function and/or biological activity of a lactic acid bacterium in a specific temperature range for a long period of time for the purpose of the present application, and more specifically, a method of existing at room temperature for 1 year or more may mean, but is not limited thereto. Specifically, the desired function and / or biological activity of the lactic acid bacterium may be maintained at 10% or more, but is not limited thereto.

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

The drying in step (d) is not particularly limited thereto, but may be air drying, spray drying, vacuum drying or freeze drying, and specifically freeze drying.

Another aspect of the present application is to provide the use of lactic acid bacteria cryoprotection of citrate.

The use of citrate, and lactic acid bacteria cryoprotection is the same as described above.

When using the composition for freeze protection of lactic acid bacteria containing citrate provided in the present application, the storage stability of the lactic acid bacteria is improved, and in particular, it is possible to maintain the activity of the lactic acid bacteria before and after freeze-drying at a high temperature to the maximum.

1 shows a method for producing a lactic acid bacterium of the present invention.

Hereinafter, the present application will be described in more detail through examples. These Examples are for describing the present application in more detail, and the scope of the present application is not limited by these Examples.

Example 1: as cryoprotectant trisodium citrate Preparation of dry powder containing lactic acid bacteria

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

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

The recovered lactic acid bacteria (solid content 30% by weight) and the composition for cryoprotection were mixed and suspended at a weight ratio of 1:2, transferred to a freeze-drying tray, and maintained for 12 to 24 hours under quick-freezing conditions (-10°C or less). After thawing in a freeze dryer, moisture was removed to obtain a lactic acid bacteria powder coated with the composition for freeze protection.

comparative example One: Trehalose and maltodextrin Preparation of dry powder containing lactic acid bacteria

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

Then, trehalose (20% by weight relative to the total weight of the composition for lactic acid bacteria cryoprotection), maltodextrin (5% by weight relative to the total weight of the composition for lactic acid bacteria cryoprotection), and water (residual amount) were mixed and sterilized. The lactic acid bacteria (solid content 30% by weight) and the cryoprotectant were mixed and suspended in a weight ratio of 1:2. The mixed solution was transferred to a freeze-drying tray, maintained for 12 to 24 hours under rapid-freezing conditions (below -10 °C), and then thawed in a freeze dryer to remove moisture.

That is, Comparative Example 1 omits soipeptone and trisodium citrate in the preparation of the coating solution compared to Example 1.

comparative example 2: Trehalose , maltodextrin and Soipeeptone Preparation of dry powder containing lactic acid bacteria

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

Then, trehalose (20% by weight relative to the total weight of the lactic acid bacteria cryoprotecting composition), maltodextrin (5% by weight relative to the total weight of the lactic acid bacteria cryoprotecting composition), soipeptone (5% by weight relative to the total weight of the lactic acid bacteria cryoprotecting composition), and water (remaining amount) ) were mixed and sterilized. The lactic acid bacteria and the cryoprotectant were mixed and suspended in a weight ratio of 1:2. The mixed solution was transferred to a freeze-drying tray, maintained for 12 to 24 hours under rapid-freezing conditions (below -10 °C), and then thawed in a freeze dryer to remove moisture.

That is, in Comparative Example 2, trisodium citrate was omitted when preparing the coating solution compared to Example 1.

Experimental example One: trisodium citrate High-temperature stability evaluation with and without (storage at 40℃ for 6 weeks)

The freeze-dried lactic acid bacteria powder gradually decreases in activity depending on the storage temperature and storage period. In general, factors affecting activity include temperature, oxygen, and moisture. First, the freeze-dried lactic acid bacteria powder has very strong hygroscopicity, so a lot of content reduction occurs at the beginning of storage. There are various methods for applying a deoxidizer or dehumidifying the packaging material to improve distribution and storage, but ultimately, there is a lot of difference in the storage period depending on the degree of coating of the lactic acid bacteria powder itself. Therefore, in order to alleviate the hygroscopicity due to the characteristics of the raw material, excipients such as glucose and dextrin are mixed and stored within the range of 1 to 10 times compared to the powder. In this experiment, an excipient in which maltodextrin and anhydrous crystalline glucose were mixed in a 1:1 ratio was mixed with powder and mixed excipient in a ratio of 1:9 and stored. In order to exclude air permeability during the storage period, they were individually packaged and stored in an aluminum pouch, and the survival rate under severe conditions was analyzed by storing them at 40°C for 4 weeks.

Specifically, the freeze-dried and powdered Comparative Examples 1, 2, and 1 samples were individually packaged and sealed in a predetermined amount in an aluminum pouch package. Each sample was stored in an incubator at 40 °C for 4 weeks. After a certain period of time, the experimental group sample was diluted in Saline Buffer at a ratio of 1:100, put in a sterile bag, and then homogenized. Samples serially diluted with Saline Buffer were plated on MRS agar plates. The plates were collected and counted after being cultured 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 represent the 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 measured before and after comparison. In Example 1 containing trisodium citrate, it was possible to recover lactic acid bacteria at a high rate even after severe conditions, and it was confirmed that the survival rate of lactic acid bacteria was significantly higher compared to Comparative Examples 1 and 2 that did not contain 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: Storage stability evaluation according to the content of trisodium citrate

Experiments in the same manner as in Experimental Example 1 were performed while changing the amount of trisodium citrate in Example 1 from 5% by weight. The contents of trehalose, maltodextrin, soipeptone and trisodium citrate were mixed as follows.

Example 2 Example 3 Example 4 Trehalose 20% by weight 20% by weight 20% by weight maltodextrin 5% by weight 5% by weight 5% by weight Soipeeptone 5% by weight 5% by weight 5% by weight trisodium citrate 0.1% by weight 1% by weight 2.5% by weight

After counting the lactic acid bacteria, the survival rate (%) compared to the initial lactic acid bacteria was calculated and 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 from Table 3, in the case of Example 1 containing the most trisodium citrate, the survival rate at week 1 did not appear to be the highest, but the number of lactic acid bacteria that died over time was the lowest, and the decrease in the survival rate was the most It was confirmed that it appeared low. Therefore, after 4 weeks, the survival rate of the lactic acid bacteria was finally shown to be the highest, and through this, it was confirmed that the storage stability of the lactic acid bacteria was the highest in Example 1, which had the highest content of trisodium citrate.

Experimental Example 3: Freeze-drying yield evaluation according to the content of trisodium citrate

Live lactic acid bacteria go through a drying process to improve storage stability, and the freeze-drying method that receives the least thermal energy and has a high drying yield is preferred. However, even in the freeze-drying process, the death of lactic acid bacteria cannot be prevented 100%, and the yield is affected depending on the composition of the cryoprotectant. The survival rate (%) of the total number of bacteria after drying compared to the total number of lactic acid bacteria before drying was calculated, and the results are shown in Table 4.

Example 2
(trisodium citrate 0.1%) Example 3
(Trisodium citrate 1%) Example 4
(trisodium citrate 2.5%) Example 1
(trisodium citrate 5%) transference number 66.6% 71.9% 88.0% 99.1%

As can be seen from Table 4, as the content of trisodium citrate increased, the survival rate of lactic acid bacteria compared before and after drying increased proportionally. That is, trisodium citrate served to prevent the death of lactic acid bacteria during freeze-drying, suggesting that as the content of trisodium citrate increases, its effect also increases.

Experimental Example 4: Storage stability evaluation with or without trisodium citrate (CJLP243)

Instead of Lactobacillus plantarum CJLP133 Except that the Lactobacillus plantarum CJLP243 strain was used, a composition having the same content as in Comparative Examples 1 and 1 was prepared and Experimental Example 1 was performed in the same manner. The survival rate (%) compared to the initial lactic acid bacteria was calculated and 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 when the experiment was performed by changing the lactic acid bacteria strain to Lactobacillus plantarum CJLP243, the survival rate of lactic acid bacteria was significantly higher in Example 1 containing trisodium citrate, and the time stability over time was also excellent was confirmed. That is, the effect of storage stability according to the content of the trisodium citrate suggests that it can be expressed regardless of the lactic acid bacteria strain.

The above results suggest that citrate can perform cryoprotection while maintaining safety regardless of the type of lactic acid bacteria strain, suggesting that it can be used as a cryoprotection application of excellent lactic acid bacteria that can maintain high lactic acid bacteria survival rate for a long time. will do

From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts thereof.

Claims (16)

citrate and a nitrogen source;
The citrate includes trisodium citrate,
The trisodium citrate is included in an amount of 1% to 5% by weight based on the total weight of the composition for cryoprotection of lactic acid bacteria,
The composition for cryoprotection comprises 15% to 50% by weight of the solid content of lactic acid bacteria,
The lactic acid bacteria are bacteria that obtain energy by fermenting sugars and produce lactic acid, a composition for freeze protection of lactic acid bacteria.
According to claim 1, wherein the citrate further comprises at least one material selected from the group consisting of sodium citrate and potassium citrate, the composition for cryoprotection of lactic acid bacteria.
The composition for cryoprotecting lactic acid bacteria according to claim 1, further comprising at least one selected from the group consisting of a cryoprotectant and a porous support.
The composition for cryoprotection of lactic acid bacteria according to claim 1, further comprising at least one selected from the group consisting of trehalose, maltodextrin and soipeptone.
According to claim 1, wherein the lactic acid bacteria are Lactobacillus genus ( Lactobacillus sp. ), Bifidobacterium genus ( Bifidobacterium sp. ), Streptococcus sp. , Lactococcus sp. , Enterococcus genus Enterococcus sp . _
delete delete According to claim 1, wherein the trisodium citrate is included to be added in an amount of 5 to 25 parts by weight based on 100 parts by weight of the lactic acid bacteria, the composition for cryoprotection of lactic acid bacteria.
(a) culturing lactic acid bacteria in a medium to recover the cells;
(b) preparing a composition comprising citrate and a nitrogen source;
(c) mixing the composition of step (b) with the lactic acid bacteria of step (a); and
(d) lyophilizing the mixture of step (c) to powder,
The citrate includes trisodium citrate,
The trisodium citrate is included in an amount of 1% to 5% by weight based on the total weight of the composition for cryoprotection of lactic acid bacteria,
The composition for cryoprotection comprises 15% to 50% by weight of the solid content of lactic acid bacteria,
The lactic acid bacteria is a bacterium that obtains energy by fermenting sugars and produces lactic acid.
The method of claim 9, wherein the citrate of step (b) further comprises one or more substances selected from the group consisting of sodium citrate and potassium citrate.
The method of claim 9, wherein the composition of step (b) further comprises at least one selected from the group consisting of a cryoprotectant and a porous support.
The method of claim 9, wherein the composition of step (b) further comprises at least one selected from the group consisting of trehalose, maltodextrin and soipeptone.
delete delete (a) culturing lactic acid bacteria in a medium to recover the cells;
(b) preparing a composition comprising citrate and a nitrogen source;
(c) mixing the composition of step (b) with the lactic acid bacteria of step (a); and
(d) freezing or drying the mixture of step (c) to powder it, wherein the citrate contains trisodium citrate,
The trisodium citrate is included in an amount of 1% to 5% by weight based on the total weight of the composition for cryoprotection of lactic acid bacteria,
The composition for cryoprotection comprises 15% to 50% by weight of the solid content of lactic acid bacteria,
The method for preserving lactic acid bacteria, wherein the lactic acid bacteria are bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid.
The method according to claim 15, wherein the drying is air drying, spray drying, vacuum drying or freeze drying.

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