COATING METHOD OF LACTIC ACID BACTERIA WITH INCREASED INTESTINAL SURVIVAL RATE

Abstract

This application relates to a coating method of lactic acid bacteria and a lactic acid bacteria complex produced by the coating method, the coating method comprising: (a) a step of culturing lactic acid bacteria in a medium including casein and coating the lactic acid bacteria with casein; (b) a step of mixing the casein-coated lactic acid bacteria with a solution comprising a coating agent, an edible oil, an extracellular polymeric substance (EPS) of Lactobacillus plantarum and alginic acid; and (c) a step of adding the mixture of step (b) to a calcium-containing solution to form alginic acid-calcium beads, wherein the alginic acid-calcium beads contain the casein-coated lactic acid bacteria, the coating agent, the edible oil, and the EPS of Lactobacillus plantarum.

Claims

1. A method for coating a lactic acid bacterium, comprising: (a) culturing a lactic acid bacterium in a medium containing casein to coat the lactic acid bacterium with the casein; (b) mixing the casein-coated lactic acid bacterium with a solution comprising a coating agent, an edible oil or fat, extracellular polymeric substances (EPSs) of Lactobacillus plantarum, and alginic acid; and (c) adding the mixture to a calcium-containing solution to form calcium alginate beads, Wherein the calcium alginate beads contain the casein-coated lactic acid bacterium, the coating agent, the edible oil or fat, and the EPSs of Lactobacillus plantarum.

2. The method for coating a lactic acid bacterium according to claim 1, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., Leuconostoc sp., and Weissella sp.

3. The method for coating a lactic acid bacterium according to claim 1, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Streptococcus faecalis, and Lactococcus lactis subsp. lactis.

4. The method for coating a lactic acid bacterium according to claim 1, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus plantarum CJLP243, Lactobacillus plantarum CJLP133 Lactobacillus plantarum CJLP136, Lactobacillus plantarum CJLP55, and Lactobacillus plantarum CJLP56.

5. The method for coating a lactic acid bacterium according to claim 1, wherein the casein-containing medium is a medium comprising defatted milk.

6. The method for coating a lactic acid bacterium according to claim 1, wherein the coating agent is selected from the group consisting of porous polymers, proteins, thickening polysaccharides, and mixtures thereof.

7. The method for coating a lactic acid bacterium according to claim 1, wherein the solution used in step (b) further comprises a prebiotic.

8. The method for coating a lactic acid bacterium according to claim 1, wherein the alginic acid is in the form of an aqueous solution of 2 wt % to 4 wt % of sodium alginate and the ratio of the weight of the alginic acid solution to the weight of the casein-coated lactic acid bacterium is from 1:1 to 10:1.

9. The method for coating a lactic acid bacterium according to claim 1, wherein the Lactobacillus plantarum is Lactobacillus plantarum CJLP243.

10. The method for coating a lactic acid bacterium according to claim 1, further comprising: freeze-drying the calcium alginate beads containing the casein-coated lactic acid bacterium, the coating agent, the edible oil or fat, and the EPSs of Lactobacillus plantarum.

11. The method for coating a lactic acid bacterium according to claim 1, wherein the solution used in step (b) further comprises a cryoprotectant.

12. A lactic acid bacterium complex comprising a casein-coated lactic acid bacterium, a coating agent, an edible oil or fat, EPSs of Lactobacillus plantarum, and calcium alginate beads.

13. The lactic acid bacterium complex according to claim 12, wherein the calcium alginate beads contain the casein-coated lactic acid bacterium and the EPSs of Lactobacillus plantarum.

14. The lactic acid bacterium complex according to claim 12, further comprising at least one additive selected from the group consisting of prebiotics and cryoprotectants.

15. The lactic acid bacterium complex according to claim 12, wherein the coating agent is selected from the group consisting of porous polymers, proteins, thickening polysaccharides, and mixtures thereof.

16. The lactic acid bacterium complex according to claim 12, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus sp., Leuconostoc sp., and Weissella sp.

17. The lactic acid bacterium complex according to claim 12, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Streptococcus faecalis, and Lactococcus lactis subsp. lactis.

18. The lactic acid bacterium complex according to claim 12, wherein the lactic acid bacterium comprises at least one bacterial species selected from the group consisting of Lactobacillus plantarum CJLP243, Lactobacillus plantarum CJLP133 Lactobacillus plantarum CJLP136, Lactobacillus plantarum CJLP55, and Lactobacillus plantarum CJLP56.

19. The lactic acid bacterium complex according to claim 12, wherein the Lactobacillus plantarum is Lactobacillus plantarum CJLP243.

Description

DESCRIPTION OF DRAWINGS

[0058] FIG. 1 is a flowchart illustrating a method for preparing a lactic acid bacterium complex according to one embodiment of the present invention.

MODE FOR INVENTION

[0059] The present invention will be explained in detail with reference to the following examples. However, these examples are merely illustrative and are not to be construed as limiting the scope of the invention.

EXAMPLE 1

Preparation of Lactic Acid Bacterium-Containing complex

[0060] Lactobacillus plantarum CJLP243 was cultured in MRS liquid medium (Difco, USA) supplemented with 0.02 wt % of defatted milk at 37 C. for 18 to 24 hours. The culture was centrifuged. The supernatant was discarded and only the casein-coated lactic acid bacterium was collected.

[0061] Thereafter, about 15 wt % of trehalose as a cryoprotectant, about 15 wt % of maltodextrin as a coating agent, about 4 wt % of defatted milk, about 0.01 wt % of xanthan gum as another coating agent, and 2 wt % of fructooligosaccharide as a prebiotic relative to the weight of the bacterium were mixed together. Water was added to the mixture and sterilized. The collected lactic acid bacterium was mixed with the sterilized solution and the mixture was suspended.

[0062] Then, 2 wt % of sodium alginate was dissolved in water to prepare an alginic acid solution. The suspension was mixed with the alginic acid solution in amounts such that the ratio of the weight of the alginic acid solution to the weight of the lactic acid bacterium was 1:4.

[0063] Solid saturated fats, including palm oil, were dissolved in water by heating to 100 C. To the solution was added 0.2 wt % of soybean lecithin relative to the weight of the solution. The resulting mixture was emulsified using a homogenizer to prepare an about 10 wt % emulsion. At the final stage of emulsification, the solid saturated fats and the soybean lecithin emulsion were added together with EPSs in a weight ratio of 1:0.5 relative to the weight of the lactic acid bacterium. The EPSs were prepared by the following procedure. First, 3 wt % of glucose was added to MRS broth medium (Difco) and sterilized. Lactobacillus plantarum CJLP243 was cultured in the medium at 30 C. until the glucose concentration was reduced to <0.01 wt %. Thereafter, the culture was centrifuged, and the filtrate was disinfected and concentrated or processed into a powder. The concentrate or culture broth was mixed with a 7- to 10-fold larger amount of dextrin based on solid content, dried, and processed into a powder before use. The concentrate or powder of the EPSs was suspended with the lactic acid bacterium in a concentration ratio of 1:0.2. The suspension was maintained at 30 C. before coating to maintain its viscosity at an appropriate level. A 100 mM calcium lactate solution was stirred in a glass beaker. A 10 mL syringe was filled with the lactic acid bacterium-containing mixture and the lactic acid bacterium-containing mixture was directly dropped into the calcium lactate solution by pressurizing the syringe. As a result of the reaction, beads were formed. After the formation of beads was finished, the bath containing the beads was cooled to 4 C. and stored for 30 min. The beads were collected through a 100-mesh sieve and washed twice with distilled water. The collected particles were transferred to and placed on a freeze-drying tray, maintained under rapid freezing conditions (40 C.) for 12 to 24 hours, and thawed in a freeze-dryer to remove moisture. As a result, a lactic acid bacterium complex was prepared in the form of a dry powder. The lactic acid bacterium complex had a structure in which the lactic acid bacterium was encapsulated in the calcium alginate beads.

COMPARATIVE EXAMPLE 1

Preparation of Freeze-Dried Lactic Acid Bacterium

[0064] Lactobacillus plantarum CJLP243 was cultured in MRS liquid medium (Difco, USA) at 37 C. for 18 to 24 hours. The culture was centrifuged. The supernatant was discarded and only the lactic acid bacterium was collected. About 15 wt % of trehalose relative to the weight of the bacterium was dissolved in water and sterilized. The lactic acid bacterium was mixed with the cryoprotectant and the mixture was suspended. The lactic acid bacterium was collected using a centrifuge. The collected lactic acid bacterium was transferred to and placed on a freeze-drying tray, maintained under rapid freezing conditions (40 C.) for 12 to 24 hours, and thawed in a freeze-dryer to remove moisture.

[0065] That is, Comparative Example 1 was distinguished from Example 1 in that the steps of culturing in the casein-containing medium, mixing with the coating agents and the prebiotic, mixing with the alginic acid solution, mixing with the edible oil or fat, mixing with the EPSs, and forming calcium alginate beads were omitted.

[0066] COMPARATIVE EXAMPLE 2

Preparation of Freeze-Dried and Casein-Coated Lactic Acid Bacterium (Including Treatment With Coating Agents)

[0067] Lactobacillus plantarum CJLP243 was cultured in MRS liquid medium (Difco, USA) supplemented with 0.02 wt % of defatted milk at 37 C. for 18 to 24 hours. The culture was centrifuged. The supernatant was discarded and only the casein-coated lactic acid bacterium was collected.

[0068] Thereafter, about 15 wt % of trehalose as a cryoprotectant, about 15 wt % of maltodextrin as a coating agent, about 4 wt % of defatted milk, and about 0.01 wt % of xanthan gum as another coating agent relative to the weight of the bacterium were mixed together. The mixture was dissolved in water and sterilized. The collected lactic acid bacterium was mixed with the sterilized solution and the mixture was suspended. The lactic acid bacterium was collected using a centrifuge. The collected lactic acid bacterium was transferred to and placed on a freeze-drying tray, maintained under rapid freezing conditions (40 C.) for about 12 to 24 hours, and thawed in a freeze-dryer to remove moisture.

[0069] That is, Comparative Example 2 was distinguished from Example 1 in that the steps of mixing with the prebiotic, mixing with the alginic acid solution, mixing with the edible oil or fat and the EPSs, and forming calcium alginate beads were omitted.

COMPARATIVE EXAMPLE 2

Preparation of Freeze-Dried Complex in Which Casein-Coated Lactic Acid Bacterium Was Present in Calcium Alginate Beads (Including Treatment With Coating Agents, Edible Oil or Fat, and Prebiotic but Without Treatment With EPSs)

[0070] Lactobacillus plantarum CJLP243 was cultured in MRS liquid medium (Difco, USA) supplemented with 0.02 wt % of defatted milk at 37 C. for 18 to 24 hours. The culture was centrifuged. The supernatant was discarded and only the casein-coated lactic acid bacterium was collected.

[0071] Thereafter, about 15 wt % of trehalose as a cryoprotectant, about 15 wt % of maltodextrin as a coating agent, about 4 wt % of defatted milk, about 0.01 wt % of xanthan gum as another coating agent, and 2 wt % of fructooligosaccharide as a prebiotic relative to the weight of the bacterium were mixed together. The mixture was dissolved in water and sterilized.

[0072] The collected lactic acid bacterium was mixed with the sterilized solution and the mixture was suspended. Then, 2 wt % of sodium alginate was dissolved in water to prepare an alginic acid solution. The suspension was mixed with the alginic acid solution in amounts such that the ratio of the weight of the alginic acid solution to the weight of the lactic acid bacterium was 1:4. Solid saturated fats, including palm oil, were dissolved in water by heating to 100 C. To the solution was added 0.2 wt % of soybean lecithin relative to the weight of the solution. The resulting mixture was emulsified using a homogenizer to prepare an about 10 wt % emulsion. The emulsion was cooled to 50 C. At the final stage of emulsification, the solid saturated fats and the soybean lecithin were added to the suspension in a weight ratio of 1:0.5 relative to the weight of the lactic acid bacterium. The suspension was maintained at 30 C. to maintain its viscosity at an appropriate level. A 100 mM calcium lactate solution was stirred in a glass beaker. A 10 mL syringe was filled with the lactic acid bacterium-containing mixture and the lactic acid bacterium-containing mixture was directly dropped into the calcium lactate solution by pressurizing the syringe. As a result of the reaction, beads were formed. After formation of beads was finished, the bath containing the beads was cooled to 4 C. and stored for 30 min. The beads were collected through a 100-mesh sieve and washed twice with distilled water. The collected particles were transferred to and placed on a freeze-drying tray, maintained under rapid freezing conditions (40 C.) for about 12 to 24 hours, and thawed in a freeze-dryer to remove moisture.

[0073] That is, Comparative Example 3 was distinguished from Example 1 in that the step of mixing with the EPSs was omitted.

[0074] The lactic acid bacterium complexes prepared in Example 1 and Comparative Example 3 and the freeze-dried products of the lactic acid bacterium prepared in Comparative Examples 1 and 2 were evaluated for intestinal survival and storage stability by the following respective procedures. Results are shown in Tables 1 to 4.

EXPERIMENTAL EXAMPLE 1

Evaluation of Intestinal Survival

[0075] Lactic acid bacteria are likely to be killed by various environmental factors in the digestive organs after being eaten. The most important factors are gastric acid from the stomach and bile acid from the duodenum. Specifically, gastric acid directly acts on bacteria to induce their death due to its strong acidity. Bile acid is involved in the killing of bacteria due to the presence of various digestive enzymes (mainly lipases) or the stress of osmotic pressure. A simple model method for evaluating the survival of living bacteria against gastric acid/bile acid is the simulated stomach duodenum passage (SSDP) test proposed by M. G. Vizoso Pinto, C. M. A. P. Franz, U. Schillinger, and W. H. Holzapfel in Lactobacillus spp. with in-vitro prebiotic properties from human faeces and traditional fermented products, International Journal of Food Microbiology, vol. 109, no. 3, pp. 205-214, 2006. According to a major SSDP test, a predetermined concentration of a living bacterium or a powder of a living bacterium was subjected to stationary culture for 1 hour on MRS medium under acidic conditions (pH conditions (pH 3.0) of the stomach after food ingestion) and for an additional 2 hours under bile acid conditions (artificial bile juice, Oxgall/salts), and survival rates of the bacterium were checked every hour. The continuously applied gastric acid-bile acid conditions in the SSDP test are severer for survival of the bacterium than the individual conditions but are more similar to the actual environment of the digestive tract. Particularly, since the dried powders of lactic acid bacterium are deactivated, they are more susceptible to the severe environment of the SSDP test but are considered more suitable as models that are finally eaten.

[0076] Each experimental sample was diluted 1:100 with saline buffer, placed in a sterile bag, and homogenized. The sample was continuously diluted with saline buffer depending on the bacterial mass and plated on MRS agar medium (Agar Plate). The plate was collected. After stationary culture at 37 C. for 24 hours under aerobic conditions, the bacterial number was counted (initial bacterial number data).

[0077] The MRS broth was completely dissolved in distilled water with stirring to a concentration of 55 g/l, adjusted to pH 3.0 with 5 M HCl with stirring, and sterilized (121 C., 15 min), thereby preparing an acidic MRS medium. 50 ml of the acidic MRS medium was plated in a sterile flask, and 1/100 equivalents of the sample was added and dissolved with sufficient shaking. The exact time when the sample was dissolved was checked. The sample was shaken at 80 rpm at 37 C. 1 hour after culture, 1 ml of the sample was continuously diluted with saline buffer and plated on an MRS agar plate. The plate was collected. After stationary culture at 37 C. for 24 hours under aerobic conditions, the bacterial number was counted (1 h data).

[0078] 10 wt % Oxgall solution was prepared by dissolving 10 wt % of Oxgall (Difco) in distilled water. The Oxgall solution was sterilized at 121 C. for 15 min. Immediately after sampling, 20 ml of the sterilized Oxgall solution was added to a flask. Subsequently, 85 ml of a buffer for the artificial bile juice was added and sufficiently shaken. The buffer for the artificial bile juice was prepared by dissolving NaHCO.sub.3 (6.4 g/l), KCl (0.239 g/l), and NaCl (1.28 g/l) in distilled water, adjusting the pH of the solution to 7.4 with 5 M HCl, and sterilizing the pH-adjusted solution at 121 C. for 15 min. Shake culture was performed at 80 rpm and 37 C. Thereafter, samples were taken every hour for 2 h, continuously diluted with the buffer, and plated on MRS agar plates. The plates were collected. After stationary culture at 37 C. for 24 hours under aerobic conditions, the bacterial numbers were counted (2 h and 3 h data).

TABLE-US-00001 TABLE 1 Conditions 2 hr. 3 hr. 1 hr. (gastric (gastric 0 hr. (gastric acid + acid + Decrement (initial) acid) bile acid) bile acid) (Log CFU/g) Comparative 12.23 7.97 6.67 6.9 5.33 Example 1 Comparative 11.02 11.01 8.76 8.69 2.33 Example 2 Comparative 10.60 10.48 10.16 10.21 0.39 Example 3 Example 1 10.44 10.45 10.02 10.16 0.28

TABLE-US-00002 TABLE 2 Conditions 2 hr. 3 hr. 1 hr. (gastric (gastric 0 hr. (gastric acid + acid + Survival (initial) acid) bile acid) bile acid) (%) Comparative 1.70E+12 9.33E+07 4.68E+06 7.94E+06 0.0005 Example 1 Comparative 1.05E+11 1.02E+11 5.75E+08 4.90E+08 0.47 Example 2 Comparative 3.98E+10 3.02E+10 1.45E+10 1.62E+10 41 Example 3 Example 1 2.75E+10 2.82E+10 1.05E+10 1.45E+10 52

[0079] Table 1 shows the log values of the experimental results and Table 2 shows the found experimental results. As for Comparative Example 1, the bacterial numbers decreased by about 4.3 log and about 1.1 log in gastric acid and bile acid, respectively. As for Comparative Example 2, the bacterium was stable in gastric acid but its number decreased by about 2.3 log in bile acid. As for Comparative Example 3, the bacterial numbers decreased by about 0.4 log in gastric acid/bile acid. However, the survival of the bacterium in the complex prepared in Comparative Example 3 was improved by about 2 log (100 times) compared to that of the freeze-dried bacterium prepared in Comparative Example 2. The survival of the bacterium in the complex prepared in Example 1 was improved by 0.1 log compared to that of the bacterium in the complex prepared in Comparative Example 3.

[0080] 2. Evaluation of Storage Stability (at 50 C. for 72 h)

[0081] Lactic acid bacteria in the form of freeze-dried powders gradually lost their activity depending on storage temperature and period. Generally, factors affecting the activity of lactic acid bacteria include temperature, oxygen, and moisture. Freeze-dried powders of lactic acid bacteria primarily undergo a significant reduction in content at the initial stage of storage due to their very high hygroscopicity. Many methods for improving the storage stability of lactic acid bacteria are known, for example, by applying oxygen absorbers to packaging materials or dehumidifying packaging materials. Ultimately, the storage period of powders of lactic acid bacteria greatly depends on how much they are coated. In attempts to reduce hygroscopicity resulting from the characteristics of raw materials, excipients (e.g., glucose and dextrin) are added in 1 to 10-fold larger amounts than powders before storage. In this experiment, a mixture of maltodextrin and anhydrous crystalline glucose (1:1) as excipients was mixed with the powder in a ratio of 3:1 before storage. To secure air tightness during storage, the samples were individually packaged in aluminum pouches before storage. The packaged samples were analyzed for survival during storage under short-term severe conditions (50 C., 3 days).

[0082] A predetermined amount of each of the samples in the form of freeze-dried powders prepared in Comparative Examples 1-3 and Example 1 was packaged and sealed in an aluminum pouch and stored in an incubator at 50 C. for 72 hours. The experimental sample was diluted 1:100 with saline buffer, placed in a sterile bag, and homogenized. The sample continuously diluted with saline buffer and plated on an MRS agar plate. The plate was collected. After stationary culture at 37 C. for 24 hours under aerobic conditions, the bacterial number was counted.

TABLE-US-00003 TABLE 3 Comparative Comparative Comparative Example 1 Example 2 Example 3 Example 1 Initial 10.95 10.72 10.68 10.52 50 C., 72 hr. 4.88 8.75 9.74 9.91 Decrement (Log) 6.07 1.97 0.94 0.61

TABLE-US-00004 TABLE 4 Comparative Comparative Comparative Example 1 Example 2 Example 3 Example 1 Initial 8.90E+10 5.25E+10 4.79E+10 3.31E+10 50 C., 72 hr. 7.60E+04 5.62E+08 5.50E+09 8.13E+09 Survival (%) 0.00009 1.1 11.5 24.6

[0083] The activities of the bacteria before and after storage under short-term severe conditions were measured and compared. Table 3 shows the log values of the experimental results and Table 4 shows the found experimental results. As for Comparative Example 2, a reduction in activity by about 2 log was observed. As for Comparative Example 3, the survival of the bacterium was improved by about 0.9 log. As for Example 1, the survival of the bacterium was further improved to a level of 0.6 log.