KR20220170701A - Use of Lactobacillus reuteri strain for the promotion of intestinal development and the recovery from damage of intestine - Google Patents

Abstract

The present invention relates to the use of a novel Lactobacillus reuteri strain. The Lactobacillus reuteri strain and a culture solution thereof of the present invention increases the size and budding structure of intestinal organoids made by differentiation from human intestinal stem cells.

Description

Use of Lactobacillus reuteri strain for the promotion of intestinal development and the recovery from damage of intestine}

The present invention relates to the use of a Lactobacillus reuteri strain for intestinal maturation and recovery from intestinal damage.

The 'intestine', composed of the small and large intestines, is an organ that plays a key role in digesting food by secreting various types of digestive enzymes, plays an important role in absorbing digested nutrients and moisture, and secretes hormones. Food that is ingested through the mouth and passed through the digestive process is absorbed into the body as it moves along the intestinal tract. The villus of the small intestine absorbs nutrients including amino acids and glucose, and absorbs most of the water in the large intestine. The length and area of the intestinal tract, which provides a passage through which food passes and a cross-section through which digested food comes into contact, and the development of structures such as intestinal villi, allow for normal digestion and absorption.

In addition to digestion and absorption functions, the intestine is an organ closely related to the microbiome. About 95% of microorganisms in the human body live in the intestine, and the human intestine contains the number of cells in the human body. There are more than 10 times as many microbes. As research results have revealed that the types of microorganisms in the intestines, the number and ratio of each species, etc. can have a significant effect on the health or physical characteristics of the human body, the correlation between the intestinal microorganisms and human diseases and health interest in is increasing.

Therefore, if there is a problem with the development or maturation of the intestine, the length of the intestine is shorter and the cross-sectional area is smaller than that of a normal person, or the development of the detailed structure of the intestine is insufficient, the digestion and absorption capacity is reduced, and there is a possibility of adversely affecting overall health. In particular, when the development of the intestine is slow in fetuses, newborns, infants, etc. whose development and growth are not completed, it adversely affects the development and overall growth of organs other than the intestine, so the need for improvement is higher. It can be said that the importance of research on methods that can help the development and maturation of the intestine in developing and growing individuals is high.

Meanwhile, Lactobacillus reuteri is a type of lactic acid bacteria living in the intestine and is known to have various functions. Among published prior patent documents, there is a report that a Lactobacillus reuteri strain has an effect of preventing and improving skin aging (Korean Patent Publication No. 2020-0028627), or a report on a Lactobacillus reuteri strain having an immunity enhancing effect. (Korean Patent Publication No. 2018-0053499), and there is a report on a strain that promotes the development of the 'nervous system' such as intestinal neuron cells (Korean Patent Publication No. 2014-0131501), but Lactobacillus reuteri strains are No prior patents disclosing that it has excellent properties for promoting development and maturation or protecting and restoring damaged intestines have not been reported.

An object of the present invention is to provide a composition using Lactobacillus reuteri, which is a lactic acid bacterium strain that has an excellent effect of promoting intestinal development or intestinal maturation and exhibits an effect of protecting and restoring damaged intestines and a culture medium thereof.

In addition, an object of the present invention is to provide a composition that can be used for the purpose of promoting the development or maturation of the intestine or promoting the recovery of intestinal damage using the lactic acid bacteria strain as described above.

In addition, an object of the present invention is to provide a pharmaceutical composition that can be used for preventing or treating intestinal developmental disorders or inflammatory bowel disease using the lactic acid bacteria strain as described above.

In addition, an object of the present invention is to provide a health functional food composition and a feed composition that can be used for preventing or improving intestinal development disorders or inflammatory bowel diseases using the lactic acid bacteria strain as described above.

One aspect of the present invention, in order to achieve the above object, provides a composition for promoting intestinal development, promoting intestinal maturation or promoting recovery from intestinal damage, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.

Another aspect of the present invention, in order to achieve the above object, provides a pharmaceutical composition for preventing or treating intestinal developmental disorders or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture solution thereof as an active ingredient.

Another aspect of the present invention, in order to achieve the above object, provides a health functional food composition for preventing or improving intestinal developmental disorder or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.

Another aspect of the present invention, in order to achieve the above object, provides a feed composition for preventing or improving intestinal developmental disorder or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture solution thereof as an active ingredient.

The Lactobacillus reuteri strain and its culture solution of the present invention increase the size of intestinal organoids made by differentiation from human intestinal stem cells, increase the budding structure, and increase the expression of mature intestinal marker genes and proteins. there is

In addition, even when the human intestinal stem cells isolated from the intestinal organoids are treated with the Lactobacillus reuteri strain and its culture medium, there is an effect of promoting the growth of intestinal stem cells.

In addition, when the Lactobacillus reuteri is co-treated with intestinal organoids together with inflammatory cytokines, it can increase the surface area of the damaged intestine again and increase the expression level of marker proteins related to barrier function and proliferation, thereby recovering intestinal damage. The promotion and improvement effect is also excellent.

Furthermore, even when the Lactobacillus reuteri strain of the present invention and its culture medium were administered to mice by oral gavage, the length, area and depth of the villi of the small intestine were increased, and the depth of the crypt of the large intestine, mucosal/submucosal It promotes intestinal development, such as by increasing the layer ratio, and increases the expression of mature intestinal marker genes and proteins.

In particular, the Lactobacillus reuteri strain of the present invention is characterized in that the above effects are more remarkably exhibited even when compared to lactic acid bacteria belonging to other species, and not only the effects shown in animals other than humans, but also produced in human intestinal models It was confirmed that the effect was shown in intestinal organoids and human intestinal stem cells, and it was newly confirmed that excellent intestinal development promotion, intestinal maturation promotion, and intestinal damage recovery promotion and improvement effect appeared in humans.

Therefore, the Lactobacillus reuteri strain of the present invention is useful for promoting intestinal development, intestinal maturation or recovery from intestinal damage, or as a medicine, food and feed for preventing, treating and improving intestinal development disorders or inflammatory bowel disease. can be used extensively, which is very useful in related industries.

Panel a of Figure 1 is B. longum, L. gasseri, L. curvatus, L. rhamnosus, and Lactobacillus reuteri DS0384 ( L. reuteri ) of the present invention. The results of microscopic examination of morphological changes (upper data, Bright field, BF) and the expression of mature intestinal marker proteins through immunofluorescence staining (lower data) are shown. Scale bars are 500 μm in black and 100 μm in white. Panel b of FIG. 1 shows intestinal organoids treated with cultures of B. longum, L. gasseri, L. curvatus, L. rhamnosus and the Lactobacillus reuteri DS0384 ( L. reuteri ) strain of the present invention, followed by intestinal organoids. In order to confirm morphological changes, it is a graph comparing the size change of intestinal organoids (left panel) and the number of budding structures (right panel) of intestinal organoids. (*: p<0.05 control group versus experimental group according to t-test, **: p<0.01 control group versus experimental group according to t-test, ***: p<0.001 control group versus experimental group according to t-test) Panels of FIG. 1 c is the expression level of mature intestinal marker genes ( CDX2, DPP4, OLFM4, DEFA5, CREB3L3, KRT20, LYZ, LCT, SLC5A1 and MUC13 ) in intestinal organoids treated with the Lactobacillus reuteri DS0384 (L. reuteri) strain of the present invention It is a graph comparing the results of the control group by confirming by qRT-PCR. (*: p<0.05 control group versus experimental group according to t-test, **: p<0.01 control group versus experimental group according to t-test)
Panel a of FIG. 2 shows intestinal organoids treated with cultures of DS0191, DS0195, DS0333, DS0354 and Lactobacillus reuteri DS0384 strains classified as Lactobacillus reuteri, and then morphological changes in intestinal organoids were confirmed under a microscope. The result (upper data, Bright field, BF) and the result (lower data) shown by comparing the expression of the mature intestinal marker protein through immunofluorescence staining. Scale bars are 500 μm in black and 100 μm in white. Panel b of FIG. 2 is a graph comparing the expression levels of mature intestinal marker genes ( CDX2, DPP4, OLFM4, DEFA5, CREB3L3, KRT20, LYZ, LCT, SLC5A1 and MUC13 ) by qRT-PCR. (*: p<0.05 control group versus experimental group according to t-test, **: p<0.01 control group versus experimental group according to t-test)
Figure 3a shows the result of confirming the morphological changes of the intestinal organoids after culturing the Lactobacillus reuteri DS0384 strain for 6 hours, 12 hours, 18 hours, and 24 hours, and then treating the obtained culture medium with the intestinal organoids. Scale bar is 500 μm. Figure 3b is a graph comparing the expression patterns of intestinal maturation marker genes in intestinal organoids treated with the culture solution of the Lactobacillus reuteri strain obtained at different times through qRT-PCR. (*: p<0.05 control group versus experimental group according to t-test, **: p<0.01 control group versus experimental group according to t-test)
Figure 4a shows the result of confirming the morphological changes of human intestinal stem cells isolated from intestinal organoids after treating the culture medium of Lactobacillus reuteri DS0384 strain. The scale bar is 1 mm in black and 200 μm in white. Figure 4b shows the relative size of the surface area of intestinal stem cell colonies treated with the culture medium of the above strain using the imageJ program. (n≥5 per group, *p < 0.05, control vs experimental group according to t-test, **p < 0.01 control vs experimental group according to t-test)
Figure 5a shows the control group in which intestinal organoids were treated with inflammatory cytokines IFNγ/TNFα for 3 days, and the intestinal organoids were treated with both the inflammatory cytokines and the culture solution of Lactobacillus reuteri DS0384 strain, followed by organoids. It is a diagram confirming the morphological change (Bright field, BF). Scale bar is 1 mm. Figure 5b shows intestinal organoids of the control group treated only with PBS, the control group treated with only inflammatory cytokines (IFNγ/TNFα), and the group treated with both inflammatory cytokines and Lactobacillus reuteri DS0384 strain culture medium. Morphological changes were confirmed by hematoxylin and eosin staining. Scale bar is 200 μm. FIG. 6C is a graph comparing changes in the surface area of intestinal organoids between the control group and the Lactobacillus reuteri DS0384 strain culture solution-treated group by quantifying them. (n≥10 per group, *p < 0.05 according to t-test, control vs experimental group)
Figure 6a shows a control (control) treated with PBS to intestinal organoids, a control group (IFNγ / TNFα) treated with inflammatory cytokines IFNγ / TNFα for 3 days, and the inflammatory cytokines and It is a diagram confirming the expression of intestinal barrier function marker ZO-1 protein and proliferation marker Ki67 protein of intestinal organoids through immunofluorescence staining after all cultures of strain Lactobacillus reuteri DS0384 were treated. Scale bars are 125 μm in white and 500 μm in yellow. Figure 6b is a graph comparing the expression levels of ZO-1 and Ki67 measured as described above. (n≥5 per group, *p < 0.05, control vs experimental group according to t-test, ***p < 0.001 control vs experimental group according to t-test)
Figure 7 shows the intestinal tissue of a mouse gavaged with a culture solution containing cells ("strain" in the figure) and a culture medium in which the cells were removed by centrifugation ("culture medium" in the figure) among the culture medium in which the Lactobacillus reuteri DS0384 strain was cultured, and PBS This is the result confirmed through histological morphology analysis by staining the intestinal tissues of mice gavaged with hematoxylin and eosin.
8 shows the results according to FIG. 7, the length and area of the villus of the small intestine jejunum, the depth of the crypt, and the depth of the crypt of the colon, mucosa/submucosa It is a graph showing the ratio change numerically and compared. (n>7 per group, *: p<0.05 control vs experimental group according to t-test, **: p<0.01 control vs experimental group according to t-test, ***: p<0.001 control vs. experimental group according to t-test) experimental group)

Hereinafter, the present invention will be described in detail.

In the present invention, the term "culture medium" refers to the culture medium itself obtained by culturing the strain, or the culture supernatant obtained by removing the strain therefrom, and a filtrate, concentrate or dried product thereof, "culture supernatant" , "conditioned medium" or "conditioned medium" may be used interchangeably.

In the present invention, the term "prevention" refers to any action that suppresses or delays the onset of a disease or adverse condition by administering a composition to a subject.

In the present invention, the term "treatment" may be any action that improves the symptoms of a disease or negative condition that has been caused by administering a composition to a subject.

In the present invention, the term "improvement" refers to all actions including improvement, suppression, or delay of symptoms, and may be used interchangeably with the prevention or treatment.

One. Lactobacillus reuteri ( Lactobacillus reuteri ) Promotes intestinal development of strains, promotes intestinal maturation or promotes recovery from intestinal damage

One aspect of the present invention, Lactobacillus reuteri ( Lactobacillus reuteri ) Provides a composition for promoting intestinal development, promoting intestinal maturation or promoting recovery from intestinal damage, comprising a strain or a culture medium thereof as an active ingredient.

The Lactobacillus reuteri strain may be a strain capable of inhabiting the intestines of humans or non-human animals, and may have various effects on the health of humans or non-human animals while living in the intestines. General microorganisms of the genus Lactobacillus are Gram-positive anaerobic bacteria, capable of fermenting in the intestines to produce lactic acid or acetic acid, suppressing the growth of harmful bacteria, or enhancing immunity of humans or non-human animals inhabited by the microorganisms, digestion It is known as a lactic acid bacteria that has effects such as promoting and improving intestinal disorders. The Lactobacillus reuteri strain is a microorganism classified in the genus Lactobacillus, and may exhibit some of the characteristics of the microorganisms of the genus Lactobacillus. The Lactobacillus reuteri strain may be a safe microorganism that does not exhibit toxicity or cause disease to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or animals other than humans in the intestine Bar, probiotics (probiotics) can function as microorganisms.

The Lactobacillus reuteri may be Lactobacillus reuteri DS0384, DS0191, DS0195, DS0333, DS0354, etc., but is not limited thereto. The Lactobacillus reuteri DS0384 strain may be deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC) as accession number KCTC 14164BP on April 6, 2020.

In the present invention, the term "intestine" refers to the region of the digestive tract extending from the stomach to the anus. In humans and other mammals, the intestine consists of two regions: the small intestine (in humans, it is further subdivided into duodenum, empty intestine, and stone intestine) and the large intestine (in humans, it is further subdivided into cecum and colon); It may have a complex intestine, and in the present invention, the intestine may include all of them.

The Lactobacillus reuteri is characterized by promoting intestinal development or intestinal maturation. The Lactobacillus reuteri strain is excellent in promoting the small or large intestine to become more mature, or promoting the maturation of the immature small or large intestine. In addition, the promotion of intestinal development or intestinal maturation is to promote the formation of a specific mature epithelium, or to promote a part of the process of increasing, increasing, growing, supporting or advancing the differentiation process of enterocytes and intestinal compartments. can

For example, the Lactobacillus reuteri is excellent in increasing the length and area of small intestine villi or increasing the depth of crypts. The Lactobacillus reuteri may increase the depth of the crypts of the large intestine or increase the mucosa/submucosa ratio of the large intestine. For example, the development or maturation of the large intestine may be that the thickness of the mucosa layer increases and the thickness of the submucosa layer does not change. As the development and maturation of the large intestine progresses, the submucosa maintains its structure, and the thickness of the mucosal layer increases, so that the ratio of the mucosa/submucosa constituting the large intestine may increase.

In addition, the Lactobacillus reuteri can increase the expression of one or more genes selected from the group consisting of CDX2, DPP4, OLFM4, DEFA5, CREB3L3, KRT20, LYZ, LCT, SLC5A1 and MUC13.

In a specific embodiment of the present invention, when a culture medium containing a metabolite produced by the Lactobacillus reuteri strain is treated with an intestinal organoid prepared as an in vitro human intestinal model, its maturation and development and increase the expression of maturation-related marker genes and proteins. In addition, when not only intestinal organoids but also intestinal stem cells isolated from the intestinal organoids were treated with a culture solution of Lactobacillus reuteri strain, it was confirmed that the surface area of stem cell colonies increased compared to the control group, and the growth of intestinal stem cells , it was also confirmed that there is an activity that promotes maturation. The intestinal organoids are prepared by differentiating human pluripotent stem cells and implement an in vitro human intestinal model, and intestinal stem cells isolated from the intestinal organoids are also human intestinal stem cells. Therefore, it was newly confirmed that Lactobacillus reuteri is a strain with excellent activity for promoting intestinal development or maturation in the human intestine as well as animals other than humans, and thus can be usefully used for promoting human intestinal development or maturation. It was confirmed that the lactic acid bacteria species were present.

In another specific embodiment of the present invention, the development of the small intestine and large intestine even when the culture medium containing the Lactobacillus reuteri strain or the metabolite produced by the strain is oral gavaged to the mouse. It was confirmed that this was promoted and the expression of the mature intestinal marker gene was increased. The Lactobacillus reuteri strain exhibited an intestinal maturation promoting effect that was not shown in other species of lactic acid bacteria, and the expression level of genes and proteins related to intestinal maturation was significantly higher, or the expression was lower in other microbial culture medium treatment groups. It was found to increase the expression of intestinal maturation related marker genes that are not increased. Therefore, it was confirmed that the Lactobacillus reuteri strain exhibits an effect of promoting intestinal maturation and intestinal development at a level that could not be expected or achieved in conventionally known lactic acid bacteria. Accordingly, the Lactobacillus reuteri strain was used for intestinal development or intestinal development. It can be effectively used for maturation promotion, prevention, treatment, and improvement of intestinal development disorders.

The Lactobacillus reuteri is characterized by promoting recovery from intestinal damage. The intestinal damage may be, for example, an inflammatory reaction in the intestine, but the cause of the intestinal damage is not limited, and if the function or structure of the intestine is damaged compared to a normal state, the Lactobacillus reuteri strain of the present invention can promote its recovery. .

Recovery of the intestinal damage may be to restore the intestine to its original state by increasing the surface area of the damaged intestine, or to reduce damage by protecting the intestine from damage. In addition, the recovery of the intestinal damage may be to increase the expression of a gene or protein marker related to barrier function or proliferation in the damaged intestine. The barrier function marker may be ZO-1 protein, and the barrier proliferation marker may be Ki67 protein, but is not limited thereto.

In a specific embodiment of the present invention, it was confirmed that the damaged surface area was restored by increasing its surface area when a culture solution containing a metabolite produced by a Lactobacillus reuteri strain was simultaneously treated with an inflammatory cytokine to intestinal organoids. In addition, as it was confirmed that the expression levels of ZO-1 protein and Ki67 protein were increased, it was confirmed that the Lactobacillus reuteri strain has an activity to prevent intestinal damage and promote recovery of damaged intestine.

The subject of application of the composition of the present invention may be newborns or infants. In addition, the newborn or infant may be a premature or immature infant. The newborn means a baby from immediately after delivery until acquiring the ability to live an independent ectopic life, and may be, for example, a baby less than 28 days old. The infant may be a baby less than 5 years old, such as less than 4 years or less than 3 years. The above-described advantages and effects of the composition of the present invention may be more remarkable when applied to newborns or infants. The premature baby refers to a baby born earlier than a normal pregnancy period, and may be, for example, a baby born between 29 and 38 weeks of pregnancy. The premature baby means a baby born with immature development of the body, for example, a baby whose birth weight is low (eg, 2.5 kg or less, 2 kg or less, or 1.5 kg or less) or a baby with reduced immune ability can The cause of premature birth and/or immaturity may be maternal disease, maternal age, stress, fetal condition, etc., but the subject of application of the composition of the present invention may be applied without being limited to the above causes. The composition may be applied in a neonatal state immediately after delivery of the premature or premature infant, or may be applied in the period when the premature or premature infant is a growing infant after birth.

Since the composition of the present invention has an excellent effect of helping the development and maturation of the intestine, it has the effect of improving immature intestinal development of the person to whom it is applied. effect may be more pronounced.

The culture medium is obtained by culturing the Lactobacillus reuteri strain, and may be the culture medium itself containing the cells of the strain, or the culture supernatant obtained by removing cells therefrom, and also a filtrate, concentrate or may be dry matter. The culture broth from which the cells are removed may contain components produced and secreted by the Lactobacillus reuteri strain, such as metabolites, and thus may have intestinal development or intestinal maturation activity.

The filtrate is obtained by removing solid particles suspended from the culture solution of the Lactobacillus reuteri strain to obtain only a water-soluble supernatant excluding the precipitate, and the particles are filtered using a filter such as cotton or nylon, for example, 0.2 μm to 5 μm, or Freezing filtration, centrifugation, etc. may be used, but is not limited thereto.

The concentrate increases the solid concentration of the culture solution, and may be a concentrate of the culture solution containing the lactic acid bacteria cells or a concentrate of the culture supernatant from which the lactic acid bacteria cells are removed. The concentrate may be concentrated by vacuum concentration, plate type concentration, thin film concentration, etc., but is not limited thereto. The content of the culture medium included in the composition of the present invention may be appropriately adjusted according to the concentration of the concentrate.

The dried product includes, but is not limited to, those dried through methods such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high frequency drying, and infrared drying.

The Lactobacillus reuteri strain may be included in the composition at a concentration of 10 ⁹ to 10 ¹² cfu/g, for example, 10 ⁹ to 10 ¹¹ cfu/g, 10 ¹⁰ to 10 ¹² cfu/g or 10 ¹⁰ to 10 ¹¹ cfu. /g concentration. When the Lactobacillus reuteri strain is included in the composition at a concentration within the above range, the Lactobacillus reuteri strain promotes intestinal development, intestinal maturation, or recovery from intestinal damage, or can sufficiently produce or secrete substances that assist in this, or the growth of the strain. Since normal metabolism may be possible without being inhibited, the composition of the present invention may exhibit sufficient effects to be used for the purpose of promoting intestinal development or intestinal maturation.

The composition may further include a cryoprotectant. The description of the cryoprotectant is the same as that described above. When using a freeze-dried product obtained by freeze-drying a culture solution containing Lactobacillus reuteri cells, there is an advantage in oral intake, formulation, packaging, and storage of a composition containing the same, and the strain can be preserved for a long period of time. There is an advantage in that the strain, which has been lyophilized in the subject to which the composition of the present invention is administered, particularly in the intestine, shows normal physiological activity and can perform growth and metabolism.

The composition containing the strain of Lactobacillus reuteri or a culture solution thereof may be formulated by using carriers, excipients and/or additives according to a method that can be easily performed by those skilled in the art to which the present invention belongs. It can be prepared in dosage form or introduced into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, gel (eg, hydrogel) or lyophilizer, and the additives include a dispersant, A stabilizer or cryoprotectant may additionally be included.

Specifically, in the case of the lyophilizer, the strain or its culture is lyophilized together with a cryoprotectant and used in the form of a powder, wherein the cryoprotectant is skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, It may be mannitol, cyclodextrin, glycerol and/or honey. In addition, it includes mixing and adsorbing with a preservation carrier, followed by drying and solidifying for use, and the preservation carrier may be diatomaceous earth, activated carbon, and/or degreasing steel.

The composition of the present invention may be prepared by mixing the strain or its culture medium with any one of the carrier, excipient or additive.

The description of the strain, carrier, excipient and additive is the same as described above. When a cryoprotectant is used as the additive, the composition containing the lactic acid bacteria is mixed with the strain and the cryoprotectant, the mixture is frozen at -45 ° C to -30 ° C, and then dried at 30 ° C to 40 ° C. It may be prepared in the form of a lyophilized powder by grinding with a blender. Specifically, the freezing process may be a process of vacuum freezing for 65 to 75 hours under a temperature condition of -45 ° C to -30 ° C and a pressure condition of 5 to 50 mTorr.

2. Use of a composition containing a Lactobacillus reuteri strain or a culture solution thereof for preventing, treating, and improving intestinal development disorders or inflammatory bowel disease

Another aspect of the present invention is a pharmaceutical composition for preventing or treating intestinal developmental disorder or inflammatory bowel disease, a health functional food composition for preventing or improving intestinal developmental disorder or inflammatory bowel disease, and intestinal developmental disorder or inflammatory bowel disease. It provides a feed composition for prevention or improvement.

The pharmaceutical composition, health functional food composition and feed composition contain a Lactobacillus reuteri strain or a culture solution thereof as an active ingredient.

Description of the Lactobacillus reuteri strain and its effects and uses are the same as those described above. Since the Lactobacillus reuteri strain is excellent in increasing the size of the intestine and increasing the expression of mature intestinal marker genes, the composition containing the strain or its culture medium as an active ingredient is suitable for preventing, treating or improving intestinal development disorders. It can be used for useful purposes. In addition, the Lactobacillus reuteri strain increases the surface area of the damaged intestine again and increases the expression of marker proteins related to barrier function and proliferation, so it is excellent in promoting recovery from intestinal damage. Therefore, a composition containing the strain or its culture medium as an active ingredient can be usefully used for preventing, treating, or improving inflammatory bowel disease.

The intestinal development disorder may be a condition in which the intestinal tract does not function at a normal level due to insufficient development of the intestinal tract compared to the intestinal tract of a human or non-human animal that has completed development, maturation, differentiation, or growth, This includes intestinal conditions of fetuses, neonates, infants, and the like who are still in their developmental or growing stages. In addition, the developmental disorder of the intestinal tract may be a state in which the degree of development of the intestinal tract is less than the average developmental level of the intestinal tract based on the same period of development or growth. The developmental disorder of the intestinal tract includes all diseases caused by insufficient development of the intestinal tract or gastrointestinal diseases related thereto. The intestinal development disorders are specifically, disorder syndrome (malabsorption syndrome), inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), irritable bowel syndrome, short bowel syndrome (SBS), necrotizing enterocolitis (NEC), It may be, but is not limited to, premature infants, premature infants and infants with radiation proctitis or immature bowel.

The inflammatory bowel disease refers to a disease caused by damage to the intestine, and specifically, it may be a state in which an inflammatory reaction occurs due to damage to the intestine. The inflammatory bowel disease may specifically include, but is not limited to, ulcerative colitis, Crohn's disease, Behcet's disease, and the like, and may include any disease in which abnormal chronic inflammation occurs in the intestine regardless of the cause of inflammation. Since the Lactobacillus reuteri strain of the present invention is excellent in promoting recovery from intestinal damage, such as promoting the proliferation of the intestine inflamed by the injury, the composition of the present invention is used for preventing, treating, or improving inflammatory bowel disease. can be usefully used.

In a specific embodiment of the present invention, it was confirmed that human intestinal organoids prepared using human stem cells and human intestinal stem cells isolated therefrom showed excellent effects when treated with Lactobacillus reuteri strain. It has been proven through experiments that the composition of the invention shows sufficient effect even when applied to humans, and can be usefully used for application to humans.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. Or it may be prepared by putting it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule or gel (eg, hydrogel), and may additionally contain a dispersing agent or stabilizer. can

In addition, the strain or its culture medium containing the pharmaceutical composition can be transported in a pharmaceutically acceptable carrier such as colloidal suspension, powder, saline solution, lipid, liposome, microspheres, or nano-spherical particles. there is. They may be complexed with or associated with the delivery vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly vinylpyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral formulations at the time of actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, Or prepared using an excipient. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose in a herbal extract or fermented herbal medicine. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The pharmaceutical composition may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or improvement of intestinal development disorders.

The concentration of the active ingredient included in the pharmaceutical composition may be determined in consideration of the purpose of treatment, the condition of the patient, the required period, and the like, and is not limited to a specific range of concentration. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition may be administered as an individual therapeutic agent, or may be administered in combination with other therapeutic agents for intestinal developmental disorders, may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered single or multiple times. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, disease type, concomitant drug, administration route, It can be increased or decreased according to the severity of obesity, gender, weight, age, etc., and for example, about 0.0001 μg to 500 mg, for example, 0.01 μg to 100 mg per 1 kg of the patient's weight per day. In addition, according to the judgment of the doctor or pharmacist, it may be administered several times a day at regular time intervals, for example, 2 to 3 times a day.

Another aspect of the present invention provides a method for preventing or treating intestinal developmental disorders comprising administering the pharmaceutical composition to a subject.

The subject may be a human or non-human animal, and may be less developed than the intestinal tract of a fully developed human or non-human animal, or may be in a developing or growing phase. In addition, the subject may be a human or non-human animal whose level of development is less than the average level of development of the intestinal tract based on the same period of development or growth in which the intestinal tract is developing.

The formulation of the pharmaceutical composition, administration method, dosage and description of the concentration of the active ingredient contained in the composition are the same as those described above.

The health functional food composition of the present invention can prevent or improve intestinal development disorders in humans or non-human animals by promoting intestinal development or intestinal maturation.

When using the health functional food composition as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient can be appropriately used depending on the purpose of its use (prevention or improvement). In general, when preparing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of health functional food. Foods to which the health functional food composition can be added may be probiotics preparations, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, and various There are soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, and fermented foods, and includes all health foods in a conventional sense. In particular, the fermented food may be yogurt (hard type, soft type, drink type), fermented milk such as lactic acid bacteria beverage, cheese or butter, but is not limited thereto, and any fermented microorganisms or lactic acid bacteria are produced by fermentation. It can include food and even products.

In addition, the health functional food composition may be prepared as a food, particularly a functional food. The functional food includes ingredients commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid It may further contain a carbonation agent used in beverages and the like. The ratio of the components to be added is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition.

The feed composition of the present invention can prevent or improve intestinal development disorders by promoting intestinal development or intestinal maturation of animals other than humans, and can be added to a feed additive composition for the purpose of preventing or improving intestinal development disorders. The feed additives correspond to supplementary feeds under the Feed Management Act.

In the present invention, the term "feed" means any natural or artificial diet, one meal, etc., or a component of the one meal for an animal to eat, ingest, and digest, or suitable therefor, and the animal is an animal other than a human. . The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feed such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to specifically illustrate the present invention, and the content of the present invention is not limited to the following examples.

All results of the following experiments are expressed as the mean ± standard error of the mean (s.e.m.), and all experiments were performed in at least three replicates. P values were determined using a two-tailed t-test, and all analyzes of statistical significance were calculated relative to controls unless otherwise specified. P values less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

Example 1. Confirmation of the effect of Lactobacillus reuteri on promoting intestinal organoid maturation

In order to confirm the effect of Lactobacillus reuteri strains or their cultures on intestinal maturation, intestinal organoids were prepared as an in vitro intestinal model and treated with four types of Lactobacillus reuteri strains or their cultures, The maturation of was confirmed.

1-1. Differentiation of human intestinal organoids

Intestinal organoids were prepared by differentiating human pluripotent stem cells. For the differentiation of human pluripotent stem cells, a method known in the art ( Nature 470, 105-109 (2011)) was used. Specifically, for the induction of true endoderm in human pluripotent stem cells (H9 (WA09); WiCell Research Institute, Madison, WI, USA), fetal bovine serum for 3 days with 100 ng/ml of Activin A (FBS, Thermo Scientific)) was treated with the stem cells, and the fetal bovine serum was treated while increasing the concentration to 0%, 0.2%, and 2%, respectively. For the formation of hindgut spheroids, 500 ng/ml of FGF4, 3 μM of CHIR 99021, and 2% fetal bovine serum were additionally cultured for 4 days using a differentiation medium. Spheroids formed by induction of differentiation are inserted into the matrigel dome, and in a three-dimensional culture environment, 1Х B27 supplement (B27 supplement, Invitrogen), 100 ng/ml EGF (R&D Systems), 100 ng/ml Noggin (R&D Systems) , and cultured in a culture medium containing 500 ng/ml R-spondin1 (R-spondin1, R&D Systems), subcultured once every 10 days, and used for experiments.

1-2. Confirmation of promotion of intestinal organoid maturation by Lactobacillus reuteri strain or its culture medium

The intestinal organoids differentiated according to Example 1-1 were treated with 5 strains of Lactobacillus reuteri, and the maturation of the intestinal organoids was confirmed.

A total of 5 types of lactic acid bacteria were treated to confirm each effect, morphological changes were confirmed in the intestinal organoids treated with the lactic acid bacteria culture medium, and the expression of markers related to intestinal maturation was examined using immunocytochemistry (ICC) and qRT. -Confirmed through PCR. The lactic acid bacteria used were Bifidobacterium longum DS0431 ( B. longum DS0431, isolated from newborn feces), Lactobacillus gasseri DS0444 ( L. gasseri DS0444, isolated from breast milk), Lactobacillus curvatus AB70 ( L. curvatus AB70, female Isolated from genital organs), Lactobacillus rhamnosus DS0979 ( L. rhamnosus DS0979, isolated from breast milk) and Lactobacillus reuteri DS0384 ( L. reuteri DS0384, isolated from newborn feces) strains of the present invention were used. The culture solution of the 5 microbial strains was centrifuged at 12,000 rpm for 10 minutes using a centrifuge, and only the supernatant was collected. The collected supernatant was pasteurized for 30 minutes on a heat block preheated to 65 ° C., and impurities were removed by filtering with a 0.22 μm syringe filter unit. The culture solution isolated in this way was treated by diluting 1/100 in the culture medium of the intestinal organoid, and subcultured twice for a total of 20 days, and changes in the intestinal organoid were observed.

First, the morphological changes of the intestinal organoids were confirmed by checking the size change of the intestinal organoids and the number of budding structures. Specifically, the size of 6 organoids for each lactic acid bacteria treatment group was measured and compared through photographs taken by observing intestinal organoids under a microscope, and the number of budding structures was 6 organoids for each lactic acid bacteria treatment group. For each organoid, the number of budding structures generated was confirmed.

As a result, compared to the case of processing the culture solution of the other four types of lactic acid bacteria, when the culture solution of the Lactobacillus reuteri DS0384 strain of the present invention was treated, larger intestinal organoids were observed and more germination structures were formed. (Data above in Fig. 1 a, Fig. 1 b). The organoid surface area in the control group was measured as 0.30±0.02 ㎟, 0.27±0.06 ㎟ for the B. longum strain culture solution treatment group, 0.24±0.05 ㎟ for L. gasseri , 0.56±0.08 ㎟ for L. curvatus, and 0.56±0.08 ㎟ for L. 0.41 ± 0.10 ㎟ for rhamnosus and 1.10 ± 0.07 ㎟ for the L. reuteri (DS0384) strain culture solution treatment group of the present invention, L. curvatus and the present invention L. reuteri (DS0384) strain culture solution treatment group , the organoid size significantly increased, and among them, the organoid surface area increased the most in the L. reuteri (DS0384) strain culture solution treatment group (left panel of FIG. 1 b). As a result of measuring the number of sprouting structures, the control group was 3.17±0.52, B. longum was 2.33±0.61, L. gasseri was 2.50±0.37, L. curvatus was 8.83±0.96, L. rhamnosus was 4.33±0.78, And L. reuteri (DS0384 strain) of the present invention was measured to have 19.67 ± 2.20 germination structures formed. The number of germination significantly increased in the L. curvatus and the L. reuteri (DS0384) strain culture medium treatment group of the present invention, and among them, it was confirmed that the greatest increase was observed in the L. reuteri (DS0384) strain culture medium treatment group (b right side of FIG. 1). panel).

In addition, the expression level of the mature intestinal marker protein expressed in the mature intestine was confirmed by immunofluorescence staining. OLFM4, a mature intestinal stem cell marker, DEFA5, a mature Paneth cell marker, KRT20, a mature intestinal structural protein marker, and MUC13, a mucus-producing cell marker, were targeted as marker proteins. Specifically, intestinal organoids treated with 5 types of lactic acid bacteria cultures as described above were fixed in 4% PFA (paraformaldehyde), cryoprotected with 10-30% sucrose solution, treated with OCT solution, and frozen. The frozen intestinal organoid tissue was cut into 10-20 μm thick slices with a microtome, treated with PBS containing 0.1% Triton X-100, permeabilized through the slices, and then treated with 4% bovine serum (BSA). It was blocked for 1 hour with PBS containing albumin). Anti-OLFM4 antibody (ab85046, abcam, Cambridge, MA, USA), anti-DEFA5 antibody (ab90802, abcam), anti-KRT20 antibody (ab76126, abcam) and anti- MUC13 antibody (ab124654, abcam) was diluted at 1:100 and reacted overnight at 4°C, followed by secondary antibody, anti-goat IgG Alexa Fluor 488, A21467, Invitrogen, anti- Rabbit antibody (anti-rabbit IgG Alexa Fluor 594, A21442, Invitrogen) and anti-mouse antibody (anti-mouse IgG Alexa Fluor 594, A21203, Invitrogen) were diluted 1:200 each and reacted at room temperature for 1 hour to obtain DAPI After staining the nuclei with staining, they were observed under a fluorescence microscope.

As a result, the expression of proteins related to intestinal maturation was not observed in the group treated with four types of lactic acid bacteria cultures other than Reuteri, whereas the OFLM4, DEFA5, and KRT20 and MUC13 protein were stained, confirming that the proteins that appear during intestinal maturation were expressed and produced (data below in a of FIG. 1).

Furthermore, the expression level of mature intestinal marker genes in each lactic acid bacteria culture medium-treated intestinal organoid was confirmed by qRT-PCR. Mature intestinal marker genes were targeted: CDX2, DPP4, OLFM4, DEFA5, CREB3L3, KRT20, LYZ, LCT, SLC5A1 and MIC13 genes. RNA was isolated and extracted from the cultured intestinal organoids using the RNeasy kit (Qiagen) according to the manufacturer's protocol, and cDNA was synthesized by reverse transcription of mRNA using a cDNA synthesis kit (Superscript IV cDNA synthesis system, Invitrogen). qRT-PCR was performed on the synthesized cDNA with a PCR machine (7500 Fast Real-time PCR system, Invitrogen) using primers according to Table 1 below.

As a result, when the culture solution of the Lactobacillus reuteri DS0384 strain was treated, the expression level of all 10 intestinal maturation marker genes including the CDX2 gene increased several to several thousand times compared to the control group, indicating a significant increase (FIG. 1 of c).

sequence number sequence name Sequence (5' -> 3') One GAPDH forward primer GAAGGTGAAGGTCGGAGTC 2 GAPDH reverse primer GAAGATGGTGATGGGATTTC 3 CDX2 forward primer CTGGAGCTGGAGAAGGAGTTTC 4 CDX2 reverse primer ATTTTAACCTGCCTCTCAGAGAGC 5 DPP4 forward primers CAAATTGAAGCAGCCAGACA 6 DPP4 reverse primer GGAGTTGGGAGACCCATGTA 7 OLFM4 forward primers ACCTTTCCCGTGGACAGAGT 8 OLFM4 reverse primer TGGACATATTCCCTCACTTTGGA 9 DEFA5 forward primer CCTTTGCAGGAAATGGACTC 10 DEFA5 reverse primer GGACTCACGGGTAGCACAAC 11 CREB3L3 forward primer ATCTCCTGTTTGACCGGCAG 12 CREB3L3 reverse primer GTCGTCAGAGTCGGGGTTTG 13 KRT20 forward primer TGGCCTACACAAGCATCTGG 14 KRT20 reverse primer TAACTGGCTGCTGTAACGGG 15 LYZ forward primer AAAACCCCAGGAGCAGTTAAT 16 LYZ reverse primer CAACCCTCTTTGCACAAGCT 17 LCT forward primer GGCAGTCTGGGAGTTTTAGG 18 LCT reverse primer ATGCCAAAATGAGGCAAGTC 19 SLC5A1 forward primer GTGCAGTCAGCACAAAGTGG 20 SLC5A1 reverse primer ATGCACATCCGGAATGGGTT 21 MUC13 forward primers CGGATGACTGCCTCAATGGT 22 MUC13 reverse primer AAAGACGCTCCCTTCTGCTC

In view of the above experimental results, among various types of lactic acid bacteria isolated from feces of newborns, breast milk, or female genital organs, the Lactobacillus reuteri strain has genes related to intestinal maturation when human intestinal organoids are treated. , it was confirmed to significantly increase the expression level of the protein, and it can be seen that it actually promotes the development and maturation of the intestine, such as increasing the size of intestinal organoids and inducing the formation of germination structures. 1-3. Confirmation of difference in the effect of promoting intestinal organoid maturation between L. reuteri strains

Through Example 1-2, it was confirmed that the Lactobacillus reuteri strain was superior in promoting intestinal maturation and development compared to lactic acid bacteria belonging to other species. Accordingly, it was tested whether various strains of Lactobacillus reuteri exhibited the maturation promoting effect of intestinal organoids.

Cultures of Lactobacillus reuteri DS0384, DS0191, DS0195, DS0333, and DS0354 strains were treated with intestinal organoids to confirm the effect. Experiments were performed using the same method as in Example 1-2, and morphological changes of intestinal organoids were confirmed after treatment with the culture medium, and the expression levels of proteins and genes used as mature intestinal markers were measured by immunofluorescence staining and qRT-PCR. confirmed through

As a result, it was confirmed that all 5 types of Lactobacillus reuteri strain-treated groups had an effect of promoting the development of intestinal organoids (upper data in Figure 2a), and immunofluorescence staining results showed that OLFM4 and DEFA5 protein expression was also observed. It could be confirmed (data below in a of FIG. 2). In the results of confirming the marker gene expression level confirmed through qRT-PCR, although there were some differences for each gene marker among the strains, it was confirmed that the expression level of the mature intestinal marker increased in all 5 Lactobacillus reuteri strain culture medium treatment groups. could (b in Fig. 2). Specifically, in the case of CDX2, CREBL3, and KRT20 genes, the expression level was significantly increased in all 5 strains of Lactobacillus reuteri, and the expression level of DPP4 gene was particularly high in strain DS0191, and the expression level was also significantly increased in the treatment group of other strains. . In the case of the Lactobacillus reuteri DS0384 strain culture solution treatment group, the expression levels of all kinds of genes were significantly increased, and the DS0354 strain showed an excellent effect of increasing the expression levels of the LYZ, LCT, SLC5A1, and MUC13 genes. The OLFM4 gene is expressed at high levels in the human small intestine and large intestine, and is known to be closely related to the development and differentiation of the intestine because it corresponds to a marker gene expressed in intestinal stem cells in particular. The DEFA5 gene is a gene encoding DEFA5 (Defensin alpha 5) protein, which is abundantly present on the surface of the intestine, and is expressed at a high level in mature Paneth cells, and thus is used as a marker.

1-4. Confirmation of intestinal organoid maturation promoting effect of strain Lactobacillus reuteri DS0384 and optimization of culture conditions

Through the experimental results according to Examples 1-3, additional experiments were performed using the Lactobacillus reuteri DS0384 strain, which showed the effect of promoting intestinal organoid maturation. Differences in effects according to culture conditions were confirmed.

Specifically, while culturing the Lactobacillus reuteri DS0384 strain for 6 hours, 12 hours, 18 hours and 24 hours, the intestinal organoids were treated using the culture solution obtained for each time, and the CDX2 gene along with the morphological changes of the intestinal organoids The expression level of 10 intestinal maturation marker genes including was measured by qRT-PCR in the same manner as in Example 1-2.

As a result, morphological changes in intestinal organoids were confirmed when the culture solution of the Lactobacillus reuteri DS0384 strain was treated, and the expression levels of the 10 intestinal maturation marker genes were significantly increased (FIG. 3). In particular, morphological changes were further promoted in the intestinal organoids treated with the culture solution obtained by culturing the Lactobacillus reuteri strain for more than 18 hours (cultivation for 18 hours or 24 hours), and in the DS0384 strain culture solution treatment group cultured for more than 18 hours, the above The expression level of the mature intestine marker gene was significantly increased.

Judging from the above experimental results, it was confirmed that the intestinal maturation promoting effect was more excellent in the culture solution obtained after culturing the Lactobacillus reuteri strain for 18 hours or more, for example, 18 hours or 24 hours.

1-5. Confirmation of human intestinal stem cell growth promoting effect of Lactobacillus reuteri strain

In addition to confirming that the Lactobacillus reuteri strain has an excellent effect of promoting the growth of intestinal organoids, when human intestinal stem cells isolated from the intestinal organoids were treated with the Lactobacillus reuteri DS0384 strain, the effect of promoting the growth of stem cells was confirmed. Check if it appears.

Specifically, intestinal stem cells were separated from the human intestinal organoids, cultured, attached to a culture dish, and the pretreated culture of the Lactobacillus reuteri DS0384 strain was diluted in a cell culture medium to treat the intestinal stem cells, Intestinal stem cells were cultured and observed for 7 to 10 days while replacing the medium every 2 days. In addition, by comparing the colony morphology (surface area) of the intestinal stem cells, the difference in growth according to the treatment of the culture solution was confirmed.

As a result, it was confirmed that the surface area of intestinal stem cell colonies significantly increased compared to the control group when the culture medium of the Lactobacillus reuteri strain was treated (FIG. 4). Accordingly, it was confirmed that the Lactobacillus reuteri strain has excellent characteristics not only in promoting intestinal maturation but also in promoting the growth of intestinal stem cells.

Example 2. Confirmation of the protective effect of the lactic acid bacteria of the present invention on intestinal organoids

In order to confirm the effect of the culture solution of the Lactobacillus reuteri strain of the present invention on intestinal protection, intestinal organoids were prepared in the same manner as used to confirm the intestinal maturation promoting effect and intestinal stem cell growth promoting effect, and then damaged. was induced and the culture solution of the Lactobacillus reuteri strain was treated to determine whether a protective effect on intestinal organoids was exhibited.

First, in order to secure intestinal organoids with damage, inflammatory cytokines IFNγ and TNFα were administered to the intestinal organoids at a concentration of 125 ng/ml each for 3 days 10 days after passage of passage 2 or 3 intestinal organoids. Intestinal organoids damaged by the treatment were secured. In addition, intestinal organoids were simultaneously treated with inflammatory cytokines and Lactobacillus reuteri DS0384 culture medium diluted 1/100 of the intestinal organoid culture medium for 3 days, and the state of the intestinal organoids was observed.

As a result of performing morphological analysis by treating the culture solution of strain DS0384 on damaged intestinal organoids, the surface area was reduced in the case of intestinal organoids treated only with inflammatory cytokines. It was confirmed that the surface area of was significantly increased and recovered (FIG. 5).

In addition, after cryosectioning the intestinal organoids, expression of barrier function marker proteins and proliferation marker proteins was confirmed by immunofluorescence staining. As a result, compared to the intestinal organoids of the control group treated only with inflammatory cytokines, in the case of intestinal organoids treated with Reuteri DS0384 strain culture, the expression levels of ZO-1, an intestinal barrier function marker protein, and Ki67, a proliferation marker protein, were significantly higher. It was confirmed that it increased (FIG. 6).

In view of the above experimental results, when the culture medium of the Lactobacillus reuteri strain is treated with intestinal organoids damaged by inflammatory cytokine treatment, there is an effect of suppressing the decrease in surface area and promoting recovery, It was confirmed that the intestinal protective effect against damage was shown by increasing the expression of barrier or proliferation marker proteins. Therefore, it was confirmed that the Lactobacillus reuteri strain has an intestinal protective effect in addition to the intestinal maturation and intestinal stem cell growth promoting effects.

Example 3. Animal intestinal maturation and development promoting effect of the lactic acid bacteria of the present invention confirmed using mouse experiments

For the in vitro intestinal model intestinal organoids and intestinal stem cells made by imitating the human intestine through Example 1, the Lactobacillus reuteri strain of the present invention is superior to other microorganisms such as reuteri in maturation and development of the intestine. Confirmed. Therefore, in order to confirm whether the effect of promoting intestinal maturation appears even when the Lactobacillus reuteri strain is actually applied to animals, animal experiments were performed on mice.

3-1. Mouse model production and animal experiment design

3-day-old male C57BL/6J mice (DBL, Eumseong, Korea) were used as mice to be used in the experiment, and all animal experiments were performed by the Institutional Animal Care and Use Committee of KRIBB (Approval No: KRIBB-AEC-19222). and Use Committee, IACUC). As a control group, the mice were given physiological saline (PBS) by oral gavage, and in the case of the group administered with the DS0384 strain, the culture solution containing the cells was administered to the mice, or the Example 1-2 In the same way as in, the cells were centrifuged and the culture medium was administered by gavage to perform the experiment.

The 3-day-old mice were stabilized for 2 days, and then samples from the experimental and control groups were gavaged to the mice for 7 days, respectively. After 7 days, the mice were humanely euthanized, and the intestines were separated to separate the length of the time point. , weight change, etc. were measured. If growth retardation occurred compared to other mice at the time of gavage, they were excluded from the experiment, and in order to secure statistical significance, 7 or more sets of repeated experiments for each condition were configured (n > 7 per group).

3-2. Confirmation of mouse intestinal development promoting effect of Lactobacillus reuteri strain

As an experimental group, the mice of Example 3-1 were gavaged with the culture medium containing the cells of Lactobacillus reuteri DS0384 and the culture medium from which the cells were excluded by centrifugation, respectively, for 7 days, gavaged with PBS for 7 days, and then examined under a microscope. Tissue specimens were prepared for observation. First, a midline incision was made in the abdomen of the euthanized mouse, and the entire digestive tract was removed. Samples were taken by dividing the isolated digestive tract tissue into the jejunum of the small intestine and the distal colon of the large intestine, fixing with 4% PFA, cryoprotecting with sucrose and freezing with OCT solution. Then, 10 μm-thick cryosections were prepared using a cryostat microtome at -20 ° C. In the case of samples that are difficult to observe with a cryosection, they were fixed with 4% PFA, embedded in paraffin, and then frozen with a microtome. It was used after being cut into 5-7 μm thick.

H&E staining (Hematoxylin and eosin staining) was performed to observe the properties of the frozen section tissue. After attaching the frozen sectioned tissue to a slide glass, it was stained with hematoxylin for 5 minutes, washed with running water, and then eosin staining was performed. Thereafter, it was dehydrated using ethanol, washed with xylene, and sealed. In the case of tissues cut from paraffin-embedded tissues with a microtome, they were adhered to slide glass, dried, hematoxylin and eosin stained, and then sealed through the same process as the frozen section tissue.

The histological morphology of the tissue was observed under a microscope, and the length and area of villus and the depth of the crypt were measured for the jejunum of the small intestine, and the depth of the crypt and the thickness of the mucosa layer were measured for the large intestine. By analyzing, the degree of development and maturation of the intestine was quantified. As the colon matures, the thickness of the mucosal layer of the colon increases, and the structure of the submucosa is maintained during normal development, so the mucosa/submucosa ratio increases. Therefore, the maturity of the colon can be confirmed by measuring the depth of the crypts and the increase in the mucosal/submucosa ratio.

As a result, when the Lactobacillus reuteri strain culture was gavaged, it was observed that the length and area of villi in the small intestine of mice increased, the depth of the crypts increased, and the depth of the crypts in the large intestine increased. In particular, when the DS0384 strain culture was treated, it was confirmed that the development of the small intestine and large intestine was significantly increased compared to the PBS administration group (FIG. 7 and FIG. 8). In the case of the culture medium of Lactobacillus reuteri, it was confirmed that the development of the intestine was significantly promoted both in the case of administration including the cells and in the case of administering after removing the cells by centrifugation.

From the above results, it was found that the Lactobacillus reuteri strain and the metabolites produced therefrom have an effect of promoting the development of the small intestine and large intestine. Therefore, the Lactobacillus reuteri strain and its culture medium are not only suitable for use for promoting intestinal maturation, but also have a far superior effect on promoting intestinal maturation compared to other conventional lactic acid bacteria known and used for various purposes. As such, it can be seen that the Lactobacillus reuteri strain is a strain having new effects and characteristics that cannot be predicted or achieved from other conventional lactic acid bacteria.

In the above, the present invention has been described in detail only for the described embodiments, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the technical idea of the present invention, and it is natural that these changes and modifications fall within the scope of the appended claims.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center KCTC14164BP 20200406

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel Lactobacillus reuteri strain and the use thereof <130> 2021DPA4164 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> artificial sequence <220> <223> GAPDH forward primer <400> 1 gaaggtgaag gtcggagtc 19 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> GAPDH reverse primer <400> 2 gaagatggtg atgggatttc 20 <210> 3 <211> 22 <212> DNA <213> artificial sequence <220> <223> CDX2 forward primer <400> 3 ctggagctgg agaaggagtt tc 22 <210> 4 <211> 24 <212> DNA <213> artificial sequence <220> <223> CDX2 reverse primer <400> 4 attttaacct gcctctcaga gagc 24 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> DPP4 forward primer <400> 5 caaattgaag cagccagaca 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> DPP4 reverse primer <400> 6 ggagttggga gacccatgta 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> OLFM4 forward primer <400> 7 acctttcccg tggacagagt 20 <210> 8 <211> 23 <212> DNA <213> artificial sequence <220> <223> OLFM4 reverse primer <400> 8 tggacatatt ccctcacttt gga 23 <210> 9 <211> 20 <212> DNA <213> artificial sequence <220> <223> DEFA5 forward primer <400> 9 cctttgcagg aaatggactc 20 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> DEFA5 reverse primer <400> 10 ggactcacgg gtagcacaac 20 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> CREB3L3 forward primer <400> 11 atctcctgtt tgaccggcag 20 <210> 12 <211> 20 <212> DNA <213> artificial sequence <220> <223> CREB3L3 reverse primer <400> 12 gtcgtcagag tcggggtttg 20 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> KRT20 forward primer <400> 13 tggcctacac aagcatctgg 20 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> KRT20 reverse primer <400> 14 taactggctg ctgtaacggg 20 <210> 15 <211> 21 <212> DNA <213> artificial sequence <220> <223> LYZ forward primer <400> 15 aaaaccccag gagcagttaa t 21 <210> 16 <211> 20 <212> DNA <213> artificial sequence <220> <223> LYZ reverse primer <400> 16 caaccctctt tgcacaagct 20 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> LCT forward primer <400> 17 ggcagtctgg gagttttagg 20 <210> 18 <211> 20 <212> DNA <213> artificial sequence <220> <223> LCT reverse primer <400> 18 atgccaaaat gaggcaagtc 20 <210> 19 <211> 20 <212> DNA <213> artificial sequence <220> <223> SLC5A1 forward primer <400> 19 gtgcagtcag cacaaagtgg 20 <210> 20 <211> 20 <212> DNA <213> artificial sequence <220> <223> SLC5A1 reverse primer <400> 20 atgcacatcc ggaatgggtt 20 <210> 21 <211> 20 <212> DNA <213> artificial sequence <220> <223> MUC13 forward primer <400> 21 cggatgactg cctcaatggt 20 <210> 22 <211> 20 <212> DNA <213> artificial sequence <220> <223> MUC13 reverse primer <400> 22 aaagacgctc ccttctgctc 20

Claims (14)

A composition for promoting intestinal development, promoting intestinal maturation or promoting recovery from intestinal damage, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.
The method of claim 1,
The Lactobacillus reuteri strain is included in the composition at a concentration of 10 ⁹ to 10 ¹² cfu / g, a composition for promoting intestinal development, promoting intestinal maturation or promoting intestinal damage recovery.
The method of claim 1,
The composition further comprises a cryoprotectant, promoting intestinal development, promoting intestinal maturation or promoting intestinal damage recovery composition.
The method of claim 1,
The intestinal development or intestinal maturation is the development or maturation of the small intestine or large intestine, promoting intestinal development, promoting intestinal maturation or promoting intestinal damage recovery composition.
The method of claim 4,
The development or maturation of the small intestine is that the length and area of the villi of the small intestine are increased, or the length of the crypt is increased, promoting intestinal development, promoting intestinal maturation, or promoting intestinal damage recovery composition.
The method of claim 4,
The development or maturation of the large intestine is to increase the length of the crypts of the large intestine or increase the ratio of the mucosa/submucosa of the large intestine, for promoting intestinal development, promoting intestinal maturation, or promoting intestinal damage recovery. composition.
The method of claim 1,
The intestinal development or intestinal maturation increases the expression of one or more genes selected from the group consisting of CDX2, DPP4, OLFM4, DEFA5, CREB3L3, KRT20, LYZ, LCT, SLC5A1 and MUC13 , promotes intestinal development, intestinal maturation A composition for promoting or promoting recovery from intestinal damage.
The method of claim 1,
The intestinal damage recovery is to increase the surface area of the damaged intestine or to increase the expression of at least one protein selected from the group consisting of ZO-1 protein and Ki67 in the intestine, promoting intestinal development, promoting intestinal maturation or intestinal damage. A composition for promoting recovery.
The method of claim 1,
The target of application of the composition is a composition for promoting intestinal development, promoting intestinal maturation or promoting recovery from intestinal damage, which is to newborns or infants.
The method of claim 9,
Wherein the newborn or infant is a premature or immature infant, promotes intestinal development, promotes intestinal maturation, or promotes recovery from intestinal damage.
A pharmaceutical composition for preventing or treating intestinal developmental disorders or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.
The method of claim 11,
The pharmaceutical composition is prepared in any one formulation selected from the group consisting of capsules, powders, granules, tablets, pills and freeze-drying agents, a pharmaceutical composition for preventing or treating intestinal developmental disorders or inflammatory bowel disease.
A health functional food composition for preventing or improving intestinal developmental disorder or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.
A feed composition for preventing or improving intestinal developmental disorders or inflammatory bowel disease, comprising a Lactobacillus reuteri strain or a culture medium thereof as an active ingredient.

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