USE OF CYSTEINE OR SALT THEREOF FOR CRYOPROTECTING LACTIC ACID BACTERIA

Abstract

The present application relates to use of cysteine or a salt thereof for the protection, growth acceleration and/or stability improvement of lactic acid bacteria.

Claims

1. A composition comprising cysteine or a salt thereof as an active ingredient.

2-3. (canceled)

4. The composition of claim 1, further comprising at least one selected from the group consisting of trehalose, maltodextrin, and soy peptone.

5-9. (canceled)

10. A method for producing a lactic acid bacteria preparation, comprising: mixing lactic acid bacteria with the composition of claim 1 to prepare a mixture; and freeze-drying the mixture to be powdered.

11. The method for producing a lactic acid bacteria preparation of claim 10, further comprising culturing bacterial cells of the lactic acid bacteria using the mixture before freeze-drying the mixture.

12. A lactic acid bacteria preparation having thermal stability, comprising: lactic acid bacteria; and cysteine or a salt thereof.

13. The lactic acid bacteria preparation of claim 12, wherein the lactic acid bacteria preparation is in the form of a granule, a powder, a powdered drug, a pellet, an infusum, an emulsion, a flow agent, a tablet, a pill, a capsule, an ointment, a suppository, an injection, an inhalant, an aerosol, a suspension, a syrup, an emulsion, a soft capsule, a hard capsule, an elixir, a troche, or a lozenge.

14. The lactic acid bacteria preparation of claim 12, wherein the lactic acid bacteria preparation is in the form of a freeze-dried powder.

15. The lactic acid bacteria preparation of any one of claim 12, wherein the survival rate of the lactic acid bacteria after the lactic acid bacteria preparation is preserved at 40° C. for 4 weeks is 45% or more.

16. A method for accelerating the growth of lactic acid bacteria, comprising culturing lactic acid bacteria in a culture medium comprising the composition of claim 1.

17. A method for increasing the thermal stability of lactic acid bacteria, comprising culturing lactic acid bacteria in a culture medium comprising the composition of claim 1.

18. The composition of claim 1, wherein the composition is for cryoprotecting lactic acid bacteria.

Description

EXAMPLE 1

[0098] A Lactobacillus plantarum CJLP133 strain was cultured at 37° C. for 18 to 24 hours using an MRS liquid medium (Difco, USA), then a supernatant was discarded using a centrifugal separator, and only lactic acid bacteria were collected.

[0099] The lactic acid bacteria cells collected as described above were mixed, in a weight ratio of 1:2, with a composition for cryoprotecting lactic acid bacteria, which was produced by mixing cysteine monohydrochloride, trehalose, maltodextrin, soy peptone, and water in the contents shown in Table 1 below and sterilizing the mixture.

TABLE-US-00001 TABLE 1 Content (wt %) Example Example Example Example Ingredient 1-1 1-2 1-3 1-4 Cysteine 0.1 0.5 1 5 Monohydrochloride Trehalose 20 20 20 20 Maltodextrin 5 5 5 5 Soy Peptone 5 5 5 5 Water Remainder Remainder Remainder Remainder Total 100 100 100 100

[0100] After the mixture was suspended, the resultant was transferred to a freeze-drying tray and maintained for 12 to 24 hours under quick freezing conditions (−40° C. or lower), and then moisture was removed while performing thawing in a freeze dryer, to obtain a lactic acid bacteria powder coated with the composition of Table 1.

EXAMPLE 2

[0101] A Lactobacillus plantarum CJLP133 strain disclosed in Korean Registered Patent Publication No. 1,486,999 was cultured at 37° C. for 18 to 24 hours using an MRS liquid medium (Difco, USA) containing 0.1 wt % of cysteine monohydrochloride, then a supernatant was discarded using a centrifugal separator, and only lactic acid bacteria were collected.

[0102] The lactic acid bacteria cells collected as described above were mixed, in a weight ratio of 1:2, with a composition for cryoprotecting lactic acid bacteria, which was produced by mixing trehalose, maltodextrin, soy peptone, and water in the contents shown in Table 2 below and sterilizing the mixture.

TABLE-US-00002 TABLE 2 Content Ingredient (wt %) Trehalose 20 Maltodextrin 5 Soy Peptone 5 Water Remainder Total 100

[0103] After the mixture was suspended, the resultant was transferred to a freeze-drying tray and maintained for 12 to 24 hours under quick freezing conditions (−40° C. or lower), and then moisture was removed while performing thawing in a freeze dryer, to obtain a lactic acid bacteria powder coated with the composition of Table 2.

EXAMPLE 3

[0104] A Lactobacillus plantarum CJLP133 strain was cultured at 37° C. for 11 hours in an MRS liquid medium (Difco, USA) containing 0.1% of cysteine monohydrochloride. The ingredients of the MRS medium containing cysteine monohydrochloride are as shown in Table 3, and the pH, which was decreased during culturing, was set to be neutralized to a pH of 5.95 through an automatic aqueous ammonia feeding system.

TABLE-US-00003 TABLE 3 Ingredient Content L-Cysteine 1 g Monohydrochloride Proteose Peptone 10 g Beef Extract 10 g Yeast Extract 5 g Dextrose 20 g Polysorbate 80 1 g Ammonium Citrate 2 g Sodium Acetate 5 g Magnesium Sulfate 0.1 g Manganese Sulfate 0.05 g Dipotassium Phosphate 2 g Water 1,000 mL

COMPARATIVE EXAMPLE 1

[0105] A Lactobacillus plantarum CJLP133 strain was cultured at 37° C. for 18 to 24 hours using an MRS liquid medium (Difco, USA), then a supernatant was discarded using a centrifugal separator, and only lactic acid bacteria were collected.

[0106] The lactic acid bacteria cells collected as described above were mixed, in a weight ratio of 1:2, with a composition for cryoprotecting lactic acid bacteria, which was produced by mixing trehalose, maltodextrin, soy peptone, and water in the contents shown in Table 2 and sterilizing the mixture.

[0107] After the mixture was suspended, the resultant was transferred to a freeze-drying tray and maintained for 12 to 24 hours under quick freezing conditions (−40° C. or lower), and then moisture was removed while performing thawing in a freeze dryer, to obtain a lactic acid bacteria powder coated with the composition of Table 2.

COMPARATIVE EXAMPLE 2

[0108] A Lactobacillus plantarum CJLP133 strain was cultured at 37° C. for 11 hours in an MRS liquid medium (Difco, USA) not containing 0.1% of cysteine monohydrochloride. The ingredients of the MRS medium are as shown in Table 4, and the pH, which was decreased during culturing, was set to be neutralized to a pH of 5.95 through an automatic aqueous ammonia feeding system.

TABLE-US-00004 TABLE 4 Ingredient Content Proteose Peptone 10 g Beef Extract 10 g Yeast Extract 5 g Dextrose 20 g Polysorbate 80 1 g Ammonium Citrate 2 g Sodium Acetate 5 g Magnesium Sulfate 0.1 g Manganese Sulfate 0.05 g Dipotassium Phosphate 2 g Water 1,000 mL

EXPERIMENTAL EXAMPLE 1

[0109] Evaluation of high-temperature stability of lactic acid bacteria powder depending on presence or absence of cysteine monohydrochloride

[0110] In order to evaluate the high-temperature stability of the lactic acid bacteria powder to which cysteine monohydrochloride was applied, the survival rates in Example 1-1, Example 2, and Comparative Example 1 under the severe conditions were analyzed. The activity of the freeze-dried lactic acid bacteria powder is gradually decreased depending on the storage temperature and the storage period. The factors, which generally affect the activity, include temperature, oxygen, moisture, and the like. The freeze-dried lactic acid bacteria powder is highly hygroscopic, and thus the content thereof is significantly reduced at the initial stage of storage. In order to improve distribution storage properties, there are various methods for applying a deoxidizer to a packing material or for dehumidification, but there are many differences in the storage period ultimately depending on the degree of stability of the lactic acid bacteria powder. Therefore, the respective samples were individually packaged and preserved in an aluminum pouch in order to alleviate the hygroscopicity caused by the properties of raw materials, and after being preserved at 40° C. for 4 weeks, the survival rate under the severe conditions was analyzed.

[0111] Specifically, certain amounts of the samples of the lactic acid bacteria powders produced in Example 1-1, Example 2, and Comparative Example 1 were put into aluminum pouch packages, the samples were individually packaged and sealed, and each sample was preserved for 4 weeks in an incubator at 40° C. After a predetermined period of time elapsed, the sample of the experimental group was diluted with a saline buffer at a ratio of 1:100, put into a sterilizing bag, and then homogenized. The sample subjected to serial dilution with the saline buffer was smeared on an MRS agar plate. The plate was collected, static culturing was performed for 24 hours under aerobic conditions at 37° C., then the number of bacterial cells was counted, and the results thereof are shown in Table 5 below. The numbers listed in Table 5 below indicate the survival rate (%) compared to the number of lactic acid bacteria at the initial stage.

TABLE-US-00005 TABLE 5 Survival Rate (%) Comparative Example Example Timing Example 1 1-1 2 Initial Stage 100.0 100.0 100.0 First Week 54.7 72.7 89.0 Second Week 35.9 64.9 91.5 Third Week 40.9 52.2 81.8 Fourth Week 38.0 51.0 77.4

[0112] As a result of applying severe conditions and measuring the activity of the lactic acid bacteria over time, it was found that in Example 1-1 in which the composition for cryoprotecting containing cysteine monohydrochloride was used, and Example 2 in which the culture medium containing cysteine monohydrochloride was used, a much higher survival rate of lactic acid bacteria was exhibited, compared to Comparative Example 1 in which cysteine monohydrochloride was not contained.

EXPERIMENTAL EXAMPLE 2

[0113] Evaluation of High-Temperature Stability of Lactic Acid Bacteria Powder According to Content of Cysteine Monohydrochloride

[0114] In order to evaluate the high-temperature stability of the lactic acid bacteria powder, to which cysteine monohydrochloride was applied, according to the content of cysteine monohydrochloride, the survival rates of the lactic acid bacteria powders produced in Example 1-1 to Example 1-4 under the severe conditions were analyzed. The severe conditions were applied in the same manner as in Experimental Example 1, and the activity of the lactic acid bacteria over time was measured, and the results thereof are shown in Table 6 below. The numbers listed in Table 6 below also indicate the survival rate (%) compared to the number of lactic acid bacteria at the initial stage.

TABLE-US-00006 TABLE 6 Survival Rate (%) Example Example Example Example Timing 1-1 1-2 1-3 1-4 Initial Stage 100.0 100.0 100.0 100.0 First Week 72.7 93.0 89.2 91.7 Second Week 64.9 75.2 82.3 88.5 Third Week 52.2 74.3 78.7 81.1 Fourth Week 51.0 73.4 77.5 75.5

EXPERIMENTAL EXAMPLE 3

[0115] Evaluation of High-Concentration Culturing of Lactic Acid Bacteria Depending on Presence or Absence of Cysteine Monohydrochloride in Culture Medium (O.D. Value)

[0116] The evaluation of the lactic acid bacteria culturing was measured by diluting a lactic acid bacteria culture solution 20 times at 600 nm using a spectrophotometer (NanoPhotometer, IMPLEN) according to the method for measuring an optical density (O.D.) value of Korean Food Additives Codex.

TABLE-US-00007 TABLE 7 Comparative Example O.D. Example 2 3  0 hr 0 0  7 hr 0.446 1.282  8 hr 0.536 1.396  9 hr 0.664 1.457 10 hr 0.792 1.528 11 hr 0.937 1.510

[0117] As can be seen in Table 7, it was found that in Example 3 in which culturing is performed in the culture medium containing cysteine monohydrochloride, the culturing rate of lactic acid bacteria was remarkably high, compared to

[0118] Comparative Example 2 in which culturing is performed in the culture medium not containing cysteine monohydrochloride. From the results, it was confirmed that the ability to accelerate the growth of lactic acid bacteria due to cysteine monohydrochloride was excellent.

EXPERIMENTAL EXAMPLE 4

[0119] Evaluation of High-Concentration Culturing of Lactic Acid Bacteria Depending on Presence or Absence of Cysteine Monohydrochloride in Culture Medium (Measurement of Number of Live Bacteria)

[0120] A lactic acid bacteria culture solution was diluted with sterilized physiological water and smeared so that 30 to 300 colonies were formed in an MRS agar plate medium, and then cultured at 37° C. for 24 hours. The number of colonies observed after the culturing was counted and calculated as the number of live bacteria per mL.

TABLE-US-00008 TABLE 8 Comparative Example CFU/ml Example 2 3  0 hr 0 0  7 hr 1.2 × 10{circumflex over ( )}.sup.9 5.9 × 10{circumflex over ( )}.sup.9  8 hr 2.0 × 10{circumflex over ( )}.sup.9 9.2 × 10{circumflex over ( )}.sup.9  9 hr 2.8 × 10{circumflex over ( )}.sup.9 1.0 × 10{circumflex over ( )}.sup.10 10 hr 3.3 × 10{circumflex over ( )}.sup.9 1.2 × 10{circumflex over ( )}.sup.10 11 hr 4.2 × 10{circumflex over ( )}.sup.9 1.2 × 10{circumflex over ( )}.sup.10

[0121] It was found that in Example 3 in which culturing is performed in the culture medium containing cysteine monohydrochloride, the number of live lactic acid bacteria was remarkably high, compared to Comparative Example 2 in which culturing is performed in the culture medium not containing cysteine monohydrochloride. From the results, it was confirmed that the ability to accelerate the growth of lactic acid bacteria due to cysteine monohydrochloride was excellent. Hereinbefore, the preferred Examples of the present application have been exemplarily described, but the scope of the present application is not limited to only the specific Examples described above, and could be appropriately modified by a person with ordinary skill in the art within the scope described in the claims of the present application.