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WO2024196040A1 - Novel lactobacillus sp. strain and use thereof - Google Patents

Novel lactobacillus sp. strain and use thereof Download PDF

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Publication number
WO2024196040A1
WO2024196040A1 PCT/KR2024/002686 KR2024002686W WO2024196040A1 WO 2024196040 A1 WO2024196040 A1 WO 2024196040A1 KR 2024002686 W KR2024002686 W KR 2024002686W WO 2024196040 A1 WO2024196040 A1 WO 2024196040A1
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Prior art keywords
strain
present
composition
intestinal
maturation
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PCT/KR2024/002686
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French (fr)
Korean (ko)
Inventor
손미영
박두상
정광보
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한국생명공학연구원
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Publication of WO2024196040A1 publication Critical patent/WO2024196040A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to a novel Lactobacillus strain and its use.
  • the intestines which consist of the small and large intestines, are organs that play a key role in the digestion of food by secreting various types of digestive enzymes, and also play an important role in absorbing digested nutrients, water, etc., and are also organs that secrete hormones.
  • Food ingested through the mouth and digested is absorbed into the body as it moves along the intestines.
  • the villi of the small intestine absorb nutrients such as amino acids and glucose, and the large intestine absorbs most of the water.
  • the length and area of the intestines which provide the passageway for food and the cross-section that digested food comes into contact with, and the development of structures such as the villi of the intestines enable normal digestion and absorption.
  • the intestines are closely related to the microbiome. Approximately 95% of microorganisms in the human body reside in the intestines, and there are more than 10 times as many microorganisms in the human intestines as cells in the human body. As research results reveal that the types of intestinal microorganisms, the number and ratio of each type of microorganism, etc. can have a significant impact on human health and physical characteristics, interest in the correlation between intestinal microorganisms and human diseases and health is increasing.
  • the digestive and absorptive abilities may decrease, which may have a negative impact on overall health.
  • the development of the intestines is slow in fetuses, newborns, infants, etc. that have not fully developed and grown, it may have a negative impact on the development of organs other than the intestines and overall growth, so the need to improve this is even greater.
  • the importance of research on methods that can help the development and maturation of the intestines in developing and growing individuals is high.
  • One object of the present invention is to provide two novel strains of the genus Lactobacillus.
  • Another object of the present invention is to provide a composition comprising the novel strain.
  • Another object of the present invention is to provide a use of a composition comprising the novel strain.
  • one aspect of the present invention provides Lactobacillus paracasei DS2766 strain deposited under the accession number KCTC15344BP.
  • another aspect of the present invention provides Lactobacillus gasseri DS2831 strain deposited under the accession number KCTC15345BP.
  • another aspect of the present invention provides a composition comprising at least one selected from the group consisting of the two strains, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
  • another aspect of the present invention provides a food composition for promoting intestinal development or maturation, a health functional food composition for promoting intestinal development or maturation, a food composition or a feed/feed additive composition, or a pharmaceutical composition for preventing or treating intestinal development or maturation disorder, comprising at least one selected from the group consisting of the two kinds of strains, a culture solution of the strains, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
  • the novel Lactobacillus paracasei and Lactobacillus gasseri strains of the present invention have the effect of increasing the budding structure and surface area of intestinal tissue, and increasing the expression of mature intestinal marker genes and proteins, and these effects were verified through organoids.
  • the budding structure and surface area of intestinal tissue increase, the length and area of villi and crypt depth of the small intestine are increased, and the mucosa/submucosal layer ratio of the large intestine is increased, thereby exhibiting an activity of promoting intestinal development and maturation. Therefore, there is an advantage that it can be usefully used for the purpose of promoting intestinal development or maturation, or as a medicine, health functional food, food, and feed for preventing, improving, or treating intestinal development or maturation disorders.
  • Figure 1 shows the results of an experiment confirming the effect of promoting germination structure formation by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
  • Figure 2 shows the results of an experiment confirming the effect of the novel Lactobacillus paracasei strain of the present invention on increasing the surface area of tissue in a human intestinal organoid.
  • Figure 3 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker genes by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
  • Figure 4 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker proteins by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
  • Figure 5 shows the results of an experiment confirming the effect of promoting germination structure formation by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
  • Figure 6 shows the results of an experiment confirming the effect of the novel Lactobacillus gasseri strain of the present invention on increasing the surface area of tissue in human intestinal organoids.
  • Figure 7 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker genes by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
  • Figure 8 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker proteins by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
  • Lactobacillus paracasei Lactobacillus paracasei
  • One aspect of the present invention provides a novel Lactobacillus paracasei strain.
  • the novel Lactobacillus paracasei strain of the present invention may include 16S rRNA having a base sequence of the sequence number: SEQ ID NO: 1, and in particular, may be the Lactobacillus paracasei DS2766 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC15344BP on March 10, 2023.
  • the Lactobacillus paracasei strain was reclassified as a scientific name through whole-genome analysis by an internationally recognized organization (Microbiology Society) on April 15, 2020, and was accordingly revised in the “Standards and Specifications for Health Functional Foods” on April 20, 2021 (Ministry of Food and Drug Safety Notice No.
  • Lactobacillus paracasei DS2766 and Lacticazeibacillus paracasei DS2766 are meant to be identical strains.
  • the novel Lactobacillus paracasei strain of the present invention may be a strain capable of living in 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.
  • the novel Lactobacillus paracasei strain of the present invention may be a safe microorganism because it does not exhibit toxicity or cause diseases to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or non-human animals in the intestines, and thus may function as a probiotic microorganism.
  • Probiotics are live microorganisms that provide health benefits, and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as probiotics, they must survive gastric acid and bile acid, reach the small and large intestines, proliferate and settle in the intestines, exhibit useful effects in the intestines, and be non-toxic and non-pathogenic.
  • the DS2766 strain of the present invention may utilize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources.
  • a culture solution of the DS2766 strain of the present invention was inoculated into a tube containing glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), and then the sugar utilization ability of the strain was measured.
  • GLU glucose
  • MAN mannitol
  • LAC lactose
  • SAC sucrose
  • MAL maltose
  • SAL salicin
  • XYL xylose
  • ARA arabinose
  • GEL gelatin
  • GLY glycerol
  • CEL
  • the strain of the present invention can utilize and metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources, but cannot metabolize gelatin.
  • the DS2766 strain of the present invention may not produce a toxic substance, and the toxic substance may be, for example, indole or urea.
  • the DS2766 strain of the present invention may not produce ⁇ -glucuronidase.
  • the ⁇ -glucuronidase refers to an enzyme that promotes hydrolysis
  • ⁇ -glucuronidase is an enzyme that hydrolyzes various alcohols, phenols, amines, etc. into compounds (glucuronides) containing glucuronic acid, and exists in many living things such as bacteria, fungi, plants, and animals.
  • glucuronic acid is contained in human sweat and is secreted, and that it is involved in the production of substances that cause glandular odor through the metabolism of bacteria that live on the skin, and in particular, it has been reported to be related to colon cancer.
  • the DS2766 strain of the present invention may not exhibit hemolysis.
  • the hemolysis refers to the property of the strain to destroy red blood cells, and in order for the strain to be safely used as a probiotic, it must not exhibit hemolysis.
  • the indole, urea, and ⁇ -glucuronidase production ability of the DS2766 strain of the present invention was analyzed.
  • the strain of the present invention was found to have no production ability for toxic substances such as indole and urea, and was unable to produce ⁇ -glucuronidase associated with colon cancer, confirming that the strain of the present invention is safe as a probiotic.
  • the DS2766 strain of the present invention was cultured in a blood medium and analyzed for ⁇ , ⁇ , and ⁇ hemolysis, and as a result, the strain of the present invention did not exhibit hemolysis, confirming its safety as a probiotic.
  • the DS2766 strain of the present invention may be acid-resistant.
  • the acid-resistant refers to a property of the strain to survive well in an acidic environment, and may refer to a property of surviving even after 1 hour or more, for example, 1.5 hours or more, 2 hours or more, 2.5 hours or more, and especially 3 hours or more, in an acidic environment of pH 5 or lower, for example, pH 4 or lower, pH 3.5 or lower, and particularly, an environment of pH 3 or lower.
  • it was confirmed that the novel strain of the present invention still survived well even when cultured for 3 hours under a condition of pH 3.0, thereby having excellent acid-resistant properties.
  • the DS2766 strain of the present invention may be cholestatic.
  • the cholestatic property refers to a property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and may refer to a property of surviving in a biliary environment where bile, such as oxgall and bile salts, exists at a concentration of 5% or less, for example, 4.5% or less, 4% or less, 3.5% or less, and especially 3% or less, for 6 hours or more, for example, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, and especially 12 hours or more.
  • a specific embodiment of the present invention it was confirmed that the novel strain of the present invention still survived well even when cultured for 24 hours under conditions containing 3% (w/v) of bile salt, thereby having excellent cholestatic property.
  • the DS2766 strain of the present invention may have intestinal adhesion.
  • the intestinal adhesion refers to a property of the strain to adhere to intestinal cells and not be detached. If the intestinal adhesion is excellent, the time for which the strain can remain in the intestines increases, and the effect of improving intestinal flora, bowel movement, and promoting intestinal health as a probiotic can be maintained for a long time.
  • the intestinal adhesion refers to a number of viable bacteria, such as 1.0% or more of the initial number of viable bacteria, for example, 1.5% or more, 2.0% or more, 2.5% or more, or 2.54% or more, of which are attached to the intestinal cells after the strain reaches the intestines and remains in the intestinal environment for 12 hours or more, for example, 15 hours or more, 18 hours or more, 21 hours or more, or especially 24 hours or more.
  • the DS2766 strain of the present invention was treated to a Caco-2 cell line and cultured using glucose as a carbon source, and after washing with a PBS solution, the intestinal adhesion rate (%) of the strain was confirmed. As a result, the strain was confirmed to have an intestinal adhesion ability by showing an intestinal adhesion rate of about 0.72%.
  • the strain may not exhibit resistance to antibiotics, or may have low resistance to the antibiotics.
  • the resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced when a resistance gene for antibiotics is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the Lactobacillus paracasei DS2766 strain of the present invention, which does not exhibit extrinsic resistance to antibiotics, does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, and therefore there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics.
  • the antibiotics to which the Lactobacillus paracasei DS2766 strain of the present invention does not exhibit resistance are not particularly limited, so long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracyclines antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, may be ampicillin, gentamicin, erythromycin, clindamycin, tetracycline, etc.
  • the antibiotic resistance of the DS2766 strain was tested using MTS TM (MIC Test Strip) (Liofilchem). As a result, it was confirmed that the strain had lower resistance to ampicillin, vancomycin, erythromycin, clindamycin, and tetracycline than that described in the EFSA guide (European Food Safety Authority antibiotic safety criteria). In particular, in Europe, it has been said for a long time that lactic acid bacteria cannot be commercialized if they can horizontally transfer antibiotic resistance genes to other bacteria, and in Korea, the Ministry of Food and Drug Safety recently inserted a clause on antibiotic resistance transfer into the criteria for judging microorganisms as food ingredients. Therefore, the Lactobacillus paracasei DS2766 strain of the present invention has an advantage in that its safety as a probiotic is recognized, making it advantageous for commercialization.
  • MTS TM MIC Test Strip
  • the DS2766 strain of the present invention may have physiological characteristics that enable it to be utilized as a probiotic.
  • the novel Lactobacillus paracasei strain of the present invention may have an activity that promotes the development or maturation of the intestine.
  • the above intestine refers to a region of the digestive tract extending from the stomach to the anus.
  • the intestine is composed of two regions: the small intestine (further subdivided into the duodenum, the small intestine, and the ileum in humans) and the large intestine (further subdivided into the cecum and the colon in humans), and other mammals may have more complex intestines, and the intestine in the present invention may include all of these.
  • the novel Lactobacillus paracasei strain of the present invention may have an activity of promoting the small intestine or the large intestine to become more mature, or may have an activity of promoting the maturation of the small intestine or the large intestine from an immature state.
  • the novel Lactobacillus paracasei strain of the present invention may enhance the expression of genes such as CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, MUC13 , etc. in cells of the small intestine or the large intestine.
  • the novel Lactobacillus paracasei strain of the present invention may increase the surface area of the intestine. For example, it may increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa.
  • the novel Lactobacillus paracasei strain of the present invention is a novel strain having an activity of promoting intestinal maturation or development, and thus the strain of the present invention can be effectively used for purposes of promoting intestinal development or intestinal maturation, or for purposes of preventing, improving, or treating intestinal development or maturation disorders.
  • Lactobacillus gasseri Lactobacillus gasseri
  • One aspect of the present invention provides a novel Lactobacillus gasseri strain.
  • the novel Lactobacillus gasseri strain of the present invention may include 16S rRNA having a base sequence of sequence number 1, and in particular, may be Lactobacillus gasseri DS2831 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023 under the accession number KCTC15345BP.
  • KCTC Korean Collection for Type Culture
  • the novel Lactobacillus gasseri strain of the present invention may be a strain capable of living in 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.
  • the novel Lactobacillus gasseri strain of the present invention may be a safe microorganism because it does not exhibit toxicity or cause diseases to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or non-human animals in the intestines, and thus may function as a probiotic microorganism.
  • Probiotics are live microorganisms that provide health benefits, and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as probiotics, they must survive gastric acid and bile acid, reach the small and large intestines, proliferate and settle in the intestines, exhibit useful effects in the intestines, and be non-toxic and non-pathogenic.
  • the DS2831 strain of the present invention may utilize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources.
  • a culture solution of the DS2831 strain of the present invention was inoculated into a tube containing glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), and then the sugar utilization ability of the strain was measured.
  • GLU glucose
  • MAN mannitol
  • LAC lactose
  • SAC sucrose
  • MAL maltose
  • SAL salicin
  • XYL xylose
  • ARA arabinose
  • GEL gelatin
  • GLY glycerol
  • CEL
  • the strain of the present invention can utilize and metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources, but cannot metabolize gelatin.
  • the DS2831 strain of the present invention may not produce a toxic substance, and the toxic substance may be, for example, indole or urea.
  • the DS2831 strain of the present invention may not produce ⁇ -glucuronidase.
  • the ⁇ -glucuronidase refers to an enzyme that promotes hydrolysis
  • ⁇ -glucuronidase is an enzyme that hydrolyzes various alcohols, phenols, amines, etc. into compounds (glucuronides) containing glucuronic acid, and exists in many living things such as bacteria, fungi, plants, and animals.
  • glucuronic acid is contained in human sweat and secreted, and that it is involved in the production of substances that cause glandular odor through the metabolism of bacteria that live on the skin, and in particular, it has been reported to be related to colon cancer.
  • the DS2831 strain of the present invention may not exhibit hemolysis.
  • the hemolysis refers to the property of the strain to destroy red blood cells, and in order for the strain to be safely used as a probiotic, it must not exhibit hemolysis.
  • the indole, urea, and ⁇ -glucuronidase production ability of the DS2831 strain of the present invention was analyzed.
  • the strain of the present invention was found to have no production ability for toxic substances such as indole and urea, and was unable to produce ⁇ -glucuronidase associated with colon cancer, confirming that the strain of the present invention is safe as a probiotic.
  • the DS2831 strain of the present invention was cultured in a blood medium and analyzed for ⁇ , ⁇ and ⁇ hemolysis, and as a result, the strain of the present invention did not exhibit hemolysis, confirming its safety as a probiotic.
  • the DS2831 strain of the present invention may be acid-resistant.
  • the acid-resistant refers to a property of the strain to survive well in an acidic environment, and may refer to a property of surviving in an acidic environment of pH 5 or lower, for example, an environment of pH 4 or lower, a pH 3.5 or lower, and particularly an environment of pH 3 or lower, for a time of 1 hour or longer, for example, 1.5 hours or longer, 2 hours or longer, 2.5 hours or longer, and particularly 3 hours or longer.
  • it was confirmed that the novel strain of the present invention still survived well even when cultured for 3 hours under a condition of pH 3.0, thereby having excellent acid-resistant properties.
  • the DS2831 strain of the present invention may be cholestatic.
  • the cholestatic property refers to a property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and may refer to a property of surviving in a biliary environment where bile, such as oxgall and bile salts, exists at a concentration of 5% or less, for example, 4.5% or less, 4% or less, 3.5% or less, and especially 3% or less, for 6 hours or more, for example, 10 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 22 hours or more, and especially 24 hours or more.
  • a specific embodiment of the present invention it was confirmed that the novel strain of the present invention still survived well even when cultured for 24 hours under conditions containing 3% (w/v) of bile salt, and thus had excellent cholestatic property.
  • the DS2831 strain of the present invention may have intestinal adhesion.
  • the intestinal adhesion refers to a property of the strain to adhere to intestinal cells and not be detached. If the intestinal adhesion is excellent, the time for which the strain can remain in the intestines increases, and the effect of improving intestinal flora, bowel movement, and promoting intestinal health as a probiotic can be maintained for a long time.
  • the intestinal adhesion refers to a number of viable cells, such as 0.5% or more of the initial number of viable cells, for example, 0.6% or more, 0.7% or more, or 0.72% or more, of which are attached to the intestinal cells after the strain reaches the intestines and remains in the intestinal environment for 12 hours or more, for example, 15 hours or more, 18 hours or more, 21 hours or more, or especially 24 hours or more.
  • the DS2831 strain of the present invention was treated to a Caco-2 cell line and cultured using glucose as a carbon source, and after washing with a PBS solution, the intestinal adhesion rate (%) of the strain was confirmed. As a result, the strain was confirmed to have an intestinal adhesion ability by showing an intestinal adhesion rate of about 0.72%.
  • the DS2831 strain may not exhibit resistance to antibiotics, or may have low resistance to the antibiotics.
  • the resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced when a resistance gene for antibiotics is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the Lactobacillus gasseri DS2831 strain of the present invention, which does not exhibit extrinsic resistance to antibiotics, does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, and therefore there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics.
  • the antibiotics to which the Lactobacillus gasseri DS2831 strain of the present invention does not show resistance are not particularly limited, so long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracyclines antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, may be ampicillin, vancomycin, erythromycin, tetracycline, etc.
  • the antibiotic resistance of the DS2831 strain was tested using MTS TM (MIC Test Strip) (Liofilchem).
  • MTS TM MIC Test Strip
  • the strain was confirmed to have lower resistance to ampicillin, vancomycin, erythromycin, and tetracycline than that described in the EFSA guide (European Food Safety Authority antibiotic safety criteria).
  • EFSA guide European Food Safety Authority antibiotic safety criteria.
  • the Lactobacillus gasseri DS2831 strain of the present invention has an advantage in that its safety as a probiotic is recognized, making it advantageous for commercialization.
  • the DS2831 strain of the present invention may have physiological characteristics that can be utilized as probiotics.
  • the novel Lactobacillus gasseri strain of the present invention may have an activity that promotes the development or maturation of the intestine.
  • the above intestine refers to a region of the digestive tract extending from the stomach to the anus.
  • the intestine is composed of two regions: the small intestine (further subdivided into the duodenum, the small intestine, and the ileum in humans) and the large intestine (further subdivided into the cecum and the colon in humans), and other mammals may have more complex intestines, and the intestine in the present invention may include all of these.
  • the novel Lactobacillus gasseri strain of the present invention may have an activity of promoting the small intestine or the large intestine to become more mature, or may have an activity of promoting the maturation of the small intestine or the large intestine from an immature state.
  • the novel Lactobacillus gasseri strain of the present invention may enhance the expression of genes such as CDX2, LYZ, CREB3L3, DPP4, SLC5A1, MUC13 , etc. in cells of the small intestine or the large intestine.
  • the novel Lactobacillus gasseri strain of the present invention may increase the surface area of the intestine. For example, it may increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa.
  • the novel Lactobacillus gasseri strain of the present invention is a novel strain having an activity of promoting intestinal maturation or development, and thus the strain of the present invention can be effectively used for purposes of promoting intestinal development or intestinal maturation, or for purposes of preventing, improving, or treating intestinal development or maturation disorders.
  • composition comprising a novel Lactobacillus strain of the present invention
  • compositions comprising, as an effective ingredient, at least one selected from the group consisting of the novel Lactobacillus paracasei strain of the present invention, the novel Lactobacillus gasseri strain, a culture solution of the Lactobacillus paracasei strain or the Lactobacillus gasseri strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
  • the above culture solution is obtained by culturing the novel Lactobacillus paracasei or Lactobacillus gasseri strain of the present invention, and may be the culture solution itself containing cells of the strain, or a culture supernatant obtained by removing cells therefrom, and may also be a filtrate, concentrate or dried product thereof.
  • the culture solution from which the cells have been removed contains components produced or secreted by the novel strain of the present invention, such as metabolites, and thus may have an excellent probiotic effect active in promoting intestinal development or maturation, or preventing or treating intestinal development or maturation disorders.
  • the above concentrate increases the solid concentration of the culture solution, and may be a concentrate of a culture solution containing cells of the novel Lactobacillus paracasei or the Lactobacillus gasseri strain of the present invention, or a concentrate of a culture supernatant from which cells of the novel Lactobacillus paracasei or the Lactobacillus gasseri strain of the present invention have been removed.
  • the concentrate may be concentrated by, but is not limited to, vacuum concentration, plate concentration, thin film concentration, etc., and may be performed at a temperature of 40° C. to 60° C., for example, using a known concentrator.
  • the content of the culture solution included in the composition of the present invention can be appropriately adjusted depending on the concentration of the concentrate.
  • the above dried product includes, but is not limited to, a product dried by a method such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high-frequency drying, or infrared drying.
  • the above extract means an extract obtained from the culture solution or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a adjusted or purified product thereof, or a fraction obtained by fractionating the same.
  • composition may additionally contain other known ingredients or lactic acid bacteria that are suitable for ingestion together with the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention and have an activity of promoting the development or maturation of the intestine when ingested.
  • composition comprising the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention can be manufactured in the form of a unit dosage or by being placed in a multi-dose container by formulating the composition using a carrier, an excipient and/or an additive according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and can be manufactured.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, gels (e.g., hydrogels) or lyophilizer, and may additionally include a dispersant, a stabilizer or a cryoprotectant as the additives.
  • the freeze-drying agent includes using the strain in the form of a powder by freeze-drying it together with a cryoprotectant, and the cryoprotectant may be skimmed milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol and/or honey.
  • the cryoprotectant includes using it by mixing it with a preservation carrier, adsorbing it, drying it and solidifying it, and the preservation carrier may be diatomaceous earth, activated carbon and/or defatted steel.
  • composition comprising the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention can be manufactured through a step of mixing at least one selected from the group consisting of a strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution with any one of the carrier, excipient, or additive.
  • the novel Lactobacillus paracasei strain of the present invention and the cryoprotectant may be mixed, the mixture may be frozen at -45 degrees Celsius to -30 degrees Celsius, dried at 30 degrees Celsius to 40 degrees Celsius, ground in a mixer, and manufactured in the form of a freeze-dried powder.
  • the freezing process may be a vacuum freezing process under temperature conditions of -45 degrees Celsius to -30 degrees Celsius and pressure conditions of 5 to 50 mTorr for 65 to 75 hours.
  • composition in the freeze-dried form can be ingested in the form of a powder, and in this case, the strain in the composition can exhibit its activity while growing or metabolizing in the body.
  • Another aspect of the present invention provides a use of a composition comprising the novel Lactobacillus paracasei or Lactobacillus gasseri strain of the present invention for promoting intestinal development or maturation, or preventing or treating intestinal development or maturation disorders.
  • the composition may be a food, a feed, or a pharmaceutical.
  • the composition may be a health functional food composition for promoting intestinal development or maturation or a general food composition
  • the composition may be a feed composition for promoting intestinal development or maturation or a feed additive composition
  • the composition may be a pharmaceutical composition for preventing or treating intestinal development or maturation disorders.
  • the health functional food composition or the food composition can enhance the expression of genes such as CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, MUC13, etc. in a cell, increase the expression of proteins such as OLFM4, DEFA5, KRT20, MUC13 , etc., increase the surface area of the intestine, increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa.
  • genes such as CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, MUC13, etc. in a cell
  • proteins such as OLFM4, DEFA5, KRT20, MUC13 , etc.
  • increase the surface area of the intestine increase the length or surface area of villi of the small intestine
  • increase the depth of crypts increase the
  • the composition comprises at least one selected from the group consisting of the novel Lactobacillus paracasei strain of the present invention, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution as an effective ingredient, it may enhance the expression of at least one gene selected from the group consisting of CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, and MUC13 in intestinal cells.
  • the composition comprises at least one selected from the group consisting of the novel Lactobacillus gasseri strain of the present invention, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution as an effective ingredient, it may enhance the expression of at least one gene selected from the group consisting of CDX2, LYZ, CREB3L3, DPP4, SLC5A1, and MUC13 in intestinal cells.
  • the health functional food composition of the present invention When used as a food additive, the health functional food composition can be added to food as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method.
  • the amount of the effective ingredient can be used appropriately depending on its intended use (prevention or improvement).
  • 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.
  • the amount can be less than the above range, and since there is no problem in terms of safety, the effective ingredient can be used in an amount greater than the above range.
  • Examples of the above health functional foods or foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, dairy products, fermented milk, etc., and include all health functional foods or foods in the conventional sense.
  • the above health functional food is a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects.
  • the above health functional food can be manufactured by a method commonly used in the technical field of the present invention, and can be manufactured by adding raw materials and ingredients commonly added in the industry.
  • the formulation of the above health functional food can be manufactured in various forms without limitation as long as it is a formulation recognized as a health functional food, and unlike general drugs, it has the advantage of having no side effects that may occur when taking drugs for a long period of time since it uses food as a raw material, and has the advantage of excellent portability.
  • the health functional food of the present invention includes ingredients that are usually added during food manufacturing, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings.
  • the natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.).
  • the flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
  • various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained.
  • the ratio of these added components is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.
  • the composition when the composition is a feed composition or feed additive composition for promoting intestinal development or maturation, the feed composition or feed additive composition can be used in the diet of animals for the purpose of promoting intestinal development or maturation.
  • the feed additive of the present invention corresponds to auxiliary feed under the Feed Management Act.
  • feed in the present invention may mean any natural or artificial diet, meal, etc., or a component of said meal, which is suitable for an animal to eat, ingest, or digest.
  • the type of said feed is not particularly limited, and feed commonly used in the relevant technical field may be used.
  • Non-limiting examples of said feed include plant-based feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; animal-based feed such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
  • the intestinal development or maturation disorder when the composition is a pharmaceutical composition for preventing or treating an intestinal development or maturation disorder, the intestinal development or maturation disorder may be a state in which the intestinal development is insufficient compared to the intestinal tract of a human or non-human animal that has completed development, maturation, differentiation, or growth, and the intestinal tract may not function at a normal level, and this includes the state of the intestinal tract of a fetus, newborn, infant, etc. that is still in the developmental or growth period.
  • the intestinal development disorder may be a state in which the degree of development is insufficient compared to the average development level of the intestinal tract based on the same period of the developmental or growth period in which the development of the intestinal tract is in progress.
  • the intestinal development disorder includes all diseases caused by the insufficient degree of development of the intestinal tract or gastrointestinal diseases related thereto.
  • the above intestinal developmental disorders may specifically include, but are not limited to, malabsorption syndrome (malabsorption syndrome), inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), irritable bowel syndrome, short bowel syndrome (SBS), necrotizing enterocolitis (NEC), radiation proctitis, or premature infants, preterm infants, and infants with immature intestines.
  • the above prevention may mean any action that blocks, suppresses or delays symptoms caused by developmental or maturational disorders of the above intestines.
  • the above treatment may mean any action that improves or benefits symptoms caused by developmental or maturational disorders of the intestines.
  • the pharmaceutical composition may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and thereby manufactured.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or stabilizer.
  • the strain contained in the pharmaceutical composition may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nano-spheres. They may form a complex with or be associated with a carrier, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.
  • a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nano-spheres. They may form a complex with or be associated with a carrier, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents,
  • pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations.
  • lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included in addition to the above ingredients. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • the pharmaceutical composition according to the present invention 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 dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing a herbal extract or a fermented herbal product with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases can include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.
  • the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of intestinal development or maturation disorders.
  • the concentration of the effective ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentrations.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent, or can be administered in combination with other therapeutic agents for the prevention and treatment of intestinal development or maturation disorders, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of intestinal development or maturation disorder, sex, weight, age, etc.
  • Another aspect of the present invention provides a method for preventing or treating a disorder of intestinal development or maturation, comprising administering to a subject the pharmaceutical composition.
  • the subject may be a human or a non-human animal, and may be a subject whose level of development is less than that of a fully developed human or non-human animal, or who is in the developmental or growth stage.
  • the subject may be a human or non-human animal whose level of development is less than that of an average level of development of an intestinal tract based on the same period of development or growth in which development of the intestinal tract is in progress.
  • Strain #143 was isolated from newborn feces. For the strain #143 isolated as described above, PCR was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCA-3': SEQ ID NO: 2) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3': SEQ ID NO: 3) to analyze the base sequence of 16S rRNA of the strain #143.
  • the 16S rRNA base sequence of the above #143 strain was the same as sequence number 1, which was confirmed to show 100% homology with the previously reported Lactobacillus paracasei standard strain.
  • sequence comparison with the above standard strain was performed on the EZ BioCloud server (https:/eabiocloud.net/identify).
  • the above #143 strain was named as shown in Table 1 below and deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023, and was assigned the accession number as shown in Table 1 below.
  • KCTC Korean Collection for Type Culture
  • the above novel strains were inoculated onto MRS (Man, Rogosa, and Sharpe) medium and cultured under anaerobic conditions at 37°C for 24 to 36 hours to reach the stationary phase.
  • the culture was centrifuged at 3,000xg for 10 minutes and the supernatant was collected, treated at 65°C for 30 minutes for low-temperature sterilization, passed through a 0.22 ⁇ m filter, and stored at -80°C for use in the experiment.
  • the whole genome of the novel strain of the present invention separated as described above was analyzed. Specifically, complete whole genome sequencing (WGS) and draft genome analysis were performed, and the identity and functionality of the strain, such as genome homology analysis with a standard strain, phylogenomic characteristic analysis, and specific gene and metabolic pathway analysis, were investigated using the genome analysis results.
  • WGS complete whole genome sequencing
  • CDS amino acid sequence of the coding sequence
  • the Lactobacillus paracasei strain of the present invention was confirmed to have a full-length genome of 3.07 Mb and to not possess resistance or toxicity genes, indicating that it can be utilized as a probiotic.
  • Example 1 To confirm whether the novel DS2766 strain isolated and identified in Example 1 above has characteristics that can be utilized as a probiotic, tests were conducted on sugar utilization, acid resistance, bile resistance, intestinal adhesion, and antibiotic resistance in MRS medium.
  • the novel strain of the present invention was spread on MRS agar medium and prepared.
  • the sugar utilization, toxic substance productivity and gelatin decomposition ability analyses were conducted by suspending the strain colonies in API 20A Medium to a turbidity of 3 McFarland or higher and inoculating them into each well according to the provided instructions using the API 20A kit (Bio-Merieux, France).
  • the productivity of the ⁇ -glucuronidase was analyzed using the API ZYM kit.
  • the hemolysis evaluation confirmed ⁇ , ⁇ and ⁇ hemolysis on a sheep blood agar plate.
  • the types of sugars confirmed in this experiment are glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), and trehalose (TRE).
  • the types of toxic substances confirmed in this experiment are indole and urea.
  • the prepared fungal solution was inoculated into the sugar tube and cultured for 24 hours. Then, one drop of BCP reagent was added to all sugar tubes (except URE, GEL, and ESC) and the reaction results were read according to the reading table. For each reaction, + was indicated if it was positive and - was indicated if it was negative. The results are shown in Table 3.
  • DS2766 DS2766 D-glucose + D-mannose + D-mannitol + D-melezitose + D-lactose + D-raffinose + sucrose + D-sorbitol + D-maltose + D-rhamnose + salicin + D-trehalose + D-xylose + Indol production - L-arabinose + Urea production - gelatin - Gelatin resolution - esculin + ⁇ -glucuronidase - glycerol + Hemolysis - D-cellobiose +
  • the novel DS2766 strain of the present invention metabolizes glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose.
  • it did not produce toxic substances such as indole and urea, does not produce ⁇ -glucuronidase, and has no ability to decompose gelatin.
  • the DS2766 strain of the present invention does not exhibit hemolysis.
  • the novel DS2766 strain of the present invention is basically resistant to an acidic environment.
  • the strain cultured in the above example [2-1] was inoculated into 10 mL of MRS medium containing 3% (w/v) of bile salt (Oxoid TM ) at a concentration of 10 6 CFU/mL, and the survival rate was confirmed after culturing for 12 hours.
  • MRS medium containing 3% (w/v) of bile salt (Oxoid TM ) at a concentration of 10 6 CFU/mL
  • the novel DS2766 strain of the present invention was confirmed to have resistance to an environment containing bile.
  • the DS2766 strain was spread on MRS agar medium and prepared.
  • An antibiotic resistance test was performed according to the manufacturer's instructions using MTS TM (MIC Test Strip) (Liofilchem) for antibiotics commonly used in the EFSA (European Food Safety Authority) guide.
  • the antibiotics used in this experiment were vancomycin, erythromycin, clindamycin, and tetracycline, and the concentration at the point where the antibiotic strip meets the inhibition zone was considered the minimum inhibitory concentration.
  • the strain of the present invention was confirmed to have lower antibiotic resistance than the standard value of the EFSA guide for ampicillin, erythromycin, clindamycin, and tetracycline. Therefore, since the strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire external resistance and cause resistance problems to antibiotics, and thus it has an advantage in that it is also advantageous for commercialization as a probiotic.
  • Caco-2 cell line was used. Specifically, Caco-2 cell line was cultured at 37°C in a 5% CO2 incubator (PHC, MCO-230AIC, Indonesia) using MEM medium containing 10% FBS and 1% penicillin-streptomycin, dispensed into a 24-well plate at a concentration of 1x105 cells/well, and cultured for 14 days. Then, the cultured cells were suspended in MEM medium to adjust the final concentration to 1x108 CFU/ml, inoculated into each well at 1ml, and cultured for 2 hours. At this time, glucose was used as the carbon source for each strain.
  • PLC 5% CO2 incubator
  • MEM medium containing 10% FBS and 1% penicillin-streptomycin
  • the cultured strain was washed five times with PBS (Phosphate Buffered Saline) solution, treated with 0.5% trypsin-EDTA to detach non-attached Caco-2 cells from the plate, diluted the detached cells with PBS and plated on MRS plate medium, and then the number of viable cells was measured after 24 hours to evaluate the intestinal adhesion ability of the strain of the present invention.
  • PBS Phosphate Buffered Saline
  • the DS2766 strain of the present invention was confirmed to have intestinal attachment ability, showing an intestinal attachment rate of about 2.54% when glucose was used as a carbon source.
  • hPSCs Human pluripotent stem cells
  • hIOs three-dimensional human intestinal organoids
  • hPSCs for true endoderm induction of hPSCs (H9 (WA09); WiCell Research Institute), the hPSCs were cultured in a medium containing 100 ng/mL of Activin A (R&D Systems) and 0%, 0.2%, and 2% fetal bovine serum (FBS, Thermo Scientific) for 3 days. Then, to form hindgut spheroids, they were additionally cultured for 4 days using differentiation medium containing 500 ng/mL of FGF4, 3 ⁇ M CHIR 99021, and 2% fetal bovine serum.
  • FBS Activin A
  • FBS fetal bovine serum
  • the spheroids formed by inducing differentiation were inserted into the Matrigel dome and cultured in a 3D culture environment in intestinal organoid differentiation medium containing 1X B27 supplement (B27 supplement, Invitrogen), 100 ng/mL EGF (R&D Systems), 100 ng/mL Noggin (R&D Systems), and 500 ng/mL R-spondin1 (R&D Systems) for a total of 14 days to produce human intestinal organoids.
  • 1X B27 supplement B27 supplement, Invitrogen
  • 100 ng/mL EGF R&D Systems
  • 100 ng/mL Noggin R&D Systems
  • 500 ng/mL R-spondin1 R&D Systems
  • Example [3-1] After separating the 3D hIOs produced in the above Example [3-1] from the Matrigel, they were washed with cold PBS, the Matrigel around the hIOs was removed as much as possible, and then physically dissociated using a surgical scalpel. The dissociated hIOs pieces were inoculated into a new 4-well dish to form a Matrigel dome, 5 pieces per well, and cultured with hIO differentiation medium.
  • Example 1 The culture solution of the novel Lactobacillus paracasei strains of the present invention isolated and identified in Example 1 was added to the hIOs prepared as described above at a concentration of 1%, and subcultured twice for a total of 20 days, after which changes in the hIOs were observed.
  • the morphological changes in hIOs were confirmed by checking the size changes in hIOs and the number of budding structures. Specifically, the number of budding structures generated in hIOs treated with the novel Lactobacillus paracasei strain of the present invention and the change in surface area of the hIOs were confirmed through photographs taken by observing hIOs treated with the novel Lactobacillus paracasei strain of the present invention under a microscope.
  • FIGS. 1 and 2 compared to the case without any treatment, it was confirmed that not only were more germination structures formed in hIOs treated with the novel Lactobacillus paracasei strain of the present invention (FIG. 1), but also the surface area was increased by more than four times (FIG. 2).
  • the expression level of marker proteins related to intestinal maturation expressed in the mature intestine such as 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, was confirmed through immunofluorescence staining.
  • the hIOs treated with the novel Lactobacillus paracasei strains were fixed in 4% PFA (paraformaldehyde), cryoprotected with a 10-30% sucrose solution, and frozen by treating with an OCT solution.
  • the frozen hIOs tissues were cut into thin sections with a thickness of 10-20 um using a microtome, and the sections were permeabilized by treating with PBS containing 0.1% Triton X-100, and then blocked with PBS containing 4% BSA (bovine serum albumin) for 1 hour.
  • PBS containing 0.1% Triton X-100 PBS containing 0.1% Triton X-100
  • BSA bovine serum 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)
  • anti-goat IgG Alexa Fluor 488, A21467, Invitrogen anti-rabbit IgG Alexa Fluor 594, A21442, Invitrogen
  • anti-mouse IgG Alexa Fluor 594, A21203, Invitrogen were used at a dilution of 1:200 each and reacted at room temperature for 1 hour to stain the nucleus with DAPI. After staining, it was observed under a fluorescence microscope.
  • a crude product of a composition comprising the novel Lactobacillus paracasei strain of the present invention was prepared.
  • Each of the novel Lactobacillus paracasei strains of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (volume/weight)% of maltodextrin, 5-50 (volume/weight)% of trehalose, or 5-50 (volume/weight)% of cellulose as a cryoprotectant was added to the concentrate, freeze-dried, and then ground to produce lactic acid bacteria powder.
  • Strain #123 was isolated from the feces of an 18-month-old infant. For the strain #123 isolated as described above, PCR was performed using the same method as in Example 1 to analyze the base sequence of 16S rRNA of the strain #123.
  • the 16S rRNA base sequence of the above #123 strain was the same as sequence number 26, which was confirmed to have 99.86% homology with the previously reported Lactobacillus gasseri standard strain.
  • sequence comparison with the above standard strain was performed on the EZ BioCloud server (https:/eabiocloud.net/identify).
  • the above #123 strain was named as shown in Table 9 below and deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023, and was assigned the accession number as shown in Table 9 below.
  • KCTC Korean Collection for Type Culture
  • the Lactobacillus gasseri strain of the present invention was confirmed to have a full-length genome of 2.04 Mb and to not possess resistance or toxicity genes, indicating that it can be utilized as a probiotic.
  • DS2831 DS2831 D-glucose + D-mannose + D-mannitol + D-melezitose + D-lactose + D-raffinose + sucrose + D-sorbitol + D-maltose + D-rhamnose + salicin + D-trehalose + D-xylose + Indol production - L-arabinose + Urea production - gelatin - Gelatin resolution - esculin + ⁇ -glucuronidase - glycerol + Hemolysis - D-cellobiose +
  • the novel DS2831 strain of the present invention metabolizes glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose.
  • it did not produce toxic substances such as indole and urea, does not produce ⁇ -glucuronidase, and has no ability to decompose gelatin.
  • the DS2831 strain of the present invention does not exhibit hemolysis.
  • the bile tolerance of the DS2831 strain was analyzed as in the above example [4-3].
  • the novel DS2831 strain of the present invention was confirmed to have resistance to an environment containing bile.
  • the antibiotics used in this experiment were ampicillin, vancomycin, erythromycin, and tetracycline, and the concentration at the point where the antibiotic strip meets the inhibition zone was considered the minimum inhibitory concentration.
  • the DS2831 strain of the present invention was confirmed to have lower antibiotic resistance than the standard values of the EFSA guide for ampicillin, vancomycin, erythromycin, and tetracycline. Therefore, since the DS2831 strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire external resistance and cause resistance problems to antibiotics, and thus it has an advantage in that it is also advantageous for commercialization as a probiotic.
  • the DS2831 strain of the present invention was confirmed to have intestinal adhesion ability, showing an intestinal adhesion rate of about 0.72% when glucose was used as a carbon source.
  • novel Lactobacillus gasseri strain of the present invention In order to confirm the effect of the novel Lactobacillus gasseri strain of the present invention on the development or maturation of the intestine, three-dimensional human intestinal organoids (hIOs) were produced as an in vitro intestine model, and the novel Lactobacillus gasseri strain of the present invention was treated to evaluate the degree of development/maturity of the intestine.
  • hIOs human intestinal organoids
  • Example [3-1] After separating the 3D hIOs produced in the above Example [3-1] from the Matrigel, they were washed with cold PBS, the Matrigel around the hIOs was removed as much as possible, and then physically dissociated using a surgical scalpel. The dissociated hIOs pieces were inoculated into a new 4-well dish to form a Matrigel dome, 5 pieces per well, and cultured with hIO differentiation medium.
  • Example 1 The culture solution of the novel Lactobacillus gasseri strains of the present invention isolated and identified in Example 1 was added to the hIOs prepared as described above at a concentration of 1%, and sub-cultured twice for a total of 20 days, after which changes in the hIOs were observed.
  • Example [3-2] the number of budding structures generated in hIOs treated with the novel Lactobacillus gasseri strain of the present invention and the change in surface area of the hIOs were confirmed.
  • FIGS. 5 and 6 compared to the case without any treatment, it was confirmed that not only were more germination structures formed in hIOs treated with the novel Lactobacillus gasseri strain of the present invention (FIG. 5), but also the surface area was increased by more than four times (FIG. 6).
  • a crude product of a composition comprising the novel Lactobacillus gasseri strain of the present invention was prepared.
  • Each of the novel Lactobacillus gasseri strains of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (volume/weight)% of maltodextrin, 5-50 (volume/weight)% of trehalose, or 5-50 (volume/weight)% of cellulose as a cryoprotectant was added to the concentrate, freeze-dried, and then ground to produce lactic acid bacteria powder.

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Abstract

The present invention relates to a novel Lactobacillus sp. strain and a use thereof.

Description

신규 락토바실러스 속 균주 및 이의 용도Novel Lactobacillus strains and their uses
[관련 출원과의 상호 인용][Cross-reference with related applications]
본 출원은 2023년 03월 21일에 출원된 한국특허출원 제10-2023-0036592호와 한국특허출원 제10-2023-0036593호에 기초한 우선권의 이익을 주장하며, 해당 한국특허출원 문헌에 개시된 모든 내용은 본 명세서의 일부로서 포함된다.This application claims the benefit of priority to Korean Patent Application No. 10-2023-0036592 and Korean Patent Application No. 10-2023-0036593, filed March 21, 2023, the entire contents of which are incorporated herein by reference.
본 발명은 신규한 락토바실러스 속 균주 및 이의 용도에 관한 것이다.The present invention relates to a novel Lactobacillus strain and its use.
소장 및 대장으로 이루어진 '장'은 여러 종류의 소화효소를 분비하여 음식물의 소화에 핵심적인 역할을 하는 기관이면서, 소화된 영양소, 수분 등을 흡수하는 중요한 역할을 하며 호르몬을 분비하는 기관이기도 하다. 입을 통해 섭취되어 소화 과정을 거친 음식물은 장관을 따라 이동하면서 체내로 흡수되는데 소장의 융모(villus)에서는 아미노산, 포도당을 비롯한 영양소들을 흡수하고 대장에서 대부분의 수분을 흡수한다. 음식물이 지나가는 통로이자 소화된 음식물이 접촉하는 단면을 제공하는 장관의 길이와 면적, 그리고 장의 융모와 같은 구조의 발달은 정상적인 소화 및 흡수를 가능케 한다. The intestines, which consist of the small and large intestines, are organs that play a key role in the digestion of food by secreting various types of digestive enzymes, and also play an important role in absorbing digested nutrients, water, etc., and are also organs that secrete hormones. Food ingested through the mouth and digested is absorbed into the body as it moves along the intestines. The villi of the small intestine absorb nutrients such as amino acids and glucose, and the large intestine absorbs most of the water. The length and area of the intestines, which provide the passageway for food and the cross-section that digested food comes into contact with, and the development of structures such as the villi of the intestines enable normal digestion and absorption.
소화 및 흡수 기능뿐만 아니라, 장은 마이크로바이옴(microbiome)과도 밀접한 관련성이 있는 기관으로, 인간의 체내에 존재하는 약 95% 정도의 미생물이 장내에 서식하고 있으며, 인간의 장내에는 인체의 세포 수의 10배가 넘는 수의 미생물이 존재한다. 장내 미생물들의 종류, 각 종의 미생물의 수와 비율 등이 인체의 건강이나 신체적 특성들에 중요한 영향을 미칠 수 있다는 연구 결과들이 밝혀짐에 따라, 장내 미생물과 인체의 질환, 건강 등과의 상관관계에 대한 관심도가 높아지고 있다. In addition to digestive and absorptive functions, the intestines are closely related to the microbiome. Approximately 95% of microorganisms in the human body reside in the intestines, and there are more than 10 times as many microorganisms in the human intestines as cells in the human body. As research results reveal that the types of intestinal microorganisms, the number and ratio of each type of microorganism, etc. can have a significant impact on human health and physical characteristics, interest in the correlation between intestinal microorganisms and human diseases and health is increasing.
따라서, 장의 발달이나 성숙에 문제가 있어 정상적인 사람에 비해 장의 길이가 짧고 단면적이 적거나, 장의 세부 구조의 발달이 미흡할 경우 소화 및 흡수 능력이 떨어지게 되며 전체적인 건강에 악영향을 줄 가능성이 있다. 특히, 발달과 성장이 완료되지 않은 태아, 신생아, 영유아 등에서 장의 발달이 더딘 경우 장 이외의 기관의 발달이나 전반적인 성장에도 좋지 않은 영향을 주어 이를 개선해야 할 필요성이 더욱 높으며, 장 발달에 장애가 있지 않더라도 발달, 성장 중인 개체에서 장의 발달과 성숙에 도움을 줄 수 있는 방법에 대한 연구의 중요성은 높다고 할 수 있다.Therefore, if there is a problem with the development or maturation of the intestines, so that the length of the intestines is shorter and the cross-sectional area is smaller than that of normal people, or if the development of the detailed structures of the intestines is insufficient, the digestive and absorptive abilities may decrease, which may have a negative impact on overall health. In particular, if the development of the intestines is slow in fetuses, newborns, infants, etc. that have not fully developed and grown, it may have a negative impact on the development of organs other than the intestines and overall growth, so the need to improve this is even greater. In addition, even if there is no disorder in the development of the intestines, it can be said that the importance of research on methods that can help the development and maturation of the intestines in developing and growing individuals is high.
본 발명의 일 목적은 신규의 락토바실러스 속 균주 2종을 제공하는 것이다.One object of the present invention is to provide two novel strains of the genus Lactobacillus.
본 발명의 다른 목적은 상기 신규 균주를 포함하는 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition comprising the novel strain.
본 발명의 또 다른 목적은 상기 신규 균주를 포함하는 조성물의 용도를 제공하는 것이다.Another object of the present invention is to provide a use of a composition comprising the novel strain.
상기의 목적을 달성하기 위하여, 상기의 일 목적을 달성하기 위하여, 본 발명의 일 측면은, 수탁번호 KCTC15344BP로 기탁된 락토바실러스 파라카제이(Lactobacillus paracasei) DS2766 균주를 제공한다.In order to achieve the above object, one aspect of the present invention provides Lactobacillus paracasei DS2766 strain deposited under the accession number KCTC15344BP.
또한, 상기의 목적을 달성하기 위하여, 본 발명의 다른 측면은 수탁번호 KCTC15345BP로 기탁된 락토바실러스 가세리(Lactobacillus gasseri) DS2831 균주를 제공한다.In addition, to achieve the above purpose, another aspect of the present invention provides Lactobacillus gasseri DS2831 strain deposited under the accession number KCTC15345BP.
또한, 상기의 목적을 달성하기 위하여, 본 발명의 다른 측면은 상기 2종의 균주, 상기 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택되는 적어도 하나를 포함하는 조성물을 제공한다.In addition, in order to achieve the above purpose, another aspect of the present invention provides a composition comprising at least one selected from the group consisting of the two strains, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
또한, 상기의 목적을 달성하기 위하여, 본 발명의 또 다른 측면은 상기 2종의 균주, 상기 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택되는 적어도 하나를 포함하는, 장의 발달 또는 성숙 촉진용 식품 조성물, 장의 발달 또는 성숙 촉진용 건강기능식품 조성물, 식품 조성물 또는 사료/사료첨가제 조성물, 또는 장관 발달 또는 성숙 장애의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, in order to achieve the above object, another aspect of the present invention provides a food composition for promoting intestinal development or maturation, a health functional food composition for promoting intestinal development or maturation, a food composition or a feed/feed additive composition, or a pharmaceutical composition for preventing or treating intestinal development or maturation disorder, comprising at least one selected from the group consisting of the two kinds of strains, a culture solution of the strains, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
본 발명의 상기 신규 락토바실러스 파라카제이 및 락토바실러스 가세리 균주는 장 조직의 발아(budding) 구조와 표면적을 증가시키고, 성숙 장관 마커 유전자 및 단백질의 발현을 증가시키는 효과가 있고, 이러한 효과가 오가노이드를 통해 검증되었다. 장 조직의 발아 구조와 표면적이 증가하게 되면, 소장의 융모의 길이, 면적 그리고 선와(crypt) 깊이를 증가시키고 대장의 점막/점막하층 비율을 증가시키는 등 장의 발달과 성숙을 촉진하는 활성을 나타내게 되는바, 장의 발달 또는 성숙을 촉진하기 위한 용도로 이용하거나, 장관 발달 또는 성숙 장애를 예방, 개선 또는 치료하기 위한 의약품, 건강기능식품, 식품 및 사료로 유용하게 이용될 수 있는 장점이 있다.The novel Lactobacillus paracasei and Lactobacillus gasseri strains of the present invention have the effect of increasing the budding structure and surface area of intestinal tissue, and increasing the expression of mature intestinal marker genes and proteins, and these effects were verified through organoids. When the budding structure and surface area of intestinal tissue increase, the length and area of villi and crypt depth of the small intestine are increased, and the mucosa/submucosal layer ratio of the large intestine is increased, thereby exhibiting an activity of promoting intestinal development and maturation. Therefore, there is an advantage that it can be usefully used for the purpose of promoting intestinal development or maturation, or as a medicine, health functional food, food, and feed for preventing, improving, or treating intestinal development or maturation disorders.
다만, 본 발명의 효과는 상기에서 언급한 효과로 제한되지 아니하며, 언급되지 않은 또 다른 효과들은 하기의 기재로부터 당업자에게 명확히 이해될 수 있을 것이다.However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.
도 1은 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 파라카제이 균주에 의한 발아 구조 형성 촉진 효과를 확인한 실험 결과를 나타낸다.Figure 1 shows the results of an experiment confirming the effect of promoting germination structure formation by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
도 2는 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 파라카제이 균주에 의한 조직의 표면적의 증가 촉진 효과를 확인한 실험 결과를 나타낸다.Figure 2 shows the results of an experiment confirming the effect of the novel Lactobacillus paracasei strain of the present invention on increasing the surface area of tissue in a human intestinal organoid.
도 3은 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 파라카제이 균주에 의한 장관 성숙 관련 마커 유전자들의 발현 변화를 확인한 실험 결과를 나타낸다.Figure 3 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker genes by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
도 4는 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 파라카제이 균주에 의한 장관 성숙 관련 마커 단백질들의 발현 변화를 확인한 실험 결과를 나타낸다.Figure 4 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker proteins by the novel Lactobacillus paracasei strain of the present invention in human intestinal organoids.
도 5는 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 가세리 균주에 의한 발아 구조 형성 촉진 효과를 확인한 실험 결과를 나타낸다.Figure 5 shows the results of an experiment confirming the effect of promoting germination structure formation by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
도 6은 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 가세리 균주에 의한 조직의 표면적의 증가 촉진 효과를 확인한 실험 결과를 나타낸다.Figure 6 shows the results of an experiment confirming the effect of the novel Lactobacillus gasseri strain of the present invention on increasing the surface area of tissue in human intestinal organoids.
도 7은 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 가세리 균주에 의한 장관 성숙 관련 마커 유전자들의 발현 변화를 확인한 실험 결과를 나타낸다.Figure 7 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker genes by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
도 8은 인간 장 오가노이드에서, 본 발명의 신규 락토바실러스 가세리 균주에 의한 장관 성숙 관련 마커 단백질들의 발현 변화를 확인한 실험 결과를 나타낸다.Figure 8 shows the results of an experiment confirming changes in the expression of intestinal maturation-related marker proteins by the novel Lactobacillus gasseri strain of the present invention in human intestinal organoids.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
1. 1. 신규 락토바실러스 파라카제이(A novel Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) 균주) strain
본 발명의 일 측면은 신규한 락토바실러스 파라카제이 균주를 제공한다.One aspect of the present invention provides a novel Lactobacillus paracasei strain.
상기 본 발명의 신규 락토바실러스 파라카제이 균주는 서열번호 서열번호 1의 염기서열을 갖는 16S rRNA를 포함하는 것일 수 있고, 특히 2023년 3월 10일자로 한국생명공학연구원 생물자원센터(Korean Collection for Type Culture, KCTC)에 수탁번호 KCTC15344BP로 기탁된 락토바실러스 파라카제이 DS2766 균주일 수 있다. 상기 락토바실러스 파라카제이 균주는 2020. 04. 15 국제 공인기관(Microbiology Society)에서 전장유전체분석을 통해 학명을 재분류함에 따라 2021. 4. 20 고시 기준 「건강기능식품의 기준 및 규격」에서도 동일하게 개정되어(식품의약품안전처 고시 제2021-168호), 락티카제이바실러스 파라카제이(Lacticaseibacillus paracasei)라고도 호칭된다. 따라서, 본 발명에서 락토바실러스 파라카제이 DS2766과 락티카제이바실러스 파라카제이 DS2766은 서로 동일한 균주인 것을 의미한다.The novel Lactobacillus paracasei strain of the present invention may include 16S rRNA having a base sequence of the sequence number: SEQ ID NO: 1, and in particular, may be the Lactobacillus paracasei DS2766 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC15344BP on March 10, 2023. The Lactobacillus paracasei strain was reclassified as a scientific name through whole-genome analysis by an internationally recognized organization (Microbiology Society) on April 15, 2020, and was accordingly revised in the “Standards and Specifications for Health Functional Foods” on April 20, 2021 (Ministry of Food and Drug Safety Notice No. 2021-168), and is also called Lacticaseibacillus paracasei . Therefore, in the present invention, Lactobacillus paracasei DS2766 and Lacticazeibacillus paracasei DS2766 are meant to be identical strains.
상기 본 발명의 신규 락토바실러스 파라카제이 균주는 인간 또는 인간을 제외한 동물의 장내에서 서식이 가능한 균주일 수 있으며, 장내에 서식하면서 인간 또는 인간을 제외한 동물의 건강에 다양한 영향을 미칠 수 있다. 상기 본 발명의 신규 락토바실러스 파라카제이 균주는 인간 또는 인간을 제외한 동물에 대해 독성을 나타내거나 질환을 유발하지 않아 안전성이 있는 미생물일 수 있으며, 장내에서 인간 또는 인간을 제외한 동물의 건강에 도움을 주는 유익균으로 작용할 수 있는바, 프로바이오틱스(probiotics) 미생물로 기능할 수 있다.The novel Lactobacillus paracasei strain of the present invention may be a strain capable of living in 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. The novel Lactobacillus paracasei strain of the present invention may be a safe microorganism because it does not exhibit toxicity or cause diseases to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or non-human animals in the intestines, and thus may function as a probiotic microorganism.
프로바이오틱스(probiotics)는 건강에 이익을 제공하는 살아있는 미생물을 일컫는 것으로, 장내 균총에서 유해균을 억제하고 유익균을 증가시키는데 중요한 역할을 하게 된다. 프로바이오틱스로 인정받기 위해서는 위산과 담즙산에서 살아남아 소장, 대장까지 도달하여 장에서 증식하고 정착하여야 하며 장관 내에서 유용한 효과를 나타내어야 하고, 독성이 없으며 비병원성이어야 한다.Probiotics are live microorganisms that provide health benefits, and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as probiotics, they must survive gastric acid and bile acid, reach the small and large intestines, proliferate and settle in the intestines, exhibit useful effects in the intestines, and be non-toxic and non-pathogenic.
본 발명의 DS2766 균주는 당 이용능에 있어서, 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스를 탄소원으로 이용하는 것일 수 있다. The DS2766 strain of the present invention may utilize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources.
본 발명의 구체적인 실시예에서는 본 발명의 DS2766 균주의 배양액을 각각 글루코오스(GLU), 만니톨(MAN), 락토오스(LAC), 수크로오스(SAC), 말토오스(MAL), 살리신(SAL), 자일로스(XYL), 아라비노스(ARA), 젤라틴(GEL), 에스쿨린(ESC), 글리세롤(GLY), 셀로비오스(CEL), 만노오스(MNE), 멜레지토오스(MLZ), 라피노오스(RAF), 소르비톨(SOR), 람노오스(RHA) 또는 트레할로오스(TRE)을 포함하는 튜브에 접종한 후, 균주의 당 이용능을 측정하였다. 그 결과, 본 발명의 균주는 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스를 탄소원으로 이용할 수 있어 대사할 수 있으나, 젤라틴을 이용하여 대사할 수는 없는 것으로 확인되었다.In a specific embodiment of the present invention, a culture solution of the DS2766 strain of the present invention was inoculated into a tube containing glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), and then the sugar utilization ability of the strain was measured. As a result, it was confirmed that the strain of the present invention can utilize and metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources, but cannot metabolize gelatin.
또한, 본 발명의 상기 DS2766 균주는 독성 물질을 생산하지 않는 것일 수 있고, 상기 독성 물질은 예컨대 인돌(Indol) 또는 요소(Urea) 등이 있을 수 있다.In addition, the DS2766 strain of the present invention may not produce a toxic substance, and the toxic substance may be, for example, indole or urea.
나아가, 본 발명의 상기 DS2766 균주는 β-글루쿠로니다아제(β-glucuronidase)를 생산하지 않는 것일 수 있다. 상기 β-글루쿠로니다아제는 가수 분해를 촉진하는 효소를 의미하는 것으로, β-글루쿠로니다아제는, 각종 알코올류, 페놀류, 아민류 등이 글루쿠론산 포함된 화합물 (글루쿠로니드) 을 가수 분해시키는 효소로서, 세균, 진균류, 식물, 동물 등 많은 생물에 존재하며, 예를 들어 인간의 땀 중에 글루쿠론산이 포함되어 분비되고, 피부에 생식하는 세균의 대사를 통하여 선취(腺臭)의 원인인 물질이 생성되는 것에 관여하는 것은 알려져 있으며, 특히 대장암과 연관성이 있는 것으로 보고되어 있다.Furthermore, the DS2766 strain of the present invention may not produce β-glucuronidase. The β-glucuronidase refers to an enzyme that promotes hydrolysis, and β-glucuronidase is an enzyme that hydrolyzes various alcohols, phenols, amines, etc. into compounds (glucuronides) containing glucuronic acid, and exists in many living things such as bacteria, fungi, plants, and animals. For example, it is known that glucuronic acid is contained in human sweat and is secreted, and that it is involved in the production of substances that cause glandular odor through the metabolism of bacteria that live on the skin, and in particular, it has been reported to be related to colon cancer.
또한, 본 발명의 상기 DS2766 균주는 용혈성을 나타내지 않는 것일 수 있다. 상기 용혈성은 균주가 적혈구를 파괴하는 성질을 의미하는 것으로, 균주가 프로바이오틱스로서 안전하게 이용되기 위해서는 용혈성을 갖지 않아야 한다.In addition, the DS2766 strain of the present invention may not exhibit hemolysis. The hemolysis refers to the property of the strain to destroy red blood cells, and in order for the strain to be safely used as a probiotic, it must not exhibit hemolysis.
본 발명의 구체적인 실시예에서는 본 발명의 DS2766 균주의 인돌, 요소 및 β-글루쿠로니다아제 생산능을 분석하였다. 그 결과, 본 발명의 균주는 인돌 및 요소와 같은 독성 물질에 대한 생산능이 없고, 대장암과 연관된 β-글루쿠로니다아제를 생산할 수 없는 것으로 나타나, 본 발명의 균주는 프로바이오틱스로서 안전성이 있음을 확인할 수 있었다.In a specific embodiment of the present invention, the indole, urea, and β-glucuronidase production ability of the DS2766 strain of the present invention was analyzed. As a result, the strain of the present invention was found to have no production ability for toxic substances such as indole and urea, and was unable to produce β-glucuronidase associated with colon cancer, confirming that the strain of the present invention is safe as a probiotic.
또한, 본 발명의 구체적인 실시예에서는 본 발명의 DS2766 균주를 혈액 배지에서 배양하여 α, β 및 γ 용혈성을 분석한 결과, 본 발명의 균주는 용혈성을 나타내지 않아, 프로바이오틱스로서 안전성이 있음을 확인하였다. In addition, in a specific embodiment of the present invention, the DS2766 strain of the present invention was cultured in a blood medium and analyzed for α, β, and γ hemolysis, and as a result, the strain of the present invention did not exhibit hemolysis, confirming its safety as a probiotic.
또한, 본 발명의 상기 DS2766 균주는 내산성을 갖는 것일 수 있다. 상기 내산성은 균주가 산성의 환경에서 잘 생존할 수 있는 성질을 의미하는 것으로서, pH 5 이하의 환경, 예컨대 pH 4 이하, pH 3.5 이하의 환경, 특히 pH 3 이하의 환경 산성 환경에서 1시간 이상, 예컨대 1.5시간 이상, 2시간 이상, 2.5시간 이상, 특히 3 시간 이상의 시간이 경과하더라도, 생존할 수 있는 특성을 의미하는 것일 수 있다. 본 발명의 구체적인 실시예에서는 상기 본 발명의 신규 균주가 pH 3.0의 조건에서 3시간 동안 배양된 경우에도 여전히 잘 생존하고 있어 우수한 내산성을 가지는 것으로 확인되었다.In addition, the DS2766 strain of the present invention may be acid-resistant. The acid-resistant refers to a property of the strain to survive well in an acidic environment, and may refer to a property of surviving even after 1 hour or more, for example, 1.5 hours or more, 2 hours or more, 2.5 hours or more, and especially 3 hours or more, in an acidic environment of pH 5 or lower, for example, pH 4 or lower, pH 3.5 or lower, and particularly, an environment of pH 3 or lower. In a specific embodiment of the present invention, it was confirmed that the novel strain of the present invention still survived well even when cultured for 3 hours under a condition of pH 3.0, thereby having excellent acid-resistant properties.
또한, 본 발명의 상기 DS2766 균주는 내담즙성을 갖는 것일 수 있다. 상기 내담즙성은 균주가 쓸개에서 분배되는 담즙이 존재하는 담즙성 환경에서 잘 생존할 수 있는 성질을 의미하는 것으로서, 옥스갈(oxgall), 담즙염(bile salt) 등과 같은 담즙이 5% 이하, 예컨대 4.5% 이하, 4% 이하, 3.5% 이하, 특히 3% 이하의 농도로 존재하는 담즙성 환경에서, 6시간 이상, 예컨대 7시간 이상, 8시간 이상, 9시간 이상, 10시간 이상, 11시간 이상, 특히 12시간 이상의 시간이 경과하더라도, 생존할 수 있는 특성을 의미하는 것일 수 있다. 본 발명의 구체적인 실시예에서는 상기 본 발명의 신규 균주가 3%(w/v)의 담즙염이 포함된 조건에서 24시간 동안 배양된 경우에도 여전히 잘 생존하고 있어 우수한 내담즙성을 가지는 것으로 확인되었다.In addition, the DS2766 strain of the present invention may be cholestatic. The cholestatic property refers to a property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and may refer to a property of surviving in a biliary environment where bile, such as oxgall and bile salts, exists at a concentration of 5% or less, for example, 4.5% or less, 4% or less, 3.5% or less, and especially 3% or less, for 6 hours or more, for example, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, and especially 12 hours or more. In a specific embodiment of the present invention, it was confirmed that the novel strain of the present invention still survived well even when cultured for 24 hours under conditions containing 3% (w/v) of bile salt, thereby having excellent cholestatic property.
또한, 본 발명의 상기 DS2766 균주는 장 부착능을 갖는 것일 수 있다. 상기 장 부착능은 균주가 장 세포에 붙어 떼어지지 않는 성질을 의미하는 것으로, 장 부착능이 우수하면, 상기 균주가 장에 머무를 수 있는 시간이 늘어나, 프로바이오틱스로서 장내 균총 개선, 배변활동 및 장 건강 상태를 증진시키는 효과가 오래 유지될 수 있는 것을 의미한다. 구체적으로, 상기 장 부착능은 균주가 장에 도달한 후 장 세포에 부착되어 장내 환경에서 12시간 이상, 예컨대 15시간 이상, 18시간 이상, 21시간 이상, 특히 24시간 이상 경과한 이후 초기 생균수의 1.0% 이상, 예컨대 1.5% 이상, 2.0% 이상, 2.5% 이상, 특히 2.54% 이상의 수의 생균이 장 세포에 부착되어 있는 것을 의미하는 것일 수 있다. In addition, the DS2766 strain of the present invention may have intestinal adhesion. The intestinal adhesion refers to a property of the strain to adhere to intestinal cells and not be detached. If the intestinal adhesion is excellent, the time for which the strain can remain in the intestines increases, and the effect of improving intestinal flora, bowel movement, and promoting intestinal health as a probiotic can be maintained for a long time. Specifically, the intestinal adhesion refers to a number of viable bacteria, such as 1.0% or more of the initial number of viable bacteria, for example, 1.5% or more, 2.0% or more, 2.5% or more, or 2.54% or more, of which are attached to the intestinal cells after the strain reaches the intestines and remains in the intestinal environment for 12 hours or more, for example, 15 hours or more, 18 hours or more, 21 hours or more, or especially 24 hours or more.
본 발명의 구체적인 실시예에서는, 본 발명의 DS2766 균주를 Caco-2 세포주에 처리하여 포도당을 탄소원으로 배양하였고, PBS 용액으로 세척한 후, 상기 균주의 장 부착율(%)을 확인하였다. 그 결과, 상기 균주는 약 0.72%의 장 부착율을 나타내어, 장 부착능을 갖는 것으로 확인되었다. In a specific embodiment of the present invention, the DS2766 strain of the present invention was treated to a Caco-2 cell line and cultured using glucose as a carbon source, and after washing with a PBS solution, the intestinal adhesion rate (%) of the strain was confirmed. As a result, the strain was confirmed to have an intestinal adhesion ability by showing an intestinal adhesion rate of about 0.72%.
또한, 상기 균주는 항생제에 내성을 나타내지 않지 않거나, 상기 항생제에 내성이 낮은 것일 수 있다. 상기 내성은 외재 내성일 수 있고, 상기 외재 내성은 항생제에 대한 내성 유전자가 플라스미드나 트랜스포존 등과 같이 이동이 가능한 수단을 통해 외부 다른 세균으로 도입되어 유발될 수 있는 내성을 의미한다. 따라서 항생제에 대한 외재 내성을 나타내지 않는 본 발명의 락토바실러스 파라카제이 DS2766 균주는 장내에 존재하는 유해 세균으로 내성 유전자를 수평 전달하지 않으므로, 장내 유해 세균이 외재 내성을 획득하여 항생제에 대한 내성 문제를 일으킬 염려가 없다. In addition, the strain may not exhibit resistance to antibiotics, or may have low resistance to the antibiotics. The resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced when a resistance gene for antibiotics is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the Lactobacillus paracasei DS2766 strain of the present invention, which does not exhibit extrinsic resistance to antibiotics, does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, and therefore there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics.
본 발명의 락토바실러스 파라카제이 DS2766 균주가 내성을 나타내지 않는 항생제는 본 발명이 속하는 기술분야에서 알려진 것이라면 특별히 제한되지 않고, 예컨대 페니실린(penicilin)계 항생제, 테트라사이클린(tetracyclines)계 항생제, 마크로라이드(macrolide)계 항생제, 설폰아미드(sulfonamides))계 항생제, 암페니콜(amphenicols)계 항생제, 아미노글리코시드(aminoglycoside)계 항생제 등일 수 있고, 구체적으로 앰피실린(ampicillin), 젠타마이신(gentamicin), 에리트로마이신(erythromycin), 클린다마이신(clindamycin) 및 테트라사이클린(tetracycline) 등일 수 있다. The antibiotics to which the Lactobacillus paracasei DS2766 strain of the present invention does not exhibit resistance are not particularly limited, so long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracyclines antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, may be ampicillin, gentamicin, erythromycin, clindamycin, tetracycline, etc.
본 발명의 구체적인 실시예에서는 상기 DS2766 균주에 대하여 MTSTM(MIC Test Strip)(Liofilchem)을 이용하여 항생제 내성을 실험하였다. 그 결과 상기 균주는 앰피실린, 반코마이신, 에리트로마이신, 클린다마이신 및 테트라사이클린에 대한 내성이 EFSA 가이드(유럽식품안전청 항생제 안전성 기준)에 기재된 것보다 낮은 것으로 확인되었다. 특히, 유럽에서는 이미 오래전부터 유산균이 항생제 내성 유전자를 다른 세균으로 수평 전달할 수 있는 경우는 상업화할 수 없으며, 우리나라도 최근에 식약처의 미생물의 식품원료 판단기준에 항생제 내성 전이에 관한 조항을 삽입하였으므로, 본 발명의 락토바실러스 파라카제이 DS2766 균주는 프로바이오틱스로서 안전성이 인정되어 상업화되기에도 유리한 장점이 있다.In a specific embodiment of the present invention, the antibiotic resistance of the DS2766 strain was tested using MTS TM (MIC Test Strip) (Liofilchem). As a result, it was confirmed that the strain had lower resistance to ampicillin, vancomycin, erythromycin, clindamycin, and tetracycline than that described in the EFSA guide (European Food Safety Authority antibiotic safety criteria). In particular, in Europe, it has been said for a long time that lactic acid bacteria cannot be commercialized if they can horizontally transfer antibiotic resistance genes to other bacteria, and in Korea, the Ministry of Food and Drug Safety recently inserted a clause on antibiotic resistance transfer into the criteria for judging microorganisms as food ingredients. Therefore, the Lactobacillus paracasei DS2766 strain of the present invention has an advantage in that its safety as a probiotic is recognized, making it advantageous for commercialization.
한편, 본 발명의 상기 DS2766 균주는 프로바이오틱스로 활용이 가능한 생리학적 특성을 가질 수 있다. 특히, 상기 본 발명의 신규 락토바실러스 파라카제이 균주는 장의 발달 또는 성숙을 촉진하는 활성을 가지는 것일 수 있다. Meanwhile, the DS2766 strain of the present invention may have physiological characteristics that enable it to be utilized as a probiotic. In particular, the novel Lactobacillus paracasei strain of the present invention may have an activity that promotes the development or maturation of the intestine.
상기 장은 위로부터 항문까지 뻗어 있는 소화관의 구역을 의미한다. 인간 및 기타 포유동물에 있어서, 장은 소장(인간에서는 십이지장, 빈창자 및 돌창자로 더욱 세분됨)과 대장(인간에서는 맹장과 결장으로 더욱 세분됨)의 2개의 구역으로 구성되어 있으며, 기타 포유동물은 더욱 복잡한 장을 지닐 수 있는데, 본 발명에서 장은 이들 모두를 포함할 수 있다.The above intestine refers to a region of the digestive tract extending from the stomach to the anus. In humans and other mammals, the intestine is composed of two regions: the small intestine (further subdivided into the duodenum, the small intestine, and the ileum in humans) and the large intestine (further subdivided into the cecum and the colon in humans), and other mammals may have more complex intestines, and the intestine in the present invention may include all of these.
구체적으로, 상기 본 발명의 신규 락토바실러스 파라카제이 균주는 소장 또는 대장이 더욱 성숙된 상태가 되도록 촉진하는 활성이 있거나, 미성숙된 상태의 소장 또는 대장의 성숙을 촉진시키는 활성이 있을 수 있다. 예를 들어, 상기 본 발명의 신규 락토바실러스 파라카제이 균주는 소장 또는 대장의 세포에서 CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, MUC13 등과 같은 유전자의 발현을 향상시키는 것일 수 있다. 또한, 상기 본 발명의 신규 락토바실러스 파라카제이 균주는 장의 표면적을 증가시키는 것일 수 있다. 예컨대, 소장의 융모(villus)의 길이나 표면적을 증가시키거나, 선와(crypt)의 깊이를 증가시키는 것이거나, 대장의 선와의 깊이를 증가시키거나 점막/점막하층(mucosa/submucosa)의 비율을 증가시키는 것일 수 있다.Specifically, the novel Lactobacillus paracasei strain of the present invention may have an activity of promoting the small intestine or the large intestine to become more mature, or may have an activity of promoting the maturation of the small intestine or the large intestine from an immature state. For example, the novel Lactobacillus paracasei strain of the present invention may enhance the expression of genes such as CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, MUC13 , etc. in cells of the small intestine or the large intestine. In addition, the novel Lactobacillus paracasei strain of the present invention may increase the surface area of the intestine. For example, it may increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa.
본 발명의 구체적인 실시예에서는, 상기 본 발명의 신규 락토바실러스 파라카제이 균주를 포함하는 배양액을, 인체의 체외 장 모델로 제작된 3차원의 인간 장 오가노이드에 처리하였을 때, 이의 발아 구조의 형성을 촉진하고 표면적으로 향상시킬 뿐만 아니라(도 1 및 도 2 참고), 장관의 성숙과 관련 마커 유전자, 단백질의 발현을 증가시키는 것을 확인하였다(도 3 및 도 4 참고).In a specific embodiment of the present invention, when a culture solution containing the novel Lactobacillus paracasei strain of the present invention was treated on a three-dimensional human intestinal organoid produced as an in vitro human intestinal model, it was confirmed that not only the formation of a germination structure was promoted and the surface area was improved (see FIGS. 1 and 2), but also the expression of marker genes and proteins related to intestinal maturation was increased (see FIGS. 3 and 4).
상기와 같은 결과로부터, 본 발명의 신규 락토바실러스 파라카제이 균주는 장의 성숙 또는 발달을 촉진하는 활성을 갖는 신규한 균주임을 알 수 있고, 이에 따라 본 발명의 상기 균주를 장 발달 또는 장 성숙 촉진 용도, 장관 발달 또는 성숙 장애의 예방, 개선 또는 치료를 위한 용도에 효과적으로 사용할 수 있다.From the above results, it can be seen that the novel Lactobacillus paracasei strain of the present invention is a novel strain having an activity of promoting intestinal maturation or development, and thus the strain of the present invention can be effectively used for purposes of promoting intestinal development or intestinal maturation, or for purposes of preventing, improving, or treating intestinal development or maturation disorders.
2. 2. 신규 락토바실러스 가세리(A new Lactobacillus gasseri ( Lactobacillus gasseriLactobacillus gasseri ) 균주) strain
본 발명의 일 측면은 신규한 락토바실러스 가세리 균주를 제공한다.One aspect of the present invention provides a novel Lactobacillus gasseri strain.
상기 본 발명의 신규 락토바실러스 가세리 균주는 서열번호 1의 염기서열을 갖는 16S rRNA를 포함하는 것일 수 있고, 특히 2023년 3월 10일자로 한국생명공학연구원 생물자원센터(Korean Collection for Type Culture, KCTC)에 수탁번호 KCTC15345BP로 기탁된 락토바실러스 가세리 DS2831 균주일 수 있다.The novel Lactobacillus gasseri strain of the present invention may include 16S rRNA having a base sequence of sequence number 1, and in particular, may be Lactobacillus gasseri DS2831 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023 under the accession number KCTC15345BP.
상기 본 발명의 신규 락토바실러스 가세리 균주는 인간 또는 인간을 제외한 동물의 장내에서 서식이 가능한 균주일 수 있으며, 장내에 서식하면서 인간 또는 인간을 제외한 동물의 건강에 다양한 영향을 미칠 수 있다. 상기 본 발명의 신규 락토바실러스 가세리 균주는 인간 또는 인간을 제외한 동물에 대해 독성을 나타내거나 질환을 유발하지 않아 안전성이 있는 미생물일 수 있으며, 장내에서 인간 또는 인간을 제외한 동물의 건강에 도움을 주는 유익균으로 작용할 수 있는바, 프로바이오틱스(probiotics) 미생물로 기능할 수 있다.The novel Lactobacillus gasseri strain of the present invention may be a strain capable of living in 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. The novel Lactobacillus gasseri strain of the present invention may be a safe microorganism because it does not exhibit toxicity or cause diseases to humans or non-human animals, and may act as a beneficial bacterium that helps the health of humans or non-human animals in the intestines, and thus may function as a probiotic microorganism.
프로바이오틱스(probiotics)는 건강에 이익을 제공하는 살아있는 미생물을 일컫는 것으로, 장내 균총에서 유해균을 억제하고 유익균을 증가시키는데 중요한 역할을 하게 된다. 프로바이오틱스로 인정받기 위해서는 위산과 담즙산에서 살아남아 소장, 대장까지 도달하여 장에서 증식하고 정착하여야 하며 장관 내에서 유용한 효과를 나타내어야 하고, 독성이 없으며 비병원성이어야 한다.Probiotics are live microorganisms that provide health benefits, and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. In order to be recognized as probiotics, they must survive gastric acid and bile acid, reach the small and large intestines, proliferate and settle in the intestines, exhibit useful effects in the intestines, and be non-toxic and non-pathogenic.
본 발명의 DS2831 균주는 당 이용능에 있어서, 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스를 탄소원으로 이용하는 것일 수 있다. The DS2831 strain of the present invention may utilize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources.
본 발명의 구체적인 실시예에서는 본 발명의 DS2831 균주의 배양액을 각각 글루코오스(GLU), 만니톨(MAN), 락토오스(LAC), 수크로오스(SAC), 말토오스(MAL), 살리신(SAL), 자일로스(XYL), 아라비노스(ARA), 젤라틴(GEL), 에스쿨린(ESC), 글리세롤(GLY), 셀로비오스(CEL), 만노오스(MNE), 멜레지토오스(MLZ), 라피노오스(RAF), 소르비톨(SOR), 람노오스(RHA) 또는 트레할로오스(TRE)을 포함하는 튜브에 접종한 후, 균주의 당 이용능을 측정하였다. 그 결과, 본 발명의 균주는 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스를 탄소원으로 이용할 수 있어 대사할 수 있으나, 젤라틴을 이용하여 대사할 수는 없는 것으로 확인되었다.In a specific embodiment of the present invention, a culture solution of the DS2831 strain of the present invention was inoculated into a tube containing glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), and then the sugar utilization ability of the strain was measured. As a result, it was confirmed that the strain of the present invention can utilize and metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources, but cannot metabolize gelatin.
또한, 본 발명의 상기 DS2831 균주는 독성 물질을 생산하지 않는 것일 수 있고, 상기 독성 물질은 예컨대 인돌(Indol) 또는 요소(Urea) 등이 있을 수 있다.In addition, the DS2831 strain of the present invention may not produce a toxic substance, and the toxic substance may be, for example, indole or urea.
나아가, 본 발명의 상기 DS2831 균주는 β-글루쿠로니다아제(β-glucuronidase)를 생산하지 않는 것일 수 있다. 상기 β-글루쿠로니다아제는 가수 분해를 촉진하는 효소를 의미하는 것으로, β-글루쿠로니다아제는, 각종 알코올류, 페놀류, 아민류 등이 글루쿠론산 포함된 화합물 (글루쿠로니드) 을 가수 분해시키는 효소로서, 세균, 진균류, 식물, 동물 등 많은 생물에 존재하며, 예를 들어 인간의 땀 중에 글루쿠론산이 포함되어 분비되고, 피부에 생식하는 세균의 대사를 통하여 선취(腺臭)의 원인인 물질이 생성되는 것에 관여하는 것은 알려져 있으며, 특히 대장암과 연관성이 있는 것으로 보고되어 있다.Furthermore, the DS2831 strain of the present invention may not produce β-glucuronidase. The β-glucuronidase refers to an enzyme that promotes hydrolysis, and β-glucuronidase is an enzyme that hydrolyzes various alcohols, phenols, amines, etc. into compounds (glucuronides) containing glucuronic acid, and exists in many living things such as bacteria, fungi, plants, and animals. For example, it is known that glucuronic acid is contained in human sweat and secreted, and that it is involved in the production of substances that cause glandular odor through the metabolism of bacteria that live on the skin, and in particular, it has been reported to be related to colon cancer.
또한, 본 발명의 상기 DS2831 균주는 용혈성을 나타내지 않는 것일 수 있다. 상기 용혈성은 균주가 적혈구를 파괴하는 성질을 의미하는 것으로, 균주가 프로바이오틱스로서 안전하게 이용되기 위해서는 용혈성을 갖지 않아야 한다.In addition, the DS2831 strain of the present invention may not exhibit hemolysis. The hemolysis refers to the property of the strain to destroy red blood cells, and in order for the strain to be safely used as a probiotic, it must not exhibit hemolysis.
본 발명의 구체적인 실시예에서는 본 발명의 DS2831 균주의 인돌, 요소 및 β-글루쿠로니다아제 생산능을 분석하였다. 그 결과, 본 발명의 균주는 인돌 및 요소와 같은 독성 물질에 대한 생산능이 없고, 대장암과 연관된 β-글루쿠로니다아제를 생산할 수 없는 것으로 나타나, 본 발명의 균주는 프로바이오틱스로서 안전성이 있음을 확인할 수 있었다.In a specific embodiment of the present invention, the indole, urea, and β-glucuronidase production ability of the DS2831 strain of the present invention was analyzed. As a result, the strain of the present invention was found to have no production ability for toxic substances such as indole and urea, and was unable to produce β-glucuronidase associated with colon cancer, confirming that the strain of the present invention is safe as a probiotic.
또한, 본 발명의 구체적인 실시예에서는 본 발명의 DS2831 균주를 혈액 배지에서 배양하여 α, β 및 γ 용혈성을 분석한 결과, 본 발명의 균주는 용혈성을 나타내지 않아, 프로바이오틱스로서 안전성이 있음을 확인하였다. In addition, in a specific embodiment of the present invention, the DS2831 strain of the present invention was cultured in a blood medium and analyzed for α, β and γ hemolysis, and as a result, the strain of the present invention did not exhibit hemolysis, confirming its safety as a probiotic.
또한, 본 발명의 상기 DS2831 균주는 내산성을 갖는 것일 수 있다. 상기 내산성은 균주가 산성의 환경에서 잘 생존할 수 있는 성질을 의미하는 것으로서, pH 5 이하의 환경, 예컨대 pH 4 이하, pH 3.5 이하의 환경, 특히 pH 3 이하의 환경 산성 환경에서 1시간 이상, 예컨대 1.5시간 이상, 2시간 이상, 2.5시간 이상, 특히 3 시간 이상의 시간이 경과하더라도, 생존할 수 있는 특성을 의미하는 것일 수 있다. 본 발명의 구체적인 실시예에서는 상기 본 발명의 신규 균주가 pH 3.0의 조건에서 3시간 동안 배양된 경우에도 여전히 잘 생존하고 있어 우수한 내산성을 가지는 것으로 확인되었다.In addition, the DS2831 strain of the present invention may be acid-resistant. The acid-resistant refers to a property of the strain to survive well in an acidic environment, and may refer to a property of surviving in an acidic environment of pH 5 or lower, for example, an environment of pH 4 or lower, a pH 3.5 or lower, and particularly an environment of pH 3 or lower, for a time of 1 hour or longer, for example, 1.5 hours or longer, 2 hours or longer, 2.5 hours or longer, and particularly 3 hours or longer. In a specific embodiment of the present invention, it was confirmed that the novel strain of the present invention still survived well even when cultured for 3 hours under a condition of pH 3.0, thereby having excellent acid-resistant properties.
또한, 본 발명의 상기 DS2831 균주는 내담즙성을 갖는 것일 수 있다. 상기 내담즙성은 균주가 쓸개에서 분배되는 담즙이 존재하는 담즙성 환경에서 잘 생존할 수 있는 성질을 의미하는 것으로서, 옥스갈(oxgall), 담즙염(bile salt) 등과 같은 담즙이 5% 이하, 예컨대 4.5% 이하, 4% 이하, 3.5% 이하, 특히 3% 이하의 농도로 존재하는 담즙성 환경에서, 6시간 이상, 예컨대 10시간 이상, 12시간 이상, 16시간 이상, 20시간 이상, 22시간 이상, 특히 24시간 이상의 시간이 경과하더라도, 생존할 수 있는 특성을 의미하는 것일 수 있다. 본 발명의 구체적인 실시예에서는 상기 본 발명의 신규 균주가 3%(w/v)의 담즙염이 포함된 조건에서 24시간 동안 배양된 경우에도 여전히 잘 생존하고 있어 우수한 내담즙성을 가지는 것으로 확인되었다.In addition, the DS2831 strain of the present invention may be cholestatic. The cholestatic property refers to a property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and may refer to a property of surviving in a biliary environment where bile, such as oxgall and bile salts, exists at a concentration of 5% or less, for example, 4.5% or less, 4% or less, 3.5% or less, and especially 3% or less, for 6 hours or more, for example, 10 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 22 hours or more, and especially 24 hours or more. In a specific embodiment of the present invention, it was confirmed that the novel strain of the present invention still survived well even when cultured for 24 hours under conditions containing 3% (w/v) of bile salt, and thus had excellent cholestatic property.
또한, 본 발명의 상기 DS2831 균주는 장 부착능을 갖는 것일 수 있다. 상기 장 부착능은 균주가 장 세포에 붙어 떼어지지 않는 성질을 의미하는 것으로, 장 부착능이 우수하면, 상기 균주가 장에 머무를 수 있는 시간이 늘어나, 프로바이오틱스로서 장내 균총 개선, 배변활동 및 장 건강 상태를 증진시키는 효과가 오래 유지될 수 있는 것을 의미한다. 구체적으로, 상기 장 부착능은 균주가 장에 도달한 후 장 세포에 부착되어 장내 환경에서 12시간 이상, 예컨대 15시간 이상, 18시간 이상, 21시간 이상, 특히 24시간 이상 경과한 이후 초기 생균수의 0.5% 이상, 예컨대 0.6% 이상, 0.7% 이상, 특히 0.72% 이상의 수의 생균이 장 세포에 부착되어 있는 것을 의미하는 것일 수 있다. In addition, the DS2831 strain of the present invention may have intestinal adhesion. The intestinal adhesion refers to a property of the strain to adhere to intestinal cells and not be detached. If the intestinal adhesion is excellent, the time for which the strain can remain in the intestines increases, and the effect of improving intestinal flora, bowel movement, and promoting intestinal health as a probiotic can be maintained for a long time. Specifically, the intestinal adhesion refers to a number of viable cells, such as 0.5% or more of the initial number of viable cells, for example, 0.6% or more, 0.7% or more, or 0.72% or more, of which are attached to the intestinal cells after the strain reaches the intestines and remains in the intestinal environment for 12 hours or more, for example, 15 hours or more, 18 hours or more, 21 hours or more, or especially 24 hours or more.
본 발명의 구체적인 실시예에서는, 본 발명의 DS2831 균주를 Caco-2 세포주에 처리하여 포도당을 탄소원으로 배양하였고, PBS 용액으로 세척한 후, 상기 균주의 장 부착율(%)을 확인하였다. 그 결과, 상기 균주는 약 0.72%의 장 부착율을 나타내어, 장 부착능을 갖는 것으로 확인되었다. In a specific embodiment of the present invention, the DS2831 strain of the present invention was treated to a Caco-2 cell line and cultured using glucose as a carbon source, and after washing with a PBS solution, the intestinal adhesion rate (%) of the strain was confirmed. As a result, the strain was confirmed to have an intestinal adhesion ability by showing an intestinal adhesion rate of about 0.72%.
또한, 상기 DS2831 균주는 항생제에 내성을 나타내지 않지 않거나, 상기 항생제에 내성이 낮은 것일 수 있다. 상기 내성은 외재 내성일 수 있고, 상기 외재 내성은 항생제에 대한 내성 유전자가 플라스미드나 트랜스포존 등과 같이 이동이 가능한 수단을 통해 외부 다른 세균으로 도입되어 유발될 수 있는 내성을 의미한다. 따라서 항생제에 대한 외재 내성을 나타내지 않는 본 발명의 락토바실러스 가세리 DS2831 균주는 장내에 존재하는 유해 세균으로 내성 유전자를 수평 전달하지 않으므로, 장내 유해 세균이 외재 내성을 획득하여 항생제에 대한 내성 문제를 일으킬 염려가 없다. In addition, the DS2831 strain may not exhibit resistance to antibiotics, or may have low resistance to the antibiotics. The resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced when a resistance gene for antibiotics is introduced into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the Lactobacillus gasseri DS2831 strain of the present invention, which does not exhibit extrinsic resistance to antibiotics, does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, and therefore there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics.
본 발명의 락토바실러스 가세리 DS2831 균주가 내성을 나타내지 않는 항생제는 본 발명이 속하는 기술분야에서 알려진 것이라면 특별히 제한되지 않고, 예컨대 페니실린(penicilin)계 항생제, 테트라사이클린(tetracyclines)계 항생제, 마크로라이드(macrolide)계 항생제, 설폰아미드(sulfonamides))계 항생제, 암페니콜(amphenicols)계 항생제, 아미노글리코시드(aminoglycoside)계 항생제 등일 수 있고, 구체적으로 앰피실린(ampicillin), 반코마이신(vancomycin), 에리트로마이신(erythromycin) 및 테트라사이클린(tetracycline) 등일 수 있다. The antibiotics to which the Lactobacillus gasseri DS2831 strain of the present invention does not show resistance are not particularly limited, so long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracyclines antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, may be ampicillin, vancomycin, erythromycin, tetracycline, etc.
본 발명의 구체적인 실시예에서는 상기 DS2831 균주에 대하여 MTSTM(MIC Test Strip)(Liofilchem)을 이용하여 항생제 내성을 실험하였다. 그 결과 상기 균주는 앰피실린(ampicillin), 반코마이신(vancomycin), 에리트로마이신(erythromycin) 및 테트라사이클린(tetracycline)에 대한 내성이 EFSA 가이드(유럽식품안전청 항생제 안전성 기준)에 기재된 것보다 낮은 것으로 확인되었다. 특히, 유럽에서는 이미 오래전부터 유산균이 항생제 내성 유전자를 다른 세균으로 수평 전달할 수 있는 경우는 상업화할 수 없으며, 우리나라도 최근에 식약처의 미생물의 식품원료 판단기준에 항생제 내성 전이에 관한 조항을 삽입하였으므로, 본 발명의 락토바실러스 가세리 DS2831 균주는 프로바이오틱스로서 안전성이 인정되어 상업화되기에도 유리한 장점이 있다.In a specific embodiment of the present invention, the antibiotic resistance of the DS2831 strain was tested using MTS TM (MIC Test Strip) (Liofilchem). As a result, the strain was confirmed to have lower resistance to ampicillin, vancomycin, erythromycin, and tetracycline than that described in the EFSA guide (European Food Safety Authority antibiotic safety criteria). In particular, in Europe, it has been said for a long time that lactic acid bacteria cannot be commercialized if they can horizontally transfer antibiotic resistance genes to other bacteria, and in Korea, the Ministry of Food and Drug Safety recently inserted a clause on antibiotic resistance transfer into the criteria for judging microorganisms as food ingredients. Therefore, the Lactobacillus gasseri DS2831 strain of the present invention has an advantage in that its safety as a probiotic is recognized, making it advantageous for commercialization.
한편, 본 발명의 상기 DS2831 균주는 프로바이오틱스로 활용이 가능한 생리학적 특성을 가질 수 있다. 특히, 상기 본 발명의 신규 락토바실러스 가세리 균주는 장의 발달 또는 성숙을 촉진하는 활성을 가지는 것일 수 있다. Meanwhile, the DS2831 strain of the present invention may have physiological characteristics that can be utilized as probiotics. In particular, the novel Lactobacillus gasseri strain of the present invention may have an activity that promotes the development or maturation of the intestine.
상기 장은 위로부터 항문까지 뻗어 있는 소화관의 구역을 의미한다. 인간 및 기타 포유동물에 있어서, 장은 소장(인간에서는 십이지장, 빈창자 및 돌창자로 더욱 세분됨)과 대장(인간에서는 맹장과 결장으로 더욱 세분됨)의 2개의 구역으로 구성되어 있으며, 기타 포유동물은 더욱 복잡한 장을 지닐 수 있는데, 본 발명에서 장은 이들 모두를 포함할 수 있다.The above intestine refers to a region of the digestive tract extending from the stomach to the anus. In humans and other mammals, the intestine is composed of two regions: the small intestine (further subdivided into the duodenum, the small intestine, and the ileum in humans) and the large intestine (further subdivided into the cecum and the colon in humans), and other mammals may have more complex intestines, and the intestine in the present invention may include all of these.
구체적으로, 상기 본 발명의 신규 락토바실러스 가세리 균주는 소장 또는 대장이 더욱 성숙된 상태가 되도록 촉진하는 활성이 있거나, 미성숙된 상태의 소장 또는 대장의 성숙을 촉진시키는 활성이 있을 수 있다. 예를 들어, 상기 본 발명의 신규 락토바실러스 가세리 균주는 소장 또는 대장의 세포에서 CDX2, LYZ, CREB3L3, DPP4, SLC5A1, MUC13등과 같은 유전자의 발현을 향상시키는 것일 수 있다. 또한, 상기 본 발명의 신규 락토바실러스 가세리 균주는 장의 표면적을 증가시키는 것일 수 있다. 예컨대, 소장의 융모(villus)의 길이나 표면적을 증가시키거나, 선와(crypt)의 깊이를 증가시키는 것이거나, 대장의 선와의 깊이를 증가시키거나 점막/점막하층(mucosa/submucosa)의 비율을 증가시키는 것일 수 있다.Specifically, the novel Lactobacillus gasseri strain of the present invention may have an activity of promoting the small intestine or the large intestine to become more mature, or may have an activity of promoting the maturation of the small intestine or the large intestine from an immature state. For example, the novel Lactobacillus gasseri strain of the present invention may enhance the expression of genes such as CDX2, LYZ, CREB3L3, DPP4, SLC5A1, MUC13 , etc. in cells of the small intestine or the large intestine. In addition, the novel Lactobacillus gasseri strain of the present invention may increase the surface area of the intestine. For example, it may increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa.
본 발명의 구체적인 실시예에서는, 상기 본 발명의 신규 락토바실러스 가세리 균주를 포함하는 배양액을, 인체의 체외 장 모델로 제작된 3차원의 인간 장 오가노이드에 처리하였을 때, 이의 발아 구조의 형성을 촉진하고 표면적으로 향상시킬 뿐만 아니라(도 4 및 도 5 참고), 장관의 성숙과 관련 마커 유전자, 단백질의 발현을 증가시키는 것을 확인하였다(도 6 및 도 7 참고).In a specific embodiment of the present invention, when a culture solution containing the novel Lactobacillus gasseri strain of the present invention was treated on a three-dimensional human intestinal organoid produced as an in vitro human intestinal model, it was confirmed that not only the formation of germination structures was promoted and the surface area was improved (see FIGS. 4 and 5), but also the expression of marker genes and proteins related to intestinal maturation was increased (see FIGS. 6 and 7).
상기와 같은 결과로부터, 본 발명의 신규 락토바실러스 가세리 균주는 장의 성숙 또는 발달을 촉진하는 활성을 갖는 신규한 균주임을 알 수 있고, 이에 따라 본 발명의 상기 균주를 장 발달 또는 장 성숙 촉진 용도, 장관 발달 또는 성숙 장애의 예방, 개선 또는 치료를 위한 용도에 효과적으로 사용할 수 있다.From the above results, it can be seen that the novel Lactobacillus gasseri strain of the present invention is a novel strain having an activity of promoting intestinal maturation or development, and thus the strain of the present invention can be effectively used for purposes of promoting intestinal development or intestinal maturation, or for purposes of preventing, improving, or treating intestinal development or maturation disorders.
3. 3. 본 발명의 신규 락토바실러스 속 균주를 포함하는 조성물Composition comprising a novel Lactobacillus strain of the present invention
본 발명의 다른 측면은 상술한 본 발명의 신규 락토바실러스 파라카제이 균주, 신규 락토바실러스 가세리 균주, 상기 락토바실러스 파라카제이 균주 또는 상기 락토바실러스 가세리 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 포함하는 조성물을 제공한다.Another aspect of the present invention provides a composition comprising, as an effective ingredient, at least one selected from the group consisting of the novel Lactobacillus paracasei strain of the present invention, the novel Lactobacillus gasseri strain, a culture solution of the Lactobacillus paracasei strain or the Lactobacillus gasseri strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
상기 배양액은 상기 본 발명의 신규 락토바실러스 파라카제이 또는 상기 락토바실러스 가세리 균주를 배양하여 수득되는 것으로, 상기 균주의 세포를 포함하는 배양액 자체이거나, 또는 이로부터 세포를 제거하여 수득된 배양 상층액일 수 있으며, 또한 이들의 여과물, 농축액 또는 건조물일 수 있다. 상기 세포가 제거된 배양액은 본 발명의 신규 균주에 의해 생산, 분비되는 성분, 예컨대 대사산물을 포함하며 이에 따라 장 발달 또는 성숙 촉진, 장관 발달 또는 성숙 장애의 예방 또는 치료에 대한 활성이 우수한 프로바이오틱(probiotic) 효과를 갖는 것일 수 있다.The above culture solution is obtained by culturing the novel Lactobacillus paracasei or Lactobacillus gasseri strain of the present invention, and may be the culture solution itself containing cells of the strain, or a culture supernatant obtained by removing cells therefrom, and may also be a filtrate, concentrate or dried product thereof. The culture solution from which the cells have been removed contains components produced or secreted by the novel strain of the present invention, such as metabolites, and thus may have an excellent probiotic effect active in promoting intestinal development or maturation, or preventing or treating intestinal development or maturation disorders.
상기 농축액은 상기 배양액의 고형분 농도를 높이는 것으로, 상기 본 발명의 신규 락토바실러스 파라카제이 또는 상기 락토바실러스 가세리 균주의 세포를 포함하는 배양액의 농축액이거나 상기 본 발명의 신규 락토바실러스 파라카제이 또는 상기 락토바실러스 가세리 균주의 세포를 제거한 배양 상층액의 농축액일 수 있다. 상기 농축액은 진공농축, 판형농축, 박막농축 등에 의해 농축된 것일 수 있으나 이에 제한되지 않으며, 예컨대 공지의 농축기를 이용하여 40 ℃ 내지 60 ℃의 온도에서 수행할 수 있다. 상기 농축액의 농도에 따라 본 발명의 조성물에 포함되는 배양액의 함량을 적절히 조절할 수 있다.The above concentrate increases the solid concentration of the culture solution, and may be a concentrate of a culture solution containing cells of the novel Lactobacillus paracasei or the Lactobacillus gasseri strain of the present invention, or a concentrate of a culture supernatant from which cells of the novel Lactobacillus paracasei or the Lactobacillus gasseri strain of the present invention have been removed. The concentrate may be concentrated by, but is not limited to, vacuum concentration, plate concentration, thin film concentration, etc., and may be performed at a temperature of 40° C. to 60° C., for example, using a known concentrator. The content of the culture solution included in the composition of the present invention can be appropriately adjusted depending on the concentration of the concentrate.
상기 건조물은 동결건조, 진공건조, 열풍건조, 분무건조, 감압건조, 분무건조, 포말건조, 고주파건조, 적외선건조 등의 방법을 통해 건조된 것을 포함하나 이에 제한되지 않는다.The above dried product includes, but is not limited to, a product dried by a method such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high-frequency drying, or infrared drying.
상기 추출물은 상기 배양액 또는 그의 농축액으로부터 추출한 것을 의미하며, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 포함할 수 있다.The above extract means an extract obtained from the culture solution or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a adjusted or purified product thereof, or a fraction obtained by fractionating the same.
또한, 상기 조성물은 상기 본 발명의 신규 락토바실러스 파라카제이 및/또는 상기 락토바실러스 가세리 균주와 함께 섭취하기에 적합하고, 섭취시 장의 발달이나 성숙을 촉진하는 활성을 가지는 다른 종류의 공지된 성분이나 유산균을 추가로 포함할 수 있다.In addition, the composition may additionally contain other known ingredients or lactic acid bacteria that are suitable for ingestion together with the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention and have an activity of promoting the development or maturation of the intestine when ingested.
상기 본 발명의 신규 락토바실러스 파라카제이 및/또는 상기 락토바실러스 가세리 균주를 포함하는 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 담체, 부형제 및/또는 첨가제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때, 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제, 젤(예컨대, 하이드로젤) 또는 동결건조제의 형태일 수도 있으며, 상기 첨가제로 분산제, 안정화제 또는 동결 보호제를 추가적으로 포함할 수 있다. The composition comprising the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention can be manufactured in the form of a unit dosage or by being placed in a multi-dose container by formulating the composition using a carrier, an excipient and/or an additive according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and can be manufactured. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, gels (e.g., hydrogels) or lyophilizer, and may additionally include a dispersant, a stabilizer or a cryoprotectant as the additives.
구체적으로, 상기 동결건조제의 경우, 상기 균주를 동결 보호제와 함께 동결건조하여 분말의 형태로 사용하는 것을 포함하며, 상기 동결 보호제는 탈지분유, 말토덱스트린, 덱스트린, 트레할로스, 말토오스, 유당, 만니톨, 사이클로덱스트린, 글리세롤 및/또는 꿀일 수 있다. 또한, 보존 담체와 혼합하여 흡착시킨 후 건조시켜 고체화하여 사용하는 것을 포함하며, 상기 보존 담체는 규조토, 활성탄 및/또는 탈지강일 수 있다.Specifically, in the case of the freeze-drying agent, it includes using the strain in the form of a powder by freeze-drying it together with a cryoprotectant, and the cryoprotectant may be skimmed milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol and/or honey. In addition, it includes using it by mixing it with a preservation carrier, adsorbing it, drying it and solidifying it, and the preservation carrier may be diatomaceous earth, activated carbon and/or defatted steel.
상기 본 발명의 신규 락토바실러스 파라카제이 및/또는 상기 락토바실러스 가세리 균주를 포함하는 조성물은 균주, 상기 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택된 1종 이상을 상기 담체, 부형제 또는 첨가제 중 어느 하나와 혼합하는 단계를 거쳐 제조될 수 있다.The composition comprising the novel Lactobacillus paracasei and/or Lactobacillus gasseri strain of the present invention can be manufactured through a step of mixing at least one selected from the group consisting of a strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution with any one of the carrier, excipient, or additive.
상기 균주, 담체, 부형제 및 첨가제에 대한 설명은 전술한 바와 같다. 상기 첨가제로 동결 보호제를 이용하는 경우, 상기 본 발명의 신규 락토바실러스 파라카제이 균주와 동결 보호제를 혼합하고, 상기 혼합물을 섭씨 -45도 내지 섭씨 -30도에서 동결하는 과정을 거친 후, 섭씨 30도 내지 섭씨 40도에서 건조하여 믹서기로 갈아 동결건조된 분말 형태로 제조되는 것일 수 있다. 구체적으로, 상기 동결하는 과정은 섭씨 -45도 내지 섭씨 -30도의 온도 조건, 5 내지 50 mTorr의 압력 조건에서 65 내지 75 시간 동안 진공동결하는 과정일 수 있다.The description of the strain, carrier, excipient, and additive is as described above. When using a cryoprotectant as the additive, the novel Lactobacillus paracasei strain of the present invention and the cryoprotectant may be mixed, the mixture may be frozen at -45 degrees Celsius to -30 degrees Celsius, dried at 30 degrees Celsius to 40 degrees Celsius, ground in a mixer, and manufactured in the form of a freeze-dried powder. Specifically, the freezing process may be a vacuum freezing process under temperature conditions of -45 degrees Celsius to -30 degrees Celsius and pressure conditions of 5 to 50 mTorr for 65 to 75 hours.
본 발명의 조성물에 동결보호제가 더 포함될 경우, 상기 조성물이 동결 건조되는 과정에서 상기 균주의 손상이나 사멸이 억제될 수 있으며, 동결건조된 형태의 조성물은 보관, 유통, 저장 과정에서 유리한 장점이 있다. 또한, 상기 동결건조된 형태의 조성물은 분말의 형태로 섭취될 수 있으며, 이 경우 체내에서 상기 조성물 내 균주가 성장 또는 대사하면서 이의 활성을 나타낼 수 있다.When a cryoprotectant is further included in the composition of the present invention, damage or death of the strain can be suppressed during the freeze-drying process of the composition, and the composition in the freeze-dried form has advantageous advantages during the storage, distribution, and preservation processes. In addition, the composition in the freeze-dried form can be ingested in the form of a powder, and in this case, the strain in the composition can exhibit its activity while growing or metabolizing in the body.
4. 4. 본 발명의 조성물의 용도Uses of the composition of the present invention
본 발명의 또 다른 측면은 상기 본 발명의 신규 락토바실러스 파라카제이 또는 상기 락토바실러스 가세리 균주를 포함하는 조성물의, 장의 발달 또는 성숙을 촉진하거나, 장관 발달 또는 성숙 장애를 예방 또는 치료하기 위한 용도를 제공한다.Another aspect of the present invention provides a use of a composition comprising the novel Lactobacillus paracasei or Lactobacillus gasseri strain of the present invention for promoting intestinal development or maturation, or preventing or treating intestinal development or maturation disorders.
특히, 상기 조성물은 식품, 사료 또는 의약품일 수 있다. 상기 조성물이 식품일 경우 장의 발달 또는 성숙 촉진용 건강기능식품 조성물이거나 또는 일반 식품 조성물일 수 있고, 상기 조성물이 사료일 경우 장의 발달 또는 성숙 촉진용 사료 조성물 또는 사료 첨가제 조성물일 수 있으며, 상기 조성물이 의약품일 경우 장관 발달 또는 성숙 장애의 예방 또는 치료용 약학적 조성물일 수 있다.In particular, the composition may be a food, a feed, or a pharmaceutical. If the composition is a food, it may be a health functional food composition for promoting intestinal development or maturation or a general food composition, if the composition is a feed, it may be a feed composition for promoting intestinal development or maturation or a feed additive composition, and if the composition is a pharmaceutical, it may be a pharmaceutical composition for preventing or treating intestinal development or maturation disorders.
본 발명의 일 구현예에서, 상기 조성물이 장의 발달 또는 성숙 촉진용 건강기능식품 조성물 또는 일반 식품 조성물인 경우, 상기 건강기능식품 조성물 또는 식품 조성물은 세포에서 CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, MUC13 등과 같은 유전자의 발현을 향상시키거나, OLFM4, DEFA5, KRT20, MUC13등과 같은 단백질의 발현을 증가시키거나, 장의 표면적을 증가시키거나, 소장의 융모(villus)의 길이나 표면적을 증가시키거나, 선와(crypt)의 깊이를 증가시키는 것이거나, 대장의 선와의 깊이를 증가시키거나 점막/점막하층(mucosa/submucosa)의 비율을 증가시킬 수 있다. 구체적으로, 상기 조성물이 본 발명의 신규 락토바실러스 파라카제이 균주, 상기 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 포함하는 경우에는, 장 세포에서 CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1 및 MUC13으로 구성되는 군에서 선택되는 적어도 하나의 유전자의 발현을 향상시키는 것일 수 있다. 상기 조성물이 본 발명의 신규 락토바실러스 가세리 균주, 상기 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 포함하는 경우에는, 장 세포에서 CDX2, LYZ, CREB3L3, DPP4, SLC5A1 및 MUC13으로 구성되는 군에서 선택되는 적어도 하나의 유전자의 발현을 향상시키는 것일 수 있다.In one embodiment of the present invention, when the composition is a health functional food composition or a general food composition for promoting intestinal development or maturation, the health functional food composition or the food composition can enhance the expression of genes such as CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, MUC13, etc. in a cell, increase the expression of proteins such as OLFM4, DEFA5, KRT20, MUC13 , etc., increase the surface area of the intestine, increase the length or surface area of villi of the small intestine, increase the depth of crypts, increase the depth of crypts of the large intestine, or increase the ratio of mucosa/submucosa. Specifically, when the composition comprises at least one selected from the group consisting of the novel Lactobacillus paracasei strain of the present invention, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution as an effective ingredient, it may enhance the expression of at least one gene selected from the group consisting of CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, and MUC13 in intestinal cells. When the composition comprises at least one selected from the group consisting of the novel Lactobacillus gasseri strain of the present invention, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution as an effective ingredient, it may enhance the expression of at least one gene selected from the group consisting of CDX2, LYZ, CREB3L3, DPP4, SLC5A1, and MUC13 in intestinal cells.
본 발명의 건강기능식품 조성물을 식품첨가물로 사용하는 경우, 상기 건강기능식품 조성물을 식품에 그대로 첨가하거나 다른 식품 또는 식품성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 양은 그의 사용 목적(예방 또는 개선)에 따라 적절하게 사용될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 건강기능식품 조성물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강을 목적으로 장기간 섭취하는 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로 사용될 수도 있다.When the health functional food composition of the present invention is used as a food additive, the health functional food composition can be added to food as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the effective ingredient can be used appropriately depending on its intended use (prevention or improvement). Generally, when manufacturing 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 case of long-term intake for health purposes, the amount can be less than the above range, and since there is no problem in terms of safety, the effective ingredient can be used in an amount greater than the above range.
상기 건강기능식품이나 식품의 종류에 특별한 제한은 없다. 상기 건강기능식품이나 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차 드링크제, 알콜 음료 및 비타민 복합제, 유제품, 발효유 등이 있으며, 통상적인 의미에서의 건강기능식품이나 식품을 모두 포함한다.There are no special restrictions on the types of the above health functional foods or foods. Examples of the above health functional foods or foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, dairy products, fermented milk, etc., and include all health functional foods or foods in the conventional sense.
특히, 상기 건강기능식품은 영양 공급 이외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품으로, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 수 있다. 상기 건강기능식품은 본 발명의 기술분야에서 통상적으로 사용되는 방법에 의하여 제조할 수 있으며, 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 상기 건강기능식품의 제형도 건강기능식품으로 인정되는 제형이면 제한 없이 다양한 형태의 제형으로 제조될 수 있으며, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고 휴대성이 뛰어난 장점이 있다.In particular, the above health functional food is a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The above health functional food can be manufactured by a method commonly used in the technical field of the present invention, and can be manufactured by adding raw materials and ingredients commonly added in the industry. In addition, the formulation of the above health functional food can be manufactured in various forms without limitation as long as it is a formulation recognized as a health functional food, and unlike general drugs, it has the advantage of having no side effects that may occur when taking drugs for a long period of time since it uses food as a raw material, and has the advantage of excellent portability.
본 발명의 건강기능식품은 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소 및 조미제를 포함한다. 예컨대, 드링크제로 제조되는 경우에는 유효성분 이외에 천연 탄수화물 또는 향미제를 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 모노사카라이드(예컨대, 글루코오스, 프럭토오스 등), 디사카라이드(예컨대, 말토스, 수크로오스 등), 올리고당, 폴리사카라이드(예컨대, 덱스트린, 시클로덱스트린 등) 또는 당알코올(예컨대, 자일리톨, 소르비톨, 에리쓰리톨 등)인 것이 바람직하다. 상기 향미제는 천연 향미제(예컨대, 타우마틴, 스테비아 추출물 등)와 합성 향미제(예컨대, 사카린, 아스파르탐 등)를 이용할 수 있다.The health functional food of the present invention includes ingredients that are usually added during food manufacturing, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the effective ingredients. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).
상기 건강기능식품 조성물 이외에 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 더 함유할 수 있다. 이러한 상기 첨가되는 성분의 비율은 크게 중요하진 않지만 본 발명의 건강기능식품 조성물 100 중량부에 대하여, 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of these added components is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.
또한, 본 발명의 다른 구현 예에서, 상기 조성물이 장의 발달 또는 성숙 촉진용 사료 조성물 또는 사료 첨가제 조성물인 경우, 상기 사료 조성물 또는 사료 첨가제 조성물은 장의 발달 또는 성숙의 촉진을 목적으로 동물의 식이에 이용될 수 있다. 본 발명의 사료첨가제는 사료관리법상의 보조사료에 해당한다.In addition, in another embodiment of the present invention, when the composition is a feed composition or feed additive composition for promoting intestinal development or maturation, the feed composition or feed additive composition can be used in the diet of animals for the purpose of promoting intestinal development or maturation. The feed additive of the present invention corresponds to auxiliary feed under the Feed Management Act.
본 발명에서 용어 "사료"는 동물이 먹고, 섭취하며, 소화시키기 위한 또는 이에 적당한 임의의 천연 또는 인공 규정식, 한끼식 등 또는 상기 한끼식의 성분을 의미할 수 있다. 상기 사료의 종류는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 사료를 사용할 수 있다. 상기 사료의 비제한적인 예로는, 곡물류, 근과류, 식품 가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류 또는 곡물 부산물류 등과 같은 식물성 사료; 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질류, 동물성 플랑크톤류 또는 음식물 등과 같은 동물성 사료를 들 수 있다. 이들은 단독으로 사용되거나 2종 이상을 혼합하여 사용될 수 있다.The term "feed" in the present invention may mean any natural or artificial diet, meal, etc., or a component of said meal, which is suitable for an animal to eat, ingest, or digest. The type of said feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; animal-based feed such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.
본 발명의 또 다른 구현예에서, 상기 조성물이 장관 발달 또는 성숙 장애의 예방 또는 치료용 약학적 조성물인 경우, 상기 장관 발달 또는 성숙 장애는, 발달, 성숙, 분화, 또는 성장이 완료된 인간 또는 인간을 제외한 동물의 장관과 비교할 때, 장관의 발달의 정도가 부족하여 장관이 정상적인 수준의 기능을 하지 못하는 상태일 수 있으며, 이는 아직 발달기 또는 성장기에 있는 태아, 신생아, 영유아 등의 장관 상태를 포함한다. 또한, 상기 장관 발달 장애는, 장관의 발달이 진행되고 있는 발달기 또는 성장기의 동일한 시기를 기준으로 하여, 평균적인 장관의 발달 수준보다 발달의 정도가 부족한 상태일 수 있다. 상기 장관 발달 장애는 장관의 발달의 정도가 부족함으로 인해 발생하는 질환 또는 이와 관련된 위장 질환을 모두 포함한다. 상기 장관 발달 장애는 구체적으로, 장애 증후군(흡수장애 증후군), 염증성 장질환(크론병, 궤양성 대장염 등), 과민성 대장 증후군, 단장 증후군(short bowel syndrome, SBS), 괴사성 장염(necrotizing enterocolitis, NEC), 방사선 직장염 또는 미성숙 장을 가진 조산아, 미숙아 및 영유아일 수 있으나, 이에 제한되지 않는다.In another embodiment of the present invention, when the composition is a pharmaceutical composition for preventing or treating an intestinal development or maturation disorder, the intestinal development or maturation disorder may be a state in which the intestinal development is insufficient compared to the intestinal tract of a human or non-human animal that has completed development, maturation, differentiation, or growth, and the intestinal tract may not function at a normal level, and this includes the state of the intestinal tract of a fetus, newborn, infant, etc. that is still in the developmental or growth period. In addition, the intestinal development disorder may be a state in which the degree of development is insufficient compared to the average development level of the intestinal tract based on the same period of the developmental or growth period in which the development of the intestinal tract is in progress. The intestinal development disorder includes all diseases caused by the insufficient degree of development of the intestinal tract or gastrointestinal diseases related thereto. The above intestinal developmental disorders may specifically include, but are not limited to, malabsorption syndrome (malabsorption syndrome), inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), irritable bowel syndrome, short bowel syndrome (SBS), necrotizing enterocolitis (NEC), radiation proctitis, or premature infants, preterm infants, and infants with immature intestines.
또한, 상기 예방은 상기 장관의 발달 또는 성숙 장애로 인한 증상을 차단하거나, 그 증상을 억제 또는 지연시키는 모든 행위를 의미하는 것일 수 있다. 또한, 상기 치료는 장관의 발달 또는 성숙 장애로 인한 증상이 호전되거나 이롭게 되는 모든 행위를 의미하는 것일 수 있다.In addition, the above prevention may mean any action that blocks, suppresses or delays symptoms caused by developmental or maturational disorders of the above intestines. In addition, the above treatment may mean any action that improves or benefits symptoms caused by developmental or maturational disorders of the intestines.
한편, 상기 약학적 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때, 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제, 캅셀제 또는 젤(예컨대, 하이드로젤) 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. Meanwhile, the pharmaceutical composition may be manufactured in a unit dosage form or may be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and thereby manufactured. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or stabilizer.
또한, 상기 약학적 조성물이 포함하는 상기 균주는 콜로이드 현탁액, 분말, 식염수, 지질, 리포좀, 미소구체(microspheres), 또는 나노 구형입자와 같은 약학적으로 허용될 수 있는 담체에 운반될 수 있다. 이들은 운반 수단과 복합체를 형성하거나 관련될 수 있고, 지질, 리포좀, 미세입자, 금, 나노입자, 폴리머, 축합 반응제, 다당류, 폴리아미노산, 덴드리머, 사포닌, 흡착 증진 물질 또는 지방산과 같은 당업계에 공지된 운반 시스템을 사용하여 생체 내 운반될 수 있다.In addition, the strain contained in the pharmaceutical composition may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nano-spheres. They may form a complex with or be associated with a carrier, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.
이 외에도, 약학적으로 허용되는 담체는 제제시 통상적으로 이용되는 락토스, 덱스트로스, 수크로스, 소르비톨, 만니톨, 전분, 아카시아, 고무, 인산칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세 결정성 셀룰로스, 폴리비닐 피로리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. 또한, 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 레밍턴의 약학적 과학(Remington's Pharmaceutical Sciences, 19th ed., 1995)에 상세히 기재되어 있다.In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included in addition to the above ingredients. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명에 따른 약학적 조성물은 임상 투여시에 경구 또는 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다. 즉, 본 발명의 약학적 조성물은 실제 임상 투여시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 생약 추출물 또는 생약 발효물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention 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 dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing a herbal extract or a fermented herbal product with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.
본 발명의 약학적 조성물은 장관 발달 또는 성숙 장애의 예방 및 치료를 위하여 단독으로, 또는 수술, 방사선치료, 호르몬치료, 화학치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of intestinal development or maturation disorders.
본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며 특정 범위의 농도로 한정되지 않는다. 본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서 '약학으로 유효한 양'은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나, 장관 발달 또는 성숙 장애의 예방 및 치료를 위한 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 동시에, 별도로, 또는 순차적으로 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The concentration of the effective ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentrations. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent, or can be administered in combination with other therapeutic agents for the prevention and treatment of intestinal development or maturation disorders, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.
구체적으로 본 발명의 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성율, 배설 속도, 질병 종류, 병용되는 약물에 따라 달라질 수 있으며, 투여 경로, 장관 발달 또는 성숙 장애의 중증도, 성별, 체중, 연령 등에 따라 증감될 수 있다.Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of intestinal development or maturation disorder, sex, weight, age, etc.
본 발명의 또 다른 측면은, 상기 약학적 조성물을 대상에게 투여하는 단계를 포함하는 장관 발달 또는 성숙 장애의 예방 또는 치료 방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating a disorder of intestinal development or maturation, comprising administering to a subject the pharmaceutical composition.
상기 대상은 인간 또는 인간을 제외한 동물일 수 있으며, 발달이 완료된 인간 또는 인간을 제외한 동물의 장관보다 발달의 정도가 부족하거나, 발생기, 성장기에 있는 대상일 수 있다. 또한, 상기 대상은 장관의 발달이 진행되고 있는 발달기 또는 성장기의 동일한 시기를 기준으로 하여, 평균적인 장관의 발달 수준보다 발달의 정도가 부족한 인간 또는 인간을 제외한 동물일 수 있다.The subject may be a human or a non-human animal, and may be a subject whose level of development is less than that of a fully developed human or non-human animal, or who is in the developmental or growth stage. In addition, the subject may be a human or non-human animal whose level of development is less than that of an average level of development of an intestinal tract based on the same period of development or growth in which development of the intestinal tract is in progress.
상기 약학적 조성물의 제형, 투여 방법, 투여량 및 조성물에 함유되는 유효성분의 농도에 관한 설명은, 상기한 것과 동일하다.The description of the formulation, administration method, dosage, and concentration of the active ingredient contained in the composition of the above pharmaceutical composition is the same as described above.
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
단, 하기 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 하기 실시예에 의해 한정되지 아니한다.However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.
[실시예 1] [Example 1]
본 발명의 신규 락토바실러스 파라카제이 균주의 확립Establishment of a novel Lactobacillus paracasei strain of the present invention
[1-1] 본 발명의 락토바실러스 파라카제이 균주의 분리 및 동정[1-1] Isolation and identification of the Lactobacillus paracasei strain of the present invention
신생아 분변으로부터 #143 균주를 분리하였다. 상기와 같이 분리된 #143 균주에 대하여, 유니버셜 프라이머인 27F (5'-AGAGTTTGATCMTGGCTCA-3': 서열번호 2)와 1492R (5'-TACGGYTACCTTGTTACGACTT-3': 서열번호 3)을 사용하여 PCR을 수행하여 상기 #143 균주의 16S rRNA의 염기서열을 분석하였다. Strain #143 was isolated from newborn feces. For the strain #143 isolated as described above, PCR was performed using universal primers 27F (5'-AGAGTTTGATCMTGGCTCA-3': SEQ ID NO: 2) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3': SEQ ID NO: 3) to analyze the base sequence of 16S rRNA of the strain #143.
그 결과, 상기 #143 균주의 16S rRNA 염기서열은 서열번호 1과 같았고, 이는 기존에 보고된 락토바실러스 파라카제이(Lactobacillus paracasei) 표준 균주와 100%의 상동성을 나타내는 것으로 확인되었다. 이때, 상기 표준 균주와의 서열 비교는 EZ BioCloud 서버(https:/eabiocloud.net/identify)에서 수행하였다.As a result, the 16S rRNA base sequence of the above #143 strain was the same as sequence number 1, which was confirmed to show 100% homology with the previously reported Lactobacillus paracasei standard strain. At this time, sequence comparison with the above standard strain was performed on the EZ BioCloud server (https:/eabiocloud.net/identify).
이에, 상기 #143 균주를 하기 표 1과 같이 명명하고, 2023년 3월 10일자로 한국생명공학연구원 생물자원센터(Korean Collection for Type Culture, KCTC)에 기탁하여 하기 표 1과 같은 기탁번호를 부여받았다. Accordingly, the above #143 strain was named as shown in Table 1 below and deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023, and was assigned the accession number as shown in Table 1 below.
균주Strain 명칭designation 기탁번호Accession number
#143#143 락토바실러스 파라카제이(Lactobacillus paracasei) DS2766 Lactobacillus paracasei DS2766 KCTC15344BPKCTC15344BP
상기와 같은 신규 균주를 MRS(Man, Rogosa, and Sharpe) 배지에 접종하고, 섭씨 37도의 혐기성 조건에서 24 내지 36시간 동안 배양하여 정지상(stationary phase)에 도달시킨 다음, 배양액을 3,000xg의 속도로 10분간 원심분리하고, 상등액을 수득하여, 저온살균을 위해 섭씨 65도에서 30분간 처리한 후, 0.22um 필터를 통과시킨 다음, -80℃에 보관하며 실험에 활용하였다.The above novel strains were inoculated onto MRS (Man, Rogosa, and Sharpe) medium and cultured under anaerobic conditions at 37°C for 24 to 36 hours to reach the stationary phase. The culture was centrifuged at 3,000xg for 10 minutes and the supernatant was collected, treated at 65°C for 30 minutes for low-temperature sterilization, passed through a 0.22 μm filter, and stored at -80°C for use in the experiment.
[1-2] 본 발명의 DS2766 균주의 전장 유전체 분석[1-2] Whole genome analysis of the DS2766 strain of the present invention
상기와 같이 분리된 본 발명의 신규 균주의 전장유전체를 분석하였다. 구체적으로, 전장 유전체 시퀀싱(complete whole genome sequencing, WGS) 및 드래프트 게놈(draft genome) 분석을 실시하였고, 유전체 분석결과를 활용하여 표준 균주와의 유전체 상동성 분석, 계통 유전체학적(phylogenomic) 특성 분석, 특정 유전자 및 대사경로 분석 등 균주의 특성(identity) 및 기능성을 조사하였다. 또한, 유전체 수준의 안정성 조사를 위하여 전장 유전체를 확보하고 코딩 서열(coding sequence, CDS)의 아미노산 서열을 추출한 다음, 유전체 내 예측된 단백질 암호화 유전자로부터 독성유전자 상동성을 탐색하여 독성유전자 보유 여부를 확인하였다. 상기 유전체 상동성 분석은 Antibiotic Resistance Genes DB, Comprehensive Antibiootic Resistance DB를 활용하여 검색하였다. 아울러, 본 발명의 균주의 기능성 유전자 탐색을 위하여 조립 유전체의 manual curation을 통하여 시퀀싱(sequencing) 결과에 대한 오류를 교정하였고, 상기 유전자의 구조 예측 및 기능성 유전자 표기(tagging)를 수행하였으며, 유전체 지도를 작성하였다. 구체적으로, GeneBank DB로부터 표준 균주 및 기능성 비교 균주를 선정하고, 유전체 서열로부터 CDS를 추출한 다음, All to All BLAST 및 Markov Cluster Algorithm (MCL)을 활용하여 예측된 단백질 서열의 이종상동성 유전자 클러스터링(orthologous gene clustering)을 수행하였다. 이후 상기 비교 대상 균주와 본 발명의 균주의 유전자를 비교 분석함으로써 본 발명의 균주의 유전적 특성을 분석하였다. The whole genome of the novel strain of the present invention separated as described above was analyzed. Specifically, complete whole genome sequencing (WGS) and draft genome analysis were performed, and the identity and functionality of the strain, such as genome homology analysis with a standard strain, phylogenomic characteristic analysis, and specific gene and metabolic pathway analysis, were investigated using the genome analysis results. In addition, in order to investigate the stability at the genome level, the whole genome was secured, the amino acid sequence of the coding sequence (CDS) was extracted, and then the homology of the toxic genes was searched from the predicted protein encoding genes in the genome to confirm whether the strain had the toxic genes. The genome homology analysis was searched using the Antibiotic Resistance Genes DB and the Comprehensive Antibiootic Resistance DB. In addition, in order to search for functional genes of the strain of the present invention, errors in the sequencing results were corrected through manual curation of the assembled genome, the structure prediction of the genes and functional gene tagging were performed, and a genome map was created. Specifically, a standard strain and a functional comparison strain were selected from GeneBank DB, CDSs were extracted from the genome sequences, and then orthologous gene clustering of the predicted protein sequences was performed using All to All BLAST and Markov Cluster Algorithm (MCL). Thereafter, the genetic characteristics of the strain of the present invention were analyzed by comparing and analyzing the genes of the strain to be compared with the strain of the present invention.
그 결과, 표 2에 기재된 바와 같이, 본 발명의 락토바실러스 파라카제이 균주는 전장 유전체가 3.07Mb이며, 내성 및 독성 유전자를 보유하지 않은 것으로 확인되어, 프로바이오틱스로서 활용가능한 점을 알 수 있었다.As a result, as described in Table 2, the Lactobacillus paracasei strain of the present invention was confirmed to have a full-length genome of 3.07 Mb and to not possess resistance or toxicity genes, indicating that it can be utilized as a probiotic.
균주 명Strain name 분리원Separation circle 전장유전체(Mb)Whole genome (Mb) 내성 및 독성 유전자Resistance and virulence genes
락토바실러스 파라카제이 DS2766Lactobacillus paracasei DS2766 분변Feces 3.073.07 없음doesn't exist
[실시예 2][Example 2]
신규 DS2766 균주의 특성 확인Characterization of the new DS2766 strain
상기 실시예 1에서 분리 및 동정한 신규 DS2766 균주가 프로바이오틱스로 활용 가능한 특성을 갖는지 확인하기 위하여, MRS 배지에서 당 이용능, 내산성, 내담즙성, 장 부착능 및 항생제 내성에 대하여 실험하였다.To confirm whether the novel DS2766 strain isolated and identified in Example 1 above has characteristics that can be utilized as a probiotic, tests were conducted on sugar utilization, acid resistance, bile resistance, intestinal adhesion, and antibiotic resistance in MRS medium.
[2-1] 당 이용능, 독성 물질 생산성, 젤라틴 분해능, β-글루쿠로니다아제의 생산성 및 용혈성 확인[2-1] Confirmation of sugar utilization, toxic substance productivity, gelatin decomposition, β-glucuronidase productivity and hemolysis
본 발명의 DS2766 균주의 당 이용능, 독성 물질 생산성, 젤라틴 분해능, β-글루쿠로니다아제(β-glucuronidase)의 생산성 및 용혈성을 확인하기 위해, MRS 한천 배지에 본 발명의 신규 균주를 도말하여 준비하였다. 상기 당 이용능, 독성 물질 생산성, 및 젤라틴 분해능 분석은 API 20A kit (Bio-Merieux, France)를 사용하여 제공된 사용 방법에 따라 균주 colony를 API 20A Medium에 3 McFarland 탁도 이상이 되도록 현탁 시킨 뒤 각 well에 접종하는 방식으로 실험을 진행하였다. 또한, 상기 β-글루쿠로니다아제는 대장암과의 연관성이 보고되어 있어, β-글루쿠로니다아제의 생산성을 API ZYM kit을 통해 분석하였다. 다음으로, 상기 용혈성 평가는 Sheep blood agar plate에서 α, β 및 γ 용혈성을 확인하였다.In order to confirm the sugar utilization, toxic substance productivity, gelatin decomposition ability, β-glucuronidase productivity and hemolysis of the DS2766 strain of the present invention, the novel strain of the present invention was spread on MRS agar medium and prepared. The sugar utilization, toxic substance productivity and gelatin decomposition ability analyses were conducted by suspending the strain colonies in API 20A Medium to a turbidity of 3 McFarland or higher and inoculating them into each well according to the provided instructions using the API 20A kit (Bio-Merieux, France). In addition, since the β-glucuronidase has been reported to be associated with colon cancer, the productivity of the β-glucuronidase was analyzed using the API ZYM kit. Next, the hemolysis evaluation confirmed α, β and γ hemolysis on a sheep blood agar plate.
본 실험에서 확인한 상기 당의 종류는 글루코오스(GLU), 만니톨(MAN), 락토오스(LAC), 수크로오스(SAC), 말토오스(MAL), 살리신(SAL), 자일로스(XYL), 아라비노스(ARA), 젤라틴(GEL), 에스쿨린(ESC), 글리세롤(GLY), 셀로비오스(CEL), 만노오스(MNE), 멜레지토오스(MLZ), 라피노오스(RAF), 소르비톨(SOR), 람노오스(RHA) 및 트레할로오스(TRE)이다. 본 실험에서 확인한 상기 독성 물질의 종류는 인돌(indol) 및 요소(urea)이다.The types of sugars confirmed in this experiment are glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SAC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), and trehalose (TRE). The types of toxic substances confirmed in this experiment are indole and urea.
준비한 균액을 당 튜브에 접종하고 24시간 배양한 후, BCP 시약을 모든 당 튜브(URE, GEL, ESC 제외)에 한 방울 넣고 리딩 테이블에 따라 발생된 반응 결과를 판독하였다. 각 반응에 대해 양성이면 +, 음성이면 -로 그 결과를 표 3에 나타내었다.The prepared fungal solution was inoculated into the sugar tube and cultured for 24 hours. Then, one drop of BCP reagent was added to all sugar tubes (except URE, GEL, and ESC) and the reaction results were read according to the reading table. For each reaction, + was indicated if it was positive and - was indicated if it was negative. The results are shown in Table 3.
DS2766DS2766 DS2766DS2766
D-glucoseD-glucose ++ D-mannoseD-mannose ++
D-mannitolD-mannitol ++ D-melezitoseD-melezitose ++
D-lactoseD-lactose ++ D-raffinoseD-raffinose ++
sucrosesucrose ++ D-sorbitolD-sorbitol ++
D-maltoseD-maltose ++ D-rhamnoseD-rhamnose ++
salicinsalicin ++ D-trehaloseD-trehalose ++
D-xyloseD-xylose ++ Indol 생산 Indol production --
L-arabinoseL-arabinose ++ Urea 생산Urea production --
gelatingelatin -- Gelatin 분해능Gelatin resolution --
esculinesculin ++ β-glucuronidaseβ-glucuronidase --
glycerolglycerol ++ HemolysisHemolysis --
D-cellobioseD-cellobiose ++
그 결과, 상기 표 3에 기재된 바와 같이, 본 발명의 신규 DS2766 균주에서는 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스의 대사가 이루어지는 것으로 확인되었다. 또한, 인돌 및 요소 등의 독성 물질을 생산하지 않고, β-글루쿠로니다아제를 생산하지 않는 것으로 나타났으며, 젤라틴에 대한 분해능이 없는 것으로 확인되었다. 아울러, 본 발명의 DS2766 균주는 용혈성을 나타내지 않는 것으로 관찰되었다. As a result, as described in Table 3 above, it was confirmed that the novel DS2766 strain of the present invention metabolizes glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose. In addition, it was confirmed that it does not produce toxic substances such as indole and urea, does not produce β-glucuronidase, and has no ability to decompose gelatin. In addition, it was observed that the DS2766 strain of the present invention does not exhibit hemolysis.
[2-2] 내산성 확인[2-2] Acid resistance check
상기 실시예 1에서 분리 및 동정한 신규 DS2766 균주를 MRS 배지에 접종하고, 섭씨 37도에서 전배양한 후, 상기 각각의 균주들의 농도를 OD600=1로 조정하였다. 그런 다음, pH 3.0으로 조정된 10mL의 MRS 배지에 상기 각각의 균주를 1%(v/v)의 농도로 접종하고, 3시간 동안 배양한 후 생존률을 확인하였다.The novel DS2766 strain isolated and identified in the above Example 1 was inoculated into MRS medium and pre-cultured at 37°C, and the concentration of each strain was adjusted to OD 600 = 1. Then, each strain was inoculated at a concentration of 1% (v/v) into 10 mL of MRS medium adjusted to pH 3.0, and the survival rate was confirmed after culturing for 3 hours.
균주Strain 0h의 총 총균수(CFU)Total number of colonies forming units (CFU) at 0h 3h의 총 균수 (CFU)Total bacterial count (CFU) at 3h 생존율(%)Survival rate (%)
락토바실러스 파라카제이 DS2766Lactobacillus paracasei DS2766 2.9 x 107 CFU2.9 x 10 7 CFU 3.5 x 107 CFU3.5 x 10 7 CFU 121121
그 결과, 상기 표 4에 기재된 바와 같이, 본 발명의 상기 신규 DS2766 균주는 기본적으로 산성 환경에 대한 내성을 가지고 있는 것으로 확인되었다.As a result, as described in Table 4 above, it was confirmed that the novel DS2766 strain of the present invention is basically resistant to an acidic environment.
[2-3] 내담즙성 확인[2-3] Confirmation of biliary tract
상기 실시예 [2-1]에서 전배양된 균주를, 3%(w/v)의 담즙염(OxoidTM)이 포함된 10mL의 MRS 배지에, 106CFU/mL의 농도로 접종하고, 12시간 동안 배양한 후 생존율을 확인하였다.The strain cultured in the above example [2-1] was inoculated into 10 mL of MRS medium containing 3% (w/v) of bile salt (Oxoid TM ) at a concentration of 10 6 CFU/mL, and the survival rate was confirmed after culturing for 12 hours.
균주Strain 0h의 총 총균수(CFU)Total number of colonies forming units (CFU) at 0h 12h의 총 균수 (CFU)Total bacterial count (CFU) at 12h 생존율(%)Survival rate (%)
락토바실러스 파라카제이 DS2766Lactobacillus paracasei DS2766 3.9 x 105 CFU3.9 x 10 5 CFU NCNC >200>200
NC=Not countable.NC=Not countable.
그 결과, 상기 표 5에 기재된 바와 같이, 본 발명의 신규 DS2766 균주는 담즙이 존재하는 환경에 대한 내성을 가지고 있는 것으로 확인되었다.As a result, as described in Table 5 above, the novel DS2766 strain of the present invention was confirmed to have resistance to an environment containing bile.
[2-4] 항생제 내성 확인[2-4] Antibiotic resistance confirmation
본 발명의 DS2766 균주의 항생제 내성을 확인하기 위해, MRS 한천 배지에 DS2766 균주를 도말하여 준비하였다. EFSA(European Food Safety Authority) 가이드에서 통용되는 항생제에 대하여 MTSTM(MIC Test Strip)(Liofilchem)을 이용하여 제조사의 지시에 따라 항생제 내성 실험을 실시하였다. To confirm the antibiotic resistance of the DS2766 strain of the present invention, the DS2766 strain was spread on MRS agar medium and prepared. An antibiotic resistance test was performed according to the manufacturer's instructions using MTS TM (MIC Test Strip) (Liofilchem) for antibiotics commonly used in the EFSA (European Food Safety Authority) guide.
본 실험에 사용한 항생제는 반코마이신, 에리트로마이신, 클린다마이신, 및 테트라사이클린이고, 항생제 스트립과 억제대가 만나는 지점의 농도를 최소억제농도로 하였다. The antibiotics used in this experiment were vancomycin, erythromycin, clindamycin, and tetracycline, and the concentration at the point where the antibiotic strip meets the inhibition zone was considered the minimum inhibitory concentration.
상기 준비한 배지에 본 발명의 DS2766 균주를 도말한 후, 항생제 스트립을 올리고, 다시 37℃에 48시간 배양하면서 생육이 저해되는 최소억제농도(MIC)를 측정하여 그 결과를 표 6에 나타내었다.After spreading the DS2766 strain of the present invention on the prepared medium, an antibiotic strip was placed, and the medium was cultured again at 37°C for 48 hours to measure the minimum inhibitory concentration (MIC) at which growth was inhibited. The results are shown in Table 6.
controlcontrol 앰피실린Ampicillin 젠타마이신Gentamicin 에리트로마이신Erythromycin 클린다마이신Clindamycin 테트라사이클린Tetracycline
EFSA guide
(μg/ml)EFSA guide
(μg/ml) 		44 3232 11 11 44
DS2766 (μg/ml)DS2766 (μg/ml) GrowthGrowth 0.380.38 3232 0.50.5 0.190.19 0.50.5
그 결과, 상기 표 6에 기재된 바와 같이, 본 발명의 균주는 앰피실린, 에리트로마이신, 클린다마이신, 및 테트라사이클린에 대해서 EFSA 가이드의 기준 수치보다 항생제 내성이 낮은 것으로 확인되었다. 따라서, 본 발명의 균주는 장내에 존재하는 유해 세균으로 내성 유전자를 수평 전달하지 않으므로, 장내 유해 세균이 외재 내성을 획득하여 항생제에 대한 내성 문제를 일으킬 염려가 없어 프로바이오틱스로서 상업화되기에도 유리한 장점이 있다. As a result, as described in Table 6 above, the strain of the present invention was confirmed to have lower antibiotic resistance than the standard value of the EFSA guide for ampicillin, erythromycin, clindamycin, and tetracycline. Therefore, since the strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire external resistance and cause resistance problems to antibiotics, and thus it has an advantage in that it is also advantageous for commercialization as a probiotic.
[2-5] 장 부착능 확인[2-5] Checking the attachment ability
본 발명의 DS2766 균주의 장 부착능을 평가하기 위하여, Caco-2 세포주를 이용하였다. 구체적으로, Caco-2 세포주를 10% FBS 및 1% 페니실린-스트렙토마이신이 함유된 MEM 배지를 사용하여 37°C의 온도로 5% CO2 인큐베이터(PHC, MCO-230AIC, Indonesia)에서 배양하였고, 1x105 cell/well의 농도로 24-well plate에 분주한 뒤 14일간 배양하였다. 그런 다음, 상기 배양된 세포를 MEM 배지로 현탁하여 최종 농도 1x108 CFU/ml가 되도록 조정하였고, 각 well당 1ml씩 접종한 뒤 2시간 동안 배양하였다. 이 때, 균주의 탄소원으로는 각각 포도당을 사용하였다. 이후, 배양된 균주를 PBS(Phosphate Buffered Saline) 용액으로 5회 세척하였고, 0.5% trypsin-EDTA를 처리하여 부착되지 않은 Caco-2 세포들을 플레이트에서 떼어내고, 떼어낸 세포들을 PBS로 희석하여 MRS 평판배지에 도말한 다음, 24시간 경과 후의 생균수를 측정함으로써 본 발명의 균주의 장 부착능을 평가하였다. In order to evaluate the intestinal adhesion ability of the DS2766 strain of the present invention, Caco-2 cell line was used. Specifically, Caco-2 cell line was cultured at 37°C in a 5% CO2 incubator (PHC, MCO-230AIC, Indonesia) using MEM medium containing 10% FBS and 1% penicillin-streptomycin, dispensed into a 24-well plate at a concentration of 1x105 cells/well, and cultured for 14 days. Then, the cultured cells were suspended in MEM medium to adjust the final concentration to 1x108 CFU/ml, inoculated into each well at 1ml, and cultured for 2 hours. At this time, glucose was used as the carbon source for each strain. Thereafter, the cultured strain was washed five times with PBS (Phosphate Buffered Saline) solution, treated with 0.5% trypsin-EDTA to detach non-attached Caco-2 cells from the plate, diluted the detached cells with PBS and plated on MRS plate medium, and then the number of viable cells was measured after 24 hours to evaluate the intestinal adhesion ability of the strain of the present invention.
그 결과, 표 7에 도시된 바와 같이, 본 발명의 DS2766 균주는 탄소원으로 포도당을 사용했을 때 약 2.54%의 장 부착율을 나타내어 장 부착능이 있는 것으로 확인되었다. As a result, as shown in Table 7, the DS2766 strain of the present invention was confirmed to have intestinal attachment ability, showing an intestinal attachment rate of about 2.54% when glucose was used as a carbon source.
균주 명Strain name 부착능(%)Adhesion (%) 표준편차(SD)Standard Deviation (SD)
락토바실러스 파라카제이 DS2766Lactobacillus paracasei DS2766 2.54*2.54* 0.3710.371
*p<0.5; **p<0.05; ***p<0.001*p<0.5; **p<0.05; ***p<0.001
[실시예 3] [Example 3]
본 발명의 신규 락토바실러스 파라카제이 균주의 장 발달/성숙 촉진 효과 확인Confirmation of the effect of promoting intestinal development/maturity of the novel Lactobacillus paracasei strain of the present invention
본 발명의 신규 락토바실러스 파라카제이 균주가 장의 발달 또는 성숙에 비치는 영향을 확인하기 위하여, 체외 장(intestine) 모델로 3차원의 인간 장 오가노이드(human intestinal organoids, hIOs)를 제조하고, 본 발명의 신규 락토바실러스 파라카제이 균주를 처리하여, 상기 장의 발달/성숙 정도를 평가하였다.In order to confirm the effect of the novel Lactobacillus paracasei strain of the present invention on the development or maturation of the intestine, three-dimensional human intestinal organoids (hIOs) were produced as an in vitro intestine model, and the novel Lactobacillus paracasei strain of the present invention was treated to evaluate the degree of development/maturity of the intestine.
[3-1] 3차원 hIOs의 제작[3-1] Fabrication of 3D hIOs
공지된 방법(Yoon et al., Molecular carcinogenesis, 55(4): 387-396 (2016), Son et al., Proteomics, 15(13): 2220-2229 (2015))으로 hPSC(human pluripotent stem cells)를 배양하고, 공지된 방법(Spence et al., Nature, 470(7332): 105-109 (2011))으로 3차원의 인간 장 오가노이드(human intestinal organoids, hIOs)를 제조하였다.Human pluripotent stem cells (hPSCs) were cultured using a known method (Yoon et al. , Molecular carcinogenesis, 55 (4): 387-396 (2016), Son et al. , Proteomics, 15 (13): 2220-2229 (2015)), and three-dimensional human intestinal organoids (hIOs) were produced using a known method (Spence et al. , Nature, 470 (7332): 105-109 (2011)).
구체적으로, hPSC(H9 (WA09); WiCell Research Institute)의 진정내배엽 유도를 위해, 100ng/mL의 액티빈 A(Activin A)(R&D Systems)과 0%, 0.2%, 2% 농도의 태아 소 혈청(FBS, Thermo Scientific)이 포함된 배지에서 상기 hPSC를 3일 동안 배양하였다. 그런 다음, 후장(hindgut) 스페로이드를 형성하기 위해, 500ng/mL의 FGF4, 3uM의 CHIR 99021 및 2% 농도의 태아 소 혈청이 포함된 분화배지를 이용하여 4일 동안 추가 배양하였다. 분화가 유도되어 형성된 스페로이드를 마트리젤 돔 내부에 삽입시켜, 3차원 배양 환경에서 1X B27 첨가제(B27 supplement, Invitrogen), 100ng/mL의 EGF(R&D Systems), 100ng/mL의 Noggin(R&D Systems) 및 500ng/mL의 R-스폰딘1(R-spondin1)(R&D Systems)이 포함된 장 오가노이드 분화 배지에서 총 14일간 배양하여, 인간 장 오가노이드를 제작하였다.Specifically, for true endoderm induction of hPSCs (H9 (WA09); WiCell Research Institute), the hPSCs were cultured in a medium containing 100 ng/mL of Activin A (R&D Systems) and 0%, 0.2%, and 2% fetal bovine serum (FBS, Thermo Scientific) for 3 days. Then, to form hindgut spheroids, they were additionally cultured for 4 days using differentiation medium containing 500 ng/mL of FGF4, 3 μM CHIR 99021, and 2% fetal bovine serum. The spheroids formed by inducing differentiation were inserted into the Matrigel dome and cultured in a 3D culture environment in intestinal organoid differentiation medium containing 1X B27 supplement (B27 supplement, Invitrogen), 100 ng/mL EGF (R&D Systems), 100 ng/mL Noggin (R&D Systems), and 500 ng/mL R-spondin1 (R&D Systems) for a total of 14 days to produce human intestinal organoids.
[3-2] 3차원 hIOs를 이용한, 장 발달/성숙 촉진 효과 확인[3-2] Confirmation of the effect of promoting intestinal development/maturity using 3D hIOs
상기 실시예 [3-1]에서 제작된 3차원 hIOs를 마트리젤로부터 분리한 후, 차가운 PBS로 세척하고 hIOs 주변의 마트리젤을 최대한 제거한 후 수술용 칼을 활용하여 물리적으로 해리하였다. 해리된 hIOs 조각은 새로운 4-웰 디쉬에 마트리젤 돔을 형성한 후 1개 웰당 5개씩 접종하여 hIO 분화 배지로 배양하였다.After separating the 3D hIOs produced in the above Example [3-1] from the Matrigel, they were washed with cold PBS, the Matrigel around the hIOs was removed as much as possible, and then physically dissociated using a surgical scalpel. The dissociated hIOs pieces were inoculated into a new 4-well dish to form a Matrigel dome, 5 pieces per well, and cultured with hIO differentiation medium.
상기와 같이 준비된 hIOs에 상기 실시예 1에서 분리 및 동정한 본 발명의 신규 락토바실러스 파라카제이 균주들의 배양액을 1%의 농도로 첨가하고, 총 20일 동안 2회 계대배양한 후, 상기 hIOs의 변화를 관찰하였다.The culture solution of the novel Lactobacillus paracasei strains of the present invention isolated and identified in Example 1 was added to the hIOs prepared as described above at a concentration of 1%, and subcultured twice for a total of 20 days, after which changes in the hIOs were observed.
[3-2-1] hIOs의 형태학적 변화 확인[3-2-1] Confirmation of morphological changes in hIOs
먼저, hIOs의 크기 변화와 발아(budding) 구조의 개수를 확인함으로써 hIOs의 형태학적 변화를 확인하였다. 구체적으로, 상기 신규 락토바실러스 파라카제이 균주들이 처리된 hIOs를 현미경으로 관찰하여 촬영한 사진을 통해, 본 발명의 신규 락토바실러스 파라카제이 균주를 처리한 hIOs에서 생성된 발아(budding) 구조의 개수와 상기 hIOs의 표면적 변화를 확인하였다.First, the morphological changes in hIOs were confirmed by checking the size changes in hIOs and the number of budding structures. Specifically, the number of budding structures generated in hIOs treated with the novel Lactobacillus paracasei strain of the present invention and the change in surface area of the hIOs were confirmed through photographs taken by observing hIOs treated with the novel Lactobacillus paracasei strain of the present invention under a microscope.
그 결과, 도 1 및 도 2에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 본 발명의 신규 락토바실러스 파라카제이 균주를 처리한 hIOs에서 발아 구조가 더욱 많이 형성되었을 뿐만 아니라(도 1), 그 표면적도 4배 이상 향상된 것으로 확인되었다(도 2).As a result, as shown in FIGS. 1 and 2, compared to the case without any treatment, it was confirmed that not only were more germination structures formed in hIOs treated with the novel Lactobacillus paracasei strain of the present invention (FIG. 1), but also the surface area was increased by more than four times (FIG. 2).
[3-2-2] hIOs에서의 분자생물학적 변화 확인[3-2-2] Confirmation of molecular biological changes in hIOs
다음으로, 상기 hIOs에서 CDX2, OLFM2, LYZ, DEFA5, KRT20, CREB3L3, DPP4, LCT, SLC5A1, MUC13 등과 같은 장관의 성숙과 관련된 마커 유전자들의 발현량의 변화를 qRT-PCR을 통해 확인하였다. 구체적으로, 상기 신규 락토바실러스 파라카제이 균주들이 처리된 hIOs에서 RNeasy 키트(Qiagen)를 이용하여 제조사의 지시에 따라 전체 RNA를 추출하였고, Superscript IV cDNA synthesis system(Invitrogen)을 이용하여 제조사의 지시에 따라 상기 전체 RNA로부터 cDNA를 합성하였다. 상기와 같이 합성된 cDNA를 대상으로 하기 표 8에 기재된 프라이머 쌍을 이용하여 qRT-PCR을 수행하였다.Next, the changes in the expression levels of marker genes related to intestinal maturation, such as CDX2, OLFM2, LYZ, DEFA5, KRT20, CREB3L3, DPP4, LCT, SLC5A1, and MUC13, in the hIOs were confirmed through qRT-PCR. Specifically, total RNA was extracted from hIOs treated with the novel Lactobacillus paracasei strains using the RNeasy kit (Qiagen) according to the manufacturer's instructions, and cDNA was synthesized from the total RNA using the Superscript IV cDNA synthesis system (Invitrogen) according to the manufacturer's instructions. qRT-PCR was performed on the cDNA synthesized as described above using the primer pairs described in Table 8 below.
서열 이름Sequence name 서열 (5'-> 3')Sequence (5'-> 3') 서열번호Sequence number
GAPDH forward primerGAPDH forward primer GAAGGTGAAGGTCGGAGTCGAAGGTGAAGGTCGGAGTC 44
GAPDH reverse primerGAPDH reverse primer GAAGATGGTGATGGGATTTCGAAGATGGTGATGGGATTTC 55
CDX2 forward primerCDX2 forward primer CTGGAGCTGGAGAAGGAGTTTCCTGGAGCTGGAGAAGGAGTTTC 66
CDX2 reverse primerCDX2 reverse primer ATTTTAACCTGCCTCTCAGAGAGCATTTTAACCTGCCTCTCAGAGAGC 77
DPP4 forward primerDPP4 forward primer CAAATTGAAGCAGCCAGACACAAATTGAAGCAGCCAGACA 88
DPP4 reverse primerDPP4 reverse primer GGAGTTGGGAGACCCATGTAGGAGTTGGGAGACCCATGTA 99
OLFM4 forward primerOLFM4 forward primer ACCTTTCCCGTGGACAGAGTACCTTTCCCGTGGACAGAGT 1010
OLFM4 reverse primerOLFM4 reverse primer TGGACATATTCCCTCACTTTGGATGGACATATTCCCTCACTTTGGA 1111
DEFA5 forward primerDEFA5 forward primer CCTTTGCAGGAAATGGACTCCCTTTGCAGGAAATGGACTC 1212
DEFA5 reverse primerDEFA5 reverse primer GGACTCACGGGTAGCACAACGGACTCACGGGTAGCACAAC 1313
CREB3L3 forward primerCREB3L3 forward primer ATCTCCTGTTTGACCGGCAGATCTCCTGTTTGACCGGCAG 1414
CREB3L3 reverse primerCREB3L3 reverse primer GTCGTCAGAGTCGGGGTTTGGTCGTCAGAGTCGGGGTTTG 1515
KRT20 forward primerKRT20 forward primer TGGCCTACACAAGCATCTGGTGGCCTACACAAGCATCTGG 1616
KRT20 reverse primerKRT20 reverse primer TAACTGGCTGCTGTAACGGGTAACTGGCTGCTGTAACGGG 1717
LYZ forward primerLYZ forward primer AAAACCCCAGGAGCAGTTAATAAAACCCCAGGAGCAGTTAAT 1818
LYZ reverse primerLYZ reverse primer CAACCCTCTTTGCACAAGCTCAACCCTCTTTGCACAAGCT 1919
LCT forward primerLCT forward primer GGCAGTCTGGGAGTTTTAGGGGCAGTCTGGGAGTTTTAGG 2020
LCT reverse primerLCT reverse primer ATGCCAAAATGAGGCAAGTCATGCCAAAATGAGGCAAGTC 2121
SLC5A1 forward primerSLC5A1 forward primer GTGCAGTCAGCACAAAGTGGGTGCAGTCAGCACAAAGTGG 2222
SLC5A1 reverse primerSLC5A1 reverse primer ATGCACATCCGGAATGGGTTATGCACATCCGGAATGGGTT 2323
MUC13 forward primerMUC13 forward primer CGGATGACTGCCTCAATGGTCGGATGACTGCCTCAATGGT 2424
MUC13 reverse primerMUC13 reverse primer AAAGACGCTCCCTTCTGCTCAAAGACGCTCCCTTCTGCTC 2525
그 결과, 도 3에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 본 발명의 신규 락토바실러스 파라카제이 균주를 처리한 hIOs에서 CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1 및 MUC13 유전자의 발현이 증가된 것으로 확인되었다.As a result, as shown in Fig. 3, it was confirmed that the expression of CDX2, OLFM2, LYZ, KRT20, CREB3L3, SLC5A1, and MUC13 genes increased in hIOs treated with the novel Lactobacillus paracasei strain of the present invention, compared to the case without any treatment.
[3-2-3] hIOs에서의 조직학적 변화 확인[3-2-3] Confirmation of histological changes in hIOs
또한, 상기 hIOs에서, 성숙 장 줄기세포 마커인 OLFM4, 성숙 파네스 세포(Paneth cell) 마커인 DEFA5, 성숙 장관 구조 단백질 마커인 KRT20, 점액 생성 세포 마커인 MUC13 등과 같이, 성숙한 장에서 발현되어 나타나는 장관의 성숙과 관련된 마커 단백질의 발현 정도를 면역형광염색을 통해 확인하였다. 구체적으로, 상기 신규 락토바실러스 파라카제이 균주들이 처리된 hIOs를 4% PFA(paraformaldehyde)에 고정시킨 후 10-30%의 수크로오스 용액으로 동결 보호 처리하고 OCT 용액을 처리하여 동결시켰다. 동결된 hIOs 조직을 마이크로톰(microtome)으로 10-20um의 두께로 절단시켜 박편을 만들고, 0.1%의 트리톤 X-100이 포함된 PBS를 처리하여 박편을 투과시킨 다음 4%의 BSA(bovine serum albumin)을 포함하는 PBS로 1시간 동안 차단시켰다. 상기 4가지 표적 마커 단백질에 대한 1차 항체인 항-OLFM4 항체(ab85046, abcam, Cambridge, MA, USA), 항-DEFA5 항체(ab90802, abcam), 항-KRT20 항체(ab76126, abcam) 및 항-MUC13 항체(ab124654, abcam)를 각각 1:100으로 희석하여 사용하여 4 ℃에서 하룻밤 동안 반응시킨 다음, 2차 항체인 항-염소 항체(anti-goat IgG Alexa Fluor 488, A21467, Invitrogen), 항-토끼 항체(anti-rabbit IgG Alexa Fluor 594, A21442, Invitrogen), 항-쥐 항체(anti-mouse IgG Alexa Fluor 594, A21203, Invitrogen) 를 각각 1:200 희석하여 사용하여 상온에서 1시간 동안 반응시켜 DAPI 염색으로 핵을 염색한 다음, 형광현미경으로 관찰하였다.In addition, in the hIOs, the expression level of marker proteins related to intestinal maturation expressed in the mature intestine, such as 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, was confirmed through immunofluorescence staining. Specifically, the hIOs treated with the novel Lactobacillus paracasei strains were fixed in 4% PFA (paraformaldehyde), cryoprotected with a 10-30% sucrose solution, and frozen by treating with an OCT solution. The frozen hIOs tissues were cut into thin sections with a thickness of 10-20 um using a microtome, and the sections were permeabilized by treating with PBS containing 0.1% Triton X-100, and then blocked with PBS containing 4% BSA (bovine serum albumin) for 1 hour. The primary antibodies for the above four target marker proteins, anti-OLFM4 antibody (ab85046, abcam, Cambridge, MA, USA), anti-DEFA5 antibody (ab90802, abcam), anti-KRT20 antibody (ab76126, abcam), and anti-MUC13 antibody (ab124654, abcam), were used at a dilution of 1:100 each and reacted at 4°C overnight, and then the secondary antibodies, anti-goat IgG Alexa Fluor 488, A21467, Invitrogen), anti-rabbit IgG Alexa Fluor 594, A21442, Invitrogen, and anti-mouse IgG Alexa Fluor 594, A21203, Invitrogen, were used at a dilution of 1:200 each and reacted at room temperature for 1 hour to stain the nucleus with DAPI. After staining, it was observed under a fluorescence microscope.
그 결과, 도 4에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 장의 성숙 시에 발현이 증가하는 OFLM4, DEFA5, KRT20 및 MUC13 단백질 중, 본 발명의 신규 락토바실러스 파라카제이 균주를 처리한 hIOs에서만 OFLM4, DEFA5, KRT20 및 MUC13의 발현이 증가되어 염색되는 것으로 확인되었다.As a result, as shown in Fig. 4, compared to the case without any treatment, among the OFLM4, DEFA5, KRT20 and MUC13 proteins whose expression increases during intestinal maturation, it was confirmed that the expression of OFLM4, DEFA5, KRT20 and MUC13 was increased and stained only in hIOs treated with the novel Lactobacillus paracasei strain of the present invention.
상기와 같은 결과로부터 본 발명의 신규 락토바실러스 파라카제이 균주들의 처리에 의해 장의 성숙 또는 발달이 촉진됨을 알 수 있다.From the above results, it can be seen that intestinal maturation or development is promoted by treatment with the novel Lactobacillus paracasei strains of the present invention.
[제조예 1][Manufacturing Example 1]
신규 락토바실러스 파라카제이 균주들을 포함하는 조성물의 원말 제조Preparation of crude compositions containing novel Lactobacillus paracasei strains
본 발명의 신규 락토바실러스 파라카제이 균주를 포함하는 조성물의 원말을 제조하였다. A crude product of a composition comprising the novel Lactobacillus paracasei strain of the present invention was prepared.
상기 본 발명의 신규 락토바실러스 파라카제이 균주 각각을 최적화된 증균배지와 배양조건을 설정하여 배양하였다. 균체 회수 후 농축액 대비 동결보호제인 말토덱스트린 5-50(부피/중량)% 또는 트레할로스 5-50(부피/중량)% 또는 셀룰로오스 5-50(부피/중량)%를 첨가하여 동결건조 후 분쇄하여 유산균 원말을 제조하였다. Each of the novel Lactobacillus paracasei strains of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (volume/weight)% of maltodextrin, 5-50 (volume/weight)% of trehalose, or 5-50 (volume/weight)% of cellulose as a cryoprotectant was added to the concentrate, freeze-dried, and then ground to produce lactic acid bacteria powder.
[실시예 4] [Example 4]
본 발명의 신규 락토바실러스 가세리 균주의 확립Establishment of a novel Lactobacillus gasseri strain of the present invention
[4-1] 본 발명의 균주의 분리 및 동정[4-1] Isolation and identification of the strain of the present invention
생후 18개월의 유아 분변으로부터 #123 균주를 분리하였다. 상기와 같이 분리된 #123 균주에 대하여, 실시예 1과 동일한 방법으로 PCR을 수행하여 상기 #123 균주의 16S rRNA의 염기서열을 분석하였다. Strain #123 was isolated from the feces of an 18-month-old infant. For the strain #123 isolated as described above, PCR was performed using the same method as in Example 1 to analyze the base sequence of 16S rRNA of the strain #123.
그 결과, 상기 #123 균주의 16S rRNA 염기서열은 서열번호 26과 같았고, 이는 기존에 보고된 락토바실러스 가세리(Lactobacillus gasseri) 표준 균주와 99.86%의 상동성을 나타내는 것으로 확인되었다. 이때, 상기 표준 균주와의 서열 비교는 EZ BioCloud 서버(https:/eabiocloud.net/identify)에서 수행하였다.As a result, the 16S rRNA base sequence of the above #123 strain was the same as sequence number 26, which was confirmed to have 99.86% homology with the previously reported Lactobacillus gasseri standard strain. At this time, sequence comparison with the above standard strain was performed on the EZ BioCloud server (https:/eabiocloud.net/identify).
이에, 상기 #123 균주를 하기 표 9와 같이 명명하고, 2023년 3월 10일자로 한국생명공학연구원 생물자원센터(Korean Collection for Type Culture, KCTC)에 기탁하여 하기 표 9와 같은 기탁번호를 부여받았다. Accordingly, the above #123 strain was named as shown in Table 9 below and deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on March 10, 2023, and was assigned the accession number as shown in Table 9 below.
균주Strain 명칭designation 기탁번호Accession number
#123 #123 락토바실러스 가세리(Lactobacillus gasseri) DS2831 Lactobacillus gasseri DS2831 KCTC15345BPKCTC15345BP
상기와 같은 신규 DS2831 균주를 실시예 1과 동일하게 보관하며 실험에 활용하였다.The novel DS2831 strain described above was stored in the same manner as in Example 1 and used in the experiment.
[4-2] 본 발명의 균주의 전장 유전체 분석[4-2] Whole genome analysis of the strain of the present invention
실시예 [1-2]와 동일한 방법으로 본 발명의 DS2831 균주의 유전적 특성을 분석하였다. The genetic characteristics of the DS2831 strain of the present invention were analyzed using the same method as in Example [1-2].
그 결과, 표 10에 기재된 바와 같이, 본 발명의 락토바실러스 가세리 균주는 전장 유전체가 2.04Mb이며, 내성 및 독성 유전자를 보유하지 않은 것으로 확인되어, 프로바이오틱스로서 활용가능한 점을 알 수 있었다.As a result, as described in Table 10, the Lactobacillus gasseri strain of the present invention was confirmed to have a full-length genome of 2.04 Mb and to not possess resistance or toxicity genes, indicating that it can be utilized as a probiotic.
균주 명Strain name 분리원Separation circle 전장유전체(Mb)Whole genome (Mb) 내성 및 독성 유전자Resistance and virulence genes
락토바실러스 가세리 DS2831Lactobacillus gasseri DS2831 분변Feces 2.042.04 없음doesn't exist
[실시예 5][Example 5]
신규 DS2831 균주의 특성 확인Characterization of the new DS2831 strain
상기 실시예 5에서 분리 및 동정한 신규 DS2831 균주가 프로바이오틱스로 활용 가능한 특성을 갖는지 확인하기 위하여, MRS 배지에서 당 이용능, 내산성, 내담즙성, 장 부착능 및 항생제 내성에 대하여 실험하였다.To confirm whether the novel DS2831 strain isolated and identified in Example 5 above has characteristics that can be utilized as a probiotic, tests were conducted on sugar utilization, acid resistance, bile resistance, intestinal adhesion, and antibiotic resistance in MRS medium.
[5-1] 당 이용능, 독성 물질 생산성, 젤라틴 분해능, β-글루쿠로니다아제의 생산성 및 용혈성 확인[5-1] Confirmation of sugar utilization, toxic substance productivity, gelatin decomposition, β-glucuronidase productivity and hemolysis
본 발명의 DS2831 균주의 당 이용능, 독성 물질 생산성, 젤라틴 분해능, β-글루쿠로니다아제(β-glucuronidase)의 생산성 및 용혈성을 실시예 2-1과 동일한 방법으로 확인하였다.The sugar utilization ability, toxic substance productivity, gelatin decomposition ability, β-glucuronidase productivity, and hemolysis of the DS2831 strain of the present invention were confirmed by the same method as Example 2-1.
DS2831DS2831 DS2831DS2831
D-glucoseD-glucose ++ D-mannoseD-mannose ++
D-mannitolD-mannitol ++ D-melezitoseD-melezitose ++
D-lactoseD-lactose ++ D-raffinoseD-raffinose ++
sucrosesucrose ++ D-sorbitolD-sorbitol ++
D-maltoseD-maltose ++ D-rhamnoseD-rhamnose ++
salicinsalicin ++ D-trehaloseD-trehalose ++
D-xyloseD-xylose ++ Indol 생산 Indol production --
L-arabinoseL-arabinose ++ Urea 생산Urea production --
gelatingelatin -- Gelatin 분해능Gelatin resolution --
esculinesculin ++ β-glucuronidaseβ-glucuronidase --
glycerolglycerol ++ HemolysisHemolysis --
D-cellobioseD-cellobiose ++
그 결과, 상기 표 11에 기재된 바와 같이, 본 발명의 신규 DS2831 균주에서는 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스의 대사가 이루어지는 것으로 확인되었다. 또한, 인돌 및 요소 등의 독성 물질을 생산하지 않고, β-글루쿠로니다아제를 생산하지 않는 것으로 나타났으며, 젤라틴에 대한 분해능이 없는 것으로 확인되었다. 아울러, 본 발명의 DS2831 균주는 용혈성을 나타내지 않는 것으로 관찰되었다. As a result, as described in Table 11 above, it was confirmed that the novel DS2831 strain of the present invention metabolizes glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose. In addition, it was confirmed that it does not produce toxic substances such as indole and urea, does not produce β-glucuronidase, and has no ability to decompose gelatin. In addition, it was observed that the DS2831 strain of the present invention does not exhibit hemolysis.
[5-2] 내산성 확인[5-2] Acid resistance check
상기 실시예 [4-2]에서와 같이 DS2831 균주의 내산성을 분석하였다.The acid resistance of the DS2831 strain was analyzed as in the above example [4-2].
균주Strain 0h의 총 총균수(CFU)Total number of colonies forming units (CFU) at 0h 3h의 총 균수 (CFU)Total bacterial count (CFU) at 3h 생존율(%)Survival rate (%)
락토바실러스 가세리 DS2831Lactobacillus gasseri DS2831 6.5 x 106 CFU6.5 x 10 6 CFU 1.4 x 107 CFU1.4 x 10 7 CFU >200>200
그 결과, 상기 표 12에 기재된 바와 같이, 본 발명의 상기 신규 DS2831 균주는 기본적으로 산성 환경에 대한 내성을 가지고 있는 것으로 확인되었다.As a result, as described in Table 12 above, it was confirmed that the novel DS2831 strain of the present invention is basically resistant to an acidic environment.
[5-3] 내담즙성 확인[5-3] Confirmation of biliary tract
상기 실시예 [4-3]에서와 같이 DS2831 균주의 내담즙성을 분석하였다.The bile tolerance of the DS2831 strain was analyzed as in the above example [4-3].
균주Strain 0h의 총 총균수(CFU)Total number of colonies forming units (CFU) at 0h 12h의 총 균수 (CFU)Total bacterial count (CFU) at 12h 생존율(%)Survival rate (%)
락토바실러스 가세리 DS2831Lactobacillus gasseri DS2831 1.5 x 106 CFU1.5 x 10 6 CFU 1.5 x 107 CFU1.5 x 10 7 CFU >200>200
그 결과, 상기 표 13에 기재된 바와 같이, 본 발명의 신규 DS2831 균주는 담즙이 존재하는 환경에 대한 내성을 가지고 있는 것으로 확인되었다.As a result, as described in Table 13 above, the novel DS2831 strain of the present invention was confirmed to have resistance to an environment containing bile.
[5-4] 항생제 내성 확인[5-4] Antibiotic resistance confirmation
본 발명의 DS2831 균주의 항생제 내성을 확인하기 위해, 실시예 [2-4]와 동일한 방법으로 항생제 내성 실험을 실시하였다. To confirm the antibiotic resistance of the DS2831 strain of the present invention, an antibiotic resistance test was conducted using the same method as in Example [2-4].
본 실험에 사용한 항생제는 앰피실린, 반코마이신, 에리트로마이신 및 테트라사이클린이고, 항생제 스트립과 억제대가 만나는 지점의 농도를 최소억제농도로 하였다. The antibiotics used in this experiment were ampicillin, vancomycin, erythromycin, and tetracycline, and the concentration at the point where the antibiotic strip meets the inhibition zone was considered the minimum inhibitory concentration.
상기 준비한 배지에 본 발명의 DS2831 균주를 도말한 후, 항생제 스트립을 올리고, 다시 37℃에 48시간 배양하면서 생육이 저해되는 최소억제농도(MIC)를 측정하여 그 결과를 표 14에 나타내었다.After spreading the DS2831 strain of the present invention on the prepared medium, an antibiotic strip was placed, and the medium was cultured again at 37°C for 48 hours to measure the minimum inhibitory concentration (MIC) at which growth was inhibited. The results are shown in Table 14.
controlcontrol 앰피실린Ampicillin 반코마이신Vancomycin 에리트로마이신Erythromycin 테트라사이클린Tetracycline
EFSA guide
(μg/ml)EFSA guide
(μg/ml) 		11 22 11 44
DS283 (μg/ml)DS283 (μg/ml) GrowthGrowth 0.380.38 1.51.5 0.50.5 1.51.5
그 결과, 상기 표 14에 기재된 바와 같이, 본 발명의 DS2831 균주는 앰피실린, 반코마이신, 에리트로마이신 및 테트라사이클린에 대해서 EFSA 가이드의 기준 수치보다 항생제 내성이 낮은 것으로 확인되었다. 따라서, 본 발명의 DS2831 균주는 장내에 존재하는 유해 세균으로 내성 유전자를 수평 전달하지 않으므로, 장내 유해 세균이 외재 내성을 획득하여 항생제에 대한 내성 문제를 일으킬 염려가 없어 프로바이오틱스로서 상업화되기에도 유리한 장점이 있다.As a result, as described in Table 14 above, the DS2831 strain of the present invention was confirmed to have lower antibiotic resistance than the standard values of the EFSA guide for ampicillin, vancomycin, erythromycin, and tetracycline. Therefore, since the DS2831 strain of the present invention does not horizontally transfer resistance genes to harmful bacteria existing in the intestines, there is no concern that harmful bacteria in the intestines will acquire external resistance and cause resistance problems to antibiotics, and thus it has an advantage in that it is also advantageous for commercialization as a probiotic.
[5-5] 장 부착능 확인[5-5] Checking the attachment ability
본 발명의 DS2831 균주의 장 부착능을 평가하기 위하여, 실시예 [2-5]와 동일한 방법으로 실험을 수행하였다. In order to evaluate the intestinal adhesion ability of the DS2831 strain of the present invention, an experiment was performed using the same method as in Example [2-5].
그 결과, 표 15에 도시된 바와 같이, 본 발명의 DS2831 균주는 탄소원으로 포도당을 사용했을 때 약 0.72%의 장 부착율을 나타내어 장 부착능이 있는 것으로 확인되었다. As a result, as shown in Table 15, the DS2831 strain of the present invention was confirmed to have intestinal adhesion ability, showing an intestinal adhesion rate of about 0.72% when glucose was used as a carbon source.
균주 명Strain name 부착능(%)Adhesion (%) 표준편차(SD)Standard Deviation (SD)
락토바실러스 가세리 DS2831Lactobacillus gasseri DS2831 0.72**0.72** 0.1180.118
*p<0.5; **p<0.05; ***p<0.001*p<0.5; **p<0.05; ***p<0.001
[실시예 6] [Example 6]
본 발명의 신규 락토바실러스 가세리 균주의 장 발달/성숙 촉진 효과 확인Confirmation of the effect of promoting intestinal development/maturity of the novel Lactobacillus gasseri strain of the present invention
본 발명의 신규 락토바실러스 가세리 균주가 장의 발달 또는 성숙에 비치는 영향을 확인하기 위하여, 체외 장(intestine) 모델로 3차원의 인간 장 오가노이드(human intestinal organoids, hIOs)를 제조하고, 본 발명의 신규 락토바실러스 가세리 균주를 처리하여, 상기 장의 발달/성숙 정도를 평가하였다.In order to confirm the effect of the novel Lactobacillus gasseri strain of the present invention on the development or maturation of the intestine, three-dimensional human intestinal organoids (hIOs) were produced as an in vitro intestine model, and the novel Lactobacillus gasseri strain of the present invention was treated to evaluate the degree of development/maturity of the intestine.
[6-1] 3차원 hIOs를 이용한, 장 발달/성숙 촉진 효과 확인[6-1] Confirmation of the effect of promoting intestinal development/maturity using 3D hIOs
상기 실시예 [3-1]에서 제작된 3차원 hIOs를 마트리젤로부터 분리한 후, 차가운 PBS로 세척하고 hIOs 주변의 마트리젤을 최대한 제거한 후 수술용 칼을 활용하여 물리적으로 해리하였다. 해리된 hIOs 조각은 새로운 4-웰 디쉬에 마트리젤 돔을 형성한 후 1개 웰당 5개씩 접종하여 hIO 분화 배지로 배양하였다.After separating the 3D hIOs produced in the above Example [3-1] from the Matrigel, they were washed with cold PBS, the Matrigel around the hIOs was removed as much as possible, and then physically dissociated using a surgical scalpel. The dissociated hIOs pieces were inoculated into a new 4-well dish to form a Matrigel dome, 5 pieces per well, and cultured with hIO differentiation medium.
상기와 같이 준비된 hIOs에 상기 실시예 1에서 분리 및 동정한 본 발명의 신규 락토바실러스 가세리 균주들의 배양액을 1%의 농도로 첨가하고, 총 20일 동안 2회 계대배양한 후, 상기 hIOs의 변화를 관찰하였다.The culture solution of the novel Lactobacillus gasseri strains of the present invention isolated and identified in Example 1 was added to the hIOs prepared as described above at a concentration of 1%, and sub-cultured twice for a total of 20 days, after which changes in the hIOs were observed.
[6-2] hIOs의 형태학적 변화 확인[6-2] Confirmation of morphological changes in hIOs
먼저, 실시예 [3-2]와 동일한 방법으로, 본 발명의 신규 락토바실러스 가세리 균주를 처리한 hIOs에서 생성된 발아(budding) 구조의 개수와 상기 hIOs의 표면적 변화를 확인하였다.First, in the same manner as Example [3-2], the number of budding structures generated in hIOs treated with the novel Lactobacillus gasseri strain of the present invention and the change in surface area of the hIOs were confirmed.
그 결과, 도 5 및 도 6에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 본 발명의 신규 락토바실러스 가세리 균주를 처리한 hIOs에서 발아 구조가 더욱 많이 형성되었을 뿐만 아니라(도 5), 그 표면적도 4배 이상 향상된 것으로 확인되었다(도 6).As a result, as shown in FIGS. 5 and 6, compared to the case without any treatment, it was confirmed that not only were more germination structures formed in hIOs treated with the novel Lactobacillus gasseri strain of the present invention (FIG. 5), but also the surface area was increased by more than four times (FIG. 6).
[6-3] hIOs에서의 분자생물학적 변화 확인[6-3] Confirmation of molecular biological changes in hIOs
다음으로, 실시예 [3-2]와 동일한 방법으로 본 발명의 신규 락토바실러스 가세리 균주로 인한 hIOs에서의 분자생물학적 변화를 분석하였다. Next, the molecular biological changes in hIOs caused by the novel Lactobacillus gasseri strain of the present invention were analyzed using the same method as in Example [3-2].
그 결과, 도 7에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 본 발명의 신규 락토바실러스 가세리 균주를 처리한 hIOs에서 CDX2, LYZ, CREB3L3, DPP4, SLC5A1 및 MUC13 유전자의 발현이 증가된 것으로 확인되었다.As a result, as shown in Fig. 7, it was confirmed that the expression of CDX2, LYZ, CREB3L3, DPP4 , SLC5A1 and MUC13 genes increased in hIOs treated with the novel Lactobacillus gasseri strain of the present invention, compared to the case without any treatment.
[6-4] hIOs에서의 조직학적 변화 확인[6-4] Confirmation of histological changes in hIOs
또한, 실시예 [3-2-3]과 동일한 방법으로 본 발명의 신규 락토바실러스 가세리 균주로 인한 hIOs에서의 조직학적 변화를 분석하였다.In addition, histological changes in hIOs caused by the novel Lactobacillus gasseri strain of the present invention were analyzed using the same method as in Example [3-2-3].
그 결과, 도 8에 도시된 바와 같이, 아무런 처리도 하지 않은 경우와 비교하여, 장의 성숙 시에 발현이 증가하는 OFLM4, DEFA5, KRT20 및 MUC13 단백질 중, 본 발명의 신규 락토바실러스 가세리 균주를 처리한 hIOs에서만 OFLM4, DEFA5, KRT20 및 MUC13의 발현이 증가되어 염색되는 것으로 확인되었다.As a result, as shown in Fig. 8, compared to the case without any treatment, among the OFLM4, DEFA5, KRT20 and MUC13 proteins whose expression increases during intestinal maturation, it was confirmed that the expression of OFLM4, DEFA5, KRT20 and MUC13 was increased and stained only in hIOs treated with the novel Lactobacillus gasseri strain of the present invention.
상기와 같은 결과로부터 본 발명의 신규 락토바실러스 가세리 균주들의 처리에 의해 장의 성숙 또는 발달이 촉진됨을 알 수 있다.From the above results, it can be seen that intestinal maturation or development is promoted by treatment with the novel Lactobacillus gasseri strains of the present invention.
[제조예 2][Manufacturing Example 2]
신규 락토바실러스 가세리 균주들을 포함하는 조성물의 원말 제조Preparation of crude compositions containing novel Lactobacillus gasseri strains
본 발명의 신규 락토바실러스 가세리 균주를 포함하는 조성물의 원말을 제조하였다. A crude product of a composition comprising the novel Lactobacillus gasseri strain of the present invention was prepared.
상기 본 발명의 신규 락토바실러스 가세리 균주 각각을 최적화된 증균배지와 배양조건을 설정하여 배양하였다. 균체 회수 후 농축액 대비 동결보호제인 말토덱스트린 5-50(부피/중량)% 또는 트레할로스 5-50(부피/중량)% 또는 셀룰로오스 5-50(부피/중량)%를 첨가하여 동결건조 후 분쇄하여 유산균 원말을 제조하였다.Each of the novel Lactobacillus gasseri strains of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (volume/weight)% of maltodextrin, 5-50 (volume/weight)% of trehalose, or 5-50 (volume/weight)% of cellulose as a cryoprotectant was added to the concentrate, freeze-dried, and then ground to produce lactic acid bacteria powder.
상기에서는 본 발명의 대표적인 실시예를 예시적으로 설명하였으나, 본 발명의 범위는 상기와 같은 특정 실시예에만 한정되지 아니하며, 해당 분야에서 통상의 지식을 가진 자라면 본 출원의 청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다.Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.
[수탁번호][Acceptance number]
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)
수탁번호 : KCTC15344BPAccession number: KCTC15344BP
수탁일자 : 20230310Date of Consignment: 20230310
기탁기관명 : 한국생명공학연구원 생물자원센터(KCTC)Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)
수탁번호 : KCTC15345BPAccession number: KCTC15345BP
수탁일자 : 20230310Date of Consignment: 20230310
Figure PCTKR2024002686-appb-img-000001
Figure PCTKR2024002686-appb-img-000001
Figure PCTKR2024002686-appb-img-000002
Figure PCTKR2024002686-appb-img-000002

Claims (9)

  1. 수탁번호 KCTC15344BP로 기탁된 락토바실러스 파라카제이(Lactobacillus paracasei) DS2766 균주. Lactobacillus paracasei strain DS2766 deposited under accession number KCTC15344BP.
  2. 수탁번호 KCTC15345BP로 기탁된 락토바실러스 가세리(Lactobacillus gasseri) DS2831 균주. Lactobacillus gasseri strain DS2831 deposited under accession number KCTC15345BP.
  3. 청구항 1 또는 2에 있어서,In claim 1 or 2,
    상기 균주는 글루코스, 만니톨, 락토오스, 수크로스, 말토오스, 살리신, 자일로스, 아라비노스, 에스쿨린, 글리세롤, 셀로비오스, 만노오스, 멜레지토오스, 라피노스, 소르비톨, 람노오스 및 트레할로스로 이루어진 군으로부터 선택되는 어느 하나 이상의 당에 대한 이용능이 있는 것인 균주.The strain is a strain having the ability to utilize at least one sugar selected from the group consisting of glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose, and trehalose.
  4. 수탁번호 KCTC15344BP로 기탁된 락토바실러스 파라카제이(Lactobacillus paracasei) DS2766 균주, 수탁번호 KCTC15345BP로 기탁된 락토바실러스 가세리(Lactobacillus gasseri) DS2831 균주, 상기 DS2766 또는 DS2831 균주의 배양액, 상기 배양액의 농축액, 상기 배양액의 건조물 및 상기 배양액의 추출물로 이루어지는 군에서 선택되는 적어도 하나를 포함하는 조성물.A composition comprising at least one selected from the group consisting of Lactobacillus paracasei DS2766 strain deposited under accession number KCTC15344BP, Lactobacillus gasseri DS2831 strain deposited under accession number KCTC15345BP, a culture solution of the DS2766 or DS2831 strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
  5. 청구항 4에 있어서,In claim 4,
    상기 조성물은 장 세포에서 CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, MUC13으로 구성되는 군에서 선택되는 적어도 하나의 유전자의 발현을 향상시키는 것인 조성물.The composition above is a composition that enhances the expression of at least one gene selected from the group consisting of CDX2, OLFM2, LYZ, KRT20, CREB3L3, DPP4, SLC5A1, and MUC13 in intestinal cells.
  6. 청구항 4에 있어서,In claim 4,
    상기 조성물은 장 세포에서 OLFM4, DEFA5, KRT20, 및 MUC13으로 구성되는 군에서 선택되는 적어도 하나의 단백질의 발현을 향상시키는 것인 조성물.The composition is a composition that enhances the expression of at least one protein selected from the group consisting of OLFM4, DEFA5, KRT20, and MUC13 in intestinal cells.
  7. 청구항 4에 있어서,In claim 4,
    상기 조성물은 장의 표면적을 증가시키는 것인 조성물.The above composition is a composition that increases the surface area of the intestine.
  8. 청구항 4 내지 청구항 7 중 어느 한 항에 있어서,In any one of claims 4 to 7,
    상기 조성물은 장의 발달 또는 성숙 촉진용 식품 조성물, 또는 장의 발달 또는 성숙 촉진용 건강기능식품 조성물인 것인 조성물.The composition above is a food composition for promoting intestinal development or maturation, or a health functional food composition for promoting intestinal development or maturation.
  9. 청구항 4 내지 청구항 7 중 어느 한 항에 있어서,In any one of claims 4 to 7,
    상기 조성물은 장관 발달 또는 성숙 장애의 예방 또는 치료용 약학적 조성물인 것인 조성물.The composition above is a pharmaceutical composition for preventing or treating intestinal development or maturation disorders.
PCT/KR2024/002686 2023-03-21 2024-02-29 Novel lactobacillus sp. strain and use thereof WO2024196040A1 (en)

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KR20080110447A (en) * 2006-08-04 2008-12-18 (주)바이오니아 Lactic acid bacteria isolated from mother's milk with probiotic activity and inhibitory activity against body weight augmentation
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