KR102845919B1 - Lacticaseibacillus paracasei strain PL1536 originated from Korean mother milk which has strong inhibitory activities on various pathogens as probiotics and the functional food composition containing it as an active ingredient - Google Patents

Abstract

The present invention provides Lacticaseibacillus paracasei strain PL1536 (KCTC 15295BP) isolated from Korean breast milk. The strain of the present invention has excellent probiotic activities such as acid-bile resistance, safety against antibiotic resistance, intestinal cell adhesion ability, growth inhibition function of intestinal pathogenic bacteria, and growth inhibition function of cariogenic bacteria, and provides dairy products, lactic acid bacteria preparations, and health functional food compositions containing the strain as an active ingredient for intestinal function, caries inhibition, and prevention and improvement of gastric ulcers.

Description

Lacticaseibacillus paracasei strain PL1536 originated from Korean mother milk which has strong inhibitory activities on various pathogens as probiotics and the functional food composition containing it as an active ingredient

The present invention relates to Lacticaseibacillus paracasei strain PL1536 derived from Korean breast milk, which has excellent performance in inhibiting the growth of harmful intestinal bacteria, and to a new use thereof, and to a food for improving the intestinal environment or a functional food composition for inhibiting cariogenic bacteria, which comprises the Lacticaseibacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.

Probiotics are live microorganisms that, when consumed in adequate amounts, are beneficial to the human body. Most known probiotics are lactic acid bacteria. Probiotics, such as lactic acid bacteria and other beneficial bacteria, survive stomach acid and bile acids in the body and reach the small intestine, where they multiply and establish themselves. Once established in the intestine, they exert beneficial effects on health. These probiotics must be non-toxic and non-pathogenic.

Approximately 80% of the bacteria in our bodies reside in the intestines. Harmful bacteria colonizing the intestines can lead to metabolic syndromes like obesity and diabetes. Conversely, when beneficial bacteria colonize the intestines, they can benefit human health and immune function. Lactobacillus is a representative beneficial bacteria that primarily resides in the intestines, where it promotes digestive health and boosts immunity.

Among solids, breast milk contains an oligosaccharide called HMO (Human milk oligosaccharide), which is the third most abundant after fat and lactose. HMO is used as prebiotics (food for intestinal microorganisms) and serves as food for beneficial bacteria that live in the baby's intestines. As human milk oligosaccharides are the main components of breast milk, they can promote the health of newborns and infants, and one of the main components of human milk oligosaccharides, 2-fucosyllactose (2-FL), is known to have functions such as promoting the growth of bifidobacteria, inhibiting the adhesion of pathogens to the intestinal mucosa, and inhibiting toxin production.

When newborns drink breast milk, the presence of human milk oligosaccharides such as 2-fucosyllactose can lead to a predominance of Bifidobacterium microorganisms, and beneficial species such as Lactobacillus and Staphylococcus can increase. In contrast, in newborns and infants who drink formula instead of breast milk, it has been reported that Bacteroides and Clostridium microorganisms are more prevalent than Bifidobacterium microorganisms. It is known that a deficiency of Bifidobacterium microorganisms in the intestines of newborns and infants increases the risk of inflammatory diseases, obesity, and immune diseases.

Meanwhile, pathogenic intestinal bacteria include Salmonella, Vibrio cholerae, Escherichia coli, and Salmonella typhi. Infection with these pathogenic bacteria can cause intestinal diseases such as food poisoning and enteritis. Antibiotics, which kill or reduce the causative pathogens, can be used as a treatment for these infectious diseases. Antibiotic-based treatments can provide immediate, short-term therapeutic effects for infectious diseases.

However, these treatments can lead to antibiotic overuse, which can increase the incidence of antibiotic-resistant pathogens. For example, Salmonella typhimurium DT104 has been reported to have multiple antimicrobial resistance patterns to antibiotics such as ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, unlike conventional Salmonella.

Furthermore, antibiotic treatment has been shown to alter the gut microbiota of humans and other mammals, increasing host susceptibility to infections with enteric pathogens such as Shigella flexneri, Salmonella enterica, Clostridium difficile, and vancomycin-resistant enterococcus. For these reasons, there is a continuous need to develop novel therapeutic compositions that can effectively inhibit enteric pathogens, have a therapeutic effect on infectious diseases caused by enteric pathogens, and reduce the use of antibiotics.

Dental caries, also known as dental caries, is one of the most common dental diseases. Dental caries is an infectious bacterial disease caused by the decay of oral food by acid-resistant streptococci and lactic acid bacteria, and the destruction of tooth components by acid. It is a multifactorial disease caused by the interaction between food and microorganisms in dental plaque. Streptococcus mutans, the main causative agent of dental plaque, attaches to the tooth surface membrane and synthesizes insoluble glucans from sucrose in food using glucosyltransferase (GTase). The synthesized glucans increase the bonds between microorganisms proliferating on the tooth surface, and S. mutans attached to the tooth surface produces organic acids such as lactic acid during carbohydrate metabolism, which decalcifies tooth enamel and is known to cause dental caries.

Conventional methods for preventing dental caries or periodontal disease include reducing sugar intake through brushing and proper diet, or using xylitol to prevent acid production by cariogenic bacteria or periodontal disease-causing bacteria. However, these methods alone do not achieve the fundamental caries prevention effect by killing cariogenic bacteria or periodontal disease-causing bacteria.

The present inventors have completed the present invention by confirming that Lacticaseibacillus paracasei strain PL1536 isolated from Korean breast milk has excellent intestinal adhesion, excellent acid and bile resistance, is stable in the intestine, has no antibiotic resistance, does not produce harmful enzymes or harmful products, does not cause hemolysis, and inhibits the growth of various intestinal pathogenic bacteria and caries-causing bacteria, and is thus suitable as a probiotic, parabiotic, or postbiotic.

The purpose of the present invention is to provide Lacticaseibacillus paracasei strain PL1536 derived from Korean breast milk, which has excellent growth inhibition performance against harmful intestinal bacteria and caries-causing bacteria, and a functional food composition containing the same as an active ingredient.

To achieve the above purpose,

The present invention provides a Lacticaseibacillus paracasei strain PL1536 having the accession number KCTC 15295BP.

In addition, the present invention provides a health functional food composition for preventing or improving diseases caused by intestinal pathogenic bacteria, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving oral diseases, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In addition, the present invention provides a health functional food composition for improving the intestinal environment, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In addition, the present invention provides a health functional food composition for enhancing immunity containing the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.

In addition, the present invention provides a feed additive for preventing or improving diseases caused by intestinal pathogenic bacteria, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In addition, the present invention provides a feed additive for preventing or improving oral diseases, which contains the Lacticazeibacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In addition, the present invention provides a feed additive for improving the intestinal environment, which contains the Lacticazeibacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In addition, the present invention provides an immune-enhancing feed additive containing the Lacticase Bacillus paracasei PL1536 strain, a dead cell of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The Korean breast milk-derived Lacticase Bacillus paracasei strain PL1536 (Accession No. KCTC 15295BP) of the present invention has excellent intestinal cell adhesion, excellent acid and bile resistance, is stable in the intestine, has no antibiotic resistance and hemolysis, produces almost no D-lactate, and does not undergo bile salt conjugation, making it suitable as a probiotic. It also has the function of inhibiting the growth of intestinal pathogenic bacteria and cariogenic bacteria, and can be usefully utilized as a probiotic, parabiotic, and postbiotic material.

Figure 1 is a diagram showing the results of a hemolysis test of Lacticazeibacillus paracasei strain PL1536.
Figure 2 is a diagram showing the pathogenic bacteria inhibition ability of breast milk-derived Lacticase Bacillus paracasei strain PL1536.
Figure 3 is a diagram showing the bile acid deconjugation activity of Lacticase Bacillus paracasei strain PL1536.
Figure 4 is a diagram showing the intestinal cell adhesion ability of Lacticase Bacillus paracasei strain PL1536.
Figure 5 is a diagram showing the 16S rRNA gene base sequence of Lacticazeibacillus paracasei strain PL1536 (accession number KCTC 15295BP).

Hereinafter, the present invention will be described in detail.

The present invention provides a Lacticaseibacillus paracasei strain PL1536 having the accession number KCTC 15295BP.

The strain of the present invention has a 16S rRNA gene base sequence described as sequence number 1.

The Lacticase Bacillus paracasei strain number PL1536 of the present invention is a safe breast milk lactic acid bacterium isolated from the breast milk of Korean women.

The above lactic acid bacteria exist in the intestinal microbial ecosystem of healthy people, and are known to have beneficial effects such as maintaining normal intestinal microbial flora by settling in the intestinal epithelium, alleviating lactose intolerance, reducing blood cholesterol, anticancer effects, immunomodulatory effects, and enhancing the nutritional value of food. Live lactic acid bacteria that perform these beneficial functions are classified as probiotics.

Probiotics are bacteria that have a beneficial effect on health. Representative strains include Lactobacillus, Bifidobacterium, and Enterococcus, which have been shown to promote intestinal health and strengthen the immune system. They are commonly found in fermented dairy products like yogurt and cheese, as well as fermented foods like kimchi and soybean paste. Recently, they have also been widely consumed as health functional foods in capsule or powder form.

Postbiotics is a term encompassing probiotic cells or their metabolites. Representative examples include organic acids, bacteriocins, butyrate, enzymes, amino acids, and peptides produced by lactic acid bacteria. Postbiotics are known to directly kill harmful bacteria, activate intestinal mucosal immunity, and protect the intestines from harmful bacteria by attaching to the intestinal mucosa.

The term "breast milk lactic acid bacteria" in the present invention refers to lactic acid bacteria derived from the breast milk of healthy women. Bifidobacterium infantis is an example of a lactic acid bacteria derived from breast milk. Bifidobacterium infantis is known to have the ability to inhibit harmful bacteria. Furthermore, previous studies have reported that the infantis strain of the genus Bifidobacterium has the activity of decomposing breast milk oligosaccharides.

The Lacticase Bacillus paracasei strain PL1536 of the present invention exhibits an effect of inhibiting the growth of intestinal pathogenic bacteria.

The above intestinal pathogenic bacteria include Vibrio cholerae, Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Salmonella cholerasuis, Salmonella derby, Staphylococcus aureus, enterotoxigenic E. coli, Listeria monocytogenes, Campylobacter jejuni, Shigella flexneri, Salmonella enterica, Clostridium difficile, and vancomycin resistant Enterococcus. At least one selected from the group consisting of Clostridium enterococcus and Clostridium perfringens.

Escherichia coli (E. coli) is a bacterium commonly found in the intestines (colon and small intestine) of warm-blooded animals. E. coli itself is not harmful to humans. While most strains of E. coli are harmless, strains such as serotype O157:H7 can cause food poisoning in humans and occasionally lead to large-scale food recalls.

Staphylococcus aureus (Staphylococcus aureus) is a species of gram-positive staphylococcus. It is commonly found in the nose, respiratory tract, and skin. While it can cause skin infections, respiratory infections, sinusitis, and food poisoning, it can also exist as a normal part of the flora, not as a pathogen.

The above Salmonella refers to the genus of bacteria that cause salmonellosis. Salmonella is a type of proteobacteria belonging to the genus Salmonella in the family Enterobacteriaceae. It is a rod-shaped bacterium, approximately 0.7 to 1.5 μm in diameter and 2 to 5 μm in length, and primarily inhabits the digestive tracts of humans and animals.

Clostridium difficile is a gram-positive, spore-forming bacterium that causes difficile infection. Clostridium difficile infection (CDI) is a symptomatic infection caused by Clostridium difficile. It is synonymous with antibiotic-associated pseudomembranous colitis, Clostridium difficile-associated disease (CDAD, or C. difficile associated diarrhea), and pseudomembranous colitis. Symptoms include diarrhea, fever, nausea, and abdominal pain. It accounts for about 20% of antibiotic-associated diarrhea cases. Complications may include pseudomembranous colitis, toxic megacolon, gastrointestinal perforation, and sepsis.

The Lacticase Bacillus paracasei strain PL1536 of the present invention exhibits an effect of inhibiting the growth of oral pathogenic bacteria.

The above oral harmful bacteria is at least one selected from the group consisting of Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus gordonii, Streptococcus sanguinis, Prevotella intermedia, Streptococcus sorbrinus, and Streptococcus mutans.

Streptococcus mutans, a type of streptococcus, is considered a major cause of tooth decay, along with Lactobacillus. While Lactobacillus causes tooth decay regardless of color change, Streptococcus mutans causes tooth decay while discoloring the teeth.

The Lacticazeibacillus paracasei strain PL1536 of the present invention does not exhibit antibiotic resistance.

Lactic acid bacteria act as a reservoir of antibiotic resistance genes, which are transferred to the human microbiome, and when diseases caused by these human bacteria break out, antibiotic treatment becomes impossible, so it is essential to use lactic acid bacteria that are not resistant to antibiotics (Chon et al., 2020, Korean Society of Dairy Science and Biotechnology, 38, 70-88). Therefore, the Ministry of Food and Drug Safety's probiotic guideline (Guide to the Safety Evaluation of Probiotics as Functional Raw Materials for Health Functional Foods (Guide for Civil Complainants, Guide-1124-01), July 2021) also stipulates that antibiotic resistance tests must be performed and only those that are susceptible are approved for use as probiotics.

The European Food Safety Authority (EFSA) guidelines stipulate that the minimum inhibitory concentration test (MIC test) should be performed on 10 antibiotics (ampicillin, vancomycin, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, tylosin, tetracycline, and chloramphenicol) as a priority for evaluating the antibiotic resistance of microorganisms.

The Lacticase Bacillus paracasei strain PL1536 of the present invention is a lactic acid bacterium with excellent acid and bile resistance.

For lactic acid bacteria to function effectively as probiotics, a large number of ingested bacteria must adhere to the intestines through the digestive system. Therefore, lactic acid bacteria must be stable against gastric acid and bile acids in the human intestinal tract. For this reason, acid and bile tolerance are essential for selecting useful probiotic strains.

The Lacticase Bacillus paracasei strain PL1536 of the present invention is a lactic acid bacterium that does not exhibit hemolysis.

Hemolysis is divided into a normal function that occurs when red blood cells are destroyed and an abnormal function that is formed by genetic defects, chemicals, and toxic substances produced by microorganisms. Hemolysis in the body mainly occurs in the spleen, liver, and bone cells, and when abnormal hemolysis occurs, it can lead to fatal results in the body due to impaired oxygen transport function of red blood cells.

The Lacticase Bacillus paracasei strain PL1536 of the present invention is a lactic acid bacterium that does not produce D-lactate.

Lactate exists as two optical isomers, L and D. Since the human body only contains L-lactate dehydrogenase (LDH), which is specific for the L form, the human metabolite is L-lactate. D-lactate, which is mainly produced through the breakdown of D-LDH or D,L-racemase, which exist in bacteria, is a metabolite of lower animals and is hardly measurable or exists only in very small amounts in the human body. Elevated D-lactate in the blood is associated with anatomical or functional abnormalities of the intestines, proliferation of D-lactate-producing bacteria in the intestines, and dysfunction of the liver and kidneys, which are the main metabolic organs.

The Lacticase Bacillus paracasei strain PL1536 of the present invention is a strain with excellent intestinal adhesion ability.

Intestinal cell adhesion is a crucial element for probiotics to pass through gastric acid and bile, attach to their final target site, the intestine, and exert their effects. Therefore, probiotics with superior cell adhesion are more effective in improving the intestinal environment.

The Lacticase Bacillus paracasei strain PL1536 of the present invention does not exhibit bile acid deconjugation activity.

In the small intestine, bile acids combine with taurine or glycine to form conjugated bile acids. Their high lipid solubility facilitates cholesterol absorption in the small intestine. However, when bile acids are deconjugated by the bile acid dehydrogenase of lactic acid bacteria, the bile acids are lost, and cholesterol is consumed as a precursor for the synthesis of new bile acids, lowering blood cholesterol levels.

The present invention provides a health functional food composition for preventing or improving diseases caused by intestinal pathogenic bacteria, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The above intestinal pathogenic bacteria-induced disease is at least one selected from the group consisting of food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic enterocolitis syndrome, small intestinal microbiota overgrowth, intestinal acute diarrhea, and ischemic enteritis.

The health functional food composition for preventing or improving an intestinal pathogenic bacteria-induced disease of the present invention may include 1 x 10 ⁶ to 1 x 10 ¹¹ CFU of the Lacticase Bacillus paracasei PL1536 strain, or may include 1 x 10 ⁷ to 1 x 10 ¹² cells in a dead state.

The culture or culture solution of the strain of the present invention refers to a substance obtained by culturing the novel strain of the present invention in a known liquid medium, and in the present invention, it is a concept including the novel Lacticazeibacillus paracasei PL1536 strain.

The lysate of the strain of the present invention may be a product obtained by lysing the strain itself by chemical or physical force.

The extract of the culture of the strain of the present invention may mean a product obtained by extracting the strain itself, a lysate of the strain, a culture of the strain, or a mixture thereof, regardless of the extraction method, extraction solvent, extracted component, or form of the extract, and is a broad concept that includes all substances that can be obtained by processing or handling by other methods such as fractionation or concentration after extraction.

The term "containing as an active ingredient" of the present invention means including an amount sufficient to achieve the efficacy or activity of the cells, dead cells, culture, lysate or extract of the Lacticase Bacillus paracasei PL1536 strain.

The composition of the present invention may contain 0.1 to 99.9 wt% of the extract of the present invention as an effective ingredient based on the total weight of the composition for administration. In addition, the composition of the present invention may additionally contain one or more effective ingredients exhibiting the same or similar function in addition to the above effective ingredients.

The term "health functional food" of the present invention refers to a food manufactured using nutrients that are easily deficient in daily meals or raw materials or ingredients (functional raw materials) that have functions useful to the human body, and that maintains and improves health by maintaining the normal functions of the human body or activating physiological functions, but is not limited thereto.

The health functional food of the present invention can be manufactured by adding cells, dead cells, a culture of the strain, a lysate of the strain, or an extract of the strain of Lacticase Bacillus paracasei PL1536 in an appropriate effective concentration range according to various manufacturing methods known in the art.

The health functional food of the present invention can be manufactured in the form of powder, granules, tablets, capsules, or liquid.

The above health functional foods are not particularly limited and can be applied to any organism, as long as they are intended to prevent or improve disease. For example, they can be applied to any non-human animal, such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, or fish.

The present invention provides a health functional food composition for preventing or improving oral diseases, which contains the Lacticase Bacillus paracasei PL1536 strain, a culture of the strain, a dead cell of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The above oral diseases include dental caries and periodontal disease.

The term "dental caries" in the present invention refers to a disease in which the enamel of a tooth is damaged, causing cavities in the underlying dentin and pulp. The cause of dental caries is plaque, a bacterial film formed on the tooth surface. When sugars, starches, and other substances remaining in the mouth are broken down by the bacteria that make up plaque, the resulting acids attack and damage the tooth enamel, resulting in dental caries.

The health functional food composition for preventing or improving oral diseases of the present invention may include 1 x 10 ⁶ to 1 x 10 ¹¹ CFU of the Lacticase Bacillus paracasei PL1536 strain, and may include 1 x 10 ⁷ to 1 x 10 ¹² cells in a dead state.

The present invention provides a health functional food composition for improving the intestinal environment, which contains the Lacticase Bacillus paracasei PL1536 strain, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

Maintaining and stabilizing beneficial gut microbiota composition is a crucial factor in reducing disease risk. The gut microbiota composition can be influenced not only by genetic factors but also by various environmental factors, such as diet, antibiotics, and medication intake, which can influence the development of various diseases. Furthermore, gut microbiota composition is known to play a crucial role in human survival, as it can induce toxicity and be associated with various diseases by participating in various decomposition and metabolic processes. Therefore, modulating gut microbiota composition can effectively treat and prevent immune-related diseases, metabolic syndrome, and related conditions.

The health functional food composition for improving the intestinal environment of the present invention may include 1 x 10 ⁶ to 1 x 10 ¹¹ CFU of the Lacticase Bacillus paracasei PL1536 strain, or may include 1 x 10 ⁷ to 1 x 10 ¹² cells in a dead state.

The present invention provides a health functional food composition for enhancing immunity, which contains the Lacticase Bacillus paracasei PL1536 strain, a dead cell of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.

The gut microbiome trains and strengthens the human immune system. When we consume food, external antigens enter the intestinal mucosa through the intestinal mucosa. Gut microbes, primarily distributed in the outer layer of the intestinal mucosa, serve as the primary defense against microbes present in food, triggering a rapid and powerful immune response. Throughout this process, gut microbes continuously interact with the human immune system, strengthening it.

The health functional food composition for enhancing immunity of the present invention may include 1 x 10 ⁶ to 1 x 10 ¹¹ CFU of the Lacticase Bacillus paracasei PL1536 strain, or may include 1 x 10 ⁷ to 1 x 10 ¹² cells in a dead state.

The present invention provides a feed additive for preventing or improving diseases caused by intestinal pathogenic bacteria, which contains the Lacticase Bacillus paracasei PL1536 strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The term "feed additive" of the present invention refers to a substance added to feed for various purposes, such as supplementing nutrients and preventing weight loss, increasing the digestibility of fiber in feed, improving milk quality, preventing reproductive disorders and improving conception rates, and preventing summer heat stress.

When supplying the above feed additive, it can be supplied alone or mixed into feed for poultry and livestock.

In addition, the feed additive of the present invention can be manufactured by adding the cells of Lacticase Bacillus paracasei PL1536, a culture of the strain, a lysate of the strain, or an extract of the strain in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

The above additives are not particularly limited and can be applied to any subject for the purpose of disease prevention or improvement. For example, they can be applied to any subject, including non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, or fish.

The present invention provides a feed additive for preventing or improving oral diseases, which contains the Lacticase Bacillus paracasei PL1536 strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The present invention provides a feed additive for improving the intestinal environment, which contains the Lacticase Bacillus paracasei PL1536 strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

The present invention provides an immune-enhancing feed additive containing the Lacticase Bacillus paracasei PL1536 strain, a culture of the strain, a lysate of the strain, or an extract thereof as an effective ingredient.

In a specific embodiment, the inventors of the present invention isolated lactic acid bacteria from breast milk and performed antibiotic resistance tests, acid and bile resistance tests, hemolysis tests (see Fig. 1), confirmation of D-lactate production, growth inhibition ability of various intestinal harmful bacteria (see Fig. 2), bile salt deconjugation activity (see Fig. 3), intestinal adhesion ability (see Fig. 4), and confirmation of non-production of various harmful enzymes and substances to select lactic acid bacteria suitable for probiotics. In addition, inhibition of growth of various pathogenic bacteria was confirmed.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are only intended to illustrate the present invention, and the present invention is not limited thereto.

<Example 1> Isolation of Lacticaseibacillus paracasei strain PL1536

Isolation of lactic acid bacteria from breast milk

Breast milk from 775 lactating women was collected and inoculated onto MRS media. Among the colonies formed, Gram-positive, catalase-negative colonies were identified by 16S rRNA sequence analysis, and a total of 253 lactic acid bacteria of 8 species were isolated.

Antibiotic resistance testing of lactic acid bacteria isolated from breast milk

The obtained lactic acid bacteria were tested for MIC (Minimal Inhibitory Concentration) against a total of nine antibiotics (AMP, ampicillin; CHL, chloramphenicol; CLI, clindamycin; ERY, erythromycin; GEN, gentamicin; STR, streptomycin; TET, tetracycline; VAN, vancomycin) using the agar dilution method (ISO 10932:2010) on MRS medium. If the MIC was below the cut-off value of EFSA (European Food Safety Authority, EFSA. 2018 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). Guidance on the characterization of microorganisms used as feed additives or as production organisms. EFSA Journal 16: 1-24), it was judged to be susceptible.

At this time, Lacticase Bacillus paracasei ATCC BAA-250 was used as a QC (quality control) strain. In order to reconfirm the MIC value measured by the agar dilution method, the liquid dilution method of ISO (International Organization for Standardization) (ISO 19032. 2010. Milk and milk products - determination of the minimal inhibitory concentration (MIC) of antimicrobials applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB)) was used to measure the minimum inhibitory concentration (MIC) for nine antibiotics. Resistance was determined according to the cut-off value of EFSA after 48 hours of incubation under anaerobic conditions at 37°C.

The liquid dilution test results for nine antibiotics showed that only a small number of Lactobacillus fermentum, Lactobacillus paracasei, and Streptococcus thermophilus were susceptible. As shown in Table 1, Lactobacillus paracasei was judged to be safe as it showed no resistance to any antibiotics except vancomycin, to which it has intrinsic resistance, and was therefore named Lactobacillus paracasei PL1536 (Accession No. KCTC 15295BP).

GEN KAN STR TET ERY CLI CHL AMP VAN L. paracasei PL1536 4 64 32 2 0.5 0.064 4 1 >128 EFSA cut-off value 16 64 64 8 1 4 4 2 n.r.

(GEN, gentamicin; KAN, kanamycin; STR, streptomycin; TET, tetracycline; ERY, erythromycin; CLI, clindamycin, CHL, chloramphenicol; AMP, ampicillin; VAN, vancomycin)

<Experimental Example 1> Lacticazeibacillus paracasei strain PL1536 Check your internal acidity and internal bile

Lacticazeibacillus paracasei of Example 1 The acid resistance and bile tolerance of strain PL1536 were measured as follows. Acid resistance experiments were performed in artificial gastric fluid containing 1,000 U/㎖ pepsin in a pH 3.0 medium to measure in an environment similar to the digestive tract conditions. Lacticazeibacillus paracasei PL1536 was cultured in MRS liquid medium for 24 hours, and then centrifuged to recover the cells and washed three times with sterile saline solution. 10 ml of artificial gastric fluid was added, and the cells were reacted for 90 minutes under the same conditions as the lactic acid bacteria culture conditions. After serial dilution, they were plated on MRS solid medium, cultured, and the number of viable cells was counted to measure the degree of acid resistance. In addition, the cholestatic test was performed in an artificial bile medium containing 0.3% bile (porcine bile extract, Sigma) and 1,000 U/㎖ trypsin in a pH 7.0 medium using each lactic acid bacteria culture treated for 90 minutes under artificial gastric fluid conditions. After centrifugation of the artificial gastric fluid-treated culture, the same amount of artificial bile as the supernatant was added, and the reaction was continued for 90 minutes. Serial dilutions were then plated on MRS solid medium, and the viable cell count (CFU) was counted to measure the degree of cholestatic activity.

Analysis of acid and bile tolerance revealed that Lacticazeibacillus paracasei PL1536 was resistant to acid (survival rate >70%) and bile (survival rate >76%) (Table 2).

The results of the acid-resistant bile test of Lacticase Bacillus paracasei strain PL1536 are shown in Table 2.

MRS Gastric juice Bile acid 0 min 90 min 180 min 90 min 90 min L. paracasei
PL1536 8.10 x 10 ⁸ 1.18 x 10 ⁹ 4.20 x 10 ⁹ 5.70 x 10 ⁸ 6.20 x 10 ⁸

<Experimental Example 2> Confirmation of hemolysis in Lacticazeibacillus paracasei PL1536

An experiment was conducted to confirm the hemolysis phenomenon that occurs when red blood cells are destroyed by microorganisms. Lacticase Bacillus paracasei PL1536 was spread on sheep blood agar medium, and hemolysis was observed after culturing at 37°C for 48 hours.

As shown in Fig. 1, it was confirmed that Lacticazeibacillus paracasei PL1536 of Example 1 was a safe strain that did not exhibit hemolysis.

<Experimental Example 3> Experiment on D-lactate production by Lacticase Bacillus paracasei PL1536

D-lactate production, one of the probiotic safety evaluation items, was tested using the ABcam kit (ab83429). Lacticase Bacillus paracasei PL1536 was cultured on MRS solid medium at 37°C under anaerobically controlled conditions for 48 hours. The cell pellet was washed three times, adjusted to an OD of 1 at 600 nm, and 100 μl was inoculated into 10 ml of MRS broth, followed by stationary culture at 37°C for 24 hours. The supernatant was collected by centrifugation at 13,000 rpm and 4°C for 10 minutes, and the experiment was performed according to the manual to determine D-lactate. The amount was calculated.

The upper normal limit of D-lactate in plasma is It is 5 mmol/L (450.4 mg/L) (Han et al., 2002, Plasma D-lactate Level and Acid-base Imbalance in Cirrhotic Patients, The Korean Journal of Nephrology, Vol.21, No. 1; 47-54),

The amount of D-lactate in the Lacticase Bacillus paracasei PL1536 culture medium of Example 1 was 4.35 mg/L, which was less than 1/100 of the normal upper limit of 450.4 mg/L in human plasma, confirming that it had no effect on the human body.

<Experimental Example 4> Pathogenic Bacterial Growth Inhibition Test

In order to confirm the growth inhibition of various pathogenic bacteria by Lacticazeibacillus paracasei PL1536 of Example 1, the following experiment was performed. Lacticazeibacillus paracasei PL1536 was cultured overnight in MRS liquid medium, centrifuged, and the supernatant was prepared. Food-borne pathogens Escherichia coli O157:H7 ATCC 43894, Listeria monocytogenes ATCC19113C3, Staphylococcus aureus ATCC 25923, Salmonella typhimurium ATCC 13311, Clostridium difficile ATCC 9689, and cariogenic Streptococcus mutans ATCC 25175 were used. Pathogenic bacteria grown overnight were adjusted to a McFaland standard of 0.5 for turbidity, inoculated onto a solid medium using a sterile cotton swab, and a hole was made with a sterile pipette tip to drop 200 μl of lactic acid bacteria culture supernatant. After overnight incubation, the size of the growth inhibition zone was measured. At this time, MRS broth was used as a negative control.

As shown in Fig. 2 and Table 3, Lacticazeibacillus paracasei PL1536 showed inhibitory activity against all six pathogenic strains.

L. paracasei
PL1536 Pathogenic bacterial growth inhibition zone (mm) C. difficle
ATCC 9689 E. coli
ATCC43894 L. monocytogenes
ATCC19113C3a Sal. typhimurium
ATCC13311 S. aureus
ATCC25923 Strep. mutans
ATCC25175 14 14 14 14 19 14

*Well diameter is 8 mm.

<Experimental Example 5> Evaluation of bile acid deconjugation activity of Lacticase Bacillus paracasei PL1536

Lacticazeibacillus paracasei PL1536 of Example 1 The strains were inoculated onto MRS solid medium supplemented with 0.5% taurodeoxycholic acid (TDCA, Sigma) and cultured at 37°C for 2 days under anaerobic conditions. In the case of strains with BSH activity (positive), opaque white colonies were formed or the medium around the colonies became cloudy.

As shown in Fig. 3, Lacticazeibacillus paracasei strain PL1536 was found to be negative.

<Experimental Example 6> Confirmation of the intestinal cell adhesion ability of Lacticase Bacillus paracasei strain PL1536.

In order to confirm the intestinal cell adhesion ability of Lacticase Bacillus paracasei strain number PL1536 of Example 1, it was added to HT-29 cells (human cololectal adenocarcinoma cell line with epithelial morphology), cultured overnight, and non-attached bacteria were removed, followed by Gram staining and observation under a microscope.

As a result, it was confirmed that Lacticase Bacillus paracasei strain number PL1536 had excellent intestinal cell adhesion ability (Fig. 4).

In addition, to count the lactic acid bacteria attached to HT-29 cells, 100 ul (1 Х 10 ⁷ CFU) of live Lacticase Bacillus paracasei PL1536 was added to HT-29 cells and treated at 5% CO ₂ for 90 minutes. The culture dish was washed three times with phosphate buffered saline (PBS) to wash away any non-attached lactic acid bacteria. Then, 1 ml trypsin-EDTA was added to detach the cells from the culture dish and mixed with 9 mL of 1 X PBS. This was serially diluted and inoculated onto MRS solid medium, and the CFU was counted.

As a result, it was found that 2 X 10 ⁵ lactic acid bacteria were attached to the cells per culture dish.

<Experimental Example 7> 16S rRNA gene sequence analysis of Lacticazeibacillus paracasei PL 1536

Lacticazeibacillus paracasei PL 1536 of Example 1 was identified as Lacticazeibacillus paracasei PL 1536 by analyzing the base sequence of the 16S rRNA gene. Specifically, PCR was performed using 16S rRNA primers (27F, 5'-AGA GTT TCM TGG CTC AG-3'; 1088R, 5'-GCTG TACTA AG-3') to amplify the 16S rRNA gene. The DNA sequence was obtained by performing sequencing PCR on a 16S rRNA gene fragment obtained by PCR at Bionix (Seoul, Korea) using an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). When this information was analyzed using the BLAST algorithm on the National Center for Biotechnology Information web server (https://www.ncbi.nlm.nih.gov), 1451 bases of Lacticazeibacillus paracasei PL1536 were confirmed to be 99% identical to Lacticazeibacillus paracasei. The 16S rRNA base sequence of Lacticazeibacillus paracasei PL1536 (accession number KCTC 15295BP) is shown in Fig. 5.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15295BP 20230117

Attach an electronic file of the sequence list

Claims (18)

Lacticaseibacillus paracasei strain PL1536, accession number KCTC 15295BP.
In the first paragraph,
The above strain is characterized by having a 16S rRNA base sequence described as sequence number 1.
In the first paragraph,
The strain is characterized in that it is isolated from breast milk.
In the first paragraph,
A strain characterized in that the strain inhibits the growth of at least one selected from the group consisting of Salmonella typhimurium, Staphylococcus aureus, enterotoxigenic E. coli, Clostridium difficile, and Listeria monocytogenes.
delete In the first paragraph,
The above strain is characterized in that it inhibits the growth of Streptococcus mutans.
delete A composition comprising the Lacticase Bacillus paracasei PL1536 strain of the first clause, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
A health functional food composition for preventing or improving intestinal diseases caused by at least one selected from the group consisting of Salmonella typhimurium, Staphylococcus aureus, enterotoxigenic E. coli, Clostridium difficile, and Listeria monocytogenes.
In paragraph 8,
The above intestinal disease is characterized by at least one selected from the group consisting of food poisoning, typhoid fever, peritonitis, ulcerative colitis, Crohn's disease, intestinal Behcet's disease, infectious diarrhea, gastroenteritis, inflammatory bowel disease, neurogenic enterocolitis syndrome, small intestinal microbiota overgrowth, acute intestinal diarrhea, and ischemic enteritis.
A health functional food composition for preventing or improving intestinal diseases caused by at least one selected from the group consisting of Salmonella typhimurium, Staphylococcus aureus, enterotoxigenic E. coli, Clostridium difficile, and Listeria monocytogenes.
In paragraph 8,
The composition is characterized in that it comprises 1 x 10 ⁶ to 1 x 10 ¹¹ CFU of Lacticase Bacillus paracasei PL1536 strain or 1 x 10 ⁷ to 1 x 10 ¹² dead cells of the strain.
A health functional food composition for preventing or improving intestinal diseases caused by at least one selected from the group consisting of Salmonella typhimurium, Staphylococcus aureus, enterotoxigenic E. coli, Clostridium difficile, and Listeria monocytogenes.
A health functional food composition for preventing or improving oral diseases caused by Streptococcus mutans, comprising the Lacticase Bacillus paracasei PL1536 strain of claim 1, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
In Article 11,
A health functional food composition for preventing or improving oral diseases caused by Streptococcus mutans, characterized in that the oral diseases include dental caries and periodontal disease.
A health functional food composition for improving the intestinal environment, comprising the Lacticase Bacillus paracasei PL1536 strain of claim 1, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
A health functional food composition for enhancing immunity, comprising the Lacticase Bacillus paracasei PL1536 strain of the first clause, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
A composition comprising the Lacticase Bacillus paracasei PL1536 strain of the first clause, a dead cell of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
A feed additive for preventing or improving intestinal diseases caused by at least one selected from the group consisting of Salmonella typhimurium, Staphylococcus aureus, enterotoxigenic E. coli, Clostridium difficile, and Listeria monocytogenes.
A feed additive for preventing or improving oral diseases caused by Streptococcus mutans, containing the Lacticase Bacillus paracasei PL1536 strain of Article 1, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.
A feed additive for improving the intestinal environment, containing as an active ingredient the Lacticase Bacillus paracasei PL1536 strain of the first clause, dead cells of the strain, a culture of the strain, a lysate of the strain, or an extract thereof.
An immune-enhancing feed additive containing the Lacticase Bacillus paracasei PL1536 strain of the first clause, a dead cell of the strain, a culture of the strain, a lysate of the strain, or an extract thereof as an active ingredient.