Novel Lactococcus lactis subsp. lactis CAB701 having anti-obesity and immune-promoting activities and uses thereof

Abstract

The present invention relates to a Lactococcus lactis subspecies lactis CAB701 strain having simultaneous anti-obesity and immune-enhancing activities, a food composition for anti-obesity or immune-enhancing containing the same, and a vegan fermented milk for anti-obesity or immune-enhancing containing the same. The CAB701 strain of the present invention has pancreatic lipase inhibitory activity and can be utilized in various ways for the prevention, improvement, or treatment of obesity and for immune enhancement. Furthermore, it can be usefully used as a starter for fermentation. In addition, since the vegan fermented milk produced with the CAB701 strain of the present invention shows a significantly slower decrease in the number of viable lactic acid bacteria over time compared to vegan fermented milk produced using conventional vegan fermented milk starter cultures, it can be seen that the CAB701 strain of the present invention is useful for the production of vegan fermented milk.

Description

Technology Field

[0001] The present invention relates to a Lactococcus lactis subspecies lactis CAB701 strain having simultaneous anti-obesity and immune-enhancing activities, a food composition for anti-obesity or immune-enhancing containing the same, and a vegan fermented milk for anti-obesity or immune-enhancing containing the same. Background Technology

[0002] As Korea’s lifestyle has evolved into that of developed Western nations alongside economic development, changes in dietary habits and a decrease in physical activity have led to an increase in body fat and metabolic imbalances, resulting in a growing obese population. Consequently, the incidence of so-called metabolic syndrome, characterized by the complex manifestation of cardiovascular diseases such as diabetes, hyperlipidemia, and thrombotic disorders, is continuously rising (Lee SJ, Park JY, Nam CM, Jee SH. 2008. The prevalence estimation of metabolic syndrome and its related factors based on data from general health medical examination: a multi-center study. J KoreanSocHealthInformationHealthStatistics33: 119-133; Frohlich J, Lear SA. 2002. Old and new risk factors for atherosclerosis and development of treatment recommendations. ClinExpPharmacolPhysiol29: 838-842).

[0003] Obesity is not a disease caused by a single factor, but a syndrome resulting from a complex interplay of genetic, environmental, and social factors. It occurs when excess energy is not consumed during metabolic activity and accumulates as triglycerides in adipose tissue (Chua, SCJr. Monogenic models of obesity. BehavGenet. 27: 277-284, 1997). In addition, increased blood fatty acid levels and abdominal obesity seen in obesity are common symptoms of hyperlipidemia and type 2 diabetes. Increased free fatty acids cause ventricular hypertrophy (DeFronzo, RA, Ferrannini, E. Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease. DiabetesCare. 14(3): 173-194, 1991), and abdominal fat accumulation has been reported to be closely associated with the induction of insulin resistance (Tamori, Y., Kasuga, M. Obesity and insulin resistance. NipponRinsho. 67(2):236-244, 2009). Consequently, these factors become causes of lifestyle diseases such as hypertension, arteriosclerosis, diabetes, and hyperlipidemia, or factors that worsen health.

[0004] While it is important to prevent health problems caused by obesity, patients require long-term and effective obesity management and treatment. Current methods for treating obesity include lifestyle modifications such as dietary therapy, exercise therapy, and behavioral therapy, as well as pharmacological and surgical treatments (Mason EE. 1992. Methods for voluntary weight loss and control. ObesSurg2: 275-276). Generally, since lifestyle modifications alone have limitations in treating obesity, pharmacological treatment is often necessary in conjunction with lifestyle changes. Although the use of drugs for obesity treatment has been attempted for a long time, due to various serious side effects, the obesity drugs currently approved for long-term use by the U.S. Food and Drug Administration (FDA) are sibutramine, which acts by inhibiting the reabsorption of norepinephrine and serotonin, and orlistat, which exerts its effect by inhibiting lipase secreted from the pancreas and digestive system (King DJ, Devaney N. 1988. Clinical pharmacology of sibutramine hydrochloride (BTS 54524), a new antidepressant, in healthy volunteers. Br J Pharmacol26: 607-611; Yanovski SZ, Yanovski JA: Obesity. N Engl J Med 346:591-602, 2002). However, all of these drugs are not widely used due to some limited approvals caused by side effects (Padwal R, Li SK, Lau DC: Long-term pharmacotherapy for overweight and obesity: a systematic review and meta-analysis of randomized controlled trials.Int J ObesRelatMetabDisord 27: 1437-1446, 2003; Thearle M, Aronne LJ: Obesity and pharmacologic therapy: EndocrinMetabClinNorthAm 32: 1005-24, 2003).

[0005] Triglycerides in food are hydrolyzed into fatty acids and monoglycerides by the action of pancreatic lipase, and the fatty acids produced by hydrolysis form micelles together with cholesterol. Consequently, fatty acids are absorbed into the body in the form of micelles. Lipase is a type of esterifying enzyme that breaks down ingested lipids into fatty acids and glycerol. Although the human body contains gastric lipase and pancreatic lipase, fat digestion hardly occurs through the aforementioned gastric lipase and is mostly carried out in the intestinal tract by pancreatic lipase (Goldstein JL, Schrott H., Hazzard E., Bierman E., Motuski A., J. Clin. Invest., 52, 1544-1568 (1973); Rodwell VW, McNamara DJ, Shapiro DJ, Adv. Enzymol., 38, 373-412 (1973); Carey MC, Small DM, Bliss CM, Annu. Rev. Physiol., 45, 651-677 (1983)).

[0006] Lipase inhibitors or lipase-inhibiting compositions act as intestinal lipase inhibitors. When lipases are inactivated, dietary fats cannot be hydrolyzed into absorbable monoglycerides and free fatty acids; consequently, undigested triglycerides are not absorbed into the body, thereby inducing a reduction in calorie intake. Therefore, these can be used for the treatment of obesity and can be utilized for weight loss and maintenance in conjunction with a low-calorie diet. In particular, the inhibition of lipases is crucial for the absorption of fatty components in the blood, such as triglycerides and cholesterol. Since an increase in blood lipids is known to be a cause of circulatory system diseases, lowering blood lipid levels is important for the prevention and treatment of such diseases. Hyperlipidemia, which occurs due to an increase in lipid components in the blood, may also be caused by abnormalities in lipoprotein metabolism, such as excessive intake of cholesterol and triglycerides, increased biosynthesis or decreased breakdown of lipoproteins, or delayed removal of lipoproteins from the plasma, in a state where the concentration of lipids in the plasma is high. In particular, hypercholesterolemia is a cause of ischemic heart disease and arterioslerosis (Lee, Kwang-Woo, Diabetes and Obesity. Diabetes, 14(1) 5-11(1990); Lee, Hong-Kyu, Diseases Related to Obesity. Journal of the Korean Society for the Study of Obesity, 1(1), 34-39 (1992); Jung, Min-Young, Comorbidities of Obesity. Journal of the Korean Society for the Study of Obesity, 1(1), 5-10 (1992); Clinical Obesity, Korean Society for the Study of Obesity. Koryo Medical. 281-283 (1995)).

[0007] Therefore, research on the development of lipase inhibitors is important both medically and nutritionally, and many researchers are focusing on developing lipase inhibitors derived from natural products (NB Cater and SM Grundy, International Journal of Obesity., 3(35), 11 (1987)). Satouchi et al. isolated a protein with lipase inhibitory activity from soybeans (K. Satouchi, T. Mori and S. Matsushita: Characterization of Inhibitor Protein for lipase in Soybean Seeds. Agr. Biol. Chem., 38(1), 97-101 (1974)). In Korea, Kim et al. investigated lipase inhibitory activity from bell peppers (Kim. BM: Lipase Inhibitors from green pepper Capsocum annuun Lin. Korean J. Food Sci. Technol., 9(3), 234-240 (1997)), and Shimura et al. reported that tannin components isolated from Erigeron annuus inhibit lipase activity (S. Shimura, W. Tsuzuki, S. Kobayashi and T. Suzuki: Nippon Shokuhin Kogyo Gakkaishi., 41(8), 561-564 (1994)).

[0008] As a result of research efforts to develop a pancreatic lipase inhibitor, the inventors confirmed that the Lactococcus lactis subspecies lactis CAB701 strain isolated from cabbage has excellent pancreatic lipase inhibitory activity and, furthermore, possesses anti-obesity and immune-enhancing activities simultaneously, thereby completing the present invention. The problem to be solved

[0009] The objective of the present invention is for a Lactococcus lactis subspecies lactis having simultaneous anti-obesity and immune-enhancing activity ( Lactococcus lactis subsp. lactis ) It is to provide the CAB701 strain.

[0010] Another object of the present invention is Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis The present invention provides a food composition for anti-obesity or immune enhancement containing the CAB701 strain as an active ingredient.

[0011] Another objective of the present invention is Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis The purpose is to provide a vegan fermented milk product containing the CAB701 strain for anti-obesity or immune enhancement.

[0012] Another objective of the present invention is to provide a plant-based milk substitute (Lactococcus lactis subspecies lactis) Lactococcus lactis subsp. lactis The present invention provides a method for producing vegan fermented milk for anti-obesity or immune enhancement, comprising the step of fermenting with the CAB701 strain. means of solving the problem

[0013] To achieve the above objective, the present invention relates to Lactococcus lactis subspecies lactis of accession number KCCM13360P having simultaneous anti-obesity and immune-enhancing activities ( Lactococcus lactis subsp. lactis ) Provides the CAB701 strain.

[0014] According to one embodiment of the present invention, the strain may have acid resistance to pH 2 to 3, bile resistance to 0.1 to 1% bile acid, pancreatic fluid resistance to 0.1 to 1% pancreatic fluid, and intestinal adhesion ability.

[0015] According to one embodiment of the present invention, the strain may have lipase enzyme inhibitory activity.

[0016] According to one embodiment of the present invention, the strain may promote the production of nitric oxide (NO), which is an immune indicator.

[0017] According to one embodiment of the present invention, the strain may include a 16S rRNA base sequence represented by SEQ ID NO. 1.

[0018] To achieve the other objectives mentioned above, the present invention (Lactococcus lactis subspecies lactis) Lactococcus lactis subsp. lactis A food composition for anti-obesity or immune enhancement is provided, comprising as active ingredients the CAB701 strain (accession number: KCCM13360P), a culture of the strain, a supernatant of the culture, a concentrate of the culture, a dried product of the culture, a crushed product of the strain, or a fermented product of the strain.

[0019] According to one embodiment of the present invention, the composition may promote the production of nitric oxide (NO), which is an immune indicator.

[0020] According to one embodiment of the present invention, the composition may increase the expression of immune indicators such as TNF-α, IL-6, IL-1β, COX-2, or iNOS genes.

[0021] According to one embodiment of the present invention, the composition may inhibit lipase activity.

[0022] According to one embodiment of the present invention, the composition may be for the prevention or improvement of diseases caused by a decline in immune function.

[0023] According to one embodiment of the present invention, the disease caused by the decrease in immune function may be one or more selected from infectious diseases, allergic diseases and chronic fatigue.

[0024] To achieve the other aforementioned objective, the present invention (Lactococcus lactis subspecies lactis) Lactococcus lactis subsp. lactis The present invention provides a vegan fermented milk product for anti-obesity or immune enhancement comprising the CAB701 strain (accession number: KCCM13360P), a culture of the strain, a supernatant of the culture, a concentrate of the culture, a dried product of the culture, a crushed product of the strain, or a fermented product of the strain.

[0025] To achieve the other objectives mentioned above, the present invention provides (1) a plant-based alternative milk containing Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactisThe present invention provides a method for producing vegan fermented milk for anti-obesity or immune enhancement, comprising: (1) a step of inoculating a CAB701 strain (accession number: KCCM13360P) at a concentration of 0.2 to 3% (v / v); and (2) a step of fermenting the plant-based alternative milk inoculated with the strain at 35 to 39°C for 12 to 24 hours to achieve a pH of 3.5 to 4.5. Effects of the invention

[0026] The CAB701 strain of the present invention has pancreatic lipase inhibitory activity and can be utilized in various ways for the prevention, improvement, or treatment of obesity and for immune enhancement. Furthermore, it can be usefully used as a starter for fermentation.

[0027] In addition, since the vegan fermented milk produced with the CAB701 strain of the present invention shows a significantly slower decrease in the number of viable lactic acid bacteria over time compared to vegan fermented milk produced using conventional vegan fermented milk starter cultures, it can be seen that the CAB701 strain of the present invention is useful for the production of vegan fermented milk. Brief explanation of the drawing

[0028] Figure 1 is a phylogenetic tree of the CAB701 strain isolated in one embodiment of the present invention. FIG. 2 is a genome map of a strain of L. lactis subsp. lactis according to an embodiment of the present invention, where FIG. 2a shows the circular chromosome and plasmid of strain CAB701 and FIG. 2b shows the chromosome of strain WiKim0124. In FIG. 2, the outermost band represents the contig, the second and third bands represent COG functions, the fourth band represents rRNA and tRNA of the genome, and the fifth band represents the GC skew metric as an indicator to identify replication locations and preceding or succeeding strands (Green: values ​​higher than average; Red: values ​​lower than average). The innermost band represents the GC ratio (Blue: values ​​higher than average; Yellow: values ​​lower than average). Figure 3 shows the CAZy classification (prediction result of genes involved in carbohydrate metabolism) through gene function annotation prediction analysis of L. lactis strains according to one embodiment of the present invention. Figure 4 is a heatmap showing the phylogenetic relationships of L. lactis strains according to one embodiment of the present invention. Figure 5 is a graph showing the genome alignment of the CAB701 strain and the WiKim0124 strain according to one embodiment of the present invention using NUCMER. Figure 6 is a graph showing the genome alignment of the CAB701 strain and the WiKim0124 strain according to one embodiment of the present invention using Mauve. FIG. 7 is a Venn diagram showing the pangenome analysis of L. lactis strains according to one embodiment of the present invention. FIG. 8 is a Pareto chart showing the standardized effects of various factors (sodium acetate, citric acid, manganese sulfate, yeast extract, magnesium sulfate, potassium phosphate, sucrose, Polysorbate 80) on the growth of a CAB701 strain according to one embodiment of the present invention. In FIG. 8, the response represents the number of viable cells (log CFU / mL). Figure 9 is a three-dimensional (a) and contour plot (b) of RSM showing the combined effect of four variables (yeast extract, citric acid, sodium acetate and manganese sulfate) on the number of viable cells of the CAB701 strain according to one embodiment of the present invention. FIG. 10 is a growth curve of a strain at different sucrose concentrations showing the effect of sucrose on the growth of a CAB701 strain according to one embodiment of the present invention. Figure 11 (a) is an image confirming the differentiation of 3T3-L1 cells into adipocytes treated with the CAB701 strain isolated in one embodiment of the present invention, and (b) is a graph showing the adipocyte differentiation rate. In Figure 11, 'MRS' refers to 3T3-L1 adipocyte progenitor cells exposed to the CAB701 strain cultured in an unoptimized MRS medium, and 'YSC' refers to 3T3-L1 adipocyte progenitor cells exposed to the CAB701 strain cultured in the optimized medium (YSC medium) of the present invention. FIG. 12 is a three-dimensional (b) and contour plot (c) of RSM showing the change in the number of surviving cells and pH over time of the CAB701 strain according to one embodiment of the present invention, and the combined effect of three variables (initial pH, temperature, and time) on the number of surviving cells. Figure 13 is a graph showing the cytotoxicity of the CAB701 strain isolated in one embodiment of the present invention against RAW 264.7 cells. Figure 14 is a graph showing the expression levels of cytokine-related genes (iNOS, COX-2, TNF-α) in RAW 264.7 cells treated with the CAB701 strain isolated in one embodiment of the present invention. Figure 15 is a graph showing the expression levels of proteins related to the MAPKs signaling pathway in RAW 264.7 cells treated with the CAB701 strain isolated in one embodiment of the present invention. Figure 16 is a graph showing the cytotoxicity of the CAB701 strain isolated in one embodiment of the present invention against 3T3-L1 adipocyte progenitor cells. FIG. 17a is an image confirming the differentiation of 3T3-L1 cells treated with the CAB701 strain isolated in one embodiment of the present invention into adipocytes, and FIG. 17b is a graph showing the adipocyte differentiation rate. FIG. 18 is a graph showing the expression levels of genes related to lipid production or lipid metabolism in 3T3-L1 cells treated with a CAB701 strain isolated in one embodiment of the present invention. Figure 19 is a graph showing the expression levels of lipid metabolism-related proteins in 3T3-L1 cells treated with a CAB701 strain isolated in one embodiment of the present invention. Figure 20 is an E-test photograph showing the antibiotic resistance of the CAB701 strain isolated in one embodiment of the present invention. Figure 21 is the result of analyzing the enzyme activity of the CAB701 strain isolated in one embodiment of the present invention using the API ZYM kit. Figure 22 is a graph showing the change in body weight, body weight gain, and epididymal adipose tissue weight of mice administered the CAB701 strain isolated in one embodiment of the present invention along with a high-fat diet for 6 weeks. Figure 23 is a graph showing serum biochemical indicators (triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol) of mice administered a CAB701 strain isolated in one embodiment of the present invention along with a high-fat diet for 6 weeks. Figure 24 is a graph showing the expression levels of lipid synthesis factor proteins (FAS, PPAR-γ) in the epididymal adipose tissue of mice administered the CAB701 strain isolated in one embodiment of the present invention along with a high-fat diet for 6 weeks. Figure 25 is an image showing the size of lipid globules observed under an optical microscope after H&E staining of the epididymal adipose tissue of a mouse administered a CAB701 strain isolated in one embodiment of the present invention along with a high-fat diet for 6 weeks. Figure 26 is a graph showing the size of lipid globules measured by an optical microscope after H&E staining of the epididymal adipose tissue of mice administered the CAB701 strain isolated in one embodiment of the present invention along with a high-fat diet for 6 weeks. Specific details for implementing the invention

[0029] The present invention will be described in detail below.

[0030] In this invention, "obesity" refers not merely to having a high body weight, but to a state in which body fat is excessively accumulated. This means that even if a person appears to have a normal weight on the outside, they can be classified as obese if their body fat percentage is high. Typically, obesity is determined using the Body Mass Index (BMI); a BMI of 23–24.9 is classified as overweight, 25–29.9 as mild obesity, 30–34.9 as moderate obesity, and 35 or higher as severe obesity. Obesity occurs due to the complex interplay of multiple factors rather than a single cause, including poor dietary habits (including Westernized eating habits), reduced physical activity, emotional factors, and genetic factors. Consequently, obesity increases the risk of developing chronic diseases such as hyperlipidemia, diabetes, and hypertension.

[0031] In the present invention, "immunostimulation" refers to a function that promotes an immune response to an antigen non-specifically during the initial activation process of immune cells, or a function that strengthens immunity by increasing the activity of cells in the immune system.

[0033] The present invention relates to Lactococcus lactis subspecies lactis of accession number KCCM13360P having simultaneous anti-obesity and immune-enhancing activities ( Lactococcus lactis subsp. lactis This concerns the CAB701 strain.

[0034] The Lactococcus lactis subspecies lactis CAB701 strain of the present invention is a novel strain of Lactococcus lactis subspecies lactis derived from cabbage. Although the Lactococcus lactis subspecies lactis CAB701 strain in the present invention was isolated and identified from cabbage, the route of obtaining it is not limited thereto.

[0035] The lactic acid bacteria strain isolated through the embodiment of the present invention was found to have the nucleotide sequence of Sequence No. 1 as a result of 16S rRNA nucleotide sequence analysis for the identification and classification of microorganisms.

[0036] Accordingly, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO. 1 is Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis It was named strain CAB701 and deposited with the Korean Culture Collection of Microorganisms on June 16, 2023 (accession number KCCM13360P).

[0037] The Lactococcus lactis subspecies CAB701 strain of the present invention is a Gram-positive bacterium and a facultative anaerobe capable of growing under both aerobic and anaerobic conditions; it does not form spores, is non-motile, and the cells take the form of cocci.

[0038] According to the experimental results of the present invention, when a high-fat diet was administered for a short or long period, body weight and body fat significantly increased. In addition, it increased blood triglyceride, blood total cholesterol, and blood LDL cholesterol concentrations, thereby inducing obesity.

[0039] In one embodiment, when the Lactococcus lactis subspecies lactis CAB701 strain of the present invention was treated to RAW 264.7 cells, the amount of NO produced increased to 86.32% of the level of the positive control group treated with LPS, specifically confirming that it has high immunostimulatory activity.

[0040] In another example, when the CAB701 strain of the present invention was treated to RAW 264.7 cells, the expression of cytokine-related genes (iNOS, COX-2, TNF-α, IL-1β, and IL-6) and the expression of proteins related to the MAPKs signaling pathway were significantly increased, specifically confirming that it has excellent immunostimulatory activity.

[0041] In another example, the pancreatic lipase inhibition rate of the CAB701 strain of the present invention was confirmed, and it was specifically confirmed that it exhibited an excellent pancreatic lipase inhibition rate (28.87%), which is the same as that of the fat absorption inhibitor Orlistat (1000 μg / mL).

[0042] In another embodiment, to confirm the anti-obesity effect of the CAB701 strain, mouse preadipocytes (3T3-L1) were cultured in an adipogenesis differentiation medium to induce differentiation into adipocytes, and after treatment with live CAB701 bacteria, the degree of adipocyte differentiation was checked using the Oil-Red O staining method. As a result, in the treatment group (CON) that did not induce differentiation into adipocytes, no staining occurred, and based on the treatment group that induced differentiation, the level of red staining decreased in a concentration-dependent manner in all experimental groups treated with live CAB701 bacteria, confirming that the differentiation of adipocytes was inhibited by 61.47%.

[0043] In another example, the inhibitory effect of the CAB701 strain on the expression of genes involved in lipid production was confirmed using preadipocytes. As a result, it was confirmed that the expression of LPL and adiponectin genes, which are transcription factors important for adipocyte differentiation, was significantly reduced in the group treated with the CAB701 strain compared to the positive control group treated only with differentiation medium without treatment with the CAB701 strain.

[0044] In another embodiment, mouse animal experiments with the CAB701 strain were conducted, wherein the normal group (NOR) was fed a normal diet, the obesity-induced group (HFD) was fed only a high-fat diet, and the CAB701 administration group was fed a high-fat diet along with oral administration of the CAB701 strain. Subsequently, the body weight and adipose tissue weight of the mice were checked, and it was confirmed that the body weight and adipose tissue weight of the CAB701 administration group were significantly reduced. In addition, it was confirmed that the blood triglyceride, total cholesterol, and LDL-cholesterol concentrations of the CAB701 administration group decreased to 56.76%, 81.94%, and 72.94%, respectively, compared to the obese control group. Furthermore, it was specifically confirmed that the size of lipid globules in the epididymal adipose tissue of the CAB701 administration group decreased to 70.19% compared to the obese control group.

[0045] Therefore, it can be seen that the CAB701 strain of the present invention possesses pancreatic lipase activity while simultaneously possessing anti-obesity and immune-enhancing activities.

[0047] In addition, the present invention relates to Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis The present invention relates to a food composition for anti-obesity or immune enhancement comprising as active ingredients the CAB701 strain (accession number: KCCM13360P), a culture of the strain, a supernatant of the culture, a concentrate of the culture, a dried product of the culture, a crushed product of the strain, or a fermented product of the strain.

[0048] As the “Lactococcus lactis subspecies lactis CAB701 strain”, “anti-obesity”, and “immunostimulation” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0049] In the present invention, "consisting of an active ingredient" or "containing an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of the CAB701 strain of the present invention, specifically meaning containing an amount sufficient to exhibit anti-obesity or immune-enhancing activity. For example, the CAB701 strain may be used at a concentration of 10 to 15,000 mg / kg, preferably 100 to 10,000 mg / kg. As another example, the CAB701 strain is 1X10 6 CFU / mL to 1X10 10 CFU / mL, preferably 1X10 8 CFU / mL to 1X10 10 It can be used at a concentration of CFU / mL. Since the above CAB701 strain is a natural product and does not cause adverse effects on the human body even when administered in excess, the upper and lower quantitative limits of the CAB701 strain included in the composition of the present invention can be selected and implemented by a person skilled in the art within an appropriate range.

[0050] The food composition of the present invention can be formulated and used as a functional food or added to various foods. Examples of foods to which the composition of the present invention can be added include beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, health supplements, etc.

[0051] The food composition of the present invention may include not only the CAB701 strain as an active ingredient but also ingredients that are typically added during food manufacturing, such as, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of the carbohydrates mentioned above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as conventional sugars like dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavorings, natural flavorings [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is manufactured into a drink and a beverage, in addition to the polysaccharide fraction derived from Acorus calamus of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

[0052] The present invention provides a health functional food comprising a food composition for anti-obesity and immune enhancement containing the above-mentioned CAB701 strain as an active ingredient. The above-mentioned health functional food refers to a food prepared by adding the CAB701 strain to food materials such as beverages, teas, spices, chewing gum, and confectionery, or by encapsulating, powdering, or suspension thereof, which brings about specific health effects when consumed. Unlike general pharmaceuticals, it has the advantage of being made from food as a raw material and thus avoiding side effects that may occur with long-term use of pharmaceuticals. The health functional food of the present invention obtained in this manner is very useful because it can be consumed on a daily basis. The amount of the CAB701 strain added to such a health functional food varies depending on the type of health functional food to be used and cannot be uniformly specified, but it should be added within a range that does not impair the original taste of the food, and is typically in the range of 0.01 to 50 weight%, preferably 0.1 to 20 weight%, relative to the food to be used. In addition, for health functional foods in the form of pills, granules, tablets, or capsules, the amount added may typically be in the range of 0.1 to 100 weight%, preferably 0.5 to 80 weight%. In one embodiment, the health functional food of the present invention may be in the form of pills, tablets, capsules, or beverages.

[0054] In addition, the present invention relates to Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis The present invention relates to a vegan fermented milk product for anti-obesity or immune enhancement comprising the CAB701 strain (accession number: KCCM13360P), a culture of the strain, a supernatant of the culture, a concentrate of the culture, a dried product of the culture, a crushed product of the strain, or a fermented product of the strain.

[0055] As the “Lactococcus lactis subspecies lactis CAB701 strain”, “anti-obesity”, and “immunostimulation” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0056] The vegan fermented milk of the present invention, by fermenting using the CAB701 strain, not only possesses anti-obesity and immune-enhancing activities, but also exhibits excellent shelf life because the decrease in the number of viable lactic acid bacteria over time is significantly slower compared to vegan fermented milk produced using conventional starter cultures.

[0058] In addition, the present invention relates to a method for producing vegan fermented milk for anti-obesity or immune enhancement, comprising: (1) a step of inoculating plant-based alternative milk with a Lactococcus lactis subsp. lactis CAB701 strain (accession number: KCCM13360P) at a concentration of 0.2 to 3% (v / v); and (2) a step of fermenting the plant-based alternative milk inoculated with the strain at 35 to 39°C for 12 to 24 hours to achieve a pH of 3.5 to 4.5.

[0059] As the “Lactococcus lactis subspecies lactis CAB701 strain”, “anti-obesity”, and “immunostimulation” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0060] The above plant-based alternative milk may be an alternative milk made from one or more plant-based ingredients selected from coconut, soy, almond, oat, and rice.

[0061] The above plant-based alternative milk may have a solid content of 4 to 15 weight%, preferably 5 to 10 weight%, and more preferably 7 to 9 weight%.

[0062] The above vegan fermented milk can be stored at 1 to 8 ℃, preferably 2 to 6 ℃, after production.

[0063] The above vegan fermented milk has a viable cell count of 130×10 immediately after production. 6 CFU / mL or more, preferably 130×10 6 CFU / mL~150×10 6It is CFU / mL, and when stored at 1 to 8 ℃, preferably 2 to 6 ℃, the viable cell count is 70×10 up to the expiration date, which is 14 days after manufacture. 6 CFU / mL or more, preferably 70×10 6 CFU / mL~120×10 6 CFU / mL, more preferably 90×10 6 CFU / mL~120×10 6 It can maintain CFU / mL.

[0065] Example 1: Isolation and Identification of Strains

[0066] 1-1: Exploration of Lactic Acid Bacteria Derived from Domestic Plant Resources

[0067] Eight types of Jeju raw materials provided by BK Bio Co., Ltd. were used to isolate lactic acid bacteria derived from domestic plant resources, and the Jeju raw materials used for the isolation of lactic acid bacteria are as follows.

[0068] - Jeju Raw Ingredients (8 types): Carrot, Prickly Pear, Broccoli, Beet, Cabbage, Hallabong, Kale, Kohlrabi

[0069] Specifically, appropriately cut Jeju raw material and 0.85% (w / v) NaCl (sterile physiological saline) were added to a Stomacher bag, and the sample was homogenized. After decimal diluting the homogenized sample with sterile physiological saline, 10 0 , 10 -1 , 10 -2 The diluted samples were plated onto BCP (bromocresol purple)-MRS agar medium and anaerobically incubated at 37°C for 20 hours. Ten yellow colonies from each sample were subcultured twice on BCP-MRS.

[0071] 1-2: Isolation and Identification of Strains

[0072] 1-1-1: Isolation of Strains

[0073] Through morphological and physiological examinations of strains presumed to be lactic acid bacteria in BCP-MRS medium, a cabbage-derived strain exhibiting Gram positivity and catalase negativity, as well as possessing both anti-obesity and immune-enhancing activities, was finally isolated. The isolated strain was subcultured at least twice in MRS medium and then used in the experiment.

[0074] 1-1-2: Identification of strains

[0075] (1)The finally selected strains were identified through 16S rRNA gene sequencing analysis. To isolate the genomic DNA of each strain, colonies formed on MRS agar plates were inoculated into MRS broth medium and cultured at 37°C for 16 hours. Subsequently, 3 mL of the lactic acid bacteria culture was centrifuged at 13,000 rpm at 4°C for 1 minute to harvest the strains. Gram-positive bacterial DNA was extracted using a DNA extraction kit (AccuPrep® Genomic DNA Extraction Kit, BIONEER, Daejeon, Korea) in accordance with the manufacturer's protocol, using lysis buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2% Triton® X-100) and lysozyme (60 mg / mL). PCR amplification for identification was performed using a PCR premix (AccuPower® PCR PreMix, BIONEER) in accordance with the manufacturer's protocol. The PCR reaction was performed using 20 ng of template DNA, 27F primers (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 1492R primers (5′-TAC GGY TAC CTT GTT ACG AC-3′) and a thermocycler (TurboCycler, Blue-ray Biotech, Taipei, Taiwan) under the conditions shown in Table 1 below. The PCR-amplified DNA was purified using the QIAquick® PCR purification kit (Qiagen, Hilden, Germany) and then sequenced by the Sanger sequencing method at Bionics Co., Ltd. The nucleotide sequences of the 16S rRNA gene were compared using BLAST (Basic Local Alignment Search Tool), available from NCBI (National Institutes of Health, Bethesda, MD, USA).

[0076] step Temperature (°C) Time (sec) Cycle count Pre-denaturation 94 120 1 Denaturation 94 30 35 Annealing 50 60 Extension 72 60 Post-extension 72 490 1

[0077] (2) As a result of analyzing the 16S rRNA gene sequence of a strain isolated from cabbage among the above Jeju raw materials that simultaneously possesses anti-obesity and immune-enhancing activities, the Lactococcus lactis subspecies lactis CAB701 strain was identified.

[0078] (3) Phylogenetic tree analysis

[0079] Phylogenetic tree analysis was performed on the above-mentioned Lactococcus lactis subspecies CAB701 strain. Lactococcus comprises 22 genera and 6 subspecies; the 16S rRNA gene sequence of the type strain was obtained from LPSN (list of prokaryotic names with standing in nomenclaturegenus, http: / / www.bacterio.net). The sequences were aligned and edited using BioEdit (Ibis Biosciences, http: / / www.mbio.ncsu.edu / bioedit / bioedit.html) and ClustalW (http: / / clustal.org). A phylogenetic tree for the above-mentioned CAB701 strain was obtained using MEGA11 (https: / / www.megasoftware.net) and the neighbor-joining method (Fig. 1).

[0080] (4) Sugar Usage Analysis

[0081] Sugar utilization was analyzed for the above Lactococcus lactis subspecies lactis CAB701 strain using the API20 STREP kit.

[0082] sugar CAB701 Glycerol - Erythritol - D-Arabinose - L-Arabinose - D-Ribose + D-Xylose + L-Xlose + D-Adonitol - Methyl-β-D-xylopyranoside - D-Galactose - D-Glucose + D-Fructose + D-Mannose + L-Sorbose + L-Rhamnose - Dulcitol - Inositol - D-Mannitol - D-Sorbitol + Methyl-α-D-mannopyranoside - Methyl-α-D-glucopyranoside - N-Acetylglucosamine - Amygdalin + Arbutin + Esculin Ferric Citrate + Salicin + D-Cellobiose + D-Maltose + D-Lactose (bovine origin) + D-Melibiose - D-Saccharose (sucrose) + D-Trehalose + Inulin + D-Melezitose - D-Raffinose - Amidon (starch) + Glycogen - Xylitol - Gentiobiose + D-Turanose - D-Lyxose - D-Tagatose - D-Fucose - L-Fucose - D-Arabitol - L-Arabitol - Potassium Gluconate + Potassium 2-ketogluconate - Potassium 5-ketogluconate -

[0084] Example 2: Genome Analysis

[0085] Experimental method

[0086] (1) Genome DNA sequencing, assembly, and annotation methods

[0087] DNA extraction and quality evaluation

[0088] Genomic DNA integrity was verified by agarose gel electrophoresis. The concentration of extracted genomic DNA was quantified using a Qubit 2.0 fluorescence analyzer (Invitrogen, Carlsbad, USA). To ensure the purity and integrity of genomic DNA, the 16S rRNA gene was sequenced using an ABI 3730 DNA sequencing system (Applied Biosystems, Foster City, CA, USA). Specifications for DNA sample quantity and quality include a concentration ≥ 10 ng / μl, a total volume of 400 ng, and an A260 / A280 ratio ≥ 1.8.

[0089] Building the MiSeq library

[0090] To generate the library, genomic DNA was fragmented into approximately 550 bp using the M220 Focused-ultrasonicator™ (Covaris Ltd, Brighton, UK). The fragmented DNA was quantified using the Bioanalyzer 2100 (Agilent, Palo Alto, USA) in conjunction with the DNA 7500 kit. Subsequently, the library was constructed according to the manufacturer's protocol using the TruSeq DNA Library LT kit (Illumina, San Diego, USA). Whole-genome sequencing was performed on an Illumina MiSeq system using 2 × 300 bp paired-end reads and a 600-cycle sequencing kit (MiSeq Reagent Kit v3).

[0091] Building the PacBio library

[0092] For library preparation, 5 μg of genomic DNA was sheared using Megaruptor3 according to the manufacturer's recommended protocol and purified with AMpureXP beads to remove small fragments. Subsequently, the SMRTbell library was constructed using the SMRTbell® Express Template Preparation Kit (100-938-900). Genome size, library size, and Qubit concentration were calculated using the PacBio calculator, and the library was pooled accordingly. DNA fragments smaller than 3 kb were removed using AMpure XP beads. For sequencing, SMRTbell templates were prepared by annealing sequencing primers v4 and binding DNA polymerase using the Sequel Binding Kit 3.0. Excess primers and polymerase were removed via AMPure purification prior to sequencing. The SMRTbell library was sequenced in the Sequel System using the Sequencing Kit v3.0 and SMRT Cell 1M v2, and data was collected using 10 hours of video data per SMRT Cell 1M v2.

[0093] Whole Genome Sequencing and Assembly

[0094] The genome of the target strain was constructed de novo using both PacBio and MiSeq sequencing data. Sequencing analysis was commissioned to CJ Bioscience Inc. (Seoul, Korea). MiSeq sequencing data was quality-controlled using Trimmomatic-0.36, and PhiX sequences were removed using BBMap 38.32. Hybrid assembly was achieved using Unicycler v 0.4.9, which integrated quality-controlled MiSeq data and PacBio long reads. When the resulting contigs from the hybrid assembly were circular, they were rearranged using Circlator 1.4.0 to start from the dnaA / repA gene or the replication origin.

[0095] Gene prediction and annotation

[0096] A gene discovery and functional annotation pipeline for whole-genome assembly was executed using the EzBioCloud genome database. Protein-coding sequences (CDS) were predicted using Prodigal 2.6.2. tRNA genes were identified using tRNAscan-SE 1.3.1, while rRNA and other non-coding RNAs were identified using covariance model searches in the Rfam 12.0 database. CRISPR detection was performed using PilerCR 1.06 and CRT 1.2. CDS were classified into functional groups based on the ortholog groups of EggNOG 4.5. Additional functional annotation was performed using the UBLAST program by comparing the predicted CDS with databases such as Swissprot (UniProt 2015), KEGG, and SEED. Furthermore, to extend the functional classification, each CDS was aligned to the COG database using BLASTp, with an e-value cutoff of 1 × 10⁻⁶. -3 The EggNOG mapper tool was used for comprehensive functional annotation, identification of direct homologous groups, and functional annotation prediction based on conserved protein domains. The dbCAN3 metaserver was utilized for automated CAZy family annotation for carbohydrate-active enzyme prediction.

[0097] (2) Comparative genomics

[0098] Data selection

[0099] For comparative genomics, complete genome sequences of strains closely related to L. lactis subsp. lactis CAB701 were searched in the NCBI GenBank database. The strains selected through the search in the said database were L. lactis subsp. lactis ATCC 19435, L. lactis subsp. hordniae NBRC 100931, L. cremoris (formerly lactis subsp. cremoris) LMG 6897, and L. cremoris subsp. tructae (formerly lactis subsp. tructae) DSM 21502. Additionally, the genome of L. lactis subsp. lactis Wikim0124 was selected based on phylogenetic proximity and functional relevance. The genome sequences of the selected strains were obtained using the EzBioCloud database (Yoon et al., 2017). Comprehensive comparative genomic analysis was performed using the comparative genomic analysis tool provided by CJ Bioscience Inc. (https: / / www.ezbiocloud.net / contents / cg).

[0100] Phylogenetic analysis

[0101] OrthoANI values ​​were calculated to elucidate the phylogenetic relationships between the analyzed strains. An unweighted paired group method (UPGMA) dendrogram using the arithmetic mean was generated using the Orthologous ANI Tool (OAT) provided by CJ Bioscience (I. Lee, Kim, Park, & Chun, 2016), which clearly visualized the evolutionary distance between strains.

[0102] Genome alignment and comparison

[0103] Genome alignment was performed using NUCMER (Delcher, Phillippy, Carlton, & Salzberg, 2002) to detect genome inversions and other genomic differences. Additionally, genome rearrangements and inversions were visualized using Mauve (v 2.4.0) (Darling, Mau, & Perna, 2010).

[0104] Pan-Genome Orthologers Group Calculation

[0105] The Pangenome Orthologers Group (POG) has an e-value threshold of 1 × 10 -6 It was determined using the combined mutual best hits (RBH) method via uBLAST (Ward & Moreno-Hagelsieb, 2014). Additionally, an ORF-independent method was applied using nucleotide sequences with a cutoff value of at least 70% gene coverage (Chun et al., 2009). Some genes grouped due to short sequence lengths were further analyzed for POG clustering using UCLUST (≥95% identity). Venn diagrams were constructed using jvenn to visualize the calculated POGs (Bardou, Mariette, Escudie, Djemiel, & Klopp, 2014).

[0106] Genetic analysis related to health benefits

[0107] Targeted genome analysis was performed on strains CAB701 and WiKim0124, focusing on genes related to immune regulation and anti-obesity functions. The CDSs of the two strains were scanned using bioinformatics tools. The focus was on identifying genes related to cell surface factors such as lipoteichoic acid (LTA) and extracellular polysaccharides (EPS), as well as genes related to short-chain fatty acids (SCFAs) and extracellular vesicles (EVs).

[0109] Experimental results

[0110] 2-1: Genome Characterization and Annotation

[0111] 2-1-1: General Characteristics of the Genome

[0112] The genomes of the CAB701 strain and the WiKim0124 strain were analyzed and compared in Table 3 below.

[0113] division CAB701 WiKim0124 Assembly type Complete Incomplete (contigs) GC content 35.0% 34.8% Chromosome length (Genome size, bp) 2,575,822 2,505,913 Mean length of intergenic region 141.4(169.6) 148.4(206.3) Mean of CDS length (sd) (bp) 911.5(747.1) 886.1(611.7) Median of CDS length (bp) 759 763.5 N50 (bp) 2,517,318 2,505,913 No. of Coding sequences (CDSs) 2,445 2,420 No. of contigs 2 1 No. of rRNA genes 19 19 No. of tRNA genes 63 70 Sequencing depth of coverage 448.01x -

[0114] The complete genome sequence of the CAB701 strain consists of a circular chromosome and a plasmid (Fig. 2). Referring to Table 3 above, the chromosome of the CAB701 strain has a length of 2,517,318 bp and a GC content of 35.00%, while the plasmid has a length of 58,504 bp and a GC content of 34.09%. Additionally, the genome encodes 2,445 CDSs along with 63 tRNA genes and 19 rRNA genes, providing a comprehensive genome profile of this strain.

[0115] Meanwhile, the genome of L. lactis subsp. lactis WiKim0124 was analyzed after being uploaded to the EzBioCloud database based on sequences obtained from NCBI. The chromosome of the WiKim0124 strain has a length of 2,505,913 bp, a GC content of 34.8%, and contains 2,420 CDSs. Additionally, the WiKim0124 strain contains 19 rRNA genes and was found to have 70 tRNA genes, which is slightly more than that of L. lactis subsp. lactis CAB701.

[0116] 2-1-2: CAZy Enzyme Notes and Classification

[0117] CAZyme annotation and classification of L. lactis strains were performed using the dbCAN3 server and are shown in Figure 3. The core enzymes of L. lactis strains were classified into five groups consisting of auxiliary activity (AA), carbohydrate binding module (CBM), carbohydrate esterase (CE), glycoside hydrolase (GH), and glycosyltransferase (GT).

[0118] Referring to Fig. 3, the CAB701 strain of the present invention was found to possess 28 GTs, 35 GHs, 4 CEs, 8 CBMs, and 1 AA, confirming that it exhibits the highest glycosyltransferase activity. Meanwhile, the WiKim0124 strain was found to possess 24 GTs, 37 GHs, 5 CEs, 9 CBMs, and 1 AA, indicating enhanced carbohydrate processing ability. Additionally, the L. lactis subsp. hordniae NBRC 100931 strain exhibited a profile with 1 AA, 8 CBMs, 3 CEs, 27 GHs, and 22 GTs. L. lactis subsp. Lactis ATCC 19435 was found to have 1 AA, 8 CBM, 5 CE, 32 GH, and 23 GT, showing a slightly improved profile in CE and GH compared to the NBRC 100931 strain.

[0119] 2-2: Comparative Genomic Analysis

[0120] 2-2-1: Phylogenetic Relationships

[0121] OrthoANI values ​​were calculated to clarify the phylogenetic relationships between L. lactis strains (Fig. 4).

[0122] Looking at Figure 4, the CAB701 strain of the present invention and the WiKim0124 strain showed a close genetic relationship with an OrthoANI value of 97.597%. The L. lactis subsp. lactis ATCC 19435 strain showed a very close genetic relationship with the CAB701 strain with a high OrthoANI value of 97.48% and with the WiKim0124 strain with a value of 98.54%, and was identified as L. lactis subsp. lactis.

[0123] 2-2-2: Genome Alignment and Comparison

[0124] The genomes of the CAB701 strain and the WiKim0124 strain of the present invention were aligned and compared using NUCMER and Mauve (Figs. 5 and 6).

[0125] As shown in Figure 5, the NUCMER alignment indicates significant homologous regions between the genomes of the two L. lactis strains, which can be seen at the diagonally aligned points. Meanwhile, deviations in the diagonal pattern, appearing as scattered points, indicate genomic rearrangements including insertions, deletions, or inversions, as well as differences between the genomes of the two strains.

[0126] As shown in Fig. 6, Mauve alignment provides insight into local collinear blocks between the genomes of two L. lactis strains. In Fig. 6, the color-coded blocks represent homologous DNA regions between the genomes of the two L. lactis strains without sequence rearrangement, indicating a high level of coherence. Meanwhile, gaps or unaligned regions within the color-coded blocks represent genomic differences between the two strains.

[0127] 2-2-3: Pangenome composition of Lactococcus lactis strains

[0128] Through pangenome analysis, the core genome of 1,963 genes shared by four L. lactis strains CAB701, WiKim0124, ATCC 19435 and NBRC 10093 was identified (Fig. 7).

[0129] In addition, as shown in Fig. 7, it was confirmed that the CAB701 strain of the present invention has 183 unique genes. These 183 unique genes may be associated with unique metabolic pathways or survival mechanisms that are not present in other strains. Furthermore, it was confirmed that the WiKim0124 strain has 29 unique genes, the ATCC 19435 strain has 245 unique genes, and the NBRC 10093 strain has 51 unique genes.

[0130] 2-3: Genetic Analysis Related to Health Benefits

[0131] As a result of genetic analysis regarding health benefits, it was confirmed that the CAB701 strain of the present invention uniquely possesses an IgA-specific serine endopeptidase gene, unlike other strains, which indicates that the CAB701 strain may be specialized in immune regulation.

[0132] Meanwhile, both the CAB701 strain and the WiKim0124 strain contain genes for diacylglycerol kinase (ATP), D-alanine poly(phosphoribitol) ligase, CDP-ribitol-ribitol phosphatase, teichoic acid poly(glycerol phosphate) polymerase, and teichoic acid translocation allowant protein TagG, all of which contribute to the composition of LTA and TA. Both strains were found to possess genes for exopolysaccharide (EPS) biosynthesis. Additionally, genes involved in the production of small peptides such as lactose and serine / threonine protein kinases were detected in both strains, and the CAB701 strain was found to have a greater number of serine / threonine protein kinases compared to the WiKim0124 strain. Regarding cell wall proteins, it was observed that both strains commonly possessed D-alanine ligase, D-aspartate ligase, and glutamate racemase genes. Additionally, both strains were found to possess penicillin-binding protein and petidoglycan synthase genes, which are important for cell wall composition and maintenance.

[0133] In addition, both strains were found to possess genes involved in the biosynthesis of short-chain fatty acids (SCFAs), specifically the acetic acid kinase gene. Furthermore, both strains had four copies of the L-lactate dehydrogenase gene, indicating the ability to utilize and convert lactate. Genes encoding formate C-acetyltransferase and [formate C-acetyltransferase] activating enzymes were also identified in the same number in both strains.

[0134] 2-4: Sintering

[0135] The present invention In a study on the gene function annotation of the CAB701 strain, repetitive annotations for specific enzymes, particularly GH1 and GT2, indicate that they play important roles in the organism's carbohydrate metabolism. Specifically, the CAB701 strain was found to have nine annotations related to the GH1 enzyme, known for its ability to hydrolyze β-glycosidic bonds in various carbohydrates such as lactose, cellobiose, and specific glycosides, suggesting efficient carbohydrate processing. Additionally, the 15 annotations related to the GT2 enzyme were found to be essential for forming glycosidic bonds that play a crucial role in synthesizing cell wall components, extracellular polysaccharides, or glycoproteins, suggesting that this biosynthetic capability allows for sophisticated carbohydrate metabolism.

[0136] In conclusion, the present invention, which has higher GH and GT activities The CAB701 strain has a relatively high complex carbohydrate fermentation efficiency, so it is expected to be a suitable candidate for probiotics that help digest certain dairy products or dietary fiber.

[0138] Example 3: Culture Optimization

[0139] Experimental method

[0140] Culture Preparation and Fermentation Conditions

[0141] The CAB701 strain of the present invention was prepared by subculturing at least twice in MRS agar before preparing the inoculum. Subsequently, a single colony of the CAB701 strain was cultured in MRS medium at 37°C for 16 hours before being used as the inoculum. To optimize initial fermentation conditions such as medium, temperature, and pH, a 1% (v / v) inoculum was added to 30 mL of MRS medium and cultured at 37°C for 10 hours, and monitored using RTS-8 Plus (Biosan, Latvia, Riga).

[0142] carbon source concentration setting

[0143] 0–5% (w / v) of sucrose was added to the optimized medium. Subsequently, growth curves were generated by monitoring optical density at 600 nm, and the effect of sucrose concentration on bacterial growth was evaluated by determining the number of viable cells at 6 and 10 hours after inoculation.

[0144] Verification of the anti-obesity efficacy of the CAB701 strain in optimized media

[0145] Lipid accumulation in 3T3-L1 adipocyte progenitor cells was analyzed using the aforementioned Oil Red O staining method. 3T3-L1 adipocytes obtained from ATCC (CL-173™) were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with newborn calf serum and antibiotics. Cells were maintained at 37°C under 5% CO2. For differentiation, cells were seeded into 6-well plates and cultured to the confluence point; they were then exposed to differentiation medium containing 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, followed by a maintenance phase in differentiation medium supplemented with insulin. Subsequently, the CAB701 strain was cultured at 1 × 10⁶ in Lactobacilli de Man, Rogosa, and Sharpe media. 8 The cells were cultured at a density of CFU / mL. Subsequently, the centrifuged and washed CAB701 strain was introduced into the 3T3-L1 cells. The control group consisted of cells exposed only to differentiation medium or maintained in growth medium without the CAB701 strain. After differentiation, the 3T3-L1 cells were stained with Oil Red O according to an established protocol. The stained cells were observed under a microscope, and the dye extracted with isopropanol was quantitatively measured at an absorbance of 500 nm.

[0147] Experimental results

[0148] 3-1: Badge Optimization

[0149] 3-1-1: Effects of Carbon and Nitrogen Sources on the Growth of CAB701 Strain

[0150] It was confirmed that optimizing the fermentation medium using the OFAT method has a significant effect on the growth of the CAB701 strain.

[0151] Specifically, when various carbon sources were added at 20 g / L, sucrose and maltose were found to be the most effective, exhibiting the highest viable cell counts of 9.44 log CFU / mL and 9.39 log CFU / mL, respectively, after 10 hours of culture. Similarly, when various nitrogen sources were added at 25 g / L, yeast extract was found to be most beneficial for cell proliferation, with the final viable cell count of the yeast extract group being 9.51 log CFU / mL after 10 hours of culture. In contrast, the group supplemented with isolated soy protein showed a relatively lower cell viability, with a final cell count of 8.54 log CFU / mL. These results indicate that the types of carbon and nitrogen sources in the growth medium have a significant impact on the proliferation of the CAB701 strain.

[0152] 3-1-2: Identification of Growth Factors of CAB701 Strain

[0153] Using a Plackett-Burman design, various factors were evaluated to determine their effects on the growth of the CAB701 strain (Fig. 8). Variables ranging from carbon sources to inorganic salts were adjusted to two levels, low (-1) and high (+1), as shown in Table 4 below. A total of 48 experiments were performed, and each condition was repeated twice to ensure the reliability of the results.

[0154] sauce independent variable Variable code Low(-1) High(+1) carbon source Sucrose X1 1 100 nitrogen source Yeast extract X2 5 100 etc Polysorbate 80 X3 0.1 2 Citric acid X4 0.5 10 sodium acetate X5 1 20 magnesium sulfate X6 0.1 2 Manganese sulfate X7 0.01 1 potassium phosphate X8 0.5 10

[0155] As shown in Figure 8, four components were identified in which the standardized effect exceeded the threshold of 2.023. These components were sodium acetate, citric acid, manganese sulfate, and yeast extract, which were found to have the greatest influence on the number of viable cells (log CFU / mL) in bacterial cultures.

[0156] 3-1-3: Badge Component Optimization

[0157] The culture medium components (yeast extract, citric acid, sodium acetate, and manganese sulfate) of the CAB701 strain were optimized using RSM (Response Surface Analysis) and ANN (Artificial Neural Network). The specific experiments were performed 90 times, including 30 basic experimental runs, each repeated 3 times. The results were presented as 3D and contour plots of the RSM showing the combined effect of the four variables on the number of viable cells of the strain (Fig. 9).

[0158] Looking at Figure 9, it can be seen that the optimal medium composition is to add 60 g / L (6%) yeast extract and 10 g / L (1%) citric acid, and not to add sodium acetate and / or manganese sulfate. The medium composition provided a predicted viable cell count of 10.37 log CFU / mL, which was close to the experimental value of 9.72 log CFU / mL with a moderate MSE of 0.419.

[0159] 3-1-4: Effect of Sucrose Concentration on Strain Growth

[0160] As a result of observing the growth of the CAB701 strain, the growth of the CAB701 strain was significantly reduced in the absence of sucrose, and the number of viable cells reached 8.95 log CFU / mL. Conversely, it was found that strain proliferation increased significantly when sucrose was included (Fig. 10).

[0161] Looking at Figure 10, the number of surviving cells was found to be 9.87 log CFU / mL at 1% sucrose concentration, 9.58 log CFU / mL at 2%, 9.87 log CFU / mL at 3%, 9.79 log CFU / mL at 4%, and 9.69 log CFU / mL at 5%, confirming that it is relatively consistent at various sucrose concentrations.

[0162] 3-1-5: Verification of the anti-obesity efficacy of the CAB701 strain in optimized media

[0163] In the above Test Examples 3-1-3 and 3-1-4, the optimal medium composition for the CAB701 strain was found to be MRS medium (YSC medium) containing 1% (w / v) sucrose, 6% (w / v) yeast extract, and 1% (w / v) citric acid.

[0164] The anti-obesity activity of the strain was verified by measuring lipid accumulation in 3T3-L1 adipocyte progenitor cells treated with the CAB701 strain using the Oil Red O staining method of Test Example 6-3 below (Fig. 11).

[0165] Looking at Figure 11, the untreated control (Negative control) showed an absorbance of 0.272 and a differentiation rate of 0%, indicating that lipid accumulation was negligible, whereas the fully differentiated positive control (Negative control) showed an absorbance of 1.189 and a differentiation rate of 100%. Additionally, 3T3-L1 adipocytes exposed to the CAB701 strain cultured in non-optimized MRS medium exhibited an absorbance of 0.579 and a differentiation rate of 33.52%. On the other hand, 3T3-L1 adipocytes exposed to the CAB701 strain cultured in the optimized medium (YSC medium) showed a significantly reduced absorbance of 0.411 and a differentiation rate of 15.14%.

[0166] 3-2: Optimization of Culture Conditions

[0167] The culture conditions (initial pH, temperature, and time) of the CAB701 strain were optimized using RSM (Response Surface Analysis) and ANN (Artificial Neural Network). Specific experiments were performed 20 times for each method. The results were presented as 3D and contour plots of RSM showing the binding effect on the number of viable cells of the strain (Fig. 12).

[0168] Looking at Figure 12, the optimal fermentation conditions were pH 7.37, temperature 34.4 ℃, and time 8.69 hours, and the predicted cell count was 9.94 log CFU / mL, which was in perfect agreement with the experimental value of 9.92 log CFU / mL (MSE 0.00).

[0169] 3-3: Sintering

[0170] In conclusion, the optimal medium composition for the CAB701 strain of the present invention, optimized using RSM and ANN, was found to be a medium comprising 1% (w / v) sucrose, 6% (w / v) yeast extract, and 1% (w / v) citric acid. When the CAB701 strain of the present invention is cultured in the above-optimized medium, strain growth is further enhanced and the anti-obesity activity of the strain is significantly improved compared to when cultured in conventional MRS medium (commercial MRS medium). Furthermore, the optimal culture conditions for the CAB701 strain of the present invention were found to be an initial pH of 7.37 and a temperature of 34.4 ℃. These culture conditions are essential for the mass production of the strain, are economical, and increase the probiotic yield.

[0172] < in vitro Functional Evaluation>

[0173] Example 4: Antioxidant activity - DPPH radical scavenging activity

[0174] The antioxidant activity of the CAB701 strain was measured using the DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich, St. Louis, MO, USA) radical scavenging assay. Standard curves were constructed using 0.0625, 0.125, 0.25, 0.5, 1, and 2 mM ascorbic acid, with 2 mM ascorbic acid used as the positive control and triple-distilled water used as the negative control.

[0175] A culture of the CAB701 strain, cultured in MRS broth at 37°C for 20 hours, was inoculated into the MRS broth to a concentration of 1% (v / v) and incubated at 37°C for 16 hours. After centrifuging the CAB701 strain culture at 17,000 rpm for 5 minutes, 50 μL of the culture supernatant or the control was transferred to a 1.5 mL tube. 0.1 mM DPPH diluted with methanol was used in the experiment. 950 μL of 0.1 mM DPPH was added to the 1.5 mL tube containing the CAB701 strain culture supernatant, and the mixture was reacted in the dark at room temperature for 30 minutes. The reacted mixture was centrifuged at 13,000 rpm for 1 minute, and 200 μL of the supernatant was dispensed into a 96-well plate. The absorbance at 517 nm was measured using a microplate reader (Epoch microplate reader, Biotek Instruments, Inc., Winooski, VT, USA). The DPPH radical scavenging activity was calculated using the formula below and converted into a relative value to the DPPH radical scavenging activity of the positive control, 2 mM ascorbic acid, and is shown in Table 5 below.

[0176] DPPH radical scavenging activity (%) = (1 - (absorbance of sample / absorbance of control)) × 100

[0177] division DPPH radical scavenging activity (%) CAB701 95.6±1.3 Lactobacillus rhamnosus GG 97.2±0.6

[0178] Looking at Table 5 above, it can be seen that the DPPH radical scavenging activity of the comparative strain Lactobacillus rhamnosus GG (LGG) is 97.2%, and the CAB701 strain isolated in the above example is 95.6%, showing high DPPH radical scavenging activity of over 90% for each.

[0180] Example 5: Immunostimulative activity

[0181] Experimental method

[0182] Cell lines and their culture conditions

[0183] Mouse macrophage cell line RAW 264.7 cells (KCLB40071) were purchased from the Korean Cell Line Bank and inoculated into DMEM (High-glucose Dulbecco's modified Eagle's medium; Gibco, Grand Island, NY, USA) containing 10% (v / v) fetal bovine serum (FBS, Gibco) and 1% (v / v) penicillin-streptomycin (P / S, Gibco). After incubation at 37°C in a 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA), cells were separated using a cell scraper (SPL, Pocheon, Korea).

[0184] Strain sample preparation

[0185] The CAB701 strain was cultured in MRS broth at 37°C for 16 hours, and 1% was inoculated into 10 mL of MRS broth and cultured at 37°C at 1×10 8 After culturing to a concentration of CFU / mL, the cell precipitate was harvested by centrifuging 5 times at 10,000 rpm for 5 minutes at 4 ℃. Subsequently, it was washed 3 times with DPBS (Dulbecco's phosphate buffered saline; Welgene, Gyeongsan, Korea). It was resuspended in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) P / S, and samples were prepared by diluting the sample to the desired MOI (Multiplicity of infection; number of viable lactic acid bacteria / number of RAW264.7 cells) based on the ratio of the number of macrophages to the number of CAB701 strain cells.

[0187] Experimental results

[0188] 5-1: Cytotoxicity of bacterial strains

[0189] We wanted to confirm the cytotoxicity against RAW 264.7 before measuring the immunomodulatory ability of the CAB701 strain.

[0190] First, 5×10 RAW 264.7 cells 4 100 μL of cells / well was seeded into 96-well plates and incubated for 20 hours at 37°C and 5% CO2. Using MOI 0 as the control, 100 μL of CAB701 strain samples with MOIs of 50, 100, and 200 were added to each well and incubated for 24 hours at 37°C and 5% CO2. Cytotoxicity was measured using the EZ-CYTOX kit (Daeillab, Korea). After removing the medium, 200 μL of DMEM supplemented with 10% FBS and 1% P / S and 20 μL of EZ-Cytox were added to each well and incubated for 30 minutes in a 37°C and 5% CO2 incubator. Cell-free wells treated in the same manner were used as blanks. To measure the viability of RAW264.7 cells, absorbance was measured at 450 nm using a microplate reader when the medium turned orange, and calculated using the following formula (Fig. 13). For the blank value, the absorbance of DMEM medium and Ez-Cytox added to wells that were not seeded with cells was used.

[0191] Cell viability (%) = [(Experimental group - blank) / (Control group + blank)] × 100

[0192] Looking at Figure 13, it can be seen that the CAB701 strain did not exhibit cytotoxicity at MOI concentrations of 200 or lower, and that the cell viability exceeded 150% even when treated at an MOI concentration of 200.

[0193] 5-2: Promoting NO production

[0194] NO is produced by macrophages or neutrophils, and an appropriate amount of NO secreted by activated immune cells acts as an immune signaler, activating immune cells to provide protection against external pathogens. By measuring the amount of NO produced by endotoxins such as cytokines and LPS, the control strain LGG ( L. rhamnosus We intended to compare the immune activity of the CAB701 strain against GG).

[0195] NO production of RAW 264.7 macrophages was measured using Griess reagent. RAW 264.7 cells were 5 × 10⁶ 5 500 μL was seeded into 96-well plates at a cell / mL density and incubated for 20 hours at 37 ℃ and 5% CO2. The CAB701 strain corresponding to an MOI of 100 (5×10⁶ 7 RAW 264.7 cells were treated with CFU, the positive control LPS (1 μg / mL), and the negative control MOI 0, respectively, and cultured for 24 hours. 100 μL of culture medium was placed in a 96-well plate, and 100 μL of an equal mixture of grease reagent A and grease reagent B was added. The mixture was reacted in the dark for 15 minutes, after which the absorbance was measured at 540 nm using a microplate reader. Standard curves for NO production and absorbance were constructed using samples in which 250 μM NaNO2 was diluted twofold with DMEM down to 7.815 μM, and the NO concentration was calculated by substituting the measured absorbance values ​​into these curves. With the NO production of the positive control treated with LPS 1 μg / mL set as 100%, the NO production of samples treated with the CAB701 strain is shown in Table 6 below.

[0196] division NO production (% of positive control) CAB701 86.3±6.8 Lactobacillus rhamnosus GG 18.5±3.2

[0197] Looking at Table 6 above, it was found that the CAB701 strain produced 86.32% of NO compared to LPS, showing a significantly higher amount of NO production compared to the NO production of the comparison strain LGG (18.47%). Through the above results, it can be seen that the CAB701 strain has excellent immunostimulatory activity.

[0198] 5-3: Increased expression of cytokine-related genes

[0199] The immune activity mechanism of the CAB701 strain was investigated by treating RAW 264.7 cells with the CAB701 strain, which showed the highest NO production in the evaluation of immune activity through the NO assay, and then confirming the relative mRNA expression levels of genes regulating cytokine production through real-time quantitative PCR (RT qPCR).

[0200] 1×10⁶ RAW 264.7 cells 6After seeding into a 6-well plate at a cells / well density, the cells were cultured for 20 hours at 37°C and 5% CO2. RAW 264.7 cells were treated with strain CAB701 at an MOI of 100, 1 μg / mL LPS as a positive control, and 0 μg / mL LPS as a negative control at 37°C and 5% CO2 for 24 hours. After culture, the cells were washed twice with DPBS. Cells were isolated using 1 mL of easy-BLUE™ Total RNA Extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea) and collected in a 1.5 mL tube. 200 μL of chloroform (Sigma-Aldrich) was added to each tube and mixed by vortexing for 10 seconds. After centrifugation at 13,000 rpm for 10 minutes at 4°C, 400 μL of the supernatant was transferred to a new 1.5 mL tube. 400 μL of 2-propanol (Sigma-Aldrich) was added and mixed, then reacted at room temperature for 10 minutes. After centrifugation at 13,000 rpm for 5 minutes at 4 ℃, the supernatant was removed, the RNA pellet was washed with 1 mL of 75% ethanol (Sigma-Aldrich), and centrifuged at 10,000 rpm for 5 minutes at 4 ℃. After drying the RNA pellet as much as possible, the RNA pellet was dissolved in 30 μL of RNA-compatible DEPC-DW (BIONEER, Daejeon, Korea).

[0201] After RNA extraction, the concentration and purity of the extracted RNA for cDNA synthesis were measured using Take3 Micro-Volume Plates (Epoch microplate reader, Biotek Instruments, Inc.). cDNA was synthesized using a PCR machine with CellScript™ cDNA Master Mix (Cellsafe, Suwon, Korea). The reaction Master Mix contains oligo(dT) primers, MIMLV reverse transcriptase (RIase), dNTPs, and ribonuclease inhibitors in optimized ratios, and the PCR conditions for cDNA synthesis are as shown in the following table. RT-qPCR was performed using GreenBlue™ 2X Green qPCR Master Mix (Cellsafe) with 500 ng of cDNA and primers added according to the manufacturer's instructions, utilizing a QuantStudio1 Real-Time PCR system (Thermo Fisher Scientific, Milan, Italy). Primer sequences are shown in Table 7, and RT-qPCR was performed according to Table 8. The GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene was used as a housekeeping gene for standardization, and the relative expression levels of cytokine mRNA were calculated using ΔΔ CT values ​​and are shown in Figure 14.

[0202] number gene Primer sequence Size (bp) 1 GAPDH F 5'- CAT GGC CTT CCG TGT TCC TAC -3' 122 R 5'- TCA GTG GGC CCT CAG ATG C -3' 2 COX-2 F 5′- CTC AGC CAT ACA GCA AAT CCT T -3′ 101 R 5′- GTC CGG GTA CAA TCG CAC TTA T -3′ 3 iNOS F 5′- CCA GCC TGC CCC TTC AAT -3′ 104 R 5′- ATC CTT CGG CCC ACT TCC T -3' 4 IL-1β F 5'- TGA CGG ACC CCA AAA GAT -3' 122 R 5'- GTG ATA CTG CCT GCC TGA AG -3' 5 IL-6 F 5′- CCG GAG AGG AGA CTT CAC AGA G -3′ 107 R 5′- TCA TTT CCA CGA TTT CCC AGA G -3′ 6 TNF-α F 5′- AGG CAC TCC CCC AAA AGA TG -3′ 122 R 5′- CAC CCC GAA GTT CAG TAG ACA GA-3′

[0203] step Temperature (°C) hour Cycle count Hold stage 50 2 min 1 95 2 min PCR stage Denaturation 95 15 sec 39 Annealing 60 30 sec Extension 72 15 sec Melting curve stage 95 15 sec 1 65 1 min 97 1 sec

[0204] As shown in Figure 14, the CAB701 strain of the present invention was found to have excellent immune-enhancing activity because it increases the expression of cytokine-related genes iNOS, COX-2, and TNF-α similarly to LPS.

[0205] 5-4: Increased expression of proteins related to the MAPK signaling pathway

[0206] We intended to investigate the mechanism of immune activity of the CAB701 strain by treating RAW 264.7 cells with the CAB701 strain, which showed high NO production in the evaluation of immune activity through the NO assay, and analyzing the expression levels of proteins related to the MAPKs signaling pathway using Western blot.

[0207] RAW 264.7 cells were treated with the CAB701 strain using the same method as the RNA isolation method for RT-qPCR described above, washed twice with DPBS (Welgene), and incubated for 30 minutes at 0°C with 130 μL of RIPA buffer (Cell Signaling Technology, Beverly, MA, USA) containing a 1% (v / v) protease inhibitor cocktail (GenDEPOT, Katy, TX, USA) and a 1% (v / v) phosphatase inhibitor cocktail (GenDEPOT). Cells in the wells were separated using a cell scraper, collected in a 1.5 mL tube, vortexed for 10 seconds, and incubated at 0°C for 5 seconds—a process repeated three times. After incubating at 0°C for 2 hours, the cell lysate was centrifuged at 17,000 rpm for 10 minutes at 4°C, and the supernatant was transferred to a new 1.5 mL tube for protein extraction. The concentration of the extracted protein was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).

[0208] 30 μg of extracted protein was separated by 12.5% ​​SDS-PAGE. For Western blotting, the separated protein was transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.) using a TE70X Semi-Dry Transfer Unit (Hoefer Inc.) at 45 mV for 2 hours. 3% (w / v) bovine serum albumin Fraction V (BSA, Roche), diluted with Tris-buffered saline (TBS-T, iNtRON Biotechnology) containing 0.5% (v / v) Tween 20 at room temperature, was used as the blocking buffer. The transferred membrane was blocked for 30 minutes with gentle stirring. The membrane was then gently stirred for 1 hour at room temperature in a primary antibody diluted 1:1000 in 3% BSA from TBS-T. p38, phospho-p38, ERK, phospho-ERK, JNK, phospho-JNK, and β-actin (Cell Signaling Technology) were used as primary antibodies. The membrane, washed four times with 6 mL TBS-T every 15 minutes, was gently stirred for 1 hour at room temperature with a secondary antibody diluted 1:1000 in 3% BSA of TBS-T. Anti-rabbit IgG (Cell Signaling Technology) was used as the secondary antibody. After reacting with the secondary antibody, the membrane was washed four times with 6 mL TBS-T every 15 minutes, then reacted with ECL solution (EzWestLumi plus, ATTO Corporation, Tokyo, Japan), and visualized using an Amersham™M imager 600 (Cytiva, Amersham, England).Protein bands were quantified using image J software (National Institutes of Health, Bethesda, MD, USA), and β-actin was used as a housekeeping protein for western blotting analysis. The protein expression level of the negative control was set to 100, and the relative expression level was calculated (Fig. 15).

[0209] Looking at Fig. 15, it can be seen that macrophages treated with the CAB701 strain of the present invention showed a significant increase in the expression levels of proteins related to the MAPKs signaling pathway, such as p-JNK / JNK (372%), p-p38 / p38 (126%), and p-ERK / ERK (107%), compared to the negative control group.

[0211] Example 6: Anti-obesity activity

[0212] 6-1: Pancreatic lipase enzyme inhibitory ability

[0213] To evaluate the body fat reduction ability of the CAB701 strain according to the present invention, pancreatic lipase enzyme inhibitory ability was investigated.

[0214] 50 mg of porcine pancreatic lipase type II (Sigma) was suspended in 10 mL of 10 mM Tris-HCl buffer adjusted to pH 8.0, vortexed for 15 minutes, and centrifuged at 4000 rpm at 18 °C for 10 minutes; the supernatant was used as the enzyme solution. As the substrate, p-nitrophenyl butyrate (Sigma-Aldrich (Schnelldorf, Germany)) dissolved in acetonitrile at 10 mM was used.

[0215] The culture supernatant of the CAB701 strain, cultured in MRS broth at 37°C for 20 hours, was obtained by centrifuging at 17,000 rpm for 5 minutes. Orlistat (Sigma-Aldrich), used as a positive control, was prepared by diluting it in DMSO to concentrations of 1,000, 500, and 250 μg / mL. Triple-distilled water was used as a negative control. A final reaction mixture consisting of 880 μL of pH 8.0 10 mM Tris-HCl buffer, 100 μL of enzyme solution, and 10 μL of the CAB701 strain culture supernatant or positive control was pre-incubated at 37°C for 5 minutes. After adding 10 μL of substrate solution and reacting at 37°C for 5 minutes, the maximum absorbance of p-nitrophenol was measured at 405 nm using a microplate reader. The pancreatic lipase inhibition rate was calculated using the following formula and is shown in Table 9 below.

[0216] Pancreatic lipase inhibition rate (%) = [(Negative control absorbance - Sample absorbance) / Negative control absorbance] × 100

[0217] division Pancreatic lipase inhibition rate (%) CAB701 28.87±4.8 1000 µg / mL Orlistat 28.55±6.2

[0218] Looking at Table 9 above, it was found that the pancreatic lipase inhibition rate of the CAB701 strain was 28.87%, which is almost identical to the inhibition rate of 1000 μg / mL Orlistat.

[0219] 6-2: Cytotoxicity

[0220] We intended to measure the viability of 3T3-L1 adipocyte precursor cells using the same method as RAW 264.7 cells, and specifically, 3Te-L1 cells at a rate of 1×10 4 Cells were inoculated at a cell density of cells / well, and CAB701 strain was treated at MOI 125, MOI 250, and MOI 500 concentrations, after which cytotoxicity was measured using Ez-cytox (Fig. 16).

[0221] Looking at Figure 16, the viability of 3T3-L1 cells treated with the LAB701 strain was found to be MOI 125 (95.8%), MOI 250 (96.7%), and MOI 500 (100.31%), confirming that there was no cytotoxicity.

[0222] 6-3: Inhibition of Adipocyte Differentiation

[0223] 3T3-L1 preadipocytes (ATCC CL-173) were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and inoculated into DMEM containing 10% BCS (Gibco) and 1% P / S, then cultured at 37°C in 5% CO2. For adipocyte differentiation, 3T3-L1 cells were placed in a 6-well plate at a density of 1 × 10⁶ 5 Cells were seeded at a density of cells / well and cultured for 2 additional days after reaching 100% confluent. The medium was changed to differentiation medium (MDI) composed of DMEM containing 10% fetal bovine serum (FBS; Corning Inc., Corning, NY, USA), 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich), 10 μM dexamethasone (Sigma-Aldrich), and 10 μg / mL insulin (Sigma-Aldrich), and cultured for 3 days. Subsequently, the medium was changed to insulin medium composed of DMEM containing 10% FBS and 10 μg / mL insulin, and cultured for 2 additional days. Then, on days 5 and 7, the medium was changed to DMEM containing 10% FBS. During the medium change, lactic acid bacteria were added at a concentration of MOI 500.

[0224] Differentiated 3T3-L1 cells were fixed with 10% formaldehyde solution for 20 minutes, then 0.3% oil red O solution (Sigma-Aldrich) was added to each well and stained in the dark for 15 minutes (Fig. 17a). After washing the cells twice with PBS, 1 mL of 100% 2-propanol was added to each well, and the absorbance was measured at 500 nm to indicate differentiated adipocytes (Fig. 17b). In this case, the negative control group consisted of 3T3-L1 cells not treated with MDI medium (differentiation medium), and the positive control group consisted of 3T3-L1 cells treated only with MDI medium (differentiation medium) without treatment with lactic acid bacteria.

[0225] Adipocyte differentiation rate (%) = [(Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control absorbance) ] × 100

[0226] Looking at Figure 17, the differentiation rate of adipocytes treated with the CAB701 strain was 38.53% compared to the positive control group, confirming that the differentiation rate of adipocytes decreased by more than 60%.

[0227] 6-4: Repression of expression of genes related to fat production

[0228] As a result of confirming the ability to inhibit adipocyte differentiation using the Oil red O staining method in Example 6-3 above, since the CAB701 strain possessed excellent ability to inhibit adipocyte differentiation, we further intended to analyze the expression of genes related to fat production.

[0229] 1×10⁶ 3T3-L1 cells 5Cells were seeded into 6-well plates at a density of cells / well, treated with lactic acid bacteria to achieve an MOI of 500, and cultured at 37°C in 5% CO2 to differentiate into mature adipocytes. RNA was extracted using the same method as in RAW 264.7 cells, and the expression levels of adipogenesis-related mRNA in 3T3-L1 cells were analyzed via RT-qPCR. The primer sequences and RT-qPCR conditions used are shown in Tables 10 and 11, respectively. The β-actin gene was standardized using the housekeeping gene, and the relative expression levels of adipogenesis-related mRNA were calculated using ΔΔCT values ​​and are shown in Figure 18.

[0230] number gene Primer sequence Size (bp) 7 β-Actin F 5'- GAG CGC AAG TAC TCT GTG TG -3' 97 R 5'- CGG ACT CAT CGT ACT CCT G -3' 8 PPARα F 5'- GTA CGG TGT GTA TGA AGC CAT CTT -3' 76 R 5'- GCC GTA CGC GAT CAG CAT -3' 9 PPARδ F 5'- GCC ATA TTC CCA GGC TGT C -3' 102 R 5'- CAG CAC AAG GGT CAT CTG TG -3' 10 CPT1 F 5'- GTG ACT GGT GGG AGG AAT AC -3' 83 R 5'- GAG CAT CTC CAT GGC GTA G -3' 11 SREBP-1c F 5'-ACG GAG CCA TGG ATT GCA CA -3' 278 R 5'- AAG GGT GCA GGT GTC ACC TT -3' 12 ACC F 5'- ATG GGC GGA ATG GTC TCT TTC -3' 148 R 5'- TGG GGA CCT TGT CTT CAT CAT -3' 13 LPL F 5'- CCA CAG CAG CAA GAC CTT C -3' 87 R 5'- AGG GCG GCC ACA AGT TTG -3' 14 FAS F 5'- TGC TCC CAG CTG CAG GC -3' 107 R 5'- GCC CGG TAG CTC TGG GTG TA -3' 15 Adiponectin F 5'- GAG ATG CAG GTC TTC TTG GTC -3' 105 R 5'- GCT CTC CTT TCC TGC CAG -3'

[0231] step Temperature (°C) hour Cycle count Hold stage 50 2 min 1 95 2 min PCR stage Denaturation 95 15 sec 35 Annealing 56 30 sec Extension 72 30 sec Melting curve stage 95 15 sec 1 65 1 min 97 1 sec

[0232] The aforementioned LPL (Lipoprotein lipase) plays a role in accumulating fat in body tissues, such as adipose tissue, by transporting fatty acids from lipoproteins to tissues. Additionally, enzymes related to fat synthesis include the FAS (fatty acid synthase) complex. Furthermore, PPARα (Peroxisome proliferator-activated receptor alpha) and CPT (carnitine palmitoyltransferase) are enzymes that promote fat oxidation, and an increase in the oxidation rate of fatty acids can serve as a mechanism for reducing body fat.

[0233] Looking at Figure 18, 3T3-L1 cells treated with the CAB701 strain showed significantly increased expression of PPARα, PPARδ, and CPT1 genes, which are enzymes that promote lipid oxidation, compared to the positive control group, while significantly decreased expression of LPL, adiponectin, and FAS genes, which are enzymes related to lipid accumulation and lipid synthesis, compared to the positive control group.

[0234] 6-5: Increased expression of lipid metabolism-related proteins

[0235] We intended to analyze the protein expression levels of lipid metabolism-related genes of the CAB701 strain according to the present invention using Western blot.

[0236] Specifically, 1×10⁶ 3T3-L1 cells 5 Cells were seeded into a 6-well plate at a concentration of cells / well, treated with lactic acid bacteria to achieve an MOI of 500, and then differentiated into mature adipocytes at 37°C and 5% CO2. Proteins were extracted in the same manner as in the RAW 264.7 cells of the above example, and the expression levels of proteins related to lipid metabolism in 3T3-L1 cells were analyzed by Western blot (Fig. 19). For reference, since FABPs (Fatty acid binding proteins) play a role in transporting fatty acids and other lipids through various cellular pathways, lipid metabolism may decrease if the expression level of FABPs decreases.

[0237] Looking at Figure 19, it was found that the expression of C / EBPα, FABP4, PPARγ, and perilipin-1 in 3T3-L1 cells treated with the CAB701 strain was significantly reduced compared to the positive control group, confirming that the CAB701 strain regulates lipid metabolism in 3T3-L1 cells, and thus it can be inferred that it will exhibit anti-obesity efficacy.

[0239] Example 7: Intestinal stability of the strain

[0240] Probiotic strains generally require acid resistance, bile resistance, pancreatic fluid resistance, and intestinal adhesion ability as they must survive the highly acidic conditions of the stomach and intestines on an empty stomach to exert sufficient physiological activity. Therefore, the intestinal stability of the CAB701 strain, selected as a functional strain related to immunity and anti-obesity, was analyzed.

[0241] 7-1: Acid resistance

[0242] Artificial gastric juice was prepared by adjusting 100 mM Glycine-HCl buffer to pH 2.5. Lactic acid bacteria seed culture, inoculated into MRS broth and cultured at 37°C for 20 hours, was inoculated into 4 mL of MRS broth at a concentration of 1% (v / v) and cultured at 37°C for 16 hours. The culture medium was centrifuged at 13,000 rpm for 1 minute, and the resulting cell precipitate was washed twice with 0.88% (w / v) NaCl (sterile physiological saline). The washed pellet was resuspended in 4 mL of the artificial gastric juice and cultured at 37°C. At the 2nd hour of culture, 1 mL of the sample was collected and centrifuged at 13,000 rpm for 1 minute to obtain a cell pellet, which was washed three times with sterile physiological saline and resuspended in 1 mL of sterile physiological saline. The samples were serially diluted with 9 mL of saline solution and plated on LAB Petrifilm. After incubation at 37°C for 1 to 2 days, the viable cell count was measured (Table 12). Acid resistance was calculated using the formula below, and the sample at 0 hours of incubation was used as a control.

[0243] Acid resistance (%) = [log(number of viable cells in sample) / log(number of viable cells in control)] × 100

[0244] CAB701 Acid resistance (%) LGG (comparison strain) Acid resistance (%) 0h (CFU / mL) 2h (CFU / mL) 0h (CFU / mL) 2h (CFU / mL) 4.60×10 8 8.90×10 3 45.6 9.30×10 8 1.90×10 7 81.2 4.20×10 8 1.92×10 4 49.6 8.30×10 8 4.50×10 7 85.8 3.10×10 8 1.07×10 4 47.5 1.01×10 9 3.00×10 7 83.0 average 47.6±2.0 average 83.3±2.3

[0245] In Table 12 above, the acid resistance of the CAB701 strain of the present invention was found to be 47.6%, and the acid resistance of the comparative strain Lactobacillus rhamnosus GG, known for its excellent acid resistance, was found to be 83.3%.

[0246] 7-2: Endobile

[0247] Artificial bile solution was prepared by adding 3% of a 10% Bacto oxgall (Becton, Dickinson and Company) solution filtered through a 0.22 μm membrane to sterilized MRS broth to achieve a final oxgall concentration of 0.3%. A CAB701 strain seed culture, inoculated into MRS broth and cultured at 37°C for 20 hours, was added to 5 mL of artificial bile solution to a concentration of 1% (v / v) and cultured at 37°C for 24 hours. 1 mL of the sample was collected, serially diluted with 9 mL of saline, plated onto LAB Petrifilm, and cultured at 37°C for 1–2 days to measure the viable cell count (Table 13). Bile tolerance was calculated using the formula below, and a sample cultured in MRS broth without added oxgall was used as a control.

[0248] Bile tolerance (%) = [log(number of viable cells in sample) / log(number of viable cells in control)] × 100

[0249] division Bile resistance (%) CAB701 88.2±0.8 LGG (comparison strain) 63.6±9.3

[0250] In Table 13 above, the bile resistance of the CAB701 strain of the present invention is 63.6%, confirming that it has excellent bile resistance.

[0251] 7-3: Intrapancreatic fluid

[0252] Artificial pancreatic fluid was prepared by adding 5% of a 10% pancreatin solution (0.85% NaCl, pH 8.0), filtered through a 0.22 μm membrane, to sterilized MRS broth to achieve a final pancreatin concentration of 0.5%. Lactic acid bacteria seed culture, inoculated into MRS broth and cultured at 37°C for 20 hours, was added to 5 mL of artificial pancreatic fluid to a concentration of 1% (v / v) and incubated at 37°C. At 3 hours of incubation, 1 mL of the sample was collected, serially diluted with 9 mL of saline, plated onto LAB Petrifilm, and incubated at 37°C for 1–2 days to measure the viable cell count (Table 14). Intrapancreatic fluid properties were calculated using the formula below, and the sample at 0 hours of incubation was used as a control.

[0253] Intrapancreatic fluid content (%) = [log(number of viable cells in sample) / log(number of viable cells in control)] × 100

[0254] CAB701 Intrapancreatic fluid (%) LGG (comparison strain) Intrapancreatic fluid (%) 0h (CFU / mL) 2h (CFU / mL) 0h (CFU / mL) 2h (CFU / mL) 1.38×10 9 2.00×10 9 101.8 1.30×10 9 3.14×10 9 104.2 1.21×10 9 1.94×10 9 102.3 1.73×10 9 3.87×10 9 103.8 1.37×10 9 1.98×10 9 101.8 1.12×10 9 2.74×10 9 104.3 average 101.9±0.3 average 104.1±0.3

[0255] In Table 14 above, the pancreatic fluid resistance of the CAB701 strain of the present invention was 101.9%, and the pancreatic fluid resistance of the LGG strain was 104.1%, confirming that both strains have high pancreatic fluid resistance exceeding 100%.

[0256] 7-4: Intestinal adhesion ability

[0257] Caco-2 cells (human colorectal adenocarcinoma cells, HTB-37™), which are intestinal cell-like cells, were purchased from ATCC and cultured in DMEM medium supplemented with 10% (v / v) FBS (Cornig) and 1% (v / v) P / S in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C under 5% CO2. 0.25% tripsin-EDTA (Gibco) was used to detach cells from the culture flask.

[0258] The above Caco-2 cells are 5×10 5500 μL of the cells were seeded into 24-well plates at a cell / well density and cultured for 3 days at 37°C under 5% CO2. The 24-well plates containing cultured Caco-2 cells were washed twice with DMEM medium supplemented with 500 μL of 10% FBS. Lactic acid bacteria were added at an MOI of 200 and cultured for 2 hours at 37°C under 5% CO2. After culture, each well was washed four times with 500 μL of DPBS to remove unattached strains. 200 μL of 0.25% trypsin-EDTA was dispensed into each well and cultured for 5 minutes at 37°C under 5% CO2. The detached Caco-2 cells and strains were collected in a 1.5 mL tube with 300 μL of DPBS, centrifuged at 13,000 rpm for 1 minute to harvest the cell pellet, and washed twice with 1 mL of DPBS. The pellet was resuspended in 1 mL of 0.1% (v / v) Triton X-100 diluted with DPBS and dispensed into 9 mL of saline. This was serially diluted, plated onto LAB Petrifilm, and incubated at 37 °C for 1–2 days to measure the viable cell count (Table 15). As a control, the above cells were suspended in DMEM medium supplemented with 10% FBS and incubated at 37 °C for 2 hours; subsequently, 500 μL of the culture solution was dispensed into 9.5 mL of saline. This was serially diluted, plated onto Petrifilm, and incubated at 37 °C for 24 hours to measure the viable cell count. Intestinal adhesion ability was calculated using the following formula.

[0259] Intestinal adhesion ability (%) = [log(number of viable cells in sample) / log(number of viable cells in control)] × 100

[0260] division Intestinal adhesion ability (%) CAB701 77.2±0.8 LGG (comparison strain) 75.5±9.3

[0261] In Table 15 above, the intestinal adhesion ability of each strain was LGG (75.5%) and CAB701 (77.2%), with no significant difference observed.

[0263] Example 8: Evaluation of the safety of the strain

[0264] 8-1: Antibiotic Resistance

[0265] Antibiotic resistance to the L. lactis CAB701 strain was evaluated using the E-test (bioMeurieux, La Balme-Les-Grottes, France). 100 μL of the CAB701 strain culture was adjusted to the turbidity of 3 McFarland standard (0.03% barium chloride, 5.97% sulfuric acid) and plated onto an antibiotic-free MRS agar plate. An E-test strip was placed in the center of the plate and incubated at 37°C for 72 hours. The number at the end of the strip indicated by the inhibition ring was defined as the minimum inhibitory concentration (MIC). The cut-off values ​​for ampicillin, vancomycin, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol followed the standards proposed by EFSA (Fig. 20 and Table 16).

[0266] Looking at Figure 20, the minimum inhibitory concentrations (MICs) of the CAB701 strain for ampicillin, vancomycin, gentamycin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol were found to be 0.094 μg / mL, 0.38 μg / mL, 3 μg / mL, 8 μg / mL, 16 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.094 μg / mL, and 1.5 μg / mL, respectively, and all were found to be below the cut-off value, confirming that it does not possess antibiotic resistance.

[0267] antibiotics Cut-off value (µg / mL) MIC (µg / mL) evaluation Ampicillin 2 0.094 allowance vancomycin 4 0.38 allowance Gentamicin 32 3 allowance kanamycin 64 8 allowance Streptomycin 2 16 allowance Erythromycin 1 0.125 allowance Clindamycin 1 0.25 allowance Tetracycline 4 0.094 allowance Chloramphenicol 8 1.5 allowance

[0268] 8-2: Whether D-lactic acid production activity is active

[0269] Lactic acid exists in two optical isomers: the L-form and the D-form. While the human body can metabolize L-lactic acid, its isomer, D-lactic acid, cannot. Probiotic strains possess DL-lactic acid racemase, an enzyme that converts L-lactic acid into D-lactic acid; therefore, if the activity of this enzyme is strong, D-lactate can accumulate in newborns, children, or patients with short bowel syndrome, potentially causing acidosis. Consequently, the WHO has recommended a daily intake of D-lactic acid of no more than 100 mg / kg body mass.

[0270] After culturing the CAB701 strain of the present invention in MRS broth at 37°C for 24 hours, the concentrations of D-lactic acid and L-lactic acid in the culture medium were measured using a D-lactic acid analysis kit (Roche).

[0271] division D-lactic acid (mM) L-lactic acid (mM) D-lactic acid / L-lactic acid CAB701 - 5.41 -

[0272] Looking at Table 17 above, it can be seen that the CAB701 strain of the present invention produces only L-lactic acid, so there is no problem with safety related to D-lactic acid production.

[0273] 8-3: Enzyme Activity - API ZYM test

[0274] Enzyme activity of the CAB701 strain was analyzed using the API ZYM kit (bioMerieux, Marcy l'Etoile, France).

[0275] Specifically, the CAB701 strain of the present invention was cultured for 24 hours and then diluted in 2 mL of sterile physiological saline to adjust the turbidity to McFarland 5–6. 5 mL of sterile physiological saline was poured into a culture box, and 65 μL of the turbidity-adjusted cells were dispensed onto an API strip and incubated at 37 ℃ for 4 hours. Subsequently, ZYM A and ZYM B were added drop by drop, and the color change was observed after 5 minutes (Fig. 21 and Table 18).

[0276] Looking at Figure 21 and Table 18, when ZYM A and ZYM B were dropped onto an API ZYM kit in which the CAB701 strain was cultured, it showed enzymatic activity against Acid Phospatase, Naphtol-AS-BI-Phosphohydrolase, and β-Glucosidase, but did not show activity against β-glucuronidase, an enzyme that converts benzopyrene into a carcinogenic substance, thus confirming that it is a safe strain.

[0277] division enzyme reaction 1 Control - 2 Alkaline phosphatase - 3 Esterase (C4) - 4 Esterase Lipase(C8) - 5 Lipase(C14) - 6 Leucine arylamdiase - 7 Valine arylamdiase - 8 Crystine arylamdiase - 9 Trypsin - 10 α-Chymotrypsin - 11 Acid phosphatase + 12 Naphtol-AS-Bl-phosphohydrolase + 13 α-Galactosidase - 14 β-Galactosidase - 15 β-Glucuronidase - 16 α-Glucosidase - 17 β-Glucosidase + 18 N-Acetyl-β-glucosaminidase - 19 α-Mannosidase - 20 α-Fucosidase -

[0278] Sintering

[0279] Among the 58 strains selected from 8 types of Jeju raw materials (carrot, prickly pear, broccoli, beet, cabbage, Hallabong, kale, and kohlrabi), the Lactococcus lactis subspecies lactis CAB701 strain derived from cabbage was finally selected, which corresponds to the strain designated by the Ministry of Food and Drug Safety as a health functional probiotic and possesses both anti-obesity and immune-enhancing activities.

[0280] The NO production-promoting activity of the selected CAB701 strain was found to be very high. In addition, the expression levels of cytokine-related genes (iNOS, COX-2, TNF-α, IL-1β, and IL-6) and proteins related to the MAPKs signaling pathway in RAW 264.7 cells of the CAB701 strain were examined, and it was confirmed that the immune-enhancing activity was excellent.

[0281] In addition, it was specifically confirmed that the above CAB701 strain has excellent pancreatic lipase enzyme inhibitory activity, and that it exhibits activity to inhibit adipocyte differentiation in 3T3-L1 cells, inhibits the expression of genes related to fat production, and increases the expression of proteins related to lipid metabolism.

[0282] In summary, it was confirmed that the CAB701 strain isolated and identified in the embodiments of the present invention has high NO production-promoting activity, increases the expression of cytokine-related genes (iNOS, COX-2, TNF-α, IL-1β, and IL-6) and proteins related to the MAPKs signaling pathway, exhibits excellent pancreatic lipase enzyme inhibitory activity, inhibits adipocyte differentiation in 3T3-L1 cells, suppresses the expression of genes related to fat production, and increases the expression of proteins related to lipid metabolism.

[0283] Accordingly, the inventors deposited the above strain with the Korean Culture Collection Center (KCCM) on June 16, 2023, and were assigned the following accession number.

[0284] Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis ) - Trustee No. KCCM13360P (also named “CAB701”)

[0286] <in vivo 기능성 평가>

[0287] Example 9: Anti-obesity activity

[0288] Experimental method

[0289] Test animals

[0290] Test animals were 5-week-old C57BL / 6 mouse models (male) obtained from Hana Bio (Korea, Gyeonggi-do). The animals were then quarantined and acclimatized for one week at the animal facility of Dongnam Chemical Research Institute (Animal Facility Registration No. 412) before the experiment. During rearing, the lighting time was set to 12 hours (07:00–19:00), and food and water were provided free of charge. The separation and treatment of experimental animal groups were designed and carried out as shown in Table 19 below. This study was conducted in accordance with the policies and regulations of the Institutional Animal Care and Use Committee (SEMI-23-003) of Dongnam Chemical Research Institute.

[0291] division number of animals administration Route of administration Obesity induction Normal 10 saline solution Oral administration (dosage 10 mL / kg) - (Jeong Sang-sik) Obesity control group 10 saline solution + (60 HFD) Test group (Sample) 10 CAB701 500 mg / kg + (60 HFD)

[0292] Sample administration

[0293] The CAB701 strain was dissolved in saline solution (0.9% NaCl) at a concentration of 500 mg / kg and administered orally for 6 weeks.

[0294] Obesity induction and test substance treatment

[0295] The normal group (hereinafter Normal group) was given free intake of the basic diet, AIN 93G. To induce obesity, all test groups except the normal group were given free intake of a 60% High fat diet (hereinafter HFD, 60% fat per kcal, Research Diets Inc., New Brunswick, NJ, USA) for 6 weeks, and the CAB701 strain sample of the present invention was simultaneously administered orally. Body weight, feed intake, and water intake were measured three times a week during the administration of the CAB701 strain sample.

[0296] Completion of test and collection of biological samples

[0297] Test animals treated with CAB701 strain samples and those with induced obesity were treated with CO2, and blood was collected from the inferior vena cava. The blood was placed in serum separate tubes (SSTs), left at room temperature for about 30 minutes, and then centrifuged at 3,500 rpm for 15 minutes to separate the serum. To compare fat content between groups, fat peripidural was excised and weighed.

[0298] Analysis of serum biochemical markers

[0299] The serum separated after blood collection was tested for lipid marker enzymes TG (triglyceride), TC (total cholesterol), HDL-C (high denesitiy lipoprotein cholesterol), and LDL-C (low denesitiy lipoprotein cholesterol) using an automated biochemical analyzer (7100, Hitachi, Japan).

[0300] Analysis of triglycerides in feces

[0301] To measure the triglyceride content of feces, extraction was performed by modifying the method of Folch et al. (1957). 0.2 g of dried feces was ground in a mortar and pestle, and 5 mL of chloroform:methanol (2:1 v / v) solution was added and extracted at 4 ℃ for 24 hours. The extract was centrifuged at 3000 rpm at 4 ℃ for 10 minutes, and the supernatant was taken, dried, dissolved in 0.5 mL of the same extraction solvent, dried, and dissolved in ethanol. Then, 0.2 mL was taken for triglyceride measurement to determine the triglyceride content in the feces.

[0302] Protein expression analysis using epididymal fat: Western blot

[0303] RIPA buffer was added to epididymal fat and homogenized using a mini-homogenizer. The supernatant was separated by centrifugation at 4°C, 12,000 rpm, for 20 minutes. The separated supernatant was quantified by the Bradford method, followed by separation by size using SDS-PAGE (Polyacrylamide Gel Electrophoresis). Proteins were transferred to a nitrocellulose membrane using a semi-dry transfer system (Biorad, USA) and incubated for 1 hour in a blocking buffer containing 5% skim milk. Primary antibodies consisting of FAS (Cat. No: sc-74540, Santacruz, USA), PPAR-γ (Cat. No: sc-7273, Santacruz, USA), and β-actin (Cat. No: sc-47778, Santacruz, USA) were administered at a ratio of 1:1000 and reacted at 4°C. After reacting with the membrane using a Western blot detection kit, the expression level of the band was observed using a Chemi-Doc (Biorad, USA) instrument and compared after correction with β-actin.

[0304] Histopathological analysis

[0305] Epididymal adipose tissue was fixed and prepared into paraffin blocks for sectioning. After deparaffinization and dehydration, H&E (Hematoxylin & Eosin) staining was performed. After mounting in mounting medium, images were taken at magnifications of 40x and 100x using a standard optical microscope (E600, Nikon, Japan), and the size of lipid globules was measured using the Image J program (Java-based image processing program, USA).

[0306] Statistical processing

[0307] Statistical tests were performed using the Statview (ver. 5.0.1) statistical program, and measurements were expressed as mean ± standard deviation. Statistical significance for each analysis item was determined by conducting an ANOVA test followed by a post-hoc test using Fisher's PLSD at the p<0.05 level.

[0309] Test results

[0310] 9-1: Weight Change and Obesity Induction

[0311] In the groups excluding the normal group, the test substance (CAB701 strain) was administered concurrently with HFD for 6 weeks. As a result of checking body weight changes, the obese control group (Control) fed HFD showed an increase in body weight compared to the normal group (Normal), while the test group (Sample) showed a significant decrease in body weight compared to the obese control group (Control) (Fig. 22 and Table 20).

[0312] division Initial body weight (g) Final weight (g) Normal 15.572±0.302 24.631±1.440 Obesity control group 15.499±0.640 34.912±2.542 * Test group (Sample) 15.486±0.592 29.623±2.501 #

[0313] 9-2: Adipose tissue weight

[0314] The weight of epididymal adipose tissue increased in all groups fed a high-fat diet compared to the normal group, and the weight of epididymal adipose tissue in the test group treated with the test substance (CAB701 strain) significantly decreased compared to the obese control group (Control) with p<0.05 (Fig. 22 and Table 21).

[0315] division number of animals Weight of epididymal adipose tissue (g) Normal 10 0.475±0.090 Obesity control group 10 2.436±0.406 * Test group (Sample) 10 1.267±0.081 #

[0316] 9-3: Analysis of Serum Biochemical Markers

[0317] Serum biochemical analysis confirmed that all serum biochemical indicators in the obesity control group, in which obesity was induced by HFD, increased significantly (p<0.05) compared to the normal group. Meanwhile, in the group treated with the test substance (CAB701 strain), it was confirmed that blood triglyceride, total cholesterol, and LDL-cholesterol concentrations, which are indicators of lipid metabolism, decreased significantly (p<0.05) compared to the obesity control group (control) (Fig. 23 and Table 22).

[0318] division number of animals Triglycerides (mg / dL) Total cholesterol (mg / dL) HDL cholesterol (mg / dL) LDL cholesterol (mg / dL) Normal 10 116.73±19.33 104.65±11.98 85.62±6.03 9.69±1.21 Obesity control group 10 157.99±22.85 * 219.17±14.40 * 115.31±3.35 * 20.40±1.96 * Test group (Sample) 10 89.67±22.16 # 179.58±14.95 # 120.35±2.95 14.88±1.36 #

[0319] 9-4: Analysis of Protein Expression of Adipogenesis Factors in Epididymal Adipose Tissue

[0320] In the obesity control group induced with HFD, the expression of FAS and PPAR-γ significantly increased compared to the normal group after 6 weeks of HFD administration, whereas in the group treated with the test substance (CAB701 strain), the expression of FAS and PPAR-γ significantly decreased (p<0.05) compared to the obesity control group (Figure 24 and Table 23).

[0321] division number of animals FAS / β-actin PPAR-γ / β-actin Normal 5 7.9±6.7 52.3±16.4 Obesity control group 5 100±11.1 * 100±7.6 * Test group (Sample) 5 67.0±14.1 # 73.9±10.0 #

[0322] 9-5: Changes in Fat Globule Size Through Histological Observation

[0323] Mouse epididymal adipose tissue was stained with H&E and observed under a light microscope to determine the size of the fat globules.

[0324] It was confirmed that the fat globule size of the obese control group, which was induced with obesity by HFD, significantly increased compared to the normal group after 6 weeks of HFD administration, whereas the fat globule size of the test group (Sample), which was treated with the test substance (CAB701 strain), significantly decreased compared to the obese control group (Figs. 25, 26 and Table 24).

[0325] division number of animals Gypsum size (units) 2 ) Normal 10 5,548.4±561.9 Obesity control group 10 26,809.3±2909.3 * Test group (Sample) 10 18,816.5±1867.5 #

[0326] 9-7: Sintering

[0327] It was confirmed that administering the CAB701 strain of the present invention has the effect of inhibiting body fat synthesis. Specifically, the test group administered the CAB701 strain of the present invention showed a significant decrease in body weight and epididymal fat weight compared to the obese control group, a significant decrease in blood triglyceride, total cholesterol, and LDL-cholesterol concentrations, a significant decrease in the expression of FAS and PPAR-γ proteins known as fat synthesis factors, and a significant decrease in the size of fat globules in the epididymal fat tissue.

[0329] Clinical Trial

[0330] Example 10: Anti-obesity effect

[0331] Experimental method

[0332] Selection of test subjects

[0333] 100 healthy individuals aged 19 to 75 years with a body mass index (BMI) of 23 to 30 kg / m² were selected as subjects (50 in the test group and 50 in the control group).

[0334] Hard capsule manufacturing

[0335] For the test group, 10×10 CAB701 strains of the present invention 10 CFU / g, 3 mg of crystalline cellulose, and 14.8 mg of lactose were mixed and filled into a gelatin capsule, and then a capsule (400 mg) was prepared according to a conventional hard capsule manufacturing method. In addition, for the control group, a capsule was prepared in the same manner as above, but using maltodextrin powder instead of the CAB701 strain.

[0336] Test method

[0337] 100 subjects were instructed to eat the same diet as much as possible every day during the test period. Among them, 50 subjects in the test group were instructed to take one capsule containing the above-mentioned CAB701 strain once a day with water after a meal for 12 weeks (84 days). In addition, 50 subjects in the control group were instructed to take the above-mentioned capsule containing the above-mentioned maltodextrin powder in the same manner.

[0338] Statistical processing

[0339] Statistical tests were performed using the Statview (ver. 5.0.1) statistical program, and measurements were expressed as mean ± standard deviation. Statistical significance for each analysis item was determined by conducting an ANOVA test followed by a post-hoc test using Fisher's PLSD at the p<0.05 level.

[0341] Test results

[0342] 10-1: Anti-obesity effect

[0343] After the subjects completed 12 weeks of intake, DEXA body fat mass, body mass index (BMI), body weight, and waist circumference were measured and are shown in Table 25 below. At this time, DEXA body fat mass was measured using DEXA (Dual Energy X-Ray Absorptimetry).

[0344] division control group Test group DEXA body fat mass (kg) 0 weeks 26.76±3.65 26.38±3.78 12 weeks elapsed 27.46±3.93 * 24.57±3.68 ## Change amount 0.70±0.91 (2.61%) -1.81±2.28 (-6.86%) DEXA Body Fat Percentage (%) 0 weeks 38.99±5.42 40.81±4.90 12 weeks elapsed 39.76±5.53 * 38.47±4.90 ## Change amount 0.76±1.13 (1.97%) -2.34±1.68 (-5.73%) Body Mass Index (BMI, kg / m²) 0 weeks 25.39±2.01 25.13±1.66 12 weeks elapsed 25.80±2.04 * 24.76±1.93 # Change amount 0.41±0.45 (1.61%) -0.37±0.80 (-1.48%) Body weight (kg) 0 weeks 69.30±9.23 64.33±6.63 12 weeks elapsed 70.91±9.60 * 63.06±7.10 # Change amount 1.61±1.31 (2.65%) -1.27±2.13 (-1.97%) Waist circumference (cm) 0 weeks 84.88±7.39 81.55±5.49 12 weeks elapsed 84.21±7.52 79.35±5.64 # Change amount -0.67±3.43 -2.20±3.45 (-2.7%) Blood triglycerides (mg / dL) 0 weeks 102.63±39.56 116.72±91.32 12 weeks elapsed 114.78±52.13 * 109.51±54.52 # Change amount 12.15±48.32 (11.84%) -7.21±68.76 (-6.18%) Blood HDL (mg / dL) 0 weeks 60.04±10.78 62.19±13.36 12 weeks elapsed 59.46±12.42 62.94±13.34 Change amount -0.59±9.03 0.74±9.06 Blood LDL (mg / dL) 0 weeks 112.30±36.14 116.21±29.13 12 weeks elapsed 107.24±28.65 112.72±29.88 Change amount -5.07±32.43 -3.49±17.83 Total blood cholesterol (mg / dL) 0 weeks 194.41±41.60 205.26±37.02 12 weeks elapsed 190.89±33.32 199.11±33.24 Change amount -3.52±35.10 -6.15±26.06 FFA (μmol / L) 0 weeks 405.80±206.60 468.91±238.83 12 weeks elapsed 470.48±201.04 * 518.74±230.39 # Change amount 64.67±153.14 (15.94%) 49.83±273.47 (10.63%) Adiponectin (pg / mL) 0 weeks 5,784±3,324 5,376±3,253 12 weeks elapsed 4,942±2,758 * 5,955±3,658 # Change amount -842±2,404 (-14.56%) 479±2,432 (10.77%)

[0345] Looking at Table 25 above, it can be seen that the test group that consumed capsules containing the CAB701 strain according to the present invention showed a significant decrease in body fat mass and body fat percentage. In addition, it can be seen that the test group that consumed capsules containing the CAB701 strain according to the present invention showed a significant decrease in body mass index, body weight, waist circumference, and blood triglyceride content, and a significant increase in blood adiponectin content.

[0346] 10-2: Sintering

[0347] It was confirmed that consuming the CAB701 strain of the present invention has the effect of reducing body fat. Specifically, the test group administered the CAB701 strain of the present invention showed a significant decrease in body mass index, body weight, waist circumference, and blood triglycerides compared to the control group, and a significant decrease in blood triglyceride, total cholesterol, and LDL-cholesterol concentrations, and a significant increase in blood adiponectin content.

[0349] Example 11: Preparation of Coconut Fermented Milk Using Strain and Fermentation Characteristics

[0350] 11-1: Preparation of Fermented Coconut Milk

[0351] (1) CAB701 strains were inoculated into MRS broth and cultured at 37°C. The lactic acid bacteria culture was then centrifuged (13,000 rpm, 1 min, 4°C) to harvest the cells. The cell precipitate was washed three times with 0.85% (w / v) NaCl (sterile physiological saline) and then suspended in 1 mL of sterile physiological saline (the concentration of the suspension was 1×10⁻⁶ 8 It is CFU / mL).

[0352] (2) 1% (v / v) of the suspended strain was inoculated into 3 mL of coconut milk with a solid content of 8% by weight and fermented at 37°C for 16 hours to produce coconut fermented milk.

[0353] 11-2: Measurement of Viable Cell Count and pH of Fermented Coconut Milk

[0354] After measuring the pH of the coconut fermented milk prepared in Example 11-1, it was serially diluted with sterile physiological saline and plated on a Petrifilm™ lactic acid bacteria count plate (3M Company, St. Paul, MN, USA), and cultured for 1 to 2 days to check the viable cell count.

[0355] division LGG CAB701 Viable cell count (log CFU / mL) 8.85±0.18 9.09±0.02 pH 3.57±0.06 4.15±0.02

[0356] Looking at Table 26 above, the pH of the coconut milk fermented with LGG was found to be low at 3.57, whereas that fermented with the CAB701 strain showed a pH between 4.1 and 4.2. In addition, the viable cell counts of the coconut milk fermented with both LGG and CAB701 strains were approximately 10 9 It was found to be CFU / mL.

[0357] 11-3: Quality Characteristics of Fermented Coconut Milk

[0358] 11-3-1: Number of lactic acid bacteria by optimal acidity

[0359] The optimal acidity of vegan fermented milk is about 2.5.

[0360] The fermentation end point of the coconut fermented milk prepared in Example 11-1 was determined based on the appropriate acidity, and the number of lactic acid bacteria in the product at the fermentation end point when the appropriate acidity was reached was measured and shown in Table 27 below. As a comparative example, the procedure was carried out in the same manner as in Example 10-1, but coconut fermented milk was prepared by fermenting it with commercial strains Y1 and Y2 (vegan certified 'vegan fermented milk starter') which are available for use in the production of fermented milk, respectively, instead of the CAB701 strain.

[0361] Sando Number of lactic acid bacteria (1×10 6 CFU / mL) CAB701 Y1 Y2 2.65 156 160 102 2.5 280 302 189

[0362] Looking at Table 27 above, it can be seen that the number of lactic acid bacteria is approximately twice that of fermented milk with an acidity of 2.65 when the acidity is 2.5. In addition, the number of lactic acid bacteria in the fermented milk fermented with Y1 was the highest, and the fermented milk fermented with the CAB701 strain of the present invention also showed a similar level of lactic acid bacteria. Meanwhile, the fermented milk fermented with Y2 met the standards for fermented milk but was found to have a relatively low number of lactic acid bacteria.

[0363] 11-3-2: Number of lactic acid bacteria according to storage period

[0364] Lactic acid bacteria starters used in the production of fermented milk are known to be the most influential factor affecting product quality, such as physicochemical characteristics and the number of lactic acid bacteria. Accordingly, changes in the number of lactic acid bacteria were observed while storing coconut fermented milk fermented using the CAB701 strain of the present invention and commercial strains Y1 and Y2 at 4°C, and the results are shown in Table 28 below.

[0365] Analysis period Number of lactic acid bacteria (1×10 6 CFU / mL) CAB701 Y1 Y2 On the day of manufacture 148 132 127 7 days elapsed since manufacture (intermediate storage day) 134 95 90 14 days after manufacture (expiration date) 92 61 41 16 days have passed since manufacturing (2 days after expiration) 76 26 18

[0366] Referring to Table 28 above, it was found that the coconut fermented milk fermented with the CAB701 strain of the present invention generally had a higher number of viable lactic acid bacteria compared to coconut fermented milk fermented using commercial strains (Y1 and Y2 strains), and it was confirmed that a high number of viable lactic acid bacteria was maintained even after the expiration of the shelf life. Meanwhile, within the shelf life (14 days after manufacturing), the number of lactic acid bacteria in the above fermented milk products exceeded the legal standard (1 × 10⁻⁶). 6 It was found to meet the CFU / mL requirement.

[0368] From the above results, it can be seen that the CAB701 strain of the present invention is useful for the production of vegan fermented milk, as the decrease in the number of viable lactic acid bacteria over the storage period is significantly slower in vegan fermented milk produced using conventional vegan fermented milk starter cultures.

[0370] Although the present invention has been described as a preferred embodiment mentioned above, various modifications and variations are possible without departing from the essence and scope of the invention. Furthermore, the appended claims include such modifications and variations that fall within the essence of the invention.

[0372] Depository Name: Korean Culture Collection Center (KCCM) Trustee Number: KCCM13360P Date of Deposit: 2023-06-16

Claims

Claim 1 Lactococcus lactis subspecies lactis of accession number KCCM13360P having simultaneous anti-obesity and immunostimulatory activities ( Lactococcus lactis subsp. lactis ) CAB701 strain. Claim 2 The Lactococcus lactis subspecies lactis CAB701 strain according to claim 1, characterized in that the strain has acid resistance to pH 2-3, bile resistance to 0.1-1% bile acid, pancreatic fluid resistance to 0.1-1% pancreatic fluid, and intestinal adhesion ability. Claim 3 In claim 1, the strain is characterized by having lipase enzyme inhibitory activity, Lactococcus lactis subspecies lactis CAB701 strain. Claim 4 In claim 1, the strain is characterized by promoting the production of nitric oxide (NO), an immune indicator, Lactococcus lactis subspecies lactis CAB701 strain. Claim 5 The Lactococcus lactis subspecies lactis CAB701 strain according to claim 1, characterized in that the strain comprises the 16S rRNA nucleotide sequence represented by SEQ ID NO.

1. Claim 6 Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis A food composition for anti-obesity or immune enhancement comprising as active ingredients the CAB701 strain (accession number: KCCM13360P), a culture of said strain, a supernatant of said culture, a concentrate of said culture, a dried product of said culture, a pulverized product of said strain, or a fermented product of said strain. Claim 7 In claim 6, the above composition is a food composition for anti-obesity or immune enhancement characterized by promoting the production of nitric oxide (NO), an immune indicator. Claim 8 In claim 6, the above composition is a food composition for anti-obesity or immune enhancement characterized by increasing the expression of immune indicator genes such as TNF-α, IL-6, IL-1β, COX-2, or iNOS. Claim 9 In claim 6, the above composition is a food composition for anti-obesity or immune enhancement characterized by inhibiting lipase activity. Claim 10 In claim 6, the above composition is characterized as being for the prevention or improvement of diseases caused by a decline in immune function, and is a food composition for anti-obesity or immune enhancement. Claim 11 A food composition for anti-obesity or immune enhancement according to claim 10, characterized in that the disease caused by the decrease in immune function is one or more selected from infectious diseases, allergic diseases, and chronic fatigue. Claim 12 Lactococcus lactis subspecies lactis ( Lactococcus lactis subsp. lactis A vegan fermented milk for anti-obesity or immune enhancement comprising the CAB701 strain (accession number: KCCM13360P), a culture of the said strain, a supernatant of the said culture, a concentrate of the said culture, a dried product of the said culture, a mashed product of the said strain, or a fermented product of the said strain. Claim 13 (1) Lactococcus lactis subspecies lactis in plant-based milk substitutes ( Lactococcus lactis subsp. lactis A method for producing vegan fermented milk for anti-obesity or immune enhancement, comprising: (1) a step of inoculating a CAB701 strain (accession number: KCCM13360P) at a concentration of 0.2 to 3% (v / v); and (2) a step of fermenting the plant-based alternative milk inoculated with the strain at 35 to 39°C for 12 to 24 hours to achieve a pH of 3.5 to 4.

5. Claim 14 A method for producing vegan fermented milk for anti-obesity or immune enhancement according to claim 13, characterized in that the plant-based alternative milk is an alternative milk produced from one or more plant-based raw materials selected from coconut, soy, almond, oat, and rice. Claim 15 A method for producing vegan fermented milk for anti-obesity or immune enhancement according to claim 13, characterized in that the plant-based alternative milk has a solid content of 4 to 15 weight%. Claim 16 In Clause 13, the above vegan fermented milk has a viable cell count of 130×10 immediately after production. 6 CFU / mL or higher, and when stored at 1 to 8 ℃, the viable cell count is 70×10 up to 14 days after manufacture 6 A method for producing vegan fermented milk for anti-obesity or immune enhancement, characterized by maintaining CFU / mL or higher.

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