Composition comprising lactic acid bacteria improved in intestinal adherence by coating with silk fibroin

Abstract

The present invention relates to a method for improving lactic acid bacteria in survival rate, storage stability, resistance to acid or bile, and adherence to intestinal epithelial cells by coating the surface of lactic acid bacteria with silk fibroin, and a lactic acid bacteria composition prepared therethrough. Conventional techniques construct only a physical protective barrier outside a lactic acid bacteria body by multi-stage coating and thus retain the limitation of being unable to identify an effect on the coherence of lactic acid bacteria to intestinal epithelial cells upon the uptake of the lactic acid bacteria. In contrast, the present invention provides a method in which lactic acid bacteria is coated with silk fibroin extracted from cocoons, whereby the lactic acid bacteria is improved in stability under a storage and distribution condition as well as having remarkably increased stability and intestinal adherence particularly under an intestinal environment.

Claims

1. A composition comprising lactic acid bacteria coated with silk fibroin.

2. The composition of claim 1, wherein the lactic acid bacteria are coated with silk fibroin and cellulose.

3. The composition of claim 1, wherein the silk fibroin is pre-treated with ethanol.

4. The composition of claim 3, wherein the ethanol has a concentration of 85% (v/v) or more.

5. The composition of claim 1, wherein the lactic acid bacteria are selected from the group consisting of the genera Lactobacillus, Lactococcus, Enterococcus, Streptococcus, and Bifidobacterium.

6. The composition of claim 5, wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Bifidobacterium bifidum, and Bifidobacterium lactis.

7. The composition of claim 6, wherein the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus CKDB007 (Accession No.: KCTC13117BP), Enterococcus faecium CKDB003 (Accession No.: KCTC13115BP), Streptococcus thermophilus CKDB021 (Accession No.: KCTC13118BP), Bifidobacterium bifidum CKDB001 (Accession No.: KCTC13114BP), and Bifidobacterium lactis CKDB005 (Accession No.: KCTC13116BP).

8. The composition of claim 1, wherein the composition is selected from the group consisting of a food composition, a probiotic composition, a pharmaceutical composition, and a feed composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a diagram showing a process for manufacturing lactic acid probiotics by using silk fibroin.

(2) FIGS. 2A and 2B show comparisons of the carbon source consumption pattern and culture characteristics when silk fibroin was used for a medium for lactic acid bacteria.

(3) FIG. 3 provides images obtained by observing the morphology of lactic acid probiotics manufactured using silk fibroin and cellulose through EBSD/FE-SEM (scanning electron microscope detector).

(4) FIG. 4 shows comparisons of zeta-potential of lactic acid probiotics coated with silk fibroin according to artificial gastric liquid and artificial intestinal liquid conditions.

(5) FIG. 5 shows a comparison of binding ability with mucin in respective coating conditions using silk fibroin and cellulose.

(6) FIG. 6 shows a comparison of binding ability with intestinal epithelial cells (HT-29) in respective coating conditions using silk fibroin and cellulose.

DETAILED DESCRIPTION

(7) Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

(8) Throughout the present specification, the term “%” used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

EXAMPLES

(9) The present inventors conducted the following tests to investigate the effects of the addition and coating of silk fibroin of the present invention on lactic acid bacteria. FIG. 1 is a process diagram showing the order and contents of the tests conducted by the present inventors.

Example 1: Isolation and Purification of Silk Fibroin Component of the Present Invention

(10) Silk is a giant protein that can be obtained from nature, and silk fibroin is composed of 18 types of amino acids among 20 types of amino acids constituting human proteins. Liquid silk, which is bio-synthesized from silk glands of silkworms consisting of 18 types of natural amino acids, has a fiber arrangement with a high degree of crystallization through silkworm vomiting and consists of two components, fibroin (approximately 75%) and sericin (approximately 25%).

(11) Methods for obtaining silk fibroin may be largely divided into (i) acid hydrolysis and (ii) protein hydrolysis by a calcium chloride solution and an enzyme. The silk fibroin used in the coating of lactic acid bacteria cell in the present invention was obtained by a refining process of separating and removing the sericin component from cocoons obtained by growing silkworms (Bombyx hori).

Example 1-1: Acid Hydrolysis

(12) In order to isolate silk fibroin, purified water was warmed (95° C.), and then sodium oleate and Na.sub.2CO.sub.3 were added and completely dissolved. Thereafter, cocoons were added thereto, followed by boiling for about 40 minutes and then dehydration, thereby refining the cocoons. Specifically, for the refining, cocoons, sodium oleate, and Na.sub.2CO.sub.3 were added to the purified water at concentrations of 1.84% (w/v), 0.0092% (w/v), and 0.0055% (w/v), respectively. 2N HCl usually used in acid hydrolysis was added thereto, followed by acid hydrolysis in a temperature condition of 110° C. for 2 hours, and then the solution obtained after the hydrolysis was filtered, and neutralized with an aqueous solution of NaOH. The salts formed during the neutralization process were removed by dialysis the tubing cellulose membrane, thereby manufacturing purified acid-hydrolyzed silk fibroin. It is known that the average molecular weight of the peptides obtained through acid hydrolysis is generally about 200-10,000, and thus silk fibroin, a relatively small peptide, can be obtained.

Example 1-2: Enzymatic Hydrolysis

(13) In order to isolate silk fibroin, the cocoons were refined by the same method as in Example 1-1. Then, Alcalase, Delvorase, Flavourzyme, or Protamax (Vision Biochem Co., Ltd.), which is known as the protease produced by Bacillus licheniformis, Bacillus stearothermophilus, or Aspergillus niger, and Papain T100 (Vision Biochem Co., Ltd.) recovered from papaya were added at a concentration of 5%, followed by the treatment of the cocoons in a temperature condition of 60-80° C. for 6-10 hours. The silk fibroin solution obtained from hydrolysis was exposed to a high-temperature condition of 95° C. for 2 hours to inactivate the protease before use in the lactic acid bacteria coating process. The silk fibroin manufactured by such enzymatic hydrolysis is known to be high in solubility and body absorption rate.

(14) In order to investigate the effects of the silk fibroin of the present invention on the promotion of culturing lactic acid bacteria cells and the cell coating effects, the silk fibroin proteins manufactured by the above methods (acid hydrolysis and enzymatic hydrolysis) were respectively recovered, and then applied to cell culture and cell coating for Enterococcus faecium CKDB003 (Accession No.: KCTC13115BP), Lactobacillus acidophilus CKDB007 (Accession No.: KCTC13117BP), Streptococcus thermophilus CKDB021 (Accession No.: KCTC13118BP), Bifidobacterium lactis CKDB005 (Accession No.: KCTC13116BP), and Bifidobacterium bifidum CKDB001 (Accession No.: KCTC13114BP)

Example 2: Use of Silk Fibroin Component of Present Invention

(15) The silk protein, which contains sericin and fibroin as main components, recovered from the cocoons, is composed of 75% fibroin protein, 25% sericin protein, and about 3% minerals and carbohydrates.

(16) Silk fibroin is classified as a protein, which has the highest purity (97%) among the components present in nature, and the silk protein is composed of peptides in which various amino acids as constituent components of the human proteins and binders of the amino acids are present together.

(17) In particular, glycine, alanine, and serine, which account for the largest proportion of silk fibroin, account for 70-80% of all the amino acids, and the above amino acid components constituting silk fibroin are known to be amino acid components constituting collagen. Therefore, this silk fibroin is strong like collagen, and the constituent amino acids may be used as important nitrogen source components in the culture of lactic acid bacteria.

Example 2-1: Use of Silk Fibroin as Component for Culturing Lactic Acid Bacteria

(18) In order to investigate the effects of silk fibroin of the present invention on the culture characteristics of lactic acid bacteria when the silk fibroin was used as a component for culturing lactic acid bacteria, optimized medium and culture conditions known for respective bacteria species were used. The lactic acid bacteria cultured in the optimal culture composition were used as a control group, and the lactic acid bacteria cultured with addition of silk fibroin in the same culture conditions were used as a test group. The carbon source consumption rate (%) and viable cell count over time were compared between the control group and the test group.

(19) The results are shown in FIGS. 2a and 7b.

(20) As shown in FIGS. 2a and 2b, the lactic acid bacteria cultured by addition of silk fibroin of the present invention showed a very fast sugar consumption rate at the early stage of culture compared with the lactic acid bacteria cultured without addition of fibroin (FIG. 2a). It was also verified that culturing of the lactic acid bacteria through addition of silk fibroin shortened the time point showing optimal culture characteristics at the latter stage of culture (FIG. 2b).

(21) It can be therefore seen from the above results that the silk fibroin component of the present invention acts as an important growth factor in the growth of lactic acid bacteria, thereby promoting the culture of lactic acid bacteria and shortening the time of culture. FIG. 1 shows the culture characteristics results of the bacterial species Bifidobacterium lactis CKDB005 (Accession No.: KCTC13116BP). The culture characteristics results of the other bacterial species are not shown, but were verified to show similar effects to the bacterial species Bifidobacterium lactis.

Example 2-2: Use of Water-Soluble Calcium and Silk Fibroin as Components for Culturing Lactic Acid Bacteria

(22) The present inventors verified that when water-soluble calcium and silk fibroin are provided together as medium components for culturing lactic acid bacteria, the culture characteristics of the lactic acid bacteria can be improved, and the lactic acid produced by the lactic acid bacteria forms salts together with water-soluble calcium and silk fibroin and then aggregate, and as a result, the lactic acid bacteria can be stably coated during culture and concentration thereof. Furthermore, in order to establish the optimal conditions for coating the surface of the lactic acid bacteria cells with silk fibroin and investigate the coating effect of the lactic add bacteria with silk fibroin on the stability of the lactic acid bacteria, the following test was conducted.

(23) First, the silk fibroin used in the culture employed the dried powder prepared through the method in Example 1, and the concentration of silk fibroin added was 0-3% (w/v) relative to the volume of the culture. The water-soluble calcium additionally added employed calcium citrate, calcium hydroxide, calcium chloride, calcium lactate, calcium phosphate dibasic, and calcium phosphate monobasic, which are allowed as food additives. The lactic acid bacteria were cultured while the addition concentration thereof was 0, 0.1, and 0.5% (w/v) each, thereby manufacturing lactic acid probiotics coated with silk fibroin.

(24) Then, comparison was conducted for the viability in freeze-drying and the viability in severe conditions (40° C. and 70-75% humidity) of the manufactured lactic acid probiotics. Cell recovery and freeze-drying were conducted by ordinary methods (cell recovery through centrifugation, quick freezing in a freezer at −40° C., and then freeze-drying in freeze-drying conditions between 0 to −45° C.), and the viability in freeze-drying was determined as a percentage of the viable cell count after freeze-drying divided by the viable cell count before freeze-drying.

(25) Meanwhile, the viability in severe conditions was determined by identifying the viable cell count after the lactic acid probiotics were stored for 4 weeks in severe conditions (40° C. and 70-75% humidity).

(26) TABLE-US-00001 TABLE 1 Viability in freeze-drying (%) Calcium concentration, % (w/v) Strain 0 0.1 1 E.faecium CKDB003 64 71 68 S.thermophilus CKDB021 50 55 51 B.lactis CKDB005 26 31 28 B.bifidum CKDB001 20 24 20 L.acidophilus CKDB007 22 30 24

(27) TABLE-US-00002 TABLE 2 Stability in severe conditions (temperature of 40° C. and 70% humidity) CFU/g Viability (%) Calcium concentration, Calcium concentration, % (w/v) % (w/v) Strain 0 0.1 0.5 0 0.1 0.5 E. faecium CKDB003 1.75E+11 2.03E+11 1.94E+11 76 81 76 S. thermophilus CKDB021 8.82E+10 1.20E+11 1.09E+11 42 45 43 B. lactis CKDB005 3.00E+10 5.25E+10 5.04E+10 27 31 28 B. bifidum CKDB001 9.00E+09 1.32E+10 1.21E+10 20 24 20 L. acidophilus CKDB007 1.65E+10 3.75E+10 3.04E+10 18 25 22

(28) As shown in Tables 1 and 2, it was verified that the addition of the silk fibroin of the present invention together with water-soluble calcium leaded to excellent viability in freeze-drying and viability in severe conditions, and especially, showed most excellent viability of lactic acid bacteria at a calcium concentration of 0.1% (w/v).

Example 2-3: Use of Silk Fibroin in Culture and Coating

(29) After the verification of the improvement in culture characteristics and stability of lactic acid bacteria when silk fibroin and water-soluble calcium were used in a medium for culturing lactic acid bacteria as confirmed in Examples 2-1 and 2-2, lactic acid probiotics were manufactured as below.

(30) For preparation of a control group (uncoated), a separate coating process is not conducted after cell culture and concentration using an optimized culture medium. For a test group, silk fibroin and water-soluble calcium were added together as medium components in the culture of lactic acid bacteria, and then the lactic acid bacteria were cultured and concentrated, and then coated with silk fibroin.

(31) The coating of lactic acid bacteria was conducted by employing an ordinary method (Example 2-2), and the lactic acid probiotics were disrupted using a disruptor, and then applied to respective test examples of Example 4.

Example 3: Manufacture of Lactic Acid Probiotics Coated with Fermented Ethanol-Pre-Treated Silk Fibroin

(32) In order to establish optimal silk fibroin for being coated on lactic acid bacteria cells and investigate the effect on the coating quality of lactic acid bacteria according to the conditions of the applied silk fibroin, the silk fibroin was pre-treated with fermented ethanol. Specifically, the silk fibroin manufactured by enzymatic hydrolysis in Example 1-2 of the present invention was mixed with fermented ethanol at a proportion of 30%, and then the mixture was homogenized in the aseptic condition and room-temperature condition (25° C.), and allowed to stand for 18-24 hours.

(33) In Example 3, the lactic acid bacteria were cultured and coated by the same method in Example 2-2 except that the silk fibroin pre-treated with fermented ethanol as above was used instead of silk fibroin undergoing no pre-treatment.

Example 4: Manufacture of Lactic Acid Probiotics Co-Coated with Silk Fibroin and Cellulose

(34) In order to improve characteristics of lactic acid probiotics coated with the silk fibroin and the resistance thereof against an intestine tract environment, lactic acid bacteria co-coated with the silk fibroin of the present invention and cellulose used as a conventional enteric coating material were manufactured.

(35) Specifically, the dried powder manufactured by the method as in Example 1 was used as the silk fibroin for coating lactic acid probiotics, and the silk fibroin treated with fermented ethanol was added at a ratio of 1-10% (w/v) relative to the volume of the lactic acid bacteria concentrate. As for the cellulose, methylcellulose, carboxymethylcellulose sodium, carboxymethylcellulose calcium, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose phthalate (HPMCP), which are allowed as food additives by the Ministry of Food and Drug Safety of Korea, were applied as a coating agent. As described in the following test examples, the results verified that when lactic acid probiotics were coated by adding HPMCP (Any Coast (medical brand name), Samsung Fine Chemicals) and carboxymethyl cellulose sodium (Samsung Fine Chemicals) at a proportion of 1-10% relative to the volume of the lactic acid concentrate, the viability in freeze-drying and the viability in severe conditions (40° C. and 70% humidity) of the lactic acid bacteria were improved.

Test Examples

(36) The present inventors conducted the following tests as below in order to investigate the characteristics of lactic acid probiotics depending on coating with the silk fibroins manufactured in Examples 2 to 4 and coating method therefor.

(37) The control and test groups used in the following test examples are shown in Table 3.

(38) TABLE-US-00003 TABLE 3 Classification Lactic acid coating method Control group Example 2-3: Non-addition (uncoated control group) Test group 1: Example 2-3: Coated with silk fibroin Test group 2: Example 3: Coated with fermented ethanol-pre-treated silk fibroin Test group 3: Example 4: Co-coated with fermented ethanol-pre-treated silk fibroin and cellulose

Test Example 1: Investigation of Coating Efficiency of Lactic Acid Probiotics Depending on Coating Conditions of Silk Fibroin

(39) The samples of the control group (Example 2-3, lactic acid probiotics of uncoated control group), test group 1 (Example 2-3, lactic acid probiotics coated with silk fibroin), test group 2 (Example 3, lactic acid probiotics coated with fermented ethanol-pre-treated silk fibroin), and test group 3 (Example 4, lactic acid probiotics coated with fermented ethanol-pre-treated silk fibroin and cellulose) as shown in Table 3 were immobilized on respective metal plates by using carbon tapes, platinum-coated by a plasma sputter, and then observed at an accelerated voltage of 10 kV using EBSD/FE-SEM (scanning electron detector).

(40) The results are shown FIG. 3.

(41) As shown in FIG. 3, it was observed that the probiotics manufactured by coating lactic acid bacteria cells with only silk fibroin (test group 1) had favorable coating morphology compared with the control group (uncoated), but had partially uncoated cells. It was also verified that the cells were overall uniformly enclosed in the silk lactic acid probiotics coated with silk fibroin manufactured by fermented ethanol pretreatment (test group 2), and the cells were coated more uniformly in the lactic acid probiotics co-coated with fermented ethanol-treated silk fibroin and cellulose (test group 3) than the lactic acid probiotic coated with only fermented ethanol-treated silk fibroin (test group 2). Therefore, it can be seen that the coating quality was most excellent in the probiotics of test group 1 rather than the control group, test group 2 rather than test group 1, and test group 3 rather than test group 2.

(42) In the present example, the treatment with the fermented ethanol is for inducing silk fibroin to have a β-sheet structure through regeneration and treatment and preventing enzymatic hydrolysis of lactic acid bacteria and silk fibroin in a coating process through inactivation of the enzyme used in the enzymatic hydrolysis.

Test Example 2: Surface Hydrophobicity of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(43) The cell surface hydrophobicity is an indicator to indirectly provide the intestinal adhesion ability of lactic acid bacteria in vitro, and is used as one of primary selection methods to identify the adhesion ability of lactic acid bacteria including Lactobacillus and Bifidobacterium. In order to investigate the hydrophobicity of the lactic acid probiotics coated with silk fibroin, the following test was conducted as below.

(44) Specifically, the uncoated lactic acid probiotics (control group) and lactic acid probiotics manufactured according to respective coating conditions (test groups) were washed two times with 1× phosphate buffered saline (PBS, pH 7.2) and then suspended in 1×PBS to OD.sub.600=0.5 of cells. The lactic acid bacteria samples prepared after the suspension were mixed with added toluene, followed by treatment in a water bath at 37° C. for 20 minutes, and then the toluene was removed, and the OD.sub.600 value of the aqueous solution layer was measured. The hydrophobicity of the lactic acid probiotics was calculated by the following equation.

(45) $\frac{\begin{array}{c}\left(\mathrm{Early}\mathrm{lactic}\mathrm{acid}\mathrm{probiotic}\mathrm{suspension}{\mathrm{OD}}_{600}\right)-\\ \left(\mathrm{lactic}\mathrm{acid}\mathrm{probiotic}\mathrm{supernatant}{\mathrm{OD}}_{600}\right)\end{array}}{\left(\mathrm{Early}\mathrm{lactic}\mathrm{acid}\mathrm{probiotic}\mathrm{suspension}{\mathrm{OD}}_{600}\right)}\times 100=\mathrm{hydrophobicity}\left(\%\right)$

(46) The results are shown in Table 4.

(47) TABLE-US-00004 TABLE 4 Cell surface hydrophobicity (%) Silk fibroin coating (addition of Silk Silk pre-treatment fibroin fibroin with fermented coating coating ethanol after (acid (enzymatic enzymatic Strain Uncoated hydrolysis) hydrolysis) hydrolysis) E.faecium 49 52 64 72 CKDB003 S.thermophilus 57 57 61 67 CKDB021 B.lactis 65 68 70 78 CKDB005 B.bifidum 35 33 38 43 CKDB001 L.acidophilus 50 48 50 52 CKDB007

(48) As shown in Table 4, it was verified that the coating of lactic acid bacteria with silk fibroin showed improved hydrophobicity of cells compared with the uncoated control group. It was also verified that the hydrophobicity of the lactic acid probiotics was overall high in the lactic acid bacteria coated with the silk fibroin manufactured by enzymatic hydrolysis compared with the silk fibroin manufactured by acid hydrolysis, and especially, the hydrophobicity of the cells was further improved when the lactic acid bacteria cells were coated with silk fibroin pre-treated with fermented ethanol.

Test Example 3: Surface Zeta-Potential of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(49) The zeta-potential also called electrokinetic potential refers to a potential difference across a fluidized bed of an electric double layer, generated due to electrodynamic phenomena. The potential of the membrane surface cannot be directly measured, but instead, the zeta potential can be measured through tests to determine electrochemical properties of the surface (Chemistry of the solid-water interface, John Wiley & Sons, Inc, 1992). The zeta potential is an important parameter to determine the stability or aggregation of dispersed particles, and may have an important meaning to determine the effects of products in the body when the lactic acid probiotics were ingested. Regarding microparticles or colloids, as the absolute value of the zeta potential increases, the repulsion between particles is stronger, leading to increased particle stability, but as the zeta potential approaches zero, the particles aggregate more easily. In the present invention, the average value of analysis results obtained from five times of measurements using a zeta potential analyzer using phase analysis light scattering (PALS) was calculated and used.

(50) Specifically, the lactic acid probiotics uncoated (control group), coated with silk fibroin (test group 1), coated with fermented ethanol-pre-treated silk fibroin (test group 2), and coated with silk fibroin and cellulose (test group 3) were manufactured in an aqueous solution phase, which is a test condition artificially simulating an intestinal tract environment, and then the zeta potential values were identified.

(51) The results are shown FIG. 4.

(52) As shown in FIG. 4, it was verified that in artificial gastric liquid conditions (an aqueous solution in conditions of 2.0 g of sodium chloride, 24.0 ml/L diluted hydrochloric acid, and pH 1.2), the lactic acid probiotics coated with silk fibroin or fermented ethanol-pre-treated silk fibroin (test groups 1 and 2) showed small zeta potential values compared with the control group, and the lactic acid probiotics co-coated with pretreated silk fibroin and cellulose showed a smaller zeta potential value (test group 3).

(53) In addition, the zeta potential values of all the test groups showed negative values in artificial intestine liquid conditions (an aqueous solution in conditions of 0.3% bile acid and pH 7.0). It was verified that the zeta potential absolute value was decreased by stages in the lactic acid probiotics coated with silk fibroin or fermented ethanol-pre-treated silk fibroin (test groups 1 and 2) compared with the control group, but the zeta potential absolute value was rather increased in the lactic acid probiotics co-coated with pre-treated silk fibroin and cellulose. These results indirectly indicate that in artificial intestine liquid conditions, the hydrophobicity of the lactic acid bacteria cells coated with silk fibroin was increased and the hydrophobicity of the lactic acid bacteria cells co-coated with silk fibroin and cellulose was decreased.

(54) Therefore, it was experimentally verified through the measurement of zeta potential values that the lactic acid probiotics co-coated with silk fibroin and cellulose of the present invention showed high viability of lactic acid bacteria cells through strong aggregation in artificial gastric liquid conditions, and when the probiotics reached the small and large intestines, the cellulose layer was eluted to lower the zeta potential absolute value, and thus the silk fibroin-coated lactic acid bacteria showed increased intestine adhesion, thereby increasing the likelihood of stable survival in the intestinal tract environment.

Test Example 4: Mucin-Binding Ability of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(55) The adhesion ability of microorganisms is associated with electrostatic balances, Van der Waals bonds, and hydrophobicity of cell walls. The hydrophobicity is known to be an important factor for bacteria cells to adhere to mucosal or epithelial cells (Environ Microbiol, 2000, Vol. 66(6), pp. 2548-2554; and International Dairy Journal, 2005, Vol. 15, pp. 1289-1297).

(56) Especially, intestinal epithelial cells produce mucin, a gel-like substance, to form a membrane protecting barriers. Since mucin, an intestinal mucosa component, enables hydrophobic binding with lactic acid bacteria, lactic acid bacteria having high hydrophobic characteristics are also expected to have excellent intestinal adhesion. In the present invention, the binding ability of lactic acid bacteria cells to mucin was investigated by evaluating the adhesion ability of the respective lactic acid bacteria to mucin, an intestinal mucosal component. The mucin adhesion ability was tested by applying Munoz-Provencio (Gastroenterology. 1998, Vol. 115, pp 874-882).

(57) First, porcine stomach mucin, type II (sigma) was dispensed 200 μL each in the Maxisorb plate known as ELISA plate, and allowed to adhere thereto at 4° C. for 24 hours. Then, as shown in Table 3, the lactic acid probiotics coated with silk fibroin and the uncoated probiotics were suspended to reach OD.sub.600=0.5, and then dispensed 200 μL each in the Maxisorb plate coated with mucin, followed by incubation at 4° C. overnight (12 hours or longer). After the incubation, the lactic acid bacteria cells not adhering to mucin were removed by washing five times using a PBS buffer, and stained with crystal violet, and then the absorbance was measured at OD.sub.620.

(58) The results are shown FIG. 5.

(59) As shown in FIG. 5, it was verified that the mucin binding ability was increased in the order of the control group, test group 1, test group 3, and test group 2; the mucin binding ability was significantly increased by pre-treatment with silk fibroin; and the mucin binding ability of the lactic acid bacteria co-treated with cellulose and silk fibroin was slightly decreased compared with the mucin binding ability of the lactic acid probiotics coated with the pre-treated silk fibroin.

Test Example 5: Intestinal Epithelial Cell Adhesion Ability of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(60) In order investigate the intestinal epithelial cell adhesion ability of the lactic acid probiotics manufactured through silk fibroin coating, the adhesion ability of the lactic acid bacteria was investigated using human colon sarcoma cell line HT-29 cells.

(61) The DMED medium supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) was used as a medium for culturing HT-29 cell line. HT-29 cells forming a monolayer in the medium were washed with PBS buffer, and dispensed at 5×10.sup.8 cells/ml in the 6-well plates. The respective lactic acid probiotics were suspended in PBS to a concentration of 1×10.sup.9/ml, seeded in the well plates, and then cultured with HT-29 cells for 2 hours in conditions of CO.sub.2 5% and 37° C. After the culture, the non-adhering lactic acid bacteria were removed by washing with PBS five times, and then treated with 0.05% trypsin-0.02% EDTA for 2 minutes to separate HT-29 cells and the lactic acid bacteria adhering to the well plates. The separated cells were diluted to a decimal scale using dilution water, and cultured in MRS or BL agar plates, and then the viable cell count was measured (Trends. Food. Sci. Technol., 1999, Vol. 10, pp 405-410; and Korean Soc. Food. Sci. Nutr., 2016, Vol. 45, pp 12-19).

(62) The epithelial cell adhesion rate was calculated using the following equation.

(63) $\mathrm{Intestin}\mathrm{adhesion}\mathrm{rate}\left(\%\right)=\frac{\mathrm{Adhering}\mathrm{cell}\mathrm{count}\left(\mathrm{CFU}\right)}{\mathrm{Initial}\mathrm{viable}\mathrm{cell}\mathrm{count}\mathrm{in}\mathrm{suspension}\left(\mathrm{CFU}\right)}\times 100$

(64) Additionally, in order to visually investigate the adhesion degree between the intestinal epithelial cells and lactic acid bacteria cells, the lactic acid bacteria cells were fluorescent-stained using the LIVE/DEAD® BacLight™ Bacteial Viability kit, and then observed by an optical microscope.

(65) The results of intestinal epithelial cell adhesion rate are shown in FIG. 6.

(66) As shown in FIG. 6, it was investigated that the adhesion ability to HT-29 cell line was increased in the coating with silk fibroin compared with the control group, and further increased in the coating with pre-treated silk fibroin. However, it was verified that the binding ability to HT-29 cells was slightly reduced in the lactic acid probiotics co-coated with pre-treated silk fibroin and cellulose together compared with the lactic acid probiotics manufactured by coating with pre-treated silk fibroin.

Test Example 6: Intestine Tract Environment Stability of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(67) In order to evaluate the intestine tract environment stability of the lactic acid probiotics depending on the coating method of silk fibroin, the resistance against acid and resistance against bile of the probiotic powder materials were tested.

(68) In order to determine the resistance against acid, the probiotics were exposed to artificial gastric liquid conditions of pH 2.5 and pH 2.0, and then the vial cell count was analyzed. Specifically, the artificial gastric liquid conditions were adjusted to final pH 2.0 and pH 2.5 by using the artificial gastric liquid conditions on the food disintegration test (2.0 g of sodium chloride, 24.0 ml/L dilute hydrochloric acid, pH 1.2), and then the probiotic powder was added at a concentration of 10% and then exposed. Considering gastric contraction, the reciprocating motion was performed 100 times per minute using the dancing machine equipment (BMS Co., Ltd.) so that the probiotics were exposed to conditions similar to the gastric tract environment, and the exposure time was set to 2 hours considering the gastric passage time. The test groups exposed to the artificial gastric liquid conditions were readjusted to pH 7.0, and then analyzed by an ordinary viable cell count measurement method.

(69) Meanwhile, for the determination of resistance against bile, a medium prepared by filtering 0.5% bile acid and aseptically adding the bile acid was used, and a probiotic powder was added to a concentration of 10%, followed by incubation for 2 hours, and then the viable cell count was measured by an ordinary method.

(70) The results are shown in Table 5 (viability in intestine tract environment (%) depending on coating method of lactic acid bacteria).

(71) TABLE-US-00005 TABLE 5 Resistance against Resistance against acid bile Lactic acid Artificial Artificial Artificial bacteria gastric gastric intestine coating liquid, liquid, liquid (0.5% Strain method pH 2.0 pH 2.5 oxigall) E. faecium Uncoated 38 49 41 CKDB003 Test group 1 42 62 62 Test group 2 51 73 65 Test group 3 78 92 72 S. thermophilus Uncoated 18 24 32 CKDB021 Test group 1 43 59 38 Test group 2 58 73 50 Test group 3 72 88 53 B. lactis Uncoated 34 45 47 CKDB005 Test group 1 58 53 65 Test group 2 64 73 78 Test group 3 81 94 73 B. bifidum Uncoated 30 41 46 CKDB001 Test group 1 46 58 58 Test group 2 48 61 64 Test group 3 61 72 65 L. acidophilus Uncoated 34 38 60 CKDB007 Test group 1 42 61 68 Test group 2 58 75 78 Test group 3 79 94 75

(72) As shown in Table 5, it was investigated that the resistance against acid and the resistance against bile of the lactic acid probiotics were increased overall by coating lactic acid bacteria with fermented ethanol-pre-treated silk fibroin. It was verified that in the pH 2.0 condition, all the S. thermophilus CKDB021, B. bifidum CKDB001, E faecium CKDB003, B. lactis CKDB005, and L. acidophilus CKDB007 strains showed an increase effect in the resistance against acid by at least two-fold in test group 3 compared with the uncoated group. In artificial intestine liquid conditions, the resistance against bile also tended to increase overall by silk fibroin coating, and the B. lactis CKDB005 and L. acidophilus CKDB007 strains showed the most excellent viability when coated in the conditions of test group 2. It was therefore verified that the resistance against acid or the resistance against bile of the lactic acid probiotics was significantly increased when the lactic acid bacteria were coated with silk fibroin or fermented ethanol-pre-treated silk fibroin or co-coated with such silk fibroin and cellulose.

Test Example 7: Storage Stability of Lactic Acid Bacteria Cells Depending on Coating Method of Silk Fibroin

(73) In lactic acid probiotics, at least a certain count of viable cells need to be maintained and preserved during a distribution period of from 12 months to 24 months.

(74) In general, diplococcus type lactic acid bacteria, such as Enterococcus-based, for example, E. faecium and E. faecalis, show high stability when exposed to high-temperature and high-humidity conditions, but most of the lactic acid bacteria show a significantly decreased trend in stability when exposed to high temperature and humidity conditions.

(75) In the present invention, in order to investigate the storage stability of the lactic acid probiotics, the lactic acid probiotics were subdivided into a polyethylene bag (inner) and an aluminum bag (outer) in 50 g each, and stored in severe conditions (4500, 75% humidity), and the samples were collected in each storage time to check the viable cell count.

(76) The results are shown in Table 6 (storage stability (%) depending on coating method of lactic acid bacteria).

(77) TABLE-US-00006 TABLE 6 Severe conditions (40° C., 75%) Lactic acid Viability by storage period bacteria coating (%) Silk fibroin method 4 weeks 8 weeks 12 weeks E.faecium Control group 76 52 43 CKDB003 Test group 1 92 81 72 Test group 2 96 92 89 Test group 3 99 96 95 S.thermophilus Control group 42 33 20 CKDB021 Test group 1 48 40 27 Test group 2 62 51 48 Test group 3 83 72 68 B.lactis Control group 27 21 18 CKDB005 Test group 1 53 48 45 Test group 2 96 95 95 Test group 3 99 96 95 B.bifidum Control group 20 18 16 CKDB001 Test group 1 34 28 20 Test group 2 61 55 52 Test group 3 89 72 54 L.acidophilus Control group 18 18 15 CKDB007 Test group 1 43 38 38 Test group 2 85 78 77 Test group 3 92 90 85

(78) As shown in Table 6, it was verified that B. lactis, E. faecium, and L. acidophilus strains showed significantly increased stability in severe conditions through coating using silk fibroin, and besides, B. bifidum and S. thermophilus stains also showed slightly improved stability in severe conditions.

(79) [Accession Numbers]

(80) Depository institution name: Korea Research Institute of Bioscience and Biotechnology

(81) Accession number: KCTC13114BP

(82) Deposit date: 2016.09.23

(83) Depository institution name: Korea Research Institute of Bioscience and Biotechnology

(84) Accession number: KCTC13115BP

(85) Deposit date: 2016.09.23

(86) Depository institution name: Korea Research Institute of Bioscience and Biotechnology

(87) Accession number: KCTC13116BP

(88) Deposit date: 2016.09.23

(89) Depository institution name: Korea Research Institute of Bioscience and Biotechnology

(90) Accession number: KCTC13117BP

(91) Deposit date: 2016.09.23

(92) Depository institution name: Korea Research Institute of Bioscience and Biotechnology

(93) Accession number: KCTC13118BP

(94) Deposit date: 2016.09.23