KR101010914B1 - Probiotic Lactobacillus plantarum having anti-obesity and brain function improvement activity - Google Patents

Abstract

The present invention relates to Lactobacillus plantarum NUC-LG42 ( lactobacillus plantarum NUC-LG42 ) lactic acid bacteria isolated from Fluke, and the strain has physiologically active functions such as weight loss and brain function improvement. More specifically, the present invention relates to lactic acid bacteria having a large number of physiologically active functions, such as weight, fat gain, blood lipid and carnitine changes, leptin and insulin content changes, liver tissue mRNA changes, and memory enhancement.

Lactobacillus Plantanium, Weight Loss, Brain Function Improvement

Description

Probiotic Lactobacillus plantarum having anti-obesity and brain function improvement activity}

The present invention is isolated Lactobacillus Plantan ( Lactobacillus) plantarum NUC-LG42 ) It is related to lactic acid bacteria, and this strain has physiologically active functions such as weight loss and brain function improvement. More specifically, the present invention relates to lactic acid bacteria having a large number of physiologically active functions, such as weight, fat gain, blood lipid and carnitine changes, leptin and insulin content changes, liver tissue mRNA changes, and memory enhancement.

The rapid change in social structure due to the development of industrial society and the improvement of economic level has an effect on dietary life regardless of individuals, groups, ages and genders. Especially, malnutrition due to inadequate eating habits causes diabetes, hypertension, heart disease, The incidence of various chronic degenerative diseases such as atherosclerosis, obesity and cancer is increasing. In particular, it is estimated that dementia affects about 10% of people over 65 years of age and about 50% of people over 80 years of age, and is expected to increase rapidly as the elderly population rapidly increases in the 21st century. Recently, efforts have been made to establish various therapeutic strategies and develop effective drugs that improve and improve the cognitive and learning functions deteriorated due to dementia.However, the memory improving drugs developed to date include acetylcholine synthetic precursors, acetyl Although there are anticholinergic inhibitors, the effects are temporary, mild, and seriously controversial due to serious side effects and toxicity.

Lactic acid bacteria (lactic acid bacteria) has been the most useful bacteria in humans for a long time, especially in fermented dairy products. Lactic acid bacteria have been reported to improve the absorption of nutrients, alleviate lactose intolerance, improve intestinal motility and have anticancer effects.

However, lactic acid bacteria-related technologies having excellent effects related to prevention of obesity and improvement of brain function have not appeared. For example, Korean Patent Publication No. 2004-0027180 (Lactobacillus fermented dairy product having a prophylactic and therapeutic effect on obesity or diabetes and its manufacturing method) relates to a lactic acid bacterium fermented dairy product having a prophylactic and therapeutic effect on obesity or diabetes and a method of manufacturing the same. More specifically, Lactobacillus ruteri PRD202 (KCTC-10301BP), and also dietary fiber, soy protein, soy dietary fiber, hibiscus, green tea extract, garcinia cambogia, glucomannan, red ginseng, kelp, polydextrose, seaweed, tea powder Although it describes a fermented dairy product having a prophylactic and therapeutic effect of obesity and diabetes, such as cheese, buttermilk, yogurt, whey, and the like, which further comprises at least one auxiliary ingredient selected from Leo Rexal. Is not satisfactory.

An object of the present invention is to provide a food composition containing the same and lactic acid bacteria having a physiologically active function, such as obesity, improving brain function.

In order to achieve the above object, Lactobacillus Plantan ( Lactobacillus) excellent in physiological activity such as obesity, brain function improvement plantarum NUC-LG42 KCCM 10940P) Provide strains.

In addition, according to another embodiment of the present invention, it was confirmed that the Lactobacillus plantantan of the present invention can be used as a starter for preparing yogurt in the production of milk and soy milk yogurt. In addition, Lactobacillus plantarum NUC-LG42 KCCM 10940P according to the present invention can be used as a fermentation strain in a variety of fermentation products that Lactobacillus is used because the genus belongs to lactic acid bacteria as Lactobacillus. Moreover, it can also be used as a probiotic lactic acid bacterium as a live microbe. It can also be used as an animal feed additive.

Lactobacillus of the present invention ( Lactobacillus plantarum NUC-LG42 KCCM 10940P) Lactobacillus has been shown to reduce body weight and fat, improve plasma and liver lipids and carnitine, reduce blood leptin, insulin levels and improve memory.

The present invention relates to a probiotic lactobacillus Lactobacillus plantarum NUC-LG42 KCCM 10940P having physiologically active functions such as weight loss and brain function improvement.

Through the following examples and experimental examples will be described in more detail the characteristics and functions of the strain. However, these Examples are only for illustrating the present invention specifically, and the scope of the present invention is not limited by these Examples.

Experimental Example 1): Isolation and Identification of Lactobacillus Planta Room NUC-LG42 KCCM 10940P

Useful strains from traditional flounder (LG) were isolated in MRS (Difco, USA) medium by Minimum bactericidal concentration (MBC) Method and incubated at 30 ° C for 16-18 hours. Colonies grown on MRS solid medium were separated and inoculated in MRS liquid medium and incubated again at 37 ° C. for 16-18 hours. The isolated lactic acid bacteria were added to the culture solution to 20% glycerol and stored at -70 ℃.

As a result, it was identified as Lactobacillus plantarum systematically through the biochemical characteristics and 16s rDNA sequencing.

1 is an optical microscope (× 800) of the Lactobacillus plantarum NUC-LG42 KCCM 10940P according to the present invention. 2 is a sugar use characteristic result of biochemical test, assimilation test and fermentation test using API KIT. Figure 3 is a partial sequencing (about 1.5kbp) result of 16s rDNA, Figure 4 is a diagram showing a pyrogenic tree of the strain.

In the following Examples will be described the characteristics of the specific strain and its efficacy.

The experimental results of all examples were expressed as mean ± standard deviation (SD), and the statistical significance test between each group was t-test at p <0.05 level using Statistical Package for Social Science (SPSS) version 12.0.1. And Duncan test.

Example 1 > Lactobacillus plantarum NUC - LG42  Weight when ingested, Dietary Intake  Changes in fat and fat

The animals used in the experiment were C57BL / 6J mice (4wks, ♂, n = 40), which induced obesity and hyperglycemia with a high fat diet. Adapted to the environment by feeding the Diet Diet (D12450B) for 1 week, then, according to the randomized block design, 4 groups, ie, NDC, HDC, and high-fat diets. + Lactic acid bacteria high concentration (HD-LG42H, LG42 1x10 ⁹ ), high fat diet + Lactic acid bacteria low concentration (HD-LG42L, LG42 1x10 ⁷ ) formula was divided into oral administration. The temperature of the feeding room was 23 ± 2 ℃, the humidity was maintained at 70 ± 2%, and the contrast was adjusted every 12 hours. The experimental diet was purchased by Research Diet (USA) D12492.

In the present invention, there was no significant difference in the initial body weight, and after 12 weeks of LG42 lactobacillus administration, although there was no significant difference in dietary intake, body weight was significantly decreased in the LG42 administration group. (Figure 5. Body weight and appearance of experimental mice)

Subsequently, the fats around the epididymis of the experimental animals ingested for 12 weeks for each diet group were measured, and the fat mass around the epididymis was measured. Showed high fat mass. The LG42 lactobacillus group showed a lower tendency, especially the HD-LG42H group was significantly lower than the HDC group. (Figure 6. Epididymal fat in mice).

As a result of measuring the back fat of the test animals, the H-C group induced by obesity due to the high fat diet showed significantly higher fat mass than the N-C group. The backfat weight of the LG 42 lactobacillus group was significantly lower than that of HDC. (Figure 7. Back fat (g) of animals in mice).

Example 2 > Lactobacillus plantarum NUC - LG42  Blood intake and Liver  Lipid change

For the experimental animals ingested 12 weeks for each diet group of Example 1, Folch et al. (Biol. Chem. 226 (1): 497- to analyze lipids of liver tissues 12 weeks after LG42 lactic acid bacteria administration). Pretreatment was carried out according to the method of 509). Total cholesterol (TC), Triglycerides (TG) and HDL-cholesterol (HDL-c) were measured using a commercially available kit (Asan Pharm. Co., LTD., Korea).

Increased serum triglycerides due to the high fat diet were significantly lower in the LG42 lactobacillus group than HDC. Serum total cholesterol level was significantly higher in the LG42 lactobacillus group. This is because the amount of HDL-cholesterol in total cholesterol tended to increase in HDC. Hepatic triglycerides were lowered in the HD-LG42H group, similar to NDC, and there was no significant difference in total cholesterol. TABLE 1 Lipid content in plasma and liver.

Control LG42 Lactobacillus NDC HDC HD-LG42H HD-LG42L Serum (mg / ㎗) TC 199.3 ± 31.1 ^c 319.1 ± 30.5 ^b 395.9 ± 32.5 ^a 390.5 ± 25.1 ^a TG 148.3 ± 28.9 ^b 186.4 ± 27.9 ^a 151.0 ± 14.3 ^b 159.6 ± 12.4 ^b HDL-c 113.3 ± 24.8 ^c 168.7 ± 33.3 ^ab 187.9 ± 27.0 ^a 135 ± 30.5 ^bc HDL-c / TG 56.8 ± 0.8 ^a 24.0 ± 1.1 ^b 47.5 ± 0.8 ^a 34.7 ± 1.2 ^ab Liver (mg / g) TC 0.16 ± 0.02 0.16 ± 0.02 0.17 ± 0.03 0.17 ± 0.02 TG 2.55 ± 0.44 ^b 5.02 ± 1.04 ^a 2.77 ± 0.44 ^b 4.08 ± 0.76 ^ab

Values are expressed as mean ± SD.

Different superscripts in the same column indicate significant differences between groups at p <0.05 by Duncan's multiple comparison test.

Example 3 > Lactobacillus plantarum NUC - LG42  Ingestion of plasma and liver Carnitine  Content investigation

Liver tissue was homogenized with 0.3M perchloric acid (PCA) and centrifuged. Three fractions of carnitine, namely nonesterified carnitine (NEC), acid soluble acylcarnitine (ASAC) and acid insoluble acylcarnitine (AIAC), modified the method of Cederblad and Lindstedt (Clin. Chim. Acta. 37: 235-243) using isotopes. Carnitine in blood and liver was measured using the method of Sachan et al. (Am. J. Clin. Nutr. 39 (5): 738-744).

Serum NEC and AIAC contents were significantly higher in HD-LG42H group than in HD group, and ASAC and total carnitine contents were significantly higher in all groups treated with LG42 lactobacillus. NEC, ASAC and total carnitine contents in liver were significantly higher in all LG42 lactobacillus groups compared to HD group (Table 2 plasma and liver carnitine contents). This may be due to the change in the concentration of carnitine associated with fatty acid oxidation due to the administration of LG42 lactic acid bacteria.

Control LG42 Lactobacillus ND HD HD-LG42H HD-LG42L Serum ( nmol / 100 μl) NEC 0.47 ± 0.08 ^b 0.45 ± 0.11 ^b 0.67 ± 0.12 ^a 0.51 ± 0.10 ^b ASAC 1.25 ± 0.20 ^a 0.95 ± 0.21 ^b 1.18 ± 0.12 ^a 1.12 ± 0.24 ^a AIAC 0.22 ± 0.04 ^b 0.23 ± 0.02 ^b 0.28 ± 0.06 ^a 0.24 ± 0.02 ^ab Total carnitine 1.88 ± 0.24 ^a 1.54 ± 0.17 ^b 2.04 ± 0.25 ^a 1.92 ± 0.24 ^a Liver ( nmol / 100mg wet weight ) NEC 11.26 ± 2.02 ^a 7.69 ± 1.22 ^b 11.93 ± 3.05 ^a 11.60 ± 1.45 ^a ASAC 6.88 ± 1.39 ^a 4.20 ± 1.23 ^b 6.88 ± 1.89 ^a 6.88 ± 1.02 ^a AIAC 0.05 ± 0.03 0.02 ± 0.02 0.04 ± 0.01 0.02 ± 0.01 Total carnitine 18.87 ± 3.36 ^a 11.59 ± 2.36 ^b 17.91 ± 4.96 ^a 18.49 ± 2.39 ^a

Values are expressed as mean ± SD.

Different superscripts in the same column indicate significant differences between groups at p <0.05 by Duncan's multiple comparison test.

Example 4 > Lactobacillus plantarum NUC - LG42  Ingestion With leptin  Insulin content changes

Immunoassay kit (R&D system, USA)를 이용하여 ELISA(enzyme-linked immunosorbent assay)로 측정하였다.For the experimental animals ingested for 12 weeks for each dietary group of Example 1, blood was collected from the arrhythmia of the rat's neck, and plasma was separated to measure blood leptin and insulin concentrations in plasma.

It was measured by an enzyme-linked immunosorbent assay (ELISA) using an immunoassay kit (R & D system, USA).

Elevated leptin and insulin due to high fat diet were lowered by LG42 lactobacillus administration (HD-LG42H, L). (FIG. 8. Blood leptin concentration, FIG. 9 blood insulin concentration).

Example 5 > Lactobacillus plantarum NUC - LG42  Ingestion Liver tissue  medium mRNA  Level change

The liver tissue was homogenized in 1 ml of Trizol-Reagent (Invitrogen, USA) and allowed to stand at room temperature (15-30 ° C.) for 5 minutes, followed by vortex for 15 seconds after adding 250ul of chloroform. After 2 to 3 minutes at room temperature again was centrifuged for 15 minutes at 4 ℃, 12,000 rpm (MICRO 17R, Hanil). Carefully transfer the supernatant to a new tube, add 500ul of isopropyl alcohol, and leave for 10 minutes at room temperature. And centrifuged for 10 minutes at 4 ℃, 12,000 rpm (MICRO 17R, Hanil). Discard supernatant and get total RNA pellet. The obtained total RNA pellet was washed with 1 ml of 75% ethanol, dried and diluted with DEPC (dietyl pyrocarbonate) water and resuspended. mRNA expression amount was used real-time PCR (7500 real time PCR system, Applied Biosystems, USA).

Real time PCR was performed to investigate mRNA level changes of lipid metabolism related enzymes ACC-β (acetyl-CoA carboxylase β) and CPT-I (Carnitine palmitoyltransferase-I). The expression level was decreased in, and the expression level of CPT-I was increased in the LG42 group. In addition, PPAR-r expression was increased in HD-LG42L. It is believed that the synthesis and decomposition of fat in the body is actively performed by LG42 lactic acid bacteria. (Fig. 10. ACC expression amount, Figure 11. CPT-I expression amount, Figure 12. PPAR-r expression amount)

Example 6 > Lactobacillus plantarum NUC - LG42  Changes in memory enhancement when ingested

Animals used in the experiment were ICR mice (4wks, ♂, n = 40). Adapted to the environment by feeding the Diet Diet (D12450B) for 1 week, then, by randomized block design, 4 groups, control group (Con), Scopolamine group (Scop, Scopolamine + DW), Scopolamine + Lactobacillus high concentration (LG42H, 1x10 ⁹ ), Scopolamine + Lactobacillus low concentration (LG42L, 1x10 ⁷ ) divided into formulas were administered orally.

Passive avoidance task and Morris water maze test were performed to improve memory.

In the passive avoidance test, the lantency time was significantly higher in the group treated with lactic acid bacteria (LG42) than in the group treated with scopolamine. 13. Manual evasion experiment)

As a result of morris water maze test, there was no difference between the groups on the 1st day of training, and on the 4th day, the time to find the pedestal was significantly faster in the LG42 lactobacillus group than in the scopolamine group. This suggests that LG42 lactic acid bacteria recovered the memory impaired by scopolamine. In addition, the swimming time (escape latancy) was significantly shorter in the LG42 lactobacillus group than the scopolamine group, and the swimming distance (distance) tended to be shorter in the lactic acid bacterium group. Significantly shortened at. This suggests that LG42 lactic acid bacteria recovered the memory impaired by scopolamine.

Example 7 > Lactobacillus Planta Room NUC - LG42 Of yogurt fermented with GABA content

In the present invention, the content of γ-aminobutyric acid (GABA), a neurotransmitter involved in the improvement of brain function, was measured in yogurt fermented with Lactobacillus plantarum NUC-LG42. Yogurt fermentation is added to commercial milk (Seoul milk) and chilled soy milk (Good morning as soy milk) by adding 0.8 ~ 1% to the weight of lyophilized powder milk of Lactobacillus plantarum NUC-LG42 and fermenting at 42 ℃ for 8 hours. Yogurt was prepared.

The same amount of water was added to the prepared yogurt, and then the supernatant sample centrifuged was derivatized with OPA (o-phthaldialdehyde), and the content of GABA was quantitatively analyzed by HPLC.

HPLC analysis conditions were HPLC system; Alliance Bio, Column; Capcell Pak C18 (Shiseido, Japan), Mobile phase; 60% CAN, Flow rate; 1.0 ml / min, Decter; Water 2487 UV (254 nm).

The content of GABA in yogurt was 99.59mg / 100g in milk yogurt and 104.17mg / 100g in soymilk yogurt, indicating that it was produced more than 0.15mg / 100g of GABA in commercial yogurt.

 The GABA-producing ability of Lactobacillus plantarum NUC-LG42 may be related to the ability to improve brain function in animal experiments.

1 is an optical microscope (× 800) of the Lactobacillus plantarum NUC-LG42 KCCM 10940P according to the present invention.

2 is a sugar use characteristic result of biochemical test, assimilation test and fermentation test using API KIT.

3 shows partial sequencing (about 1.5 kbp) of 16s rDNA.

4 is a diagram showing a pyrogenic tree of the strain.

5 shows the weight change of mice.

Figure 6 shows the amount of fat increase around the epididymis of the mouse.

7 shows dorsal fat increase in mice.

8 shows leptin concentration in blood.

9 shows the blood insulin concentration.

10 shows the amount of ACC expression in liver tissue.

Figure 11 shows the amount of CPT-I expression in liver tissue.

12 shows the amount of PPAR-r expression in liver tissue.

Figure 13 shows the results of passive avoidance experiments of mice.

Claims (2)

Lactobacillus plantarum NCL 42 strain for use in any one of the group consisting of weight and fat loss, plasma and hepatic lipid and carnitine improvement, blood leptin, insulin concentration reduction, and memory enhancement (Accession No .: KCCM) 10940P). Any one selected from the group consisting of Lactobacillus Plantarum NUC LG 42 strain (Accession Number: KCCM 10940P), weight loss and fat reduction, plasma and hepatic lipid and carnitine improvement, blood leptin, insulin concentration reduction, and memory enhancement Food composition for use in the application.

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