Composition comprising lactic acid bacteria having improved intestinal colonization by coating with silk fibroin

Abstract

The present invention relates to a method for improving the survival rate, storage stability, acid tolerance, or bile tolerance and intestinal epithelial cell attachment ability of lactic acid bacteria by applying fibroin to the surface of the lactic acid bacteria, and a lactic acid bacteria composition prepared thereby. The prior art establishes a physical barrier only on the outside of the lactic acid bacteria cell by a multi-step application, and thus has the limitation that the effect of the lactic acid bacteria on the intestinal epithelial cell attachment ability upon ingestion cannot be confirmed, but the present invention is a method for applying lactic acid bacteria using fibroin extracted from a cocoon, thereby not only improving the stability under storage and circulation conditions, but also significantly increasing the stability and colonization ability in the intestinal environment.

Description

Technical Field

[0001] This invention was completed with the support of the Rural Revitalization Agency under project number PJ01128701. The Rural Revitalization Agency was the specialized agency responsible for the research and management of the aforementioned project. The project name was "New Generation Bio-Green 21 Project," and the research topic was "Development of Industrial Production Processes for Cholesterol-Lowering Health Functional Food Materials." The supervising agency was Chungken-do Biotechnology Co., Ltd., and the research period was from January 15, 2015 to December 31, 2017.

[0002] This patent application claims priority to Korean Patent Application No. 10-2017-0045326, filed with the Korean Intellectual Property Office on April 7, 2017, the disclosure of which is incorporated herein by reference.

[0003] This invention relates to a method for improving the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria by coating the surface of lactic acid bacteria with silk fibroin, and to lactic acid bacteria compositions prepared thereby. Background Technology

[0004] Lactic acid bacteria are bacteria that produce lactic acid using sugars as an energy source. They are found in the digestive tract, mouth, and vagina of humans and other mammals, and are widely distributed in nature, for example, in various fermented products. Lactic acid bacteria are among the most widely used microorganisms by humans, and are purely functional microorganisms that do not produce substances harmful to the intestines of humans or animals and have the function of preventing intestinal putrefaction.

[0005] Currently, lactic acid bacteria are classified into 12 genera (Lactobacillus, Carnobacterium, Atopobium, Lactococcus, Pediococcus, Tetragenococcus, Leuconostoc, Weisella, Oenococcus, Enterococcus, Streptococcus, and Vagococcus). Generally, the lactic acid bacteria we use include Lactobacillus sp. (as bacilli), Lactococcus sp. (as cocci), Streptococcus sp. (as cocci), Leuconostoc sp. (as cocci), and Pedicoccus sp. (as cocci) (BioWave, 2009, Vol.11, No.7, pp.1-20).

[0006] On the other hand, probiotics refer to live microorganisms or foods containing live microorganisms that, when ingested in appropriate amounts, are beneficial to the host (FAO / WHO 2001). They help maintain and regulate the normal levels of gut microbiota in the human body. Representative probiotics include lactic acid bacteria and bifidobacteria. In addition, yeasts (Saccharomyces cerevisiae, Saccharomyces boulardii) and filamentous fungi (Aspergillus oryzae) are also included in probiotics.

[0007] As is well known, the representative effects of probiotics include antibacterial activity, improvement of antibiotic-associated diarrhea, reduction of lactose intolerance, anti-cancer effects, lowering of blood cholesterol, inhibition of Helicobacter pylori in the stomach, allergic colitis, Crohn's disease, reduction of ulcerative colitis, and regulation of immune function (International Dairy Journal, 2007, Vol. 17, pp. 1262-1277). Probiotics can be divided into intestinal regulators or lactic acid bacteria preparations used as human medicines, probiotics used as feed additives, and lactic acid bacteria foods used as health foods. Lactic acid bacteria foods refer to foods in which probiotics such as lactobacillus, lactococcus, and bifidobacteria are mixed and made into powders, granules, tablets, capsules, etc., to make them stable and easy to consume.

[0008] The preparation process of this lactic acid bacteria food includes lactic acid bacteria cultivation, cell recovery, freeze drying, and product commercialization via pulverizer. During the preparation of this lactic acid bacteria food, the lactic acid bacteria are exposed to various physical and chemical stresses. Specifically, the lactic acid bacteria are affected by the osmotic pressure based on concentration during cell recovery. During freeze drying, they are affected by both temperature and osmotic pressure due to ice crystal formation and dehydration. Furthermore, during pulverization and product commercialization, they may be exposed to high temperatures and high pressures. Exposure to air leads to lipid oxidation of the cell membrane, resulting in a decrease in the survival rate of the lactic acid bacteria (Comprehensive Reviews in Food Science and Food Safety, 2004, Vol.3, pp.117-124; Curr. Issues Intest. Microbiol., 2004, Vol.5, pp.1-8; Letters in Applied Microbiology, 1996, Vol.22, No.1, pp.3438).

[0009] Furthermore, when lactic acid bacteria probiotics are exposed to high temperature, humidity, and aerobic conditions during storage or distribution, it has a significant impact on the survival and growth of probiotics (Journal of Functional Foods, 2014, Vol.9, pp.225-241).

[0010] On the other hand, unlike other industrial microbial fermentation products, probiotic products, due to the use of probiotics, are exposed to various stresses in the human body before reaching the intestines after ingestion. For example, they are exposed to a highly acidic environment of around pH 2 and various digestive enzymes in the gastrointestinal tract, and are affected by digestive enzymes and bile acids secreted by the small intestine when they reach the small intestine. Furthermore, even if the ingested probiotic lactic acid bacteria reach the intestines, their growth is inhibited by various harmful components and reactive oxygen species in the intestines, and they must compete with the various existing microorganisms colonizing the intestines and attach to the intestinal epithelial cells (Immunology and Cell Biology, 2000, Vol.78, pp.8088; American Journal of Clinical Nutrition (AJCN), 2001, Vol.73, pp.393S398S(suppl); Probiotic Bacteria and Enteric Infections, 2011, Chap.2, pp.41-63).

[0011] Therefore, there is an urgent need for methods to minimize the decrease in the stability of probiotics due to environmental factors such as osmotic pressure, temperature, pressure, humidity, or exposure to air, and to minimize the bacteria killed by stress caused by exposure to the intestinal environment after ingestion, and to solve the problem of growth inhibition in the gut.

[0012] To address the problems mentioned above, various methods for coating lactic acid bacteria have been developed. Initially, methods utilizing enteric coatings from capsules, microencapsulation methods using gelatin, polysaccharides, and gels were developed. However, these methods suffer from the use of expensive coating agents or the need for additional steps. To improve these issues, methods for preparing jelly with a dual structure containing a high concentration of viable lactic acid bacteria, or methods using a double coating of proteins and polysaccharides (Korean Patent Application No. 10-2003-0020375, Korean Patent Application No. 10-2001-0010397), or a triple coating method involving adding nanoparticles to the protein and polysaccharide coating (Korean Patent Application No. 10-2008-0008267) have been introduced. However, the lactic acid bacteria coating techniques described above do not completely coat the surface of the lactic acid bacteria, thus the heat resistance, acid resistance, and bile resistance of the lactic acid bacteria remain insufficient. Furthermore, competitive methods have been developed, such as adding edible oils to a three-coat layer for multi-coat application (Korean Patent Application No. 10-2011-0093074), or adding a coating agent containing water-soluble polymers, hyaluronic acid, porous particles, and proteins for a four-coat layer (Korean Patent Application No. 10-2011-0134486). However, these lactic acid bacteria coating technologies require a multi-step process of mixing the coating agent composition with the recovered lactic acid bacteria cultured by conventional methods, which makes it difficult to perform aseptic operations with probiotics. In particular, it leads to a decrease in competitiveness in large-scale industrial production. To address this, Korean Patent Application No. 10-1605516 proposes an effective method to increase the storage stability, acid resistance, and bile resistance of a product by adding proline in the preparation process. However, there is a problem that the effectiveness of the lactic acid bacteria after ingestion, which is related to the adhesion ability of intestinal epithelial cells, cannot be confirmed. To address these issues, the inventors have dedicated themselves to developing a method for improving the adhesion ability of lactic acid bacteria to intestinal epithelial cells. This method not only enhances the stability of the prepared probiotic lactic acid bacteria under processing and distribution conditions, but also maximizes the physiological activity of the probiotic lactic acid bacteria in the intestinal environment after ingestion.

[0013] Throughout this specification, numerous academic papers and patent documents are cited and their references are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, and this clarifies the level of technical expertise to which this invention pertains and the content of this invention. Summary of the Invention

[0014] Technical issues

[0015] The object of the present invention is to provide a composition comprising lactic acid bacteria coated with silk fibroin.

[0016] Another object of the present invention is to provide a composition comprising lactic acid bacteria coated with silk fibroin and cellulose.

[0017] Another object of the present invention is to provide a method for promoting the cultivation of lactic acid bacteria, including the step of culturing lactic acid bacteria in a medium supplemented with silk fibroin.

[0018] Another object of the present invention is to provide a method for increasing the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria.

[0019] Other objects and advantages within this invention will become more apparent from the following detailed description of the invention and the scope of the claims.

[0020] Solution to the problem

[0021] The present invention provides the following compositions or methods.

[0022] 1. A composition comprising lactic acid bacteria coated with silk fibroin.

[0023] 2. The composition according to 1. above, characterized in that the lactic acid bacteria are coated with silk fibroin and cellulose.

[0024] 3. The composition according to 1. and 2. above, characterized in that the silk fibroin is silk fibroin pretreated with ethanol.

[0025] 4. The composition according to 3. above, characterized in that the concentration of the ethanol is 85% (v / v) or higher.

[0026] 5. The composition according to 1. to 4. above, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus, Lactococcus, Enterococcus, Streptococcus and Bifidobacterium.

[0027] 6. The composition according to 1. to 5. above, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus delbrueckii ssp bulgaricus, Lactococcus lactis, Enterococcus faecium, Enterococcus fecalis, Streptococcus thermophilus, Bifidobacterium bifidum, and Bifidobacterium lactis.

[0028] 7. The composition according to 1 to 6, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus CKDB007 (accession number: KCTC13117BP), Enterococcus faecalis CKDB003 (accession number: KCTC13115BP), Streptococcus thermophilus CKDB021 (accession number: KCTC13118BP), Bifidobacterium bifidum CKDB001 (accession number: KCTC13114BP) and Bifidobacterium lactis CKDB005 (accession number: KCTC13116BP).

[0029] 8. The composition according to 1. to 7. above, characterized in that the composition is selected from the group consisting of food compositions, probiotic compositions, pharmaceutical compositions and feed compositions.

[0030] 9. A method for promoting the cultivation of lactic acid bacteria, comprising the step of culturing lactic acid bacteria in a culture medium supplemented with silk fibroin.

[0031] 10. A method for preparing a lactic acid bacteria probiotic coated with silk fibroin, comprising the step of culturing lactic acid bacteria in a culture medium containing silk fibroin.

[0032] 11. The method according to 10 above, characterized in that the culture medium for culturing lactic acid bacteria further contains water-soluble calcium.

[0033] 12. The method according to 11. above, characterized in that, relative to the volume of the culture medium for culturing lactic acid bacteria, the above-mentioned water-soluble calcium is added at a concentration of 0.01% (w / v) to 5% (w / v).

[0034] 13. The method according to 10 to 12 above, characterized in that the culture medium for culturing lactic acid bacteria further comprises cellulose.

[0035] 14. A method for increasing the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria, comprising the step of culturing lactic acid bacteria in a culture medium containing silk fibroin.

[0036] 15. The method according to 9., 10. or 14. above, characterized in that the culture medium containing the silk fibroin or the culture medium for lactic acid bacteria containing silk fibroin further contains water-soluble calcium.

[0037] 16. The method according to 15. above, characterized in that, relative to the volume of the culture medium for culturing lactic acid bacteria, the above-mentioned water-soluble calcium is added at a concentration of 0.01% to 5% by weight.

[0038] 17. The method according to 14. above, characterized in that the culture medium for culturing lactic acid bacteria further comprises cellulose.

[0039] 18. The method according to 10 to 17 above, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus, Lactococcus, Enterococcus, Streptococcus and Bifidobacterium.

[0040] 19. The method according to 10 to 18 above, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactococcus lactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Bifidobacterium bifidum and Bifidobacterium lactis.

[0041] 20. The method according to 10 to 19 above, characterized in that the lactic acid bacteria are selected from the group consisting of Lactobacillus acidophilus CKDB007 (accession number: KCTC13117BP), Enterococcus faecalis CKDB003 (accession number: KCTC13115BP), Streptococcus thermophilus CKDB021 (accession number: KCTC13118BP), Bifidobacterium bifidum CKDB001 (accession number: KCTC13114BP) and Bifidobacterium lactis CKDB005 (accession number: KCTC13116BP).

[0042] 21. A method for increasing the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria, comprising the step of culturing lactic acid bacteria in a culture medium containing silk fibroin.

[0043] In one embodiment of the invention, the invention provides a composition comprising lactic acid bacteria coated with silk fibroin.

[0044] In another embodiment of the invention, the invention provides a composition comprising lactic acid bacteria coated with silk fibroin and cellulose.

[0045] The inventors have confirmed that adding silk fibroin to the lactic acid bacteria culture process can promote the culture of lactic acid bacteria and shorten the culture time. Furthermore, they have confirmed that adding silk fibroin during or after the lactic acid bacteria culture process can improve the freeze-drying survival rate and stability under harsh conditions.

[0046] Furthermore, the inventors have confirmed that if the cultured bacteria are recovered after the lactic acid bacteria culture is completed and coated with silk fibroin optimized for various treatment conditions, the acid resistance and bile resistance, which are indicators of resistance to stress, are greatly increased, and the adhesion ability of intestinal epithelial cells is greatly improved.

[0047] In this specification, the term "silk fibroin" refers to an amino acid complex organism prepared from live silkworms through extrusion processing. Silk fibroin has a molecular weight of approximately 84,000 g / mol, and its amino acids, glycine, alanine, and serine, are distributed in a 3:2:1 ratio, accounting for 70-80% of all amino acids. Furthermore, tyrosine, valine, aspartic acid, glutamic acid, and other amino acids account for approximately 13% of all amino acids. This silk fibroin is approved by the Food and Drug Administration (FDA) and is widely used in various fields, such as surgical sutures, drug carriers, histological applications, burn treatment, and immobilization.

[0048] In one embodiment of the present invention, the silk fibroin may be prepared by acid hydrolysis or enzymatic decomposition of silkworm cocoons; however, methods commonly used in the art for preparing silk fibroin may be used without limitation.

[0049] In another example of the present invention, the aforementioned silk fibroin may be silk fibroin pretreated with 85% (v / v) or higher ethanol. Specifically, the 85% or higher ethanol is fermented alcohol. According to the Alcohol Tax Law, fermented alcohol is obtained by fermenting starchy or sugary materials to distill alcohol to 85% or higher. This is achieved by adding saccharifying enzymes to starchy (cereals, sweet potatoes, cassava) or sugary (sugar-) raw materials for fermentation, followed by continuous distillation, and is used as a raw material for diluted spirits.

[0050] Many proteins possess both α-helix and β-sheet sites within a single polypeptide chain. Silk fibroin also consists of two types: type I and type II filaments. Type I filaments are composed of α-helix structures, while type II filaments are composed of β-sheet structures. Furthermore, it is known that the strength and elasticity of silk proteins increase when they rearrange themselves into β-sheet structures in an amorphous state. Based on this, various methods for pretreating silk proteins have been investigated (Nature, 2003, Vol. 424, pp. 1057-1061).

[0051] In this invention, silk fibroin is pretreated by fermenting alcohol to induce its structure into a β-sheet, thereby adding a step to improve the stability and hydrophobicity of the silk fibroin. As a result, lactic acid bacteria probiotics with improved colonization ability against intestinal mucosal cells and improved stability are prepared by improving the hydrophobicity of the lactic acid bacteria probiotics.

[0052] In this invention, in order to apply lactic acid bacteria probiotics, silk fibroin is added at a ratio of 1 to 10% (w / v) relative to the volume of the lactic acid bacteria concentrate, but is not limited thereto.

[0053] In this specification, the term "cellulose" refers to the most abundant macromolecule on Earth, a high-molecular-weight polysaccharide formed by glucose through β-1,4 bonds. Cellulose is currently used in a variety of fields, including papermaking and spinning, and is also used as a tourniquet, skin replacement agent, and dietary fiber, among others. (Biowave, 2007, Vol.9, No.7, pp.1-11).

[0054] Food additives approved by the Ministry of Food and Drug Safety include methylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, and hydroxypropylcellulose. Since 1971, hydroxymethyl phthalate (HPMCP), which is widely used as an enteric coating material, has been used as a pharmaceutical additive.

[0055] Abroad, cellulose has been widely used as an enteric coating for tablets, granules, and capsules, and as an additive in pharmaceuticals and health foods. In particular, hydroxymethyl phthalate (HMK) is a pH-dependent solubility polymer prepared by chemical synthesis using natural pulp as raw material. The carboxyl group substituted in the cellulose ring is characterized by not disintegrating and eluting in gastric juice with low pH, but rapidly disintegrating and eluting in intestinal juice with a near-neutral pH.

[0056] In this specification, cellulose is compounded onto lactic acid bacteria cells coated with silk fibroin, so that they do not disintegrate and elute under simulated gastric fluid conditions, but rapidly disintegrate and elute under neutral pH intestinal fluid conditions.

[0057] In one embodiment of the present invention, the present invention provides a method for promoting the cultivation of lactic acid bacteria in a culture medium containing added silk fibroin.

[0058] The lactic acid bacteria culture of the present invention is carried out under conventional lactic acid bacteria culture media and culture conditions known in the past.

[0059] Silk fibroin is classified as a highly pure protein (97%) found in nature, and it is composed of peptides, which are composed of various amino acids and their conjugates, typical of human proteins. In particular, glycine, alanine, and serine, the most abundant amino acids in silk fibroin, account for 70-80% of all amino acids. Tyrosine, valine, aspartic acid, glutamic acid, and other amino acids make up the remaining proportions, making it an important nitrogen source for lactic acid bacteria culture.

[0060] In one embodiment of the present invention, based on the volume of the culture medium, the silk fibroin content of the silk fibroin-added culture medium of the present invention can be 0.001% (w / v) to 5% (w / v), 0.001% (w / v) to 4% (w / v), 0.001% (w / v) to 3% (w / v), 0.001% (w / v) to 2% (w / v), 0.001% (w / v) to 1% (w / v), 0.01% (w / v) to 2% (w / v), 0.001% (w / v) to 1% (w / v), 0.01% (w / v) to 3% (w / v), 0.0 ... % (w / v) to 5% (w / v), 0.01% (w / v) to 4% (w / v), 0.01% (w / v) to 3% (w / v), 0.01% (w / v) to 2% (w / v), 0.01% (w / v) to 1% (w / v), 0.01% (w / v) to 0.5% (w / v), 0.01% (w / v) to 0.4% (w / v), 0.01% (w / v) to 0.3% % (w / v), and may contain 0.01% (w / v) to 0.2% (w / v), 0.1% (w / v) to 5% (w / v), 0.1% (w / v) to 4% (w / v), 0.1% (w / v) to 3% (w / v), 0.1% (w / v) to 2% (w / v), 0.1% (w / v) to 1% (w / v), 1% (w / v) to 5% (w / v), 1% (w / v) to Concentrations of 4% (w / v), 1% (w / v) to 3% (w / v), 1% (w / v) to 2% (w / v), 2% (w / v) to 5% (w / v), 2% (w / v) to 4% (w / v), 2% (w / v) to 3% (w / v), 3% (w / v) to 5% (w / v), 3% (w / v) to 4% (w / v) or 4% (w / v) to 5% (w / v), but not limited thereto.

[0061] As demonstrated by the examples below, lactic acid bacteria cultured using the method of the present invention promote growth and shorten culture time.

[0062] In one embodiment of the present invention, the culture medium for culturing lactic acid bacteria may further comprise water-soluble calcium. The water-soluble calcium may be calcium citrate, calcium hydroxide, calcium chloride, calcium lactate, dicalcium phosphate, or monocalcium phosphate, all permitted as food additives. Furthermore, the concentration of the water-soluble calcium may be 0.001% (w / v) to 5% (w / v), 0.001% (w / v) to 4% (w / v), 0.001% (w / v) to 3% (w / v), 0.001% (w / v) to 2% (w / v), 0.001% (w / v) to 1% (w / v), 0.01% (w / v) to 5% (w / v), 0.01% (w / v) to 4% (w / v), or 0.01% (w / v) to 5% (w / v). 0.01% to 2% (w / v), 0.01% to 1% (w / v), 0.01% to 0.5% (w / v), 0.01% to 0.4% (w / v), 0.01% to 0.3% (w / v), specifically 0.01% to 0.2% (w / v), most specifically 0.1% (w / v), but not limited thereto.

[0063] Furthermore, in another embodiment of the present invention, the culture medium for culturing lactic acid bacteria may also contain cellulose. The cellulose may be methylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxymethyl phthalate (hereinafter referred to as HPMCP), all of which are permitted as food additives by the Ministry of Food and Drug Safety, but are not limited thereto.

[0064] Furthermore, in another embodiment of the present invention, the present invention provides a method for increasing the survival rate, storage stability, acid resistance or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria, including the step of culturing lactic acid bacteria in a culture medium containing silk fibroin.

[0065] In this invention, when the above-mentioned water-soluble calcium is added together with silk fibroin, which is a component of the culture medium for culturing lactic acid bacteria, the lactic acid produced by the lactic acid bacteria forms a salt and coagulates together with the water-soluble calcium and silk fibroin, thereby allowing for more stable coating of silk fibroin during the cultivation and concentration steps of lactic acid bacteria.

[0066] In specific examples of the present invention, it can be confirmed that the increased cell hydrophobicity, mucin adhesion ability, and intestinal epithelial cell adhesion ability of the lactic acid bacteria coated with the silk fibroin of the present invention are achieved. When the lactic acid bacteria probiotics prepared by this method are ingested, the lactic acid bacteria are stably attached to the intestinal epithelial cells, thereby enhancing the physiological activity of the lactic acid bacteria.

[0067] In another specific example of the present invention, it can be confirmed that the lactic acid bacteria coated with a composite of silk fibroin and cellulose have excellent cell hydrophobicity, mucin adhesion ability, and intestinal epithelial cell adhesion ability.

[0068] In another specific embodiment of the present invention, the lactic acid bacteria composition of the present invention exhibits excellent stability (survival rate) under acid resistance and bile resistance test conditions for determining the intestinal environment stability of existing lactic acid bacteria probiotics, as well as freeze-drying survival rate and harsh test conditions for determining storage stability.

[0069] In this invention, "resistance against acid" refers to the property of the lactic acid bacteria of this invention to tolerate acids with a pH below 7. Specifically, in this specification, acid resistance refers to the resistance of the lactic acid bacteria to artificial gastric juice with a pH of 1 to 3, for example, artificial gastric juice with a pH of 2.0 or 2.5. The survival rate can be determined by comparing the number of probiotics of lactic acid bacteria before and after contact with the artificial gastric juice. In one example of this invention, the aforementioned artificial gastric juice is prepared by adjusting the pH of a 1N hydrochloric acid (HCl) aqueous solution to 2.0 or 2.5. If necessary, it is prepared by adding a certain concentration (1 to 2%) of pepsin, which is a secreted enzyme in the stomach, but it is not limited thereto.

[0070] In this specification, "bile resistance" refers to the resistance of the lactic acid bacteria of the present invention to bile (bile fluid). Specifically, in this specification, bile resistance refers to the resistance of the lactic acid bacteria to artificial intestinal fluid (artificial bile fluid), for example, artificial intestinal fluid containing 0.1% to 1% oxigall acid. The survival rate of the lactic acid bacteria can be determined by comparing the number of probiotics before and after contact with the artificial intestinal fluid. In one example of the present invention, the artificial intestinal fluid is prepared by adding 0.5% oxigall acid to a liquid culture medium, and also by adding a bile extract, but is not limited thereto.

[0071] In this specification, "freeze-drying" refers primarily to a freeze-drying method used for the long-term preservation of lactic acid bacteria, generally carried out at temperatures ranging from -20°C to -80°C. During the freeze-drying process described above, the activity and survival rate of lactic acid bacteria cells decrease due to physical and biochemical stress. The "freeze-drying survival rate" is determined by comparing the number of probiotics in the lactic acid bacteria before and after freeze-drying. In one example of this invention, the freeze-drying survival rate is determined by comparing the number of lactic acid bacteria (CFU) in a sample that has been recovered and frozen at temperatures ranging from 0°C to -45°C, specifically from -20°C to -45°C, and then pulverized and pulverized before being cultured again in a culture medium, with the number of lactic acid bacteria in the sample before freeze-drying. However, this method is not limited to this.

[0072] In this specification, "severe test conditions" refers to the conditions used to determine the resistance of lactic acid bacteria to external conditions, primarily resistance to high temperature and high humidity. In one embodiment of the invention, the test can be conducted under the aforementioned severe test conditions of 40°C and 70-75% humidity, but is not limited thereto.

[0073] The adhesion ability of the aforementioned lactic acid bacteria to intestinal epithelial cells is influenced by the survival rate of the lactic acid bacteria, the adhesion ability of the host cells, and their relationship with other bacterial strains. In particular, it is known that the cell surface characteristics of the lactic acid bacteria have a significant impact. The cell surface characteristics affecting the adhesion ability of lactic acid bacteria are largely related to the electrostatic balance, van der Waals forces, and hydrophobicity of the bacterial surface. In particular, cell surface hydrophobicity is an indicator related to the adhesion ability between bacterial cells and intestinal epithelial cells and is used as an important indicator to confirm the adhesion ability of lactic acid bacteria strains, including Lactobacillus and Bifidobacterium (Letters in Applied Microbiology, 1998, Vol. 27, pp. 307-310).

[0074] In this invention, hydrophobicity is imparted to the cell surface of lactic acid bacteria by coating them with silk fibroin. Specifically, the silk fibroin is pretreated with fermented alcohol to induce a β-sheet structure, thereby enhancing its adhesion to intestinal mucosal cells by increasing the hydrophobicity of the surface of the silk fibroin-coated lactic acid bacteria. Compared to the α-helix structure, the β-sheet structure is highly thermally sensitive, thus improving stability and providing hydrophobic properties when used as a coating agent.

[0075] There are no particular limitations on the lactic acid bacteria used in this invention. For example, the lactic acid bacteria can be selected from the group consisting of Lactobacillus, Lactococcus, Enterococcus, Streptococcus and Bifidobacterium.

[0076] In another embodiment of the present invention, specifically, the lactic acid bacteria mentioned above may be selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactococcus lactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Bifidobacterium bifidum and Bifidobacterium lactis.

[0077] In another embodiment of the present invention, most specifically, the lactic acid bacteria mentioned above may be selected from the group consisting of Lactobacillus acidophilus CKDB007 (accession number: KCTC13117BP), Enterococcus faecalis CKDB003 (accession number: KCTC13115BP), Streptococcus thermophilus CKDB021 (accession number: KCTC13118BP), Bifidobacterium bifidum CKDB001 (accession number: KCTC13114BP) and Bifidobacterium lactis CKDB005 (accession number: KCTC13116BP).

[0078] In another embodiment of the present invention, the composition characterized by the above-described lactic acid bacteria of the present invention may be selected from the group consisting of food compositions, probiotic compositions and feed compositions.

[0079] When the compositions of the present invention are prepared into food compositions, the active ingredients may include not only the aforementioned lactic acid bacteria, but also ingredients commonly added during food preparation. For example, these added ingredients may include proteins, carbohydrates, fats, nutrients, flavorings, and flavorings. The aforementioned carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., common sugars such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc.). Natural flavorings (such as sematriol, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic sweeteners (such as saccharin, aspartame, etc.) may be used.

[0080] For example, when the food composition of the present invention is prepared into a beverage, in addition to the strains mentioned above that are the effective ingredients of the present invention, it may also contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract or licorice extract, etc.

[0081] The food compositions of this invention include all processed forms of natural materials such as food, functional food, nutritional supplement, health food, and food additives. These types of food compositions can be prepared into various forms according to conventional methods known in the art.

[0082] For example, as a health food, the aforementioned lactic acid bacteria can be prepared into the form of tea, juice, and beverages for consumption, or they can be ingested through granulation, encapsulation, and powdering. Furthermore, as a food, it can be prepared by adding the lactic acid bacteria of the present invention to beverages (including alcoholic beverages), fruits and their processed products (e.g., canned fruits, canned foods, jams, citrus marmalade, etc.), fish, meat and their processed products (e.g., ham, sausages, corn, beef, etc.), breads and noodles (e.g., udon noodles, soba noodles, ramen, pasta, macaroni, etc.), juices, various beverages, biscuits, maltose, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, artificial yellows, plant proteins, steamed or boiled foods, frozen foods, and various seasonings (e.g., miso, soy sauce, sauces, etc.). Moreover, in order to use the lactic acid bacteria of the present invention as a food additive, it can be prepared into powder or concentrated liquid form for use.

[0083] When the compositions of the present invention are prepared into pharmaceutical compositions, the pharmaceutical compositions of the present invention contain a chemically acceptable carrier. Commonly used formulations containing a chemically acceptable carrier in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may also contain lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0084] The pharmaceutical compositions of the present invention can be administered orally or non-orally, preferably by oral administration. The pharmaceutical compositions of the present invention can be formulated into various oral or non-oral administration forms, but are not limited thereto.

[0085] As dosage forms for oral administration, such as tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, and elixirs, in addition to the aforementioned active ingredients, these dosage forms may use one or more commonly used fillers, expanders, wetting agents, disintegrants, glidants, binders, surfactants, or other diluents or excipients. Disintegrants may include agar, starch, alginate or its sodium salt, and dicalcium phosphate. Glidants may include silica, talc, stearic acid or its magnesium or calcium salts, and polyethylene glycol. Binders may include magnesium aluminum silicate, starch paste, gelatin, astragalus gum, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, and low-substituted hydroxypropyl cellulose. Furthermore, lactose, glucose, sucrose, mannitol, sorbitol, cellulose, and glycine may be used as diluents, and, depending on the circumstances, may be used in conjunction with commonly known boiling mixtures, absorbents, colorants, flavorings, and sweeteners.

[0086] The above composition may be sterile or contain preservatives, stabilizers, hydrating agents or emulsifying agents, salts for adjusting osmotic pressure, buffers and other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulation and application.

[0087] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate and reaction sensitivity.

[0088] The pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable carriers and / or excipients in a manner readily practiced by those skilled in the art, thereby preparing them in unit dosage form or incorporating them into multi-dosage containers. The dosage form can be a solution, suspension, syrup, or emulsion in an oily or aqueous medium, or an extract, powder, granule, tablet, or capsule, and may also include dispersants or stabilizers.

[0089] This invention can be cross-applied to lactic acid bacteria culture media by adding lactic acid bacteria coated with the silk fibroin described above, to compositions containing the silk fibroin, to promote lactic acid bacteria culture, to prepare lactic acid bacteria coated with silk fibroin, and to increase the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria. In the case of lactic acid bacteria compositions prepared by the techniques described above, they can be used as food compositions, health functional foods, and pharmaceutical compositions. To avoid complexity in the specification, descriptions of repetitive scope have been omitted.

[0090] The effects of the invention

[0091] The features and advantages of this invention are as follows:

[0092] (i) The present invention provides a composition comprising lactic acid bacteria coated with silk fibroin or lactic acid bacteria coated with silk fibroin and cellulose.

[0093] Furthermore, the present invention provides a method for promoting the cultivation of lactic acid bacteria, including the step of culturing lactic acid bacteria in a culture medium containing added silk fibroin.

[0094] Furthermore, the present invention provides a method for increasing the survival rate, storage stability, acid or bile resistance, and intestinal epithelial cell adhesion ability of lactic acid bacteria.

[0095] (ii) The present invention utilizes silk fibroin prepared by various processing methods to coat lactic acid bacteria cells, thereby improving the survival rate and storage stability of lactic acid bacteria cells, and confirming the effects of excellent acid resistance or bile resistance and increased adhesion to intestinal epithelial cells. Attached Figure Description

[0096] Figure 1 This diagram illustrates the process of preparing lactic acid bacteria probiotics using silk fibroin.

[0097] Figure 2a and Figure 2b The graph shows the carbon source consumption pattern and culture performance when silk fibroin is used as a culture medium for lactic acid bacteria.

[0098] Figure 3 This image shows the characteristics of lactic acid bacteria probiotics prepared from silk fibroin and cellulose observed using electron backscatter diffraction / field emission scanning electron microscopy (EBSD / FE-SEM).

[0099] Figure 4 A graph showing the zeta potential under conditions of artificial gastric and intestinal fluids based on probiotic lactic acid bacteria coated with silk fibroin is presented for comparison.

[0100] Figure 5 The graph shows the binding ability of silk fibroin and cellulose to mucin under various coating conditions for comparison.

[0101] Figure 6 The graph shows the binding ability of silk fibroin and cellulose to intestinal epithelial cells (HT-29) under various application conditions for comparison. Detailed Implementation

[0102] The present invention will be further described in detail below through embodiments. It will be apparent to those skilled in the art that these embodiments are merely for illustrating the invention more specifically, and the spirit and scope of the invention are not limited by these examples.

[0103] Throughout this instruction manual, unless otherwise stated, the percentages used to indicate the concentration of a particular substance are as follows: solid / solid is (weight / weight) percentage, solid / liquid is (weight / volume) percentage, and liquid / liquid is (volume / volume) percentage.

[0104] Example

[0105] In order to confirm the effect of the addition and application of the silk fibroin of the present invention on lactic acid bacteria, the inventors conducted the following experiments. Figure 1 A process diagram illustrating the sequence and content of the experiments conducted by the inventors.

[0106] Example 1: Isolation and purification of silk fibroin components according to the present invention

[0107] Silk is a massive protein that can be obtained from nature. Fibroin is composed of 18 of the 20 amino acids that make up human proteins. The liquid-phase silk, biosynthesized by the silk glands of silkworms from these 18 natural amino acids, forms a highly crystalline array of fibers in the silkworm's silkworm soil. It consists of approximately 75% fibroin and approximately 25% sericin.

[0108] Silk fibroin can be broadly classified into two methods: (i) acid hydrolysis and (ii) protein hydrolysis using calcium chloride solution and enzymes. In this invention, silk fibroin for use in lactic acid bacteria cell coating is obtained by raising silkworms (Bombyx Hori) and obtaining cocoons from them, followed by a refining process to separate and remove the aforementioned sericin components.

[0109] Example 1-1. Acid hydrolysis method

[0110] To separate silk fibroin, purified water was heated (95°C), and sodium oleate and Na₂CO₃ were added until completely dissolved. Silkworm cocoons were then added and boiled for approximately 40 minutes, followed by a dehydration process to refine the cocoons. Specifically, refining was performed relative to purified water by adding 1.84% (w / v) of the aforementioned silkworm cocoons, 0.0092% (w / v) of sodium oleate, and 0.0055% (w / v) of Na₂CO₃. During acid hydrolysis, commonly used 2N HCl was added, and acid hydrolysis was carried out at 110°C for 2 hours. The dissolved product was filtered and neutralized with an aqueous NaOH solution. Salts generated during neutralization were removed using a dialysis tubing cellulose membrane, thus preparing purified acid-hydrolyzed silk fibroin. Generally, the average molecular weight of peptides obtained by acid hydrolysis is approximately 200–10,000, and smaller peptides of silk fibroin are known to be obtained.

[0111] Examples 1-2. Enzymatic Decomposition Method

[0112] To separate silk fibroin, silkworm cocoons were refined using the same method as in Examples 1-1 above. Next, 5% concentrations of alkaline protease, Delvorase, flavorzyme, Protamax (BISION Bio company), and papain T100 (BISION Bio company), known proteolytic enzymes produced by Bacillus licheniformis, Bacillus stearothermophilus, or Aspergillus niger, were added, and the mixture was treated at 60–80°C for approximately 6–10 hours. Before using the decomposed silk fibroin solution in the lactic acid bacteria coating process, it was exposed to a high temperature of 95°C for 2 hours to inactivate the proteolytic enzymes. Silk fibroin prepared by the enzymatic decomposition method described above is known to have high solubility and high in vivo absorption rate.

[0113] To confirm the culture-promoting effect and coating effect of the lactic acid bacteria cells on the silk fibroin of the present invention, the silk fibroin prepared by the above method (acid hydrolysis and enzymatic decomposition) was recovered and applied to the culture medium coating process of Enterococcus faecalis CKDB003 (accession number: KCTC13115BP), Lactobacillus acidophilus CKDB007 (accession number: KCTC13117BP), Streptococcus thermophilus CKDB021 (accession number: KCTC13118BP), Bifidobacterium lactis CKDB005 (accession number: KCTC13116BP) and Bifidobacterium bifidum CKDB001 (accession number: KCTC13114BP) cells described below.

[0114] Example 2. Utilization of the silk fibroin component of the present invention

[0115] Silk protein, the main component of sericin and silk protein recovered from silkworm cocoons, consists of 75% silk protein, 25% sericin, and about 3% inorganic carbohydrates.

[0116] Silk fibroin is classified as a protein with high purity (97%) found in nature, and is composed of peptides consisting of various amino acids that are components of human proteins and their conjugates.

[0117] In particular, glycine, alanine, and serine, which constitute the largest proportion of amino acids in silk fibroin, make up 70-80% of all amino acids. These amino acids are known to be the same as those that make up collagen. Therefore, these silk fibroin proteins possess the same robust properties as collagen, and their constituent amino acids can serve as an important nitrogen source in lactic acid bacteria culture.

[0118] Example 2-1. Uses of silk fibroin as a component in lactic acid bacteria culture

[0119] When the silk fibroin of the present invention is used as a component for lactic acid bacteria culture, known optimized culture media and culture conditions for each bacterial species are used to confirm its effect on the culturability of lactic acid bacteria. Lactic acid bacteria cultured in the above-mentioned optimal culture medium composition are used as the control group, and lactic acid bacteria cultured under the same culture conditions with the addition of silk fibroin are used as the experimental group, thereby comparing the carbon source consumption rate (%) and the number of viable cells over time.

[0120] The results are as follows Figure 2a and Figure 2b As shown.

[0121] like Figure 2a and Figure 2b As shown, when lactic acid bacteria are cultured with the addition of the silk fibroin of the present invention, the rate of sugar consumption in the early stage of culture is confirmed to be very fast compared with that in the case of culture without the addition of silk fibroin. Figure 2a Furthermore, when culturing with the addition of the silk fibroin of the present invention, the time point at which maximum culturability was achieved in the later stages of culture was also shortened. Figure 2b ).

[0122] Therefore, the results above show that the silk fibroin component of the present invention acts as an important growth factor for lactic acid bacteria, thereby promoting the cultivation of lactic acid bacteria and shortening the cultivation time. Figure 1 The results show the culturing results for Bifidobacterium lactis CKDB005 (accession number: KCTC13116BP), and the results for other strains are shown, but it is confirmed that they have similar effects to the above-mentioned Bifidobacterium lactis strains.

[0123] Example 2-2. Use of water-soluble calcium and silk fibroin as components for lactic acid bacteria culture.

[0124] The inventors have confirmed that when water-soluble calcium is provided along with silk fibroin as a culture medium component for culturing lactic acid bacteria, not only is the culturability of lactic acid bacteria improved, but the lactic acid produced by the lactic acid bacteria also forms a salt with the water-soluble calcium and silk fibroin and coagulates, thereby allowing for more stable coating of silk fibroin during the cultivation and concentration of lactic acid bacteria. Furthermore, optimal conditions for coating silk fibroin onto the surface of lactic acid bacteria cells were established, and the following experiments were conducted to confirm the effect of silk fibroin coating on the stability of the lactic acid bacteria.

[0125] First, the silk fibroin used for cultivation was a dried powder prepared by the method described in Example 1 above, added at a concentration of 0-3% (w / v) of the culture medium volume. Furthermore, as additionally added water-soluble calcium, calcium citrate, calcium hydroxide, calcium chloride, calcium lactate, dicalcium phosphate, and monocalcium phosphate, all permitted as food additives, were added at concentrations of 0% (w / v), 0.1% (w / v), and 0.5% (w / v), respectively. Lactic acid bacteria probiotics coated with silk fibroin were prepared by culturing lactic acid bacteria.

[0126] Then, the freeze-drying survival rate of the prepared lactic acid bacteria probiotics and the survival rate under harsh conditions (temperature of 40℃ and humidity of 70-75%) were compared. The freeze-drying process for bacterial cell recovery partially applied conventional methods (after centrifuging to recover the bacterial cells, rapid freezing in a refrigerator at -40℃ and freeze-drying under freeze-drying conditions between 0℃ and -45℃). The freeze-drying survival rate was determined as the percentage of the number of probiotics after freeze-drying divided by the number of probiotics before freeze-drying.

[0127] On the other hand, the survival rate under harsh conditions was confirmed after the probiotics were stored under harsh conditions (temperature of 40°C and humidity of 70-75%) for 4 weeks.

[0128] Table 1

[0129]

[0130]

[0131] Table 2

[0132]

[0133] As shown in Tables 1 and 2 above, when water-soluble calcium is added together with the silk fibroin of the present invention, the freeze-drying survival rate and the survival rate under harsh conditions are excellent. In particular, it was confirmed that the survival rate of lactic acid bacteria is the best when the calcium concentration is 0.1% (w / v).

[0134] Examples 2-3. Application in the culture and coating of silk fibroin

[0135] As confirmed in Examples 2-1 and 2-2 above, when silk fibroin and water-soluble calcium are used as culture media for culturing lactic acid bacteria, the culturability and stability of lactic acid bacteria are improved. The preparation of probiotic lactic acid bacteria is as follows.

[0136] To prepare a control group (uncoated), an additional coating process was performed after culturing and concentrating the bacteria using the optimal culture medium. In the case of the experimental group, water-soluble calcium was added as a culture medium component along with silk fibroin during the cultivation of lactic acid bacteria, and the coating was performed using silk fibroin after cultivation and concentration.

[0137] The lactic acid bacteria application process uses conventional methods (Examples 2-2), and the lactic acid bacteria probiotics are pulverized using a pulverizer and then applied to the experimental cases in Example 4.

[0138] Example 3: Preparation of silk fibroin coating-lactic acid bacteria probiotics using fermented alcohol pretreatment

[0139] The inventors have discovered the optimal silk fibroin for coating lactic acid bacteria cells, and to confirm the effect of the conditions of the applicable silk fibroin on the coating quality of lactic acid bacteria, the silk fibroin was pretreated with fermented alcohol. Specifically, in the silk fibroin prepared by the enzymatic degradation method of Examples 1-2 of the present invention, fermented alcohol was mixed in a 30% ratio, homogenized under sterile conditions and at room temperature (25°C), and allowed to stand for 18-24 hours.

[0140] In this Example 3, except that silk fibroin pretreated with fermented alcohol as described above was used instead of untreated silk fibroin, lactic acid bacteria cells were cultured and coated in the same manner as in Examples 2-2.

[0141] Example 4: Preparation of lactic acid bacteria probiotics with composite coating of silk fibroin and cellulose

[0142] In order to improve the characteristics of lactic acid bacteria probiotics coated with the above-mentioned silk fibroin and their resistance to the intestinal environment, the inventors prepared a lactic acid bacteria probiotic that was co-coated with the silk fibroin of the present invention and used as a cellulose in conventional enteric coatings.

[0143] Specifically, the silk fibroin used for coating the probiotic lactic acid bacteria was a dried powder prepared by the method described in Example 1 above. As described in Example 3, 1 to 10% (w / v) of pretreated fermented alcohol silk fibroin was added relative to the volume of the lactic acid bacteria concentrate. In the case of cellulose, methylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxymethyl phthalate (hereinafter referred to as HPMCP) were used as coating agents as food additives permitted by the Ministry of Food and Drug Safety. As a result, as described in the experimental examples below, when hydroxymethyl phthalate (pharmaceutical brand name (Any Coast), Samsung Fine Chemicals) and sodium carboxymethylcellulose (Samsung Fine Chemicals) were added at a ratio of 1 to 10% relative to the volume of the lactic acid bacteria concentrate to coat the probiotic lactic acid bacteria, it was confirmed that the freeze-drying survival rate of the lactic acid bacteria and the survival rate under harsh conditions (temperature of 40°C and humidity of 70%) were improved.

[0144] Experimental Example

[0145] In order to confirm whether the silk fibroin prepared according to Examples 2 to 4 above was coated and the characteristics of the lactic acid bacteria probiotics produced by the method, the inventors conducted the following experiments.

[0146] The control groups and experimental groups used in the following experimental examples are shown in Table 3 below.

[0147] Table 3

[0148]

[0149]

[0150] Experiment Example 1: Confirming the application efficiency of lactic acid bacteria and probiotics based on silk fibroin application conditions

[0151] As shown in Table 3 above, samples from the control group (Examples 2-3, uncoated lactic acid bacteria probiotics), experimental group 1 (Examples 2-3, silk fibroin coated - lactic acid bacteria probiotics), experimental group 2 (Example 3, silk fibroin pretreated with fermented alcohol coated - lactic acid bacteria probiotics), and experimental group 3 (Example 4, silk fibroin and cellulose composite coated with fermented alcohol and cellulose coated - lactic acid bacteria probiotics) were fixed onto metal plates using carbon ribbons. After platinum plating by platinum sputtering, they were observed using an electron backscatter diffraction / field emission scanning electron microscope (scanning electron detector) at an accelerating voltage of 10 kV.

[0152] The results are as follows Figure 3 As shown.

[0153] like Figure 3 As shown, in the case of probiotics prepared by coating lactic acid bacteria cells with only silk fibroin (experimental group 1), the coating coverage was better than that of the control group (uncoated), but uncoated areas were observed. Furthermore, in the case of probiotics prepared by adding fermented alcohol and coating with silk fibroin (experimental group 2), the shape of the coating uniformly surrounding the bacteria was confirmed. In the case of probiotics prepared by combining fermented alcohol-pretreated silk fibroin and cellulose (experimental group 3), the coating was more uniform than that of probiotics coated with only silk fibroin (experimental group 2). Therefore, it can be concluded that experimental group 1 is superior to the control group, experimental group 2 is superior to experimental group 1, and compared to experimental group 2, the coating quality of probiotics in experimental group 3 is the best.

[0154] In the embodiments, the pretreatment of the fermented alcohol is to induce the regeneration of silk fibroin and process it into a β-sheet structure, and to inactivate the enzyme used in the enzymatic decomposition process, so as to prevent the lactic acid bacteria and silk fibroin from being decomposed by the enzyme during the coating process.

[0155] Experimental Example 2: Surface hydrophobicity of lactic acid bacteria cells based on the silk fibroin coating method

[0156] Cell surface hydrophobicity is an indicator that can indirectly determine the ability of lactic acid bacteria to adhere to the intestinal tract in vitro, and is used as one of the primary methods for confirming the adhesion ability of lactic acid bacteria, including Lactobacillus and Bifidobacterium. In this invention, experiments were conducted to confirm the hydrophobicity of lactic acid bacteria probiotics coated with silk fibroin, using the following method.

[0157] Specifically, after washing the uncoated probiotic control group and the probiotic experimental groups prepared according to each coating condition twice with 1X phosphate-buffered saline (PBS, pH 7.2), the bacterial cells were resuspended in 1X phosphate-buffered saline to achieve an OD of [missing information]. 600 =0.5. The lactic acid bacteria sample prepared in suspension was mixed with toluene and then treated in a constant temperature water bath at 37°C for 20 minutes. After removing the toluene, the OD of the aqueous solution was measured. 600 The hydrophobicity of lactic acid bacteria probiotics is calculated using the following formula.

[0158]

[0159] The results are shown in Table 4.

[0160] Table 4

[0161]

[0162] As shown in Table 4, when silk fibroin was applied to lactic acid bacteria, the hydrophobicity of the bacterial cells was improved compared to the uncoated control group. Furthermore, compared to silk fibroin prepared by acid hydrolysis, the hydrophobicity of lactic acid bacteria probiotics was generally higher when silk fibroin prepared by enzymatic degradation was applied to the bacterial cells. In particular, when silk fibroin pretreated with fermented alcohol was used to apply to the lactic acid bacteria cells, the hydrophobicity of the cells was further improved.

[0163] Experimental Example 3: Surface zeta potential of lactic acid bacteria cells based on the silk fibroin coating method

[0164] The zeta potential, also known as electrokinetic potential, refers to the potential difference in the flow layer across the electrochemical bilayer caused by electrokinetic phenomena. While the potential at the membrane surface cannot be directly measured, the electrochemical properties of the surface can be understood through experimental measurement of the zeta potential (Chemistry of the solid-water interface, John Wiley & Sons, Inc., 1992). This zeta potential is an important parameter for judging the stability or aggregation of dispersed samples, and it can be significant in confirming the efficacy of probiotics in vivo. In the case of fine particles or colloids, an increase in the absolute value of the zeta potential increases the repulsive force between particles, thereby increasing particle stability; however, if the zeta potential approaches zero, the particles are prone to aggregation. This invention calculates and uses the average of five analytical results measured using a zeta potential analyzer that utilizes phase analysis light scattering (PALS) technology.

[0165] Specifically, lactic acid bacteria probiotics were prepared in an aqueous solution as an experimental condition to artificially mimic the intestinal environment, including uncoated (control area), coated silk fibroin (experimental area 1), coated silk fibroin pretreated with fermented alcohol (experimental area 2), and a compound coated silk fibroin and cellulose (experimental area 3), and the zeta potential value was confirmed.

[0166] The results are as follows Figure 4 As shown.

[0167] like Figure 4As shown, under simulated gastric fluid conditions (2.0 g sodium chloride, 24.0 ml / L dilute hydrochloric acid, and an aqueous solution at pH 1.2), compared with the control group, the zeta potential values ​​of lactic acid bacteria probiotics coated with silk fibroin or pretreated with fermented alcohol were confirmed to be reduced (experimental group 1, experimental group 2), and even lower in lactic acid bacteria probiotics with combined coating of pretreated silk fibroin and cellulose (experimental group 3).

[0168] Furthermore, under simulated intestinal fluid conditions (an aqueous solution containing 0.3% bile acids at pH 7.0), the zeta potential of all experimental groups was negative. Compared to the control group, the absolute value of the zeta potential gradually decreased in probiotics coated with silk fibroin or pretreated with fermented alcohol (experimental group 1, experimental group 2), but the absolute value of the zeta potential increased in probiotics with a combined coating of pretreated silk fibroin and cellulose. These results indirectly indicate that under simulated intestinal fluid conditions, the hydrophobicity of probiotic cells coated with silk fibroin increases, while the hydrophobicity decreases when combined with cellulose coating.

[0169] Therefore, by measuring the zeta potential value, it was experimentally confirmed that the lactic acid bacteria probiotics coated with the silk fibroin and cellulose composite of the present invention can improve the survival rate of lactic acid bacteria cells through strong agglutination under artificial gastric juice conditions. At the time point when they reach the small intestine and large intestine, the absolute value of the zeta potential decreases because the cellulose layer is washed away. Therefore, the intestinal colonization of the silk fibroin coated lactic acid bacteria is increased, and the possibility of stable survival in the intestinal environment is high.

[0170] Experiment Example 4: The mucin-binding ability of lactic acid bacteria cells based on the silk fibroin coating method

[0171] The adhesion ability of microorganisms is related to the electrostatic balance of the cell wall, van der Waals bonds, and hydrophobicity. Hydrophobicity is known to be an important factor in cell adhesion to mucous membranes or epithelial cells (Environ Microbiol, 2000, Vol.66(6), pp.2548-2554; International Dairy Journal, 2005, Vol.15, pp.1289-1297).

[0172] In particular, intestinal epithelial cells form a protective membrane for the intestinal wall by producing a gel-like substance called mucin. Mucin, a component of the intestinal mucosa, can hydrophobically bind to lactic acid bacteria; therefore, lactic acid bacteria with high hydrophobicity are expected to have excellent intestinal adhesion ability. To this end, in this invention, the binding ability of mucin to lactic acid bacteria cells is confirmed by evaluating the adhesion ability of mucin, a component of the intestinal mucosa, to the aforementioned lactic acid bacteria. The mucin adhesion ability test method used is Munoz-Provencio (Gastroenterology. 1998, Vol. 115, pp 874-882).

[0173] First, 200 μL of porcine stomach mucin (type II) (Sigma) was inoculated into Maxisorb plates, a known ELISA plate, and incubated at 4°C for 24 hours. Then, as shown in Table 3, the silk fibroin-coated probiotics and uncoated probiotics were suspended at OD200. 600 =0.5, then 200 μL was inoculated into Maxisorb plates coated with mucin and reacted overnight (at least 12 hours) at 4°C. After the reaction, the cells were washed five times with phosphate buffer to remove lactic acid bacteria cells not attached to mucin, and then stained with gentian violet before measuring OD. 620 The absorbance at that location.

[0174] The results are as follows Figure 5 As shown.

[0175] like Figure 5 As shown, the mucin binding capacity increased in the order of control group, experimental group 1, experimental group 3 and experimental group 2. Pretreatment of silk fibroin significantly increased the mucin binding capacity. Compared with the mucin binding capacity of lactic acid bacteria probiotics coated with silk fibroin prepared through the pretreatment process, it decreased slightly when combined with cellulose.

[0176] Experimental Example 5: Adhesion ability of lactic acid bacteria cells to intestinal epithelial cells based on silk fibroin coating method

[0177] To confirm the adhesion ability of probiotic lactic acid bacteria prepared by silk fibroin coating to intestinal epithelial cells, the adhesion ability of the related lactic acid bacteria was confirmed using the human colon sarcoma cell line HT-29.

[0178] 10% fetal bovine serum (FCS) and antibiotics (100 U / ml penicillin and 100 U / ml streptomycin) were added to DMED medium to prepare HT-29 cell line culture medium. After washing HT-29 cells that had formed a monolayer in this medium with phosphate buffer, they were sputtered at 5 × 10⁻⁶ ppm. 8 Cells were seeded at a rate of 1 × 10⁶ cells / ml in 6-well plates. 9 Each probiotic strain was suspended in phosphate buffer at a concentration of / ml and inoculated into wells of a plate. The plates were then incubated with HT-29 cells at 37°C and 5% CO2 for 2 hours. After incubation, the plates were washed five times with phosphate buffer to remove any unattached probiotics. Unattached HT-29 cells and probiotics were then separated by treating the plates with 0.05% trypsin and 0.02% EDTA for 2 minutes. The separated cells were diluted with water using a decimal method and cultured on MRS or BL agar plates before probiotic counts were determined (Trends. Food. Sci. Technol., 1999, Vol. 10, pp 405-410; Korean Soc. Food. Sci. Nutr., 2016, Vol. 45, pp 12-19).

[0179] The epithelial cell adhesion rate is calculated using the following formula.

[0180]

[0181] Furthermore, to visually confirm the degree of adhesion of intestinal epithelial cells and lactic acid bacteria, the LIVE / DEAD BacLight bacterial cell viability assay kit was used. BacLight TM Lactic acid bacteria cells were stained with fluorescent dye using the BacterialViability kit and then observed under an optical microscope.

[0182] Results of intestinal epithelial cell adhesion rate as follows Figure 6 As shown.

[0183] like Figure 6 As shown, compared with the control group, the adhesion ability of HT-29 cell lines was confirmed to increase when coated with silk fibroin, and this increase was further increased when coated with silk fibroin that had undergone a pretreatment process. However, compared with lactic acid bacteria probiotics prepared by coating with silk fibroin that had undergone an appropriate pretreatment process, the adhesion ability of HT-29 cells coated with cellulose in combination with silk fibroin that had undergone an appropriate pretreatment process was slightly reduced.

[0184] Experimental Example 6: Intestinal Environment Stability of Lactic Acid Bacteria Cells Based on Silk Fiber Coating Method

[0185] To evaluate the intestinal environment stability of probiotic lactic acid bacteria prepared using the silk fibroin coating method, experiments were conducted to test the acid resistance and bile resistance of each raw material.

[0186] To determine acid tolerance, probiotics were exposed to simulated gastric fluid conditions at pH 2.5 and pH 2.0, and probiotic counts were analyzed. Specifically, the simulated gastric fluid conditions used in food disintegration tests (2.0 g sodium chloride, 24.0 ml / L dilute hydrochloric acid, pH 1.2) were adjusted to final pH 2.0 and pH 2.5 before exposure to 10% probiotic powder. Taking into account gastric contraction movements, a dancing machine (BMS Co., Ltd.) was used to perform 100 reciprocating movements per minute to expose the probiotics to conditions similar to the gastrointestinal environment. The exposure time was over 2 hours, taking into account transit time. After adjusting the pH to 7.0, the experimental group exposed to the simulated gastric fluid conditions was analyzed according to standard probiotic count methods.

[0187] On the other hand, bile resistance was tested using a sterile filtered medium containing 0.5% bile acids, with 10% probiotic powder added. After reacting for 2 hours, the number of probiotics was determined according to standard methods.

[0188] The results are shown in Table 5 (intestinal environment survival rate (%) based on lactic acid bacteria application method).

[0189] Table 5

[0190]

[0191]

[0192] As shown in Table 5, coating lactic acid bacteria with fermented alcohol-based silk fibroin generally increased their acid and bile resistance. Under pH 2.0 conditions, compared to uncoated experimental group 3, the acid resistance of *Streptococcus thermophilus* strain CKDB021, *Bifidobacterium bifidum* strain CKDB001, *Enterococcus faecium* strain CKDB003, *Bifidobacterium lactis* strain CKDB005, and *Lactobacillus acidophilus* strain CKDB007 increased by more than two times. Under simulated intestinal fluid conditions, bile resistance also tended to increase overall with silk fibroin coating. For *Bifidobacterium lactis* strain CKDB005 and *Lactobacillus acidophilus* strain CKDB007, the survival rate was best when coated under the conditions of experimental group 2. Therefore, coating with silk fibroin, pre-treated silk fibroin with fermented alcohol, or a combination of these with cellulose significantly increased the acid and bile resistance of lactic acid bacteria probiotics.

[0193] Experimental Example 7: Storage stability of lactic acid bacteria cells based on silk fibroin coating method

[0194] In the case of lactic acid bacteria and probiotics, a certain number of probiotics should be maintained and preserved in circulation devices for a period of 12 to 24 months.

[0195] When lactic acid bacteria, such as *Enterococcus faecium* and *Enterococcus faecalis*, are produced, they exhibit high stability when exposed to high temperature and high humidity conditions. However, in the case of most lactic acid bacteria, their stability is significantly reduced when exposed to high temperature and high humidity conditions.

[0196] In this invention, in order to confirm the storage stability of lactic acid bacteria probiotics, 50g units were divided into polyethylene bags (inner) and aluminum bags (outer) and stored under harsh conditions (temperature 45°C, humidity 75%), and samples were collected according to the storage period to confirm the number of probiotics.

[0197] The results are shown in Table 6 (Storage stability (%) based on lactic acid bacteria coating method).

[0198] Table 6

[0199]

[0200] As shown in Table 6, in the cases of Bifidobacterium lactis, Enterococcus faecium, and Lactobacillus acidophilus, it was confirmed that the stability of the coating process using silk fibroin was significantly increased under harsh conditions. In addition, the stability of Bifidobacterium bifidum and Streptococcus thermophilus was also slightly increased under harsh conditions.

[0201] [Collection Number]

[0202] Name of the depository: Korea Institute of Life Sciences

[0203] Accession number: KCTC13114BP

[0204] Date of preservation: September 23, 2016

[0205] Name of the depository: Korea Institute of Life Sciences

[0206] Accession number: KCTC13115BP

[0207] Date of preservation: September 23, 2016

[0208] Name of the depository: Korea Institute of Life Sciences

[0209] Accession number: KCTC13116BP

[0210] Date of preservation: September 23, 2016

[0211] Name of the depository: Korea Institute of Life Sciences

[0212] Accession number: KCTC13117BP

[0213] Date of preservation: September 23, 2016

[0214] Name of the depository: Korea Institute of Life Sciences

[0215] Accession number: KCTC13118BP

[0216] Date of preservation: September 23, 2016

[0217] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0218] International Forms

[0219] Issued in accordance with Article 7.1 of the Implementing Rules

[0220] For the original preserved receipt

[0221] Depositor:

[0222] Jonggundang BIO Co., Ltd.

[0223] Jonggundang BIO Co., Ltd.

[0224] 292 Shinwon-ro (15604), Danwon-gu, Ansan-si, Gyeonggi-do, South Korea

[0225]

[0226] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0227] International Forms

[0228] Issued in accordance with Article 7.1 of the Implementing Rules

[0229] For the original preserved receipt

[0230] Depositor:

[0231] Jonggundang BIO Co., Ltd.

[0232] Jonggundang BIO Co., Ltd.

[0233] 292 Shinwon-ro (15604), Danwon-gu, Ansan-si, Gyeonggi-do, South Korea

[0234]

[0235] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0236] International Forms

[0237] Issued in accordance with Article 7.1 of the Implementing Rules

[0238] For the original preserved receipt

[0239] Depositor:

[0240] Jonggundang BIO Co., Ltd.

[0241] Jonggundang BIO Co., Ltd.

[0242] 292 Shinwon-ro (15604), Danwon-gu, Ansan-si, Gyeonggi-do, South Korea

[0243]

[0244] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0245] International Forms

[0246] Issued in accordance with Article 7.1 of the Implementing Rules

[0247] For the original preserved receipt

[0248] Depositor:

[0249] Jonggundang BIO Co., Ltd.

[0250] Jonggundang BIO Co., Ltd.

[0251] 292 Shinwon-ro (15604), Danwon-gu, Ansan-si, Gyeonggi-do, South Korea

[0252]

[0253] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings

[0254] International Forms

[0255] Issued in accordance with Article 7.1 of the Implementing Rules

[0256] For the original preserved receipt

[0257] Depositor:

[0258] Jonggundang BIO Co., Ltd.

[0259] Jonggundang BIO Co., Ltd.

[0260] 292 Shinwon-ro (15604), Danwon-gu, Ansan-si, Gyeonggi-do, South Korea

[0261]

Claims

1. A composition, which increases survival rate, storage stability, and resistance to acid or bile of a lactic acid bacterium, which increases mucin-binding ability of a lactic acid bacterium, and / or which increases enterocyte-attachment ability of a lactic acid bacterium, the composition comprising a lactic acid bacterium coated with silk fibroin and cellulose, wherein the silk fibroin is silk fibroin pre-treated with ethanol, wherein the concentration of the ethanol is 85% (v / v) or more, and wherein the lactic acid bacterium is selected from the group consisting of: Lactobacillus acidophilus CKDB007 having accession number KCTC 13117BP, Enterococcus faecium CKDB003 having accession number KCTC 13115BP, Streptococcus thermophilus CKDB021 having accession number KCTC 13118BP, Bifidobacterium bifidum CKDB001 having accession number KCTC 13114BP, and Bifidobacterium lactis CKDB005 having accession number KCTC 13116BP. The composition is selected from the group consisting of a food composition, a probiotic composition, a pharmaceutical composition, and a feed composition.

3. A method of increasing survival rate, storage stability, and resistance to acid or bile of a lactic acid bacterium, increasing mucin-binding ability of a lactic acid bacterium, and / or increasing enterocyte-attachment ability of a lactic acid bacterium, the method comprising the step of coating a lactic acid bacterium with silk fibroin and cellulose, wherein the silk fibroin is silk fibroin pre-treated with ethanol, and wherein the concentration of the ethanol is 85% (v / v) or more. The lactic acid bacterium is selected from the group consisting of Lactobacillus, Lactococcus, Enterococcus, Streptococcus, and Bifidobacterium. characterized in that The lactic acid bacterium is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus, Bifidobacterium bifidum, and Bifidobacterium lactis. The lactic acid bacterium is selected from the group consisting of: Lactobacillus acidophilus CKDB007 having accession number KCTC 13117BP, Enterococcus faecium CKDB003 having accession number KCTC 13115BP, Streptococcus thermophilus CKDB021 having accession number KCTC 13118BP, Bifidobacterium bifidum CKDB001 having accession number KCTC 13114BP, and Bifidobacterium lactis CKDB005 having accession number KCTC 13116BP.

7. Use of silk fibroin and cellulose for coating a lactic acid bacterium for the following purposes: increasing survival rate, storage stability, and resistance to acid or bile of the lactic acid bacterium, increasing mucin-binding ability of the lactic acid bacterium, and / or increasing enterocyte-attachment ability of the lactic acid bacterium, wherein the silk fibroin is silk fibroin pre-treated with ethanol, wherein the concentration of the ethanol is 85% (v / v) or more, and wherein the lactic acid bacterium is selected from the group consisting of: Lactobacillus acidophilus CKDB007 having accession number KCTC 13117BP, Enterococcus faecium CKDB003 having accession number KCTC 13115BP, Streptococcus thermophilus CKDB021 having accession number KCTC 13118BP, Bifidobacterium bifidum CKDB001 having accession number KCTC 13114BP, and Bifidobacterium lactis CKDB005 having accession number KCTC 13116BP. ​ ​ ​ ​ ​ ​ 2. The composition of claim 1, wherein, ​ ​ ​ ​ ​ ​ ​ ​ 4. The method of claim 3, wherein, ​ 5. The method of claim 4, wherein, ​ 6. The method of claim 5, wherein, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Enterococcus faecium CKDB003 having the accession number KCTC 13115BP, Streptococcus thermophilus CKDB021 having the accession number KCTC 13118BP, Bifidobacterium bifidum CKDB001 having the accession number KCTC 13114BP, and Bifidobacterium lactis CKDB005 having the accession number KCTC 13116BP.

Images