Novel Lactobacillus Fermentum LM1016 Strain, And Composition For Preventing Or Treating Cardiovascular Diseases

Abstract

The present invention relates to a novel strain of Lactobacillus fermentum LM1016 deposited with the Korean Culture Center of Microorganisms under accession number KCCM12468P. The novel strain of the present invention has an effect of treating or preventing cardiovascular diseases, obesity, fatty liver or diabetes mellitus.

The present invention relates to a composition comprising the strain, and the composition may be used in a pharmaceutical product, a health functional food, a dairy product, a fermented product, a food additive and an animal feed and the like.

Claims

1-10. (canceled)

11. A method of treating cardiovascular diseases, obesity, diabetes mellitus or fatty liver diseases, the method comprising administering a Lactobacillus fermentum LM1016 strain (KCCM12468P) to a subject in need thereof.

12. The method of claim 11, for treating cardiovascular diseases in a subject in need thereof.

13. The method of claim 12, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces the level of CRP (C-reactive protein), neutral fat or cholesterol, which is a risk factor for cardiovascular diseases.

14. The method of claim 11, for treating obesity, diabetes mellitus or fatty liver disease in a subject in need thereof.

15. The method of claim 14, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces inflammation in subcutaneous fat.

16. The method of claim 11, for treating obesity in a subject in need thereof, and wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces inflammation in subcutaneous fat.

17. The method of claim 14, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces the level of glucose, insulin or leptin in blood.

18. The method of claim 11, for treating diabetes mellitus in a subject in need thereof, and wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces the level of glucose, insulin or leptin in blood.

19. The method of claim 14, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces the weight of the liver or the amount of neutral fat in the liver.

20. The method of claim 11, for fatty liver disease in a subject in need thereof, and wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) reduces the weight of the liver or the amount of neutral fat in the liver.

21. The method of claim 11, wherein the subject is a human.

22. The method of claim 11, wherein the subject is a non-human animal.

23. The method of claim 11, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) is provided in a pharmaceutical composition.

24. The method of claim 11, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) is provided in a food composition.

25. The method of claim 21, wherein the food composition is a health functional food, a dairy product, a fermented product or a food additive.

26. The method of claim 11, wherein the Lactobacillus fermentum LM1016 strain (KCCM12468P) is provided in an animal feed composition.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1 shows the the levels of C-reactive protein, neutral fat, and total cholesterol in the serum of mice of Groups 1 to 3.

[0042] FIG. 2 shows the change in the cumulative amount of body weight gain of Groups 1 to 3.

[0043] FIG. 3 shows the amount of subcutaneous fat, epididymal fat, and brown fat in mice of Groups 1 to 3.

[0044] FIG. 4 shows the size of subcutaneous fat, epididymal fat, and brown fat in mice.

[0045] FIG. 5 shows the number of adipose tissue and the size of fat cells by staining with H&E stain in epididymal fat and brown fat in each group.

[0046] FIG. 6 shows the shape of the fat and the inflammatory response between the cells by treating epididymal fat of Group 2 and Group 3 by immunochemical staining.

[0047] FIG. 7 shows the blood glucose level in mice of Groups 1 to 3.

[0048] FIG. 8 shows the levels of leptin and insulin in serum.

[0049] FIG. 9 shows the weight of the liver and the amount of neutral fat in the liver.

[0050] FIG. 10 shows the results of observing the presence or absence of fatty liver by staining liver tissue with H&E stain.

DETAILED DESCRIPTION FOR WORKING THE INVENTION

[0051] The present inventors have found a novel LM1016 strain of Lactobacillus fermentum.

[0052] In addition, the novel LM1016 strain reduces CRP, neutral fat and cholesterol concentrations in blood, which are risk factors for cardiovascular diseases, thereby having an effect of preventing and treating cardiovascular diseases such as hypertension, hyperlipidemia and arteriosclerosis.

[0053] In addition, the novel LM1016 strain inhibits the accumulation of fat in the liver tissue, which is another cause of cardiovascular diseases, thereby having an effect of preventing and treating fatty liver.

[0054] In addition, the novel LM1016 strain has an effect of inhibiting the accumulation of body fat, reducing the size of fat cells, and inhibiting inflammatory response in fat cells, thereby having an effect of preventing and treating obesity and a disease caused by obesity.

[0055] In particular, the Lactobacillus fermentum LM1016 strain of the present invention rapidly reduces the blood glucose concentration and reduces the blood insulin concentration, thereby ameliorating insulin resistance and thus exhibiting an effect of preventing or treating diabetes mellitus, which may be a cause of cardiovascular diseases.

[0056] Therefore, the novel LM1016 strain of the present invention has an excellent effect of treating and preventing cardiovascular diseases, fatty liver, obesity and diabetes mellitus at the same time without side effects, and as such, probiotics ( Lactobacillus fermentum) having an effect of preventing and treating several chronic diseases at the same time have not been previously known.

[0057] Therefore, the present invention relates to a novel strain of Lactobacillus fermentum LM1016.

[0058] In addition, the present invention relates to a composition having an effect on cardiovascular diseases, fatty liver, obesity or diabetes mellitus, comprising a Lactobacillus fermentum LM1016.

[0059] The composition of the present invention may be used in a pharmaceutical product, a health functional food, a dairy product, a fermented product, a food additive or an animal feed or the like.

[0060] Specifically, the microorganism of the present invention may be administered in a unit form such as a tablet or capsule obtained by mixing with a carrier, excipient or diluent or the like selected according to the route of administration.

[0061] Carriers, excipients and diluents suitable for the composition of the present invention may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gum tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methylcellulose, methyl and propyl hydroxybenzoate, talc, magnesium stearate or mineral oil or the like.

[0062] The “composition” in the present specification does not necessarily mean only approved as a pharmaceutical product, but is a concept including a general functional food or health supplement food.

[0063] As used herein, “cardiovascular illness” or “cardiovascular disease” refers to an illness affecting the heart and/or blood vessels. In particular, cardiovascular diseases include arrhythmia; arteriosclerosis; angina; dysrhythmia; myocardial ischemia; myocardial infarction; heart or vascular aneurysm; vasculitis; stroke; peripheral obstructive arteriopathy of a limb, organ or tissue; reperfusion injury due to ischemia of the brain, heart, kidney, or other organ or tissue; endotoxicity; surgical or traumatic shock; hypertension; valvular disease of heart; heart failure; abnormal blood pressure; shock; vasoconstriction; vascular abnormality, dysfunction limited to a single organ or tissue.

[0064] In another embodiment, the present invention provides a method for preventing, ameliorating or treating cardiovascular diseases, fatty liver, obesity or diabetes mellitus in a subject in need thereof, comprising administering an effective amount of a Lactobacillus fermentum LM1016 strain to the subject in need of preventing, ameliorating or treating cardiovascular diseases, fatty liver, obesity or diabetes mellitus.

[0065] As used herein, the term “subject” includes human and non-human animals. Non-human animals include all vertebrates, for example mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, horses and the like.

[0066] In another embodiment, the present invention provides the use of a Lactobacillus fermentum LM1016 strain for preventing, ameliorating or treating cardiovascular diseases, fatty liver, obesity or diabetes mellitus.

[0067] Hereafter, the present invention will be described in more detail with reference to the following examples. It is intended that these examples illustrate the present invention in more detail and the scope of the present invention is not limited to these examples.

Example 1

Experiment of Inhibitory Action of LM1016 on Cardiovascular Disease Factor

[0068] In order to evaluate an influence of a Lactobacillus fermentum LM1016 strain deposited with the Korean Culture Center of Microorganisms, the Korea Research Institute of Bioscience and Biotechnology, under accession number KCCM12468P on cardiovascular disease factor, 5 week-old C57BL6/J mice were tamed for 1 week and then were divided into 3 groups, and the experiment was conducted.

[0069] Group 1 was a group that was administered with a normal diet (ND, 3.1 kcal/g) for 12 weeks, Group 2 was a group that was administered with ND for the first 2 weeks and with a high fat diet (HFD, 5.2 kcal/g) for remaining 10 weeks, and Group 3 was a group that was orally administered with LM1016 (10.sup.9 CFU/mouse) 5 times a week for 12 weeks while being administered with the same diet as in Group 2.

[0070] After the 12-week administration was completed, blood was collected from the heart of mice, and the CRP concentration, neutral fat and cholesterol concentration in the blood were measured. The results are shown in Table 1 below and FIG. 1.

TABLE-US-00001 Group 1 Group 2 Group 3 CRP (ng/ml) 4.3 5.4 4.6 Neutral fat (mg/dL) 62.0 79.0 74.9 Total cholesterol (mg/dL) 119.4 176.0 129.3

[0071] As shown in Table 1 above, it was confirmed that the CRP concentration in the blood was remarkably reduced to 4.6 ng/ml in Group 3 administered with LM1016 as compared with 5.4 ng/mg in Group 2, which was similar level to Group 1 (4.33 ng/ml) administered with a normal diet.

[0072] In addition, the concentration of neutral fat in the blood was 62.0 mg/dL in Group 1, 79.0 mg/dL in Group 2, and 74.9 mg/dL in Group 3, and there was no significant difference in levels between Group 2 and Group 3. However, the total cholesterol concentration was 176.0 mg/dL in Group 2 and 129.3 mg/dL in Group 3, and it was confirmed that the total cholesterol concentration of Group 3 was lowered by 46.7 mg/dL as compared with that of Group 2 and was similar level to that of Group 1.

[0073] In the end, it can be seen that the Lactobacillus fermentum LM1016 strain has an excellent effect of restoring to normal level by reducing the CRP concentration and the total cholesterol concentration.

Example 2

Inhibitory Effect of LM1016 on Body Weight Gain Rate, Accumulation of Body Fat and Inflammatory Response

[0074] The body weight was measured weekly while conducting the animal experiment for cardiovascular diseases of Example 1. At week 12, the mice were anatomized, and each of the main organs was extracted, and the weight thereof was measured.

1. Reduction of Body Weight Gain Rate

[0075] The cumulative amount of body weight gain over time was measured based on the body weight at week 0, and is shown in Table 2 below and FIG. 2.

[0076] As shown in FIG. 2, the amount of body weight gain for 2 weeks before administration of the high fat diet showed a gentle cumulative increase in all three groups. In Group 2 and Group 3, as the high-fat diet was administered, the difference in the amount of body weight gain gradually began to appear and showed a distinct difference from week 5.

TABLE-US-00002 Group 1 Group 2 Group 3 Amount of body weight gain at week 11 (g) 9.35 23.77 17.89 Amount of body weight gain at week 12 (g) 9.50 23.92 18.84

[0077] As shown in Table 2 above, it was confirmed that at week 11, the amount of body weight gain of Group 2 was 23.77 g, and the amount of body weight gain of Group 3 was 17.89 g, and the amount of body weight gain of Group 3 was reduced by 5.88 g as compared with that of Group 2.

[0078] At week 12, it was confirmed that the amount of body weight gain of Group 2 was 23.92 g, and the amount of body weight gain of Group 3 was 18.84 g, and the amount of body weight gain of Group 3 was reduced by 5.08 g as compared with that of Group 2, and thus, the body weight gain rate of Group 3 was reduced by 17% or more as compared with that of Group 2.

[0079] In the end, as a result of comparing Group 2 administered with the high fat diet and Group 3 administered with the high fat diet in combination with LM1016 at the same time, it can be seen that the difference in the body weight gain rate was shown since week 1, and the difference in the body weight gain rate was remarkably increased over time. Therefore, it can be seen that LM1016 has an effect of reducing body weight.

2. Reduction of Subcutaneous Fat, Epididymal Fat, and Brown Adipose Tissue

[0080] The weights of subcutaneous fat, epididymal fat, and brown adipose tissue for 12 weeks were measured, and the results are shown in Table 3 below, and FIGS. 3 and 4.

[0080] TABLE-US-00003 Group 1 Group 2 Group 3 Subcutaneous fat (g) 0.64 1.99 1.44 Epididymal fat (g) 0.80 2.63 2.05 Brown adipose tissue (g) 0.06 0.12 0.08

[0081] As shown in Table 3 above, it was confirmed that the increased amount of all subcutaneous fat, epididymal fat, and brown adipose tissue of Group 3 administered with the high fat diet in combination with LM1016 was reduced as compared with that of Group 2 administered with the high fat diet.

[0082] In addition, the epididymal fat was stained with H&E stain, and the results are shown in FIG. 5. As shown in FIG. 5, it was confirmed that the size of the fat cells of Group 2 was the largest, and the size of the fat cells of Group 3 was reduced to less than half as compared with that of Group 2.

[0083] The size of the fat globule was measured, and the results are shown in Table 4 below.

TABLE-US-00004 Group 1 Group 2 Group 3 Size of fat globule of subcutaneous fat (.Math.m.sup.2) 15,896 62,114 35,652

[0084] As shown in Table 4 above, the size of the fat globule of Group 1 was 15,896 .Math.m.sup.2, that of Group 2 was 62,114 .Math.m.sup.2, and that of Group 3 was 35,652 .Math.m.sup.2.sup..

3. Reduction of Inflammatory Response in Subcutaneous Fat

[0085] The inflammatory response in subcutaneous fat was confirmed by immunochemical staining using the F4/80 antibody. As a result, as shown in FIG. 6, it was confirmed that the shape of the cells of the subcutaneous fat of the mice administered with the high fat diet was distorted, and a blue spot that appeared to be inflammation between these cells was confirmed, but in the group administered with LM1016, the cell shape was maintained clean, and almost no inflammatory response occurred.

Example 3

Antidiabetic Effect of LM1016

1. Reduction of Blood Sugar

[0086] In order to measure the glucose tolerance of the mice, glucose was administered 11 weeks after the start of administration to each group, and then the change in blood sugar over time was measured, and the results are shown in Table 5 and FIG. 7.

[0086] TABLE-US-00005 Time (min) Group 1 Blood sugar (mg/dL) Group 2 Blood sugar (mg/dL) Group 3 Blood sugar (mg/dL) 0 122.83 262.50 197.50 15 316.33 559.83 478.83 30 299.00 575.17 544.83 60 193.50 552.17 415.67 120 140.00 410.67 234.67

[0087] As shown in Table 5 above, it was confirmed that the blood sugar level in Group 2 administered with the high fat diet was the highest from the initial administration to 120 minutes. On the other hand, the blood sugar level in Group 3 administered with the high fat diet in combination with LM1016 was increased during the initial 30 minutes, but the blood sugar level of Group 3 was lower than the blood sugar level of Group 2.

[0088] In addition, it was confirmed that from 30 minutes after administration, the blood sugar of Group 3 was reduced sharply, and after 120 minutes, the blood sugar of Group 3 was reduced to a level similar to that of Group 1.

[0089] Therefore, it can be seen that LM1016 has an effect of reducing the glucose tolerance due to a high fat diet by reducing the blood sugar.

2. Reduction of Levels of Insulin and Leptin

[0090] The levels of insulin and leptin in each group for 12 weeks were measured, and the results are shown in Table 6 below and FIG. 8.

[0090] TABLE-US-00006 Group 1 Group 2 Group 3 Insulin (ng/mL) 0.36 2.09 1.19 Leptin (ng/mL) 2.73 25.78 17.92

[0091] As shown in Table 6 above, it was confirmed that the insulin level of Group 3 was remarkably reduced to about half of the insulin level of Group 2.

[0092] In the case of obesity, the insulin resistance is increased, resulting in the increase of the insulin secretion. It can be seen that LM1016 has an effect of reducing the insulin resistance due to a high fat diet by reducing the insulin concentration.

[0093] In addition, the leptin level of Group 2 was 25.78 ng/mL, and the leptin level of Group 3 was 17.92 ng/mL. It was found that the leptin level of Group 3 was remarkably reduced as compared with the leptin level of Group 2.

[0094] Leptin is an appetite-regulating hormone secreted from fat cells. When neutral fats accumulate in adipose tissue and the size of fat cells increases, the synthesis of leptin is promoted and the concentration of leptin increases. Therefore, it can be seen that the concentration of leptin of Group 3 is remarkably reduced as compared with that of Group 2 because neutral fats and fat cells are reduced due to the administration of LM1016.

Example 4

Effect of Ameliorating Fatty Liver

1.Weight of Liver and Amount of Neutral Fat in Liver

[0095] After 12 weeks of administration, the liver of mice was extracted in each group, and the weight of the liver and the amount of neutral fat in the liver tissue were measured, and the results are shown in Table 7 below and FIG. 9.

[0095] TABLE-US-00007 Group 1 Group 2 Group 3 Weight of liver (g) 0.92 1.29 0.88 Amount of neutral fat (nmol/whole liver) 5.78 9.33 4.20

[0096] As shown in Table 7 above, it was confirmed that the weight of the liver of Group 3 was remarkably reduced as compared with that of Group 2, and it was also reduced as compared with that of Group 1.

[0097] In addition, as a result of measuring the amount of neutral fat in the liver tissue, it was found that the amount of neutral fat in the liver tissue of Group 2 was 9.33 nmol/whole liver, and that of Group 3 was 4.20 nmol/whole liver. It can be seen that the amount of neutral fat of Group 3 was remarkably reduced to less than half of that of Group 2. In addition, the amount of neutral fat of Group 2 was lower than that of Group 1.

[0098] Therefore, fat metabolism in the body occurs abnormally as the person becomes overweight, and lipid substances are accumulated in the liver, resulting in an increase in the weight of the liver and the amount of neutral fat in the liver tissue. Thus, it can be seen that the LM1016 strain is involved in lipid metabolism in the liver and has an effect of inhibiting the accumulation of fats in the hepatocytes.

2. Reduction of Fatty Liver

[0099] The liver was stained with H&E stain, and the presence or absence of a macro fat vacuole (X20 magnifications and X40 magnifications) was observed and is shown in FIG. 10.

[0100] As shown in FIG. 10, the liver tissues of Group 1 and Group 3 were densely packed, but white portions, which appear to be macro fat vacuoles, were observed throughout the liver tissues of Group 2. Therefore, it can be seen that the LM1016 strain has an excellent effect of inhibiting fatty liver.