KR102323673B1 - Manufacturing method for HtrA(high temperature requirement A) chaperone probiotics and composition manufactured through thereof having the effect of controlling the intestinal microbiome for the treatment of inflammatory bowel disease - Google Patents

Abstract

The present invention relates to a method for producing HtrA chaperone probiotics and a composition for treating inflammatory bowel disease having an effect of regulating the intestinal microbiome prepared therethrough. In previous studies, in order to induce chaperones of lactic acid bacteria, a method of applying a high-strength shock such as treatment with a low temperature, high temperature, high concentration of salt or organic solvent for a short time was used. While there are limitations in industrial application, the present invention induces the expression of HtrA chaperone without loss of cells during the closed system culture process of lactic acid bacteria, thereby improving resistance to external environmental stress, and inflammatory bowel disease using lactic acid bacteria as a single probiotic agent. It is possible to develop a next-generation chaperone-based pharmacobiotic formulation that can treat and further induce changes in the intestinal microbiome.

Description

Manufacturing method for HtrA chaperone probiotics and a composition for treating inflammatory bowel disease having the effect of controlling the intestinal microbiome manufactured therethrough microbiome for the treatment of inflammatory bowel disease

The present invention relates to a method for producing HtrA chaperone probiotics and a composition for treating inflammatory bowel disease having an effect of regulating the intestinal microbiome prepared therethrough.

In more detail, the present invention develops an industrial production method for inducing lactic acid bacteria in the lactic acid bacteria culture step by discovering stress factors that can induce HtrA chaperone, a defense mechanism of external environmental factors, among intestinal microorganisms. When ingested into the human body using one lactic acid bacteria raw material, the gastrointestinal tract survival rate of lactic acid bacteria, their ability to adhere to the intestinal mucosa as live bacteria, and the existing flora are secreted from the competitive exclusion with the existing flora to exclude foreign strains from adhesion to the intestinal mucosa. Therapeutic effect (competitive exclusion) in an inflammatory bowel disease animal model that confirmed whether there is resistance to the alcoholic antagonist used and further induced the criteria necessary for using the HtrA chaperone-derived probiotic as a pharmaceutical treatment with a pathogenic strain Through the therapeutic effect), it is possible to derive a manufacturing method that can be used as a single live cell treatment for HtrA chaperone Pharmabiotics.

Probiotics, meaning 'for life' in Latin, were defined by the WHO/FAO in 2002 as living microorganisms that help the health of the host when consumed in an appropriate amount. Representative species of probiotics include Lactobacillus sp., Bifidobacterium sp., and Streptococcus sp. They are classified as lactic acid bacteria because they produce organic acids of

Pharmabiotics is a compound word of pharmaceutical and probiotics, and is defined as a microorganism or a metabolite produced by a microorganism having a proven pharmaceutical role for health or disease (Hill, 2010) . For example, in recent years, probiotics correct dysbiosis of the intestinal microflora and based on this, the possibility of developing a therapeutic agent for the control of allergic diseases is highly evaluated. is losing (Im, 2018).

In order for probiotics to be used as pharmabiotics, the following conditions must be met as prerequisites. First, as a drug, it must be possible to prescribe it as a single agent. In pharmaceuticals, qualitative and quantitative analysis results of each ingredient for effective effects should be presented, but in foreign product cases, there are cases in which the therapeutic effect of a specific disease is advertised using a homogenous strain and multi-strains. . In the case of using the same type of lactic acid bacteria in the product, qualitative and quantitative analysis of each lactic acid bacteria is not possible. Second, ingestion stability must be secured. Ingestion stability must be secured by improving resistance to stressors because the death of strains caused by various stress factors in the intestine, such as gastric acid and bile acid, causes an insufficient number of intestinal bacteria and lowers the potential as a therapeutic agent.

Third, it should be possible to settle in the intestine through securing the ability to adhere to the intestine. Currently, intestinal obligate anaerobic strains are being used to develop therapeutics based on their excellent intestinal adhesion ability.

The probiotic activity in the intestinal tract of lactic acid bacteria is largely divided into a microbe to microbe relationship and a microbe to tissue relationship. The relationship between bacteria (菌) and bacteria (菌) is a functional indication that the proliferation of lactic acid bacteria and the inhibitory effect of harmful bacteria are managed as a function notified by the Ministry of Food and Drug Safety. The relationship between bacteria and tissues is based on the mechanism of interaction with the host human immune cell, and suppresses the invasion of harmful bacteria through the strengthening of tight junctions, and dendritic cells It stimulates the secretion of anti-inflammatory cytokines to relieve inflammation in the intestinal tract and inhibits the adhesion of harmful bacteria, thereby maintaining the balance of the intestinal flora, ultimately playing a role in realizing human health.

In order to treat inflammatory bowel disease induced by pathogenic bacteria based on the competitive exclusion of bacteria in the intestine, it is necessary to consume more than a certain number of live bacteria. It is difficult to expect the effect as it is not achieved. In addition, pathogenic bacteria that cause inflammatory bowel disease secrete toxin substances and disrupt intestinal ion channels, thereby causing diarrhea in the host. Diarrhea caused by the influx of water from the lumen in the large intestine loses the opportunity for live lactic acid bacteria to attach to the intestinal mucosa, ultimately lowering the potential as a therapeutic agent.

In the case of using lactic acid bacteria as a solution to treat inflammatory bowel disease, three methods have been used in the industry to solve the problem of killing the probiotics in the gastrointestinal tract during oral administration.

The first is to kill the lactic acid bacteria to solve the death of bacteria when passing through the gastrointestinal tract, and to make the particle size of the dead cells smaller than the size of the live cells to avoid competitive exclusion with the flora in the intestinal flora, similar to the toll of dendritic cells in the intestinal mucosa. It is a treatment that is expected to have a mechanism to suppress inflammation by increasing intestinal immunity by making it react directly to a toll-like receptor. However, although the original drug for this dead cell treatment was lacteol from Dongwha Pharm, the item was canceled because the dead cell strain was arbitrarily changed without notification to the Ministry of Food and Drug Safety. Since it was deleted from the Codex outside the Pharmacopoeia, the lactic acid bacteria dead cell raw material cannot be used as a raw material for health functional food as well as medicine, and its industrial value as a food raw material is low. In particular, the dead cell quantification method has not been established not only in Korea but also abroad, so it has been pointed out as the biggest problem in the development of a therapeutic agent for this formulation.

The second is a therapeutic approach using multiple types of microorganisms, which are Streptococcus thermophilus , Bifidobacterium breve , Bifidobacterium animalis ssp. lactis , Lactobacillus acidophilus , Lactobacillus plantarum , Lactobacillus paracasei , and Lactobacillus helveticus were developed in a way to help diarrhea caused by intestinal immune abnormalities by manufacturing a combination drug. However, as shown in the composition, this combination drug is a combination of the same type of lactic acid bacteria, so it cannot be used as a medicine because there is no method to quantify and analyze the content of each same type of lactic acid bacteria in the pharmaceutical formulation oriented to single formulation prescription. Even if it is specially used as When prescribing in an amount, it is difficult to confirm whether it has an effective action, which lowers the effectiveness of product development.

The third is the development of therapeutic agents using intestinal obligate anaerobes related to the microbiome , mainly using Akkermensia muciniphila and Faecalibacterium prausnitzii and researching their potential. However, these strains cannot use the general production process of lactic acid bacteria exposed to air due to the strain characteristics that die when exposed to oxygen. As it is being developed as a user interface, there is an aspect that user inconvenience is aggravated. In addition, the most important problem that is pointed out as the most difficult problem in the development of therapeutic agents using these strains is that the obligate anaerobic enterobacterium has excellent interaction with the intestinal mucosa, so it can have an effective effect in treating inflammatory diseases, but it is still recognized as a GRAS strain. Safety issues such as multiple reports of side effects have not been resolved.

In the field of life science, chaperone refers to molecules that protect proteins from various types of stress, and is a protein component that can be consumed by the human body. Conventional techniques for inducing chaperones in microorganisms such as lactic acid bacteria apply a high-intensity shock of the external environment within a short time. The resulting chaperones have properties that resist specific environmental stresses. A typical stress is heat shock, and DnaK, GroEL, etc. are representative of the chaperones generated by this, and the microorganisms produced by this chaperone have higher thermal stability than that of a wild type strain. However, although such heat shock therapy can be used in the laboratory, there is a limit to its industrial application. This is because there must be a process of instantaneously giving high heat for a few seconds at a temperature that does not kill microorganisms, then taking the heat off and lowering it to a normal temperature. However, since the fermenter for fermenting microorganisms is a closed system designed to prevent contamination of external microorganisms, it takes a long time to release the heat energy supplied from the outside, and the heat maintained within this time causes death of the fermenting microorganisms. This makes it difficult to produce a normal probiotic culture. In more detail, since the internal medium temperature is controlled by water in the outer jacket of the fermenter, it takes more than 1 hour to lower the raised internal medium, and all lactic acid bacteria that ferment can be killed, so it cannot be applied industrially. In addition, when chaperones are induced through a stress source of weak strength in order to reduce the level of lactic acid bacteria apoptosis, it is difficult to expect the effect of improving the stability of lactic acid bacteria through induction of chaperone expression because it indicates a limit to the induction of chaperone expression.

In addition, studies have been reported to induce the expression of chaperones through solvent shock using an organic solvent such as alcohol. It induces the death of microorganisms, and there is a difficulty in industrial application due to a decrease in productivity and residual problems in organic solvents.

In the present invention, lava seawater is used as culture water in a large-capacity fermenter classified as a closed system, which has been pointed out as a technical limitation of the prior art, and the HtrA chaperone expression is induced through substitution with a medium containing high concentration of sugar so that the live cell productivity of lactic acid bacteria does not fall. The mass production method was presented for the first time, and furthermore, the possibility of induced HtrA chaperone probiotics as a solution as a therapeutic pharmaceutical solution was demonstrated in a disease animal model. After exposing high-concentration sugar to the fermentation process of lactic acid bacteria as a stress source, it induces the expression of HtrA chaperone, thereby securing acid resistance and bile acid resistance related to intake stability. It has the characteristics of resistance to alcohol, and ultimately, a next-generation chaperone-based pharmacobiotic design method that can cure inflammatory bowel disease even with one lactic acid bacteria and induce changes in the intestinal microbiome was derived.

Republic of Korea Patent Registration No. 10-2188020 (Title of the invention: Lactobacillus paracasei strain and its use, Applicant: Gobio Lab Co., Ltd., Registration date: December 01, 2020)

Foucaud-Scheunemann C, Poquet I. (2003) HtrA is a key factor in the response to specific stress conditions in Lactococcus lactis. FEMS Microbiol Lett. 224(1):53-59. Mustapha M, Mohd AB, Synan FA, Khaled AE, Reyad SO, Tareq MO, Anas AA, Mark ST, Nagendra PS, Mutamed M A. (2020) Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyzes. Compr Rev Food Sci Food Saf. 19(3), 1110-1124.

It is an object of the present invention to provide a method for producing HtrA chaperone probiotics and a composition for treating inflammatory bowel disease having an effect of regulating the intestinal microbiome prepared therethrough. That is, through the present invention, the gastrointestinal tract survival rate is improved, the intestinal mucosa adhesion is excellent, and the resistance to alcoholic antagonists is improved, so that the HtrA chaperone has the characteristics of a pharmaceutical such as a therapeutic agent for inflammatory bowel disease with an excellent intestinal microbiome control function. Probiotics may be provided.

The present invention relates to a method for producing high temperature requirement A (HtrA) chaperone probiotics.

More preferably, the manufacturing method comprises:

(Step 1) preparing a medium for culturing lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water;

(Second step) inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria to obtain a culture solution containing primary-HtrA chaperone probiotics; and,

(Step 3) Substituting a medium containing sugar for the primary HtrA chaperone probiotics obtained by centrifuging the culture solution and then culturing to prepare secondary-HtrA chaperone pharmabiotics;

may include.

The medium for culturing lactic acid bacteria contains glucose, yeast extract, soy peptone and casein, and is a medium for culturing lactic acid bacteria prepared by dissolving the components in water containing 30 to 70% (v/v) lava seawater and sterilizing it. have. The medium is based on a total volume of 1 liter, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soipetone 0.5-5% (w/v), casein 0.5-3% (w) /v) may be a medium for culturing lactic acid bacteria prepared by dissolving each component in water containing 30 to 70% (v/v) of lava seawater and sterilizing it. The medium sterilization is preferably performed at 121 ~ 123 ℃ 30 ~ 60 minutes.

In the present invention, when probiotics are cultured in a medium containing a high concentration of sugar, glucose, fructose, sucrose, maltose, or a mixture of two or more of glucose, fructose, sucrose, maltose, and trehalose can be used to more remarkably increase the expression enhancing effect of HtrA chaperone. can

The lactic acid bacteria are Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Pediococcus genus ( Pediococcus sp.) and Weissella sp. It is characterized in that it is selected from the group consisting of.

It is preferable that the sugar addition concentration is 10 g/L to 100 g/L, and the sugar is preferably a mixture of sucrose and trehalose, and the mixed weight ratio thereof is that the content of trehalose is 20 to 50% (w/w). desirable. The culturing process after the sugar addition is not limited thereto, but may preferably be performed for 15 minutes to 2 hours.

The HtrA chaperone probiotics prepared in the present invention are preferably freeze-dried. For freeze-drying, a cryoprotectant and a coating agent may be treated, and the cryoprotectant is selected from at least one selected from powdered skim milk, soy flour, amino acids, peptides, gelatin, glycerol, sugar alcohol, whey, alginic acid, ascorbic acid and yeast extract. can be It is possible to use the cryoprotectant in an appropriate combination in consideration of changes in flavor, texture, etc. or product production cost. The coating agent is chitosan, malto-dextrin, indigestible dextrin, xanthan gum (XG), guar gum (GG), carboxymethyl cellulose (CMC). ), hydroxyethylcellulose (HEC), polyvinylpyrroridone (PVP), carbopol, sodium alginate, propylene glycol alginate, alginate ), polyethylene glycol (PEG), triacetin, propylene glycol, acetyl triethyl citrate, and hydroxypropyl methyl cellulose (HPMC) have.

The composition of the present invention can be provided as a pharmaceutical composition. The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of the skilled artisan, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. Since the composition of the present invention has almost no toxicity and side effects, it is a drug that can be safely used even when taken for a long period of time for prophylactic purposes.

In addition, the composition of the present invention provides a health functional food containing a food supplementary additive that is acceptable. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquids, and the food to which the composition of the present invention can be added includes, for example, various drinks, meat, sausage, bread, candy, Snacks, noodles, ice cream, dairy products, soups, ionized beverages, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

The present invention relates to a method for producing HtrA chaperone probiotics and a composition for treating inflammatory bowel disease having an effect of regulating the intestinal microbiome prepared therethrough. In the present invention, by inducing the expression of HtrA chaperone during culture through lava seawater-based culture of lactic acid bacteria, acid resistance and bile resistance, which are indicators of intestinal stability, are significantly increased. Provided are a method for producing probiotics that significantly improve adhesion ability and resistance to alcoholic antagonists, and HtrA chaperone probiotics prepared by the method. In previous studies, in order to induce chaperones of lactic acid bacteria, a method of applying a high-strength shock such as treatment with a low temperature, high temperature, high concentration of salt or organic solvent for a short time was used. While there is a limit to industrial application, the present invention induces the expression of HtrA chaperone during the culturing process of lactic acid bacteria to improve resistance to external environmental stress, thereby treating inflammatory bowel disease using lactic acid bacteria as a single probiotic and further It is possible to develop a next-generation chaperone-based pharmacobiotic formulation that can induce changes in the intestinal microbiome.

1 is a result confirming the change in the HtrA chaperone expression level and the cell mass of HtrA chaperone probiotics according to the added sugar type when the high-concentration sugar medium for inducing HtrA chaperone is replaced.
2 is a result confirming the change in the HtrA chaperone expression level and the cell mass of HtrA chaperone probiotics according to the sucrose concentration when the medium is replaced with a high-concentration sugar for inducing HtrA chaperone.
3 is a result confirming the change in the HtrA chaperone expression level and the cell mass of the HtrA chaperone probiotics according to the trehalose concentration when the medium is replaced with a high-concentration sugar for inducing HtrA chaperone.
4 is a result confirming the change in the HtrA chaperone expression level and the cell mass of the HtrA chaperone probiotics according to the mixing ratio of trehalose by weight in the added sucrose and trehalose mixture when the high-concentration sugar medium for inducing HtrA chaperone is replaced.
5 is a schematic diagram of a method for producing HtrA chaperone probiotics of the present invention.
Figure 6 is a result of colonic contraction relief by ingestion of HtrA chaperone pharmabiotics in an inflammatory bowel disease (IBD) mouse model.
7 is a result of changes in colon length and myeloperoxidase (MPO) activity by ingestion of HtrA chaperone pharmabiotics in an inflammatory bowel disease (IBD) mouse model.
8 is an inflammatory bowel disease (IBD) mouse model of HtrA chaperone by ingestion of Pharmabiotics inflammatory-non-inflammatory cytokine expression results of the colon.
9 is a result of α-diversity of changes in the intestinal microbiome by ingestion of HtrA chaperone Pharmabiotics in an inflammatory bowel disease (IBD) mouse model.
10 is a β-diversity result of changes in the intestinal microbiome by ingestion of HtrA chaperone Pharmabiotics in an inflammatory bowel disease (IBD) mouse model.
11 is a result of the correlation between changes in the intestinal microbiome and changes in colon cytokine expression caused by ingestion of HtrA chaperone pharmabiotics in an inflammatory bowel disease (IBD) mouse model.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

< manufacturing example 1. Method of manufacturing and culturing basic medium for lactic acid bacteria>

As a basic medium for culturing lactic acid bacteria in the present invention, Lactobacilli MRS Broth (BD) was prepared using constant water and sterilized at 121° C. for 15 minutes. In this sterilized basic medium, the lactic acid bacteria seed culture was inoculated into the sterile medium of the fermenter and cultured at 100rpm, 37°C for 20 hours. Thereafter, centrifugation was performed at 13,000 rpm and 4° C. for 10 minutes, and after removal of the supernatant, the same amount of PBS (phosphate buffered saline, phosphate buffer solution) was added and suspended, and used as a sample.

* Lactobacillus seed culture medium: Lactobacilli MRS Broth (BD) cultured for 24 hours at 37°C, hereinafter referred to as lactobacillus seed culture medium.

<Example 1. Preparation of HtrA chaperone probiotics using lava seawater>

In order to induce improvement of HtrA chaperone expression in Pharmabiotics, water containing 30% (v/v) lava seawater was prepared as culture water, and was used instead of constant water in the basic medium preparation conditions used in Preparation Example 1. After sterilization, the prepared medium was inoculated with a lactic acid bacteria culture medium and cultured for 20 hours at 100 rpm and 37° C. conditions. Thereafter, centrifugation was performed at 13,000 rpm and 4° C. for 10 minutes, and after removal of the supernatant, the same amount of PBS (phosphate buffered saline, phosphate buffer solution) was added and suspended, and used as a sample.

<Example 2. Preparation of HtrA chaperone probiotics using additional culture per high content after culturing in lava seawater>

After culturing lactic acid bacteria using water containing 30% (v/v) lava seawater, the HtrA chaperone expression was induced through a higher level of osmotic stress by substituting it with a high-concentration sugar-based suspension medium. In more detail, as in Example 1, water containing 30% (v/v) lava seawater was used as culture water to inoculate a lactic acid bacteria seed culture solution, and cultured for 20 hours at 100rpm, 37°C conditions. Thereafter, the cells were concentrated, and 5 types of sugars including glucose, glycerol, maltose, sucrose, and trehalose were added to each of 30 g/L, and incubated at 100 rpm and 37° C. for 30 minutes. Thereafter, centrifugation was performed at 13,000 rpm and 4° C. for 10 minutes, and after removal of the supernatant, the same amount of PBS buffer was added and suspended for use in subsequent experiments. Alternatively, as each saccharide, sucrose was added in a concentration range of 0-100 g/L, or complex saccharide culture conditions such as a mixture of trehalose and sucrose were variously tested.

<Experimental Example 1. Check the number of viable cells>

The culture solution of Preparation Example 1 and Example 1 was diluted, 1 ml was dispensed in a petridish, and 20 ml of sterilized MRS agar (BD) medium was added to harden. The number of viable cells was confirmed by counting colonies of Petri dishes cultured for 48 hours in a stationary incubator at 37°C. According to the results of [Table 1], it can be confirmed that the lactic acid bacteria cultured using the lava seawater of Example 1 does not decrease the number of viable cells compared to Preparation Example 1, which is the control.

Number of viable cells after culture completion (CFU/ml) Preparation Example 1 1.10 x 10 ⁸ Example 1 1.35 x 10 ⁸

< Experimental example 2. HtrA chaperone expression OK>

To confirm the effect of lava seawater-based culture on the mRNA expression level of HtrA chaperone, real time quantitative PCR was performed. For this, 1 ml of the suspension was centrifuged at 13,000 rpm, 4° C. for 5 minutes, and the centrifuged RNeasy extraction kit (Qiagen) was used to extract total RNA according to the manufacturer's instructions. The extracted total RNA was suspended in 50 μl of RNase-free water, and the RNA concentration of the suspended solution was confirmed using an ND-1000 spectrophotometer. A primer specific to the HtrA chaperone gene (htrA) and a primer for the control gyrA gene were designed using a primer design program (GenScript, Real-time PCR (TaqMan) Primer and Probes Design Tool), and the primers for each gene were The forward and reverse primer pairs shown in Table 2 below were used.

primer order SEQ ID NO: htrA forward 5’-CCGGAACGACCAAGATTTCC-3’ One reverse 5’-AAAGTATCCAGCCCATCGCT-3’ 2 gyrA forward 5’-GGTCTGATTGCGCTCAACTT-3’ 3 reverse 5’-ACCCATTGACCGAACATCCT-3’ 4

The cDNA was synthesized using 1 μg total RNA and a cDNA synthesis kit (Bio-Rad). SYBR Green-based quantitative PCR amplification was performed using CFX Connect Real-Time system (Bio-Rad) after mixing 2 μl of cDNA and 10 pmol of primer with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The PCR program is as follows. After holding at 95°C for 3 minutes, 10 seconds at 95°C and 30 seconds at 55°C were repeated 40 times. Thereafter, temperature dissociation curves (Melting curves) of the PCR product were prepared by holding at 95°C for 1 minute, at 55°C for 30 seconds, and at 95°C for 30 seconds. All experiments were repeated three times, and PCR data were analyzed according to the 2-ΔΔCt method (Livak and Schmittgen, 2001). At this time, the expression level of the HtrA chaperone of the lactic acid bacteria cultured by Preparation Example 1 was used as a control, and the difference was confirmed by comparing the gyrA-normalized 2-ΔΔCt data. As a result, as shown in [Table 3], the expression of the HtrA chaperone was not confirmed in Preparation Example 1, a control group, but when salt osmotic stress was induced during culture using lava seawater, the mRNA expression of the HtrA chaperone was not reduced without a decrease in cell production. It was confirmed that this was induced.

classification HtrA chaperone expression ^** Preparation Example 1 - Example 1 + -: HtrA chaperone non-expression, +: HtrA chaperone expression confirmation

< Experimental example 3. High-concentration sugar medium replacement HtrA chaperone Expression check>

The amount of HtrA chaperone expression according to various high-concentration sugar medium substitution conditions performed in Example 2 was confirmed. After culturing in lava seawater, each real-time quantitative PCR was performed under conditions in which HtrA chaperone expression was induced through a high level of osmotic stress by substituting with a high-concentration sugar-based suspension medium. did. PCR conditions were the same as in Experimental Example 2.

As a result, as shown in [Table 4] and [Fig. 1], there was a difference in the induction of HtrA chaperone expression according to the type of sugar substituted at a high concentration. When galactose, xylose, mannose, lactose, glycerol, etc. were added, no improvement in expression of HtrA chaperone was confirmed compared to the lava seawater cultured lactic acid bacteria of Example 1, but high concentrations of specific sugars such as glucose, fructose, sucrose, maltose, and trehalose In the case of lactic acid bacteria cultured by substituting the suspension medium, it was confirmed that HtrA chaperone expression was induced at a high level. Preferably, it showed the highest expression rate when sucrose was added. Through this, it was confirmed that the expression level of the HtrA chaperone was significantly enhanced by replacing the medium with a high concentration of sugar for inducing the HtrA chaperone.

classification HtrA chaperone expression ^** Example 1 Lava seawater culture + monosaccharides glucose ++ fruit sugar ++ galactose + xylose + Mannose + saccharose sucrose +++ maltose ++ lactose + Trehalose +++ etc glycerol + ^** +: HtrA chaperone expression induction confirmation and expression level similar to Example 1, ++: HtrA chaperone expression improved by 30% or more compared to Example 1, +++: HtrA chaperone expression increased by 60% or more compared to Example 1

Next, in order to confirm whether the induction of HtrA chaperone expression according to various high-concentration medium substitution conditions performed in Example 2 is also applied to other lactic acid bacteria, each of the four lactic acid strains including Lactobacillus plantarum was cultured, and HtrA expression induction for this Real time quantitative PCR (real time quantitative PCR) was performed to confirm whether the As a result, as shown in [Table 5], it was confirmed that the expression of HtrA protein was induced in all strains used in the experiment.

classification strain HtrA chaperone expression ^** Preparation Example 1 Lactobacillus plantarum - Lactobacillus Fermentum - Lactobacillus paracasei - Lactococcus lactis - Example 1 Lactobacillus plantarum + Lactobacillus Fermentum + Lactobacillus paracasei + Lactococcus lactis + Example 2 Lactobacillus plantarum +++ Lactobacillus Fermentum ++ Lactobacillus paracasei ++ Lactococcus lactis +++ ^** -: HtrA chaperone non-expression, +: HtrA chaperone expression induction confirmation, ++: HtrA chaperone expression improved by 30% or more compared to Example 1, +++: HtrA chaperone expression improved by 60% or more compared to Example 1

< Experimental example 4. sucrose and Trehalose according to concentration HtrA chaperone expression level>

In order to confirm the optimal concentrations of sucrose and trehalose in which HtrA expression was induced at high levels, the culture medium was concentrated and then treated with sucrose and trehalose compared to the concentrate, and then HtrA expression levels were compared.

As a result of comparing the HtrA expression level and the number of viable cells by adding sucrose and trehalose in a concentration range of up to 100 g/L, the HtrA chaperone expression was induced to a high level and the number of viable cells was increased when added to 50 g/L. On the other hand, when 100 g/L was added, it was confirmed that the cell mass, HtrA chaperone expression, and the number of viable cells decreased slightly [ FIGS. 2 and 3 ].

Through this, it was confirmed that the increase in the amount of sugar added to induce expression of the HtrA chaperone does not unconditionally increase the chaperone expression level, but also affects the amount of cell production.

<Experimental Example 5. HtrA chaperone expression level according to the use of complex sugar>

When a mixture of sucrose, a general sugar, and trehalose, a special sugar, that induces high-level expression of HtrA chaperone was used as a complex sugar for inducing expression of HtrA chaperone, trehalose was added at each mixing ratio to determine the effect of HtrA chaperone expression. Induction levels were compared.

Trehalose is a peat sugar to which two glucose molecules are bound. It is known as a substance produced by itself to protect cells when they are attacked by various stressors, and it is reported to play a role in preventing the degradation and denaturation of intracellular proteins. A mixture of sucrose and trehalose was used as a complex sugar for inducing HtrA chaperone expression, and the effect on HtrA chaperone expression was investigated (Jain and Roy, 2009).

To this end, a mixture of sucrose and trehalose of 50 g/L was prepared and used as a high-concentration sugar substitution medium for inducing HtrA expression, and the trehalose addition ratio was set to 0 to 100% by weight.

According to [Fig. 4], it was confirmed that the HtrA chaperone expression level was improved when trehalose was increased to 50% by weight based on the total weight of saccharides and replaced with a mixed complex sugar medium, and 20% by weight (sucrose: trehalose = 4: 1(w:w)) showed the highest HtrA chaperone expression level when substituted with a mixed sugar medium. When the mixing ratio of trehalose was increased to more than 50% by weight, the expression level of the HtrA chaperone did not increase compared to when each sugar was used alone. Through this, it was confirmed that the expression of the HtrA chaperone was induced at a higher level when trehalose was added at a certain ratio than when sucrose and trehalose were used alone when preparing a high-concentration sugar-substituted medium.

Therefore, in subsequent ingestion and intestinal stability evaluation experiments, sucrose and trehalose 50 g/L added group mixed at 4:1 (w:w) by weight, which is a culture condition optimized for HtrA chaperone protein expression, was used as HtrA chaperone probiotics. .

<Experimental Example 6. Improvement of intake stability of HtrA probiotics>

After oral ingestion, lactic acid bacteria are exposed to various stress factors in the human body before they settle in the intestine and show medicinal effects. evaluated.

Experimental Example 6-1. Acid resistance evaluation

Lactic acid bacteria are exposed to gastric acid after ingestion. Acid resistance was evaluated by creating a similar in vitro environment, and comparing the survival rate according to the induction of HtrA chaperone expression induced through culture after substitution of high-concentration sugar medium. More specifically, the pH of 100 ml of Lactobacilli MRS broth (BD) is adjusted to 3.0 using hydrochloric acid and then sterilized, and 0.4 g of pepsin (Sigma P7000, 250 Units/mg) is mixed with 100 ml of sterilized MRS broth to produce artificial gastric juice. Medium was prepared. In 100 ml of the prepared artificial gastric juice medium, each of the lactic acid bacteria cultures before spray drying prepared in Preparation Examples 1 and 2 were diluted 100 times and mixed. To check the initial number of viable cells, each mixture was immediately taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed in a Petri dish, and 20 ml of sterilized MRS agar (BD) medium was added to harden. Colonies of Petri dishes cultured for 48 hours in a stationary incubator at 37°C were counted. Thereafter, each mixture was exposed to artificial gastric juice at 37° C. for 2 hours, then the exposed mixture was diluted with physiological saline, 1 ml of the diluted solution was dispensed in a Petri dish, and 20 ml of sterilized MRS (BD) medium was added to harden. The number of viable cells was confirmed by counting colonies of Petri dishes cultured for 48 hours in a stationary incubator at 37°C. Each result is shown in [Table 6] below.

division acid resistance Initial (CFU/ml) After incubation (CFU/ml) Survival rate (%) Preparation Example 1 1.10x10 ⁸ 2.31x10 ⁷ 15 Example 1 1.35x10 ⁸ 4.46x10 ⁷ 33 Example 2 1.67x10 ⁸ 9.18x10 ⁷ 55

According to the results of [Table 6], the lactic acid bacteria cultured in lava seawater cultured with HtrA chaperone expression of Examples 1 and 2 and lactic acid bacteria cultured through lava seawater culture and high-concentration sugar medium substitution were acid-resistant compared to Preparation Example 1. It can be seen that it is very excellent (in Example 2, a survival rate of about 55% was confirmed, 40% increase compared to Preparation Example 1).

Experimental Example 6-2. Assessment of bile tolerance

Tolerance to bile acids was evaluated by creating an in vitro environment similar to bile acids exposed after ingestion of lactic acid bacteria expressing HtrA chaperone. An artificial bile medium was prepared by mixing 0.3 ml of sterilized oxgall solution with 100 ml of sterilized Lactobacilli MRS broth (BD). Thereafter, 1% (v/v) of the lactic acid bacteria cultured according to Preparation Examples 1 and 2 were inoculated into 100 ml of the prepared artificial bile medium, and the survival rate after exposure to bile acid for 5 hours was confirmed, and this was shown in [Table 7] shown in

division biliary tolerance Initial (CFU/ml) After incubation (CFU/ml) Survival rate (%) Preparation Example 1 1.03x10 ⁸ 1.34x10 ⁸ 130 Example 1 1.15x10 ⁸ 2.07x10 ⁸ 180 Example 2 1.29x10 ⁸ 3.03x10 ⁸ 235

As a result, it can be confirmed that the lactic acid bacteria cultured by Example 2 and induced to express the HtrA chaperone has improved bile resistance by 105% compared to Preparation Example 1.

<Experimental Example 7. Evaluation of intestinal stability of HtrA chaperone probiotics>

Experimental Example 7-1. Assessment of cell hydrophobicity and mucin adhesion

The lactic acid bacteria cell surface has a different polarity for each strain to facilitate interaction with the intestinal mucosa. In general, since intestinal mucosal cells exhibit hydrophobicity, the lactic acid bacteria cell surface must exhibit high hydrophobicity in order to have a good reaction binding force. More specifically, the improvement of the hydrophobicity of lactic acid bacteria affects the reactivity with mucin having hydrophobic characteristics, which are intestinal mucosal cells, and serves to increase intestinal adhesion. Therefore, the purpose of this study was to evaluate the effect of the induction of HtrA chaperone expression on the hydrophobicity of lactic acid bacteria and the change in mucin adhesion. Samples obtained through Preparation Examples 1 and 2 _{were diluted with PBS to OD 600nm} = 0.5±0.02. Then, 3 ml of the diluent and 1 ml of the solvent (Xylene) were evenly suspended, and then left at room temperature for 30 minutes. After standing, the hydrophobicity was calculated based on the OD (optical density, absorbance) value measured by collecting the water portion of the still solution and shown in [Table 8]. As a result, the hydrophobicity of the probiotics in which the expression of the HtrA chaperone prepared in Example 2 was induced was improved by about 50% compared to that of Preparation Example 1.

Cell surface hydrophobicity calculation formula

Cell surface hydrophobicity (%) = (1-(absorbance value after 30 minutes / initial absorbance value)) x 100

division Hydrophobicity (%) Preparation Example 1 33 Example 1 51 Example 2 83

In order to evaluate the mucin adhesion ability of HtrA chaperone Pharmabiotics with improved cell surface hydrophobicity, the following experiment was performed. 200 μl of 10 mg/ml mucin solution was dispensed into the wells of an immunoplate (Nunc) and reacted at 4°C for 18 hours or more to attach mucin to the wells. The mucin solution from each well was removed, and 200 μl of PBS was dispensed and washed once. 100 μl of the suspensions of Preparation Examples 1 and 2 were dispensed into each mucin well prepared in advance, and allowed to stand at 37° C. for 2 hours. The supernatant of the well was removed, and the cells that did not adhere to mucin were washed 5 times with 200 μl of PBS to remove them. 100 μl of 0.1% Triton X-100 was dispensed, covered with a lid, and automatically stirred at 1,000 rpm for 20 minutes. Cells attached to mucin were recovered by pipetting from the wells after stirring. The recovered solution was serially dilution with 0.85% (w/v) NaCl aqueous solution, and the number of attached bacteria was measured with a hemocytometer, which is shown in [Table 9].

Adhesion Calculation Formula

Adhesion (%) = (Number of adherent bacteria)/(Number of inoculum) x 100

In the above calculation formula, the number of bacteria is confirmed as cells/ml.

division Mucin adhesion (%) Preparation Example 1 8 Example 1 38 Example 2 59

Looking at the results of [Table 9], as the expression of the HtrA chaperone is induced, it is determined that the hydrophobicity of the lactic acid bacteria increases and the adhesion ability to the mucin having a hydrophobic surface increases, and through this, compared with Preparation Example 1 prepared by a general method Thus, it can be confirmed that the mucin adhesion ability, which is a prerequisite for generating actual immune activity, is improved by increasing it by about 50%. According to previous studies, the expression level of the HtrA chaperone present in the cell membrane affects the expression level of various membrane proteins present on the cell surface. It has been reported that adhesion can be improved by helping folding (Biswas and Biswas, 2005). Therefore, it was confirmed that the lactic acid bacteria in which the expression of the HtrA chaperone prepared in the present invention is induced has a high possibility of enhancing the immune response in the body through the improvement of the mucin adhesion ability constituting the mucin of the intestinal immune cells.

Experimental Example 7-2. Alcohol tolerance assessment

Considering the intestinal situation where numerous microorganisms coexist, research on the interaction between microorganisms is very important, and resistance to metabolites produced by harmful microorganisms in the intestines has a great impact on the intestinal settlement of Pharmabiotics. Therefore, the resistance of HtrA chaperone probiotics to alcohols, which is an important antagonist for the competitive exclusion of Mycobacteriaceae, was evaluated. In 100 ml of MRS broth containing 10% (v/v) of ethanol, 1% of the lactic acid bacteria culture medium of Preparation Example 1 and the high-concentration sugar medium for inducing HtrA chaperone expression of Example 2 was inoculated. The number of viable cells after 30 minutes of stationary culture compared to the initial number of viable cells was confirmed, and it is shown in [Table 10].

division alcohol tolerance Initial (CFU/ml) After incubation (CFU/ml) Survival rate (%) Preparation Example 1 1.14x10 ⁸ 3.64x10 ⁷ 32 Example 1 1.16x10 ⁸ 8.12x10 ⁷ 70 Example 2 1.20x10 ⁸ 1.32x10 ⁸ 110

Looking at the results of [Table 10], as the expression of the HtrA chaperone is induced, the resistance to alcohol is improved by about 70% or more compared to Preparation Example 1, which is secreted by the flora and exogenous strains from adhesion to the intestinal mucosa. It was confirmed that the resistance of lactic acid bacteria to the alcoholic antagonist used for exclusion was improved, and ultimately improved the potential as a therapeutic agent.

<Experimental Example 8. Mass production of HtrA chaperone probiotics>

The mass production process of HtrA chaperone probiotics was performed in order to find out whether the culture process inducing the expression of HtrA chaperone is applicable to the mass production process through the closed system fermenter [FIG. 5]. The production scale was cultured at 37°C for more than 16 hours in a 3 ton fermentation tank.

As a basic medium for this purpose, each component was prepared so that glucose 3% (w/v), yeast extract 2% (w/v), soipeptone 2% (w/v), and casein 1% (w/v) per 1 L The components were dissolved in water containing 30% (v/v) lava seawater and sterilized at 121°C for 30 minutes. In this sterilized medium, the lactic acid bacteria seed culture solution is inoculated into the sterile medium of the fermenter and cultured, while an aqueous solution in which 40% (w/v) glucose and 40% (w/v) sodium hydroxide are mixed in equal amounts is dripped at regular time intervals. The culture was maintained at a pH of 6.0 to 7.5. The culture solution under the above conditions was centrifuged to concentrate the cells, and 50 g/L of glucose or a mixture of sucrose and trehalose mixed in a ratio of 4:1 was added, followed by incubation at 50 rpm with a stirrer for 60 minutes to induce expression of the HtrA chaperone. . Then, about 100 kg of the recovered probiotics 50% by weight and hydroxypropylmethylcellulose 40% by weight (dry weight, dissolved in 5 times water) were stirred at 80 rpm for 20 minutes, followed by 10 weight of cryoprotectant % was mixed and then freeze-dried to obtain a dried powder. In this case, as the cryoprotectant, powdered skim milk was used. The control group, HtrA inactive probiotics, was prepared according to a conventional lactic acid bacteria fed-batch culture process using constant water for culture.

In order to confirm whether the mass-produced HtrA chaperone probiotics induce HtrA chaperone expression, real time quantitative PCR was performed under the same conditions as in Experimental Example 2. As a result, as shown in [Table 11], it can be seen that HtrA chaperone expression is induced without a decrease in the number of raw cells even in high-concentration sugar medium-substituted HtrA chaperone probiotics for inducing the expression of HtrA chaperone made in mass production. It can be confirmed that the culture process based on sugar medium substitution is applicable not only in the laboratory but also in industrial applications.

classification Raw material number of viable cells (CFU/g) HtrA chaperone expression ^*** HtrA Chaperone Inactive Probiotics 3.3x10 ⁹ - HtrA Chaperone Probiotics 2.7x10 ¹¹ + ^*** -: HtrA chaperone non-expression, +: HtrA chaperone expression confirmation

On the other hand, as a cryoprotectant during freeze-drying of mass-produced probiotics, instead of skim milk powder, when one selected from soy flour, amino acids, peptides, gelatin, glycerol, sugar alcohol, whey, alginic acid, ascorbic acid and yeast extract is used, similar raw materials The number of viable cells and the expression level of HtrA chaperone were confirmed.

<Experimental Example 9. Therapeutic Effect of HtrA Chaperone Pharmabiotics in Inflammatory Bowel Disease (IBD)>

The following experiment was conducted to confirm the therapeutic effect of HtrA chaperone pharmabiotics, which is HtrA chaperone probiotics with pharmabiotic properties, in inflammatory bowel disease (IBD).

The Saline healthy mice, the first groups were divided into five groups of mice, and the remaining four groups were was induced by pathogenic bacteria Klebsiella oxytoca the owner colitis by orally administered once a day five days ssikeul 10 ⁹ CFU causing intestinal inflammation. Colitis induced by oral administration of K. oxytoca Physiological saline, Sulfasalazine 50mg/kg, HtrA chaperone Pharmabiotics low concentration treatment group of ^{Example 2 (1x10 8} CFU), HtrA chaperone Pharmabiotics high concentration treatment group of Example 2 (1x10 ⁹ CFU) in 4 groups of mice, respectively It was administered orally once a day for 14 days.

After sacrificing an experimental animal, the colon tissue was separated and the length was measured, homogenized in cold RIPA lysis buffer, centrifuged at 10,000 x g for 10 minutes, and the supernatant was used as a crude enzyme solution. After reacting the reaction solution containing hydrogen peroxide and tetramethylbenzidine with a crude enzyme solution, absorbance was measured at 650 nm to measure myeloperoxidase activity.

In order to confirm the expression of inflammatory cytokines, a portion of the colon tissue suspension homogenized in RIPA lysis buffer was treated with protease inhibitor, and then centrifuged to obtain a supernatant. Expression of cytokines TNF-α and IL-10 was confirmed using an ELISA kit (Ebioscience).

As a result, as in [Fig. 6] and [Fig. 7], the length of the colon tissue contracted in the colitis-induced group (negative control group) by K. oxytoca administration was reduced in Example 2-low concentration and Example 2-high concentration. Each recovered to the level of the colitis treatment Sulfasalazine group (positive control group). In addition, as in [Fig. 7], myeloperoxidase (MPO) activity mainly found in neutrophil granules, which is an indicator of inflammatory colitis, increased more than twice in the negative control group compared to the normal group, but Example 2-HtrA chaperone Pharmabiotics low concentration, In the results of the high concentration treatment group, the inflammatory enzyme activity was lowered to the level of the normal group together with the positive control group.

In particular, the expression level of the cytokine TNF-α as an inflammatory index decreased to the level of the normal group compared to the negative control group in Example 2 low concentration, Example 2 high concentration treatment group, whereas the expression of the anti-inflammatory cytokine IL-10 was rather increased did. In addition, as in [Fig. 8], the negative control group significantly decreased the expression level of IL-10 compared to TNF-α compared to the normal group, but the positive control group and Example 2-HtrA chaperone Pharmabiotics low concentration, high concentration treatment group had reduced expression The ratio was recovered and increased to the level of the normal group, and in particular, Example 2-HtrA chaperone Pharmabiotics high concentration treatment group showed a higher expression rate than the normal group. Through the results, it was confirmed that the inflammatory colitis symptoms induced by K. oxytoca administration were alleviated by regulating the inflammatory enzyme activity and the expression rate of inflammatory cytokines through the administration of Example 2-low concentration and Example 2-high concentration.

<Experimental Example 10. Intestinal microbiome control effect of HtrA chaperone Pharmabiotics single agent>

The feces secreted by the C57BL/6 colitis model mice were recovered before (day 0) and after ingestion (day 14) of HtrA chaperone pharmabiotics, respectively, to confirm changes in the intestinal microbiome. Bacterial DNA was extracted from mouse feces with QIAamp DNA stool mini kit, QIAGEN), and an amplicon was prepared using the V4 barcoded primer of the bacterial 16S rRNA gene. Each amplicon was sequenced using an Illumina iSeq 100 instrument. A meta-genetic analysis of the microbial community was performed using the software of PIRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) based on OTUs from the amplified genes. Linear discriminant analysis (LDA) and cladogram analysis were performed using LDA effect size (LefSe). The primer sequences used are shown in [Table 12].

primer order SEQ ID NO: 515F 5'-GTGCCAGCMGCCGCGGTAA-3' 5 806R 5'-GGACTACHVGGGTWTCTAAT-3' 6

Accordingly, as a result of analyzing the change in the diversity of the microbial flora in the group as shown in [Fig. 9], it was confirmed that the diversity of the intestinal flora was significantly reduced by the intake of K. oxytoca compared to the normal group. However, since the decreased diversity was not increased in the low and high concentration treatment groups and the positive control group of Example 2-HtrA chaperone pharmabiotics, there was no effect on the α-diversity level. On the other hand, the β-diversity value comparing the diversity of the microbiota between groups increased when Example 2-HtrA chaperone Pharmabiotics were ingested. In addition, as shown in [Fig. 10] , the ratio of Verrucomicrobia phyla (Phylum) significantly increased in the negative control group compared to the normal group, and the Cyanobacteria phylum and Firmicutes phyla decreased, but the positive control group changed to a level similar to that of the normal group. Example 2- In the low- and high-concentration treatment groups of HtrA chaperone pharmabiotics, the increased Verrucomicrobia phyla in the colitis model decreased, and the decreased ratio of Cyanobacteria phyla increased, changing to a composition similar to that of the normal group. Erysipelotrichaceae at the level of the microbial family (Family) had a tendency to decrease in the negative control group, but increased in the Example 2-HtrA chaperone Pharmabiotic intake group. PAC001112_g and PAC000198_g belonging to Muribaculaceae at the classification genus level were decreased in the negative control group, but the ratio increased above the normal group level when the high concentration of Example 2-HtrA chaperone Pharmabiotics was treated. PAC0001068_g, which showed an increasing trend in the negative control group, showed a tendency to decrease to the level of the normal group in Example 2-HtrA chaperone Pharmabiotics intake group. When colitis was induced at the species level, PAC001070_s group increased, PAC001797_s decreased, but the ratio in Example 2-HtrA chaperone Pharmabiotics treatment group was recovered similar to the normal group level. As a result, in the K. oxytoca -induced colitis mouse model, the group that received Example 2-HtrA chaperone Pharmabiotics restored the proportion of some flora of the microbiome changed by K. oxytoca intake similar to that of the normal group. was confirmed to be

Next, when the correlation between the anti-inflammatory effect and changes in the intestinal microbiome was analyzed, the result was shown as [Fig. 11]. In other words, the change in the ratio of the anti-inflammatory cytokine IL-10 to the inflammatory cytokine TNF-α (IL-10/TNF-α ratio) showed a correlation at all levels of microbial classification, genus, and species. As the IL-10/TNF-α ratio increases, Erysipelotrichaceae (family), PAC001112_g (genus), PAC000198_g (genus), PAC001066_g (genus), PAC001267_s (species), PAC001066_s (species), PAC001075_s (species), PAC001094_s (species) While the proportion of the intestinal microflora increased, Helicobacter (genus), PAC001070_s_group (species), and PAC001064_s (species) decreased. Accordingly, in a mouse model of colitis induced through oral intake of K. oxytoca , inflammatory cytokine expression is increased and changes in the intestinal microbiome are induced along with intestinal contraction, but the intestinal contraction is achieved through oral administration of Example 2-HtrA chaperone Pharmabiotics. And it was confirmed that it was effective in treating colitis by alleviating the symptoms of colitis of the inflammatory reaction and restoring the intestinal microbiome similar to that of the normal group.

As described above, the present invention has been described with reference to the above preparation examples and examples, but these are merely exemplary, and those of ordinary skill in the art to which the present invention pertains to various modifications and equivalent implementations therefrom. It will be appreciated that examples are possible. Therefore, the true technical protection scope of the present invention will have to be determined by the technical matters of the appended claims.

Claims (13)

(Step 1) preparing a medium for culturing lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water;
(Second step) obtaining a culture solution containing primary-HtrA chaperone probiotics by inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria; and,
(Step 3) Primary-HtrA chaperone probiotics obtained by centrifugation of the culture medium are used alone or in a mixture of two or more of glucose, fructose, sucrose, maltose, and trehalose. Preparing a secondary-HtrA chaperone Pharmabiotics by culturing after substitution with the contained medium;
HtrA (high temperature requirement A) chaperone comprising a A method for producing probiotics. According to claim 1,
The culture medium for lactic acid bacteria contains glucose, yeast extract, soy peptone and casein as components, and is a medium prepared by dissolving the components in water containing 30-70% (v/v) lava seawater and sterilizing them. A method for producing HtrA chaperone probiotics. delete delete According to claim 1,
As the two mixture sugars, a mixture sugar of sucrose and trehalose is selected, and the proportion of trehalose in the mixture sugar is 20 to 50% by weight of the total sugar. According to claim 1,
A method, characterized in that incubation is performed for 15 minutes to 2 hours after adding sugar for substitution with a high-concentration sugar medium for inducing expression of HtrA chaperone. According to claim 1,
A method further comprising the step of treating a cryoprotectant and a coating agent after substitution with a high-concentration sugar medium for inducing expression of HtrA chaperone. delete HtrA chaperone probiotics, characterized in that obtained by the method of claim 1. A food containing the HtrA chaperone probiotic powder of claim 9. 11. The method of claim 10,
The food is a food, characterized in that it is a health functional food for improving bowel activity or for regulating immunity. The composition for treating inflammatory bowel disease, characterized in that it contains the HtrA chaperone probiotics of claim 9. A health functional food for improving inflammatory bowel disease, characterized in that it contains the HtrA chaperone probiotics of claim 9.

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