KR20130067429A - Products containing lactobacillus curvatus hy7601 and lactobacillus plantarum ky1032 having improving liver function, hyperinsulinemia, hyperglycemia and hypertriglyceridemia as effective component - Google Patents

Abstract

PURPOSE: A product containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 as active ingredients is provided to prepare a functional food and a pharmaceutical composition which improves the liver function and treats hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. CONSTITUTION: A pharmaceutical composition for improving liver function contains Lactobacillus curvatus HY7601(deposit number KCTC 11456BP) and Lactobacillus plantarum KY1032(deposit number KCCM10430) as active ingredients. A fermented milk for improving liver function contains Lactobacillus curvatus HY7601(deposit number KCTC 11456BP) and Lactobacillus plantarum KY1032(deposit number KCCM10430) as active ingredients. A drink for improving liver function contains Lactobacillus curvatus HY7601(deposit number KCTC 11456BP) and Lactobacillus plantarum KY1032(deposit number KCCM10430) as active ingredients. A health functional food for improving liver function contains Lactobacillus curvatus HY7601(deposit number KCTC 11456BP) and Lactobacillus plantarum KY1032(deposit number KCCM10430) as active ingredients. [Reference numerals] (AA) Liver somatic index(IU/L); (BB) General diet group; (CC) High fat diet group; (DD) Lactobacillus supplementary group

Description

Lactobacillus curvatus HY7601 containing lactobacillus corbetters achwai 7601 and lactobacillus plantarum kwai 1032 as an active ingredient having the effect of improving liver function and improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia {Products containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 having improving liver function, hyperinsulinemia, hyperglycemia and hypertriglyceridemia as effective component}

The present invention relates to a product containing, as an active ingredient, Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032, which have an effect of improving liver function and improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia, , The expression of HMGCR, ACAT1, and ACAT2, genes related to fatty acid synthesis, FAS, ME, ACC1, and cholesterol synthesis, which are involved in the fatty acid synthesis, and improve lipid metabolism caused by accumulation of neutral lipids and cholesterol in the liver Lactobacillus carvitus HY7601 and Lactobacillus plantarum KY1032, which are effective in reducing hepatic AST and ALT, and reducing the levels of blood insulin, ritestine, glucose, C-peptide and triglycerides, A fermented milk, a beverage, and a health functional food.

The liver is the largest organ in the human body. The main function of the liver is to process various substances introduced from the body or produced in the body and synthesize and supply important substances. There are also several proteins in the blood that play an important role in our body, about 90% of which are made in the liver. In addition, the liver, detoxification and the role of the immune system, such as the various drugs and harmful substances in our body from liver to harmful substances are changed through urine or bile is excreted. In particular, the liver contains immune cells called Cooper cells, which are able to ingest bacteria, toxins or foreign substances coming from outside the body and then dissolve them and discharge them out of the body. As the liver performs a variety of important functions, it causes various problems when the liver function is seriously degraded. Liver disease is caused by viral hepatitis, alcoholic liver disease caused by alcohol, toxic liver disease caused by drug, autoimmune liver disease caused by abnormality of human immune system, fatty liver accumulating liver fat, etc., depending on the root cause of the disease Respectively.

In particular, fatty liver is a common lesion associated with obesity, resulting in excess liver fat and cholesterol accumulation in the liver resulting in liver damage. If such liver damage is chronicized, liver fibrosis, cirrhosis, and liver cancer occur regardless of the cause. Liver fibrosis, liver cirrhosis, and liver cancer are diseases that do not have clear treatments and treatments at present and their mortality rates are also high. Therefore, prevention and treatment of liver damage before liver damage is chronic is crucial to inhibiting progression to liver fibrosis, cirrhosis, or liver cancer.

Aspartate aminotransferase (ALT) or alanine aminotransferase (ALT), which is used as an index of liver function, is an enzyme involved in amino acid metabolism in hepatocytes. The fact that the enzymes that should be present in such original hepatocytes leak from the cells and continue to exist in the blood means that the breakdown of the liver is continuing.

On the other hand, according to the regulations of the American Diabetes Association and the World Health Organization, type 2, noninsulin dependent diabetes mellitus (hereinafter referred to as "NIDDM") is a hormone that regulates glucose metabolism Refers to a hyperglycemic state that results from a decrease in insulin action. It also refers to hyperinsulinemia and concomitant metabolic disorders resulting from increased resistance to insulin. NIDDM, which accounts for 80% of diabetic patients, is also referred to as adult type diabetes and predominantly occurs after age 40. Diabetes mellitus such as overweight (BMI greater than 25 kg / m ² ), family history of diabetes, habitual physical inactivity, hypertension (over 140/90 mmHg in adults), or gestational diabetes or delivery of a child over 4 kg The person with the factor has a high incidence of NIDDM according to the surrounding environment conditions. Recently, the westernization of dietary habits has shifted from low - fat diets to high - fat diets, and due to changes in lifestyle, the obesity population is increasing due to the reduced amount of exercise compared to high nutrient intake. Obesity increases insulin requirements and, as a result, the function of the cells in the pancreas is gradually diminished, leading to diabetes. Furthermore, fat cells that are enlarged by obesity increase insulin resistance by over-secreting ricin (hormone) hormones. Experiments have shown that administration of ritestin to normal mice results in increased insulin resistance and, conversely, removal of ridestine hormone in obese mice improves insulin resistance. As such, riticus is considered to be an important link between obesity and diabetes. In addition to high-fat diets, Koreans with rice stocks are also overexposed to the risk of hyperinsulinemia, hyperglycemia and hypertriglyceridemia due to persistent high-carbohydrate diets. Studies supporting this have already revealed that hypertriglyceridemia, hyperglycemia and hypertriglyceridemia are induced in rats fed a high fructose diet. C-peptides that are cleaved as the insulin precursor matures into insulin are also used as an index to diagnose NIDDM, and in the case of hyperinsulinemia, the concentration of C-peptide is also high.

These NIDDM preventive and therapeutic methods include 1) dietary control, 2) exercise therapy, 3) oral hypoglycemic agents, and 4) insulin preparations. Among them, sulfonylurea sulfonylurea-based drugs and biguanide-based drugs can be used. Also, metformin and acarbose compounds have recently been used to treat NIDDM patients. However, such oral hypoglycemic agents have been limited in their use due to side effects such as liver toxicity, weight gain, and low blood glucose. Failure to treat NIDDM can be exacerbated by cardiovascular death and other diabetic complications including retinopathy, nephropathy and peripheral neuropathy.

Accordingly, the present inventors have found that Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 reduce liver weight gain caused by obesity, improve neutral lipid and cholesterol accumulation in liver, , The genes involved in the synthesis of fatty acids FAS, ME, ACC1, and the genes associated with cholesterol synthesis HMGCR, ACAT1, ACAT2 expression and liver AST and ALT are reduced by reducing liver function is excellent and the fact that blood insulin, The present invention has been accomplished by discovering that it is excellent in improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia by reducing the levels of ritestine, glucose, C-peptide and triglyceride.

DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide a method for reducing liver weight by obesity, improving fatty liver symptoms caused by accumulation of neutral lipids and cholesterol in the liver, And cholesterol synthesis by reducing the expression of HMGCR, ACAT1, and ACAT2, decreasing hepatic AST and ALT levels, and reducing the levels of insulin, ritestin, glucose, C-peptide and triglyceride in the blood, A pharmaceutical composition containing as an active ingredient Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 having an effect of improving blood pressure , hyperglycemia and hypertriglyceridemia, fermented milk, beverage, health It is intended to provide a functional food.

In order to accomplish the above object, the present invention provides a method for reducing liver weight by obesity, improving fatty liver symptoms caused by accumulation of fat in the liver, lowering the content of triglyceride and cholesterol in the liver, (3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase), ACAT1 (Acetyl-CoA Acetyltransferase 1) associated with cholesterol synthesis, Fatty Acid Synthase, ME (Malic Enzyme) , Decreases the expression of ACAT2 (Acetyl-CoA Acetyltransferase 2), decreases the levels of Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and decreases the levels of insulin, ritestin, glucose, C-peptide and triglyceride liver function improved and hyperinsulinemia, hyperglycemia and high Lactobacillus Cinque's Better (Lactobacillus curvatus) HY7601 and Lactobacillus Planta Room (Lacto having the effect of improving hypertriglyceridemia bacillus plantarum ) KY1032 as an active ingredient, a fermented milk, a beverage, and a health functional food.

Hereinafter, the present invention will be described in detail.

In order to isolate the strain according to the present invention, kimchi prepared in a domestic home was added to an MRS liquid medium containing 0.02% sodium azide and cultured at 37 ° C for 24 hours, , The culture was taken and plated on a medium containing 0.02% sodium azide and cultured at 37% for 48 hours. The size and morphology of the isolates were observed by light microscopy (SAMWON, CSB-FEI) through Cowan (1974). Biochemical characteristics of the isolates were determined by Cowan and Steel (1984) and Macfaddin . The results of this study were as follows: 1) The characteristics of the strains were classified according to Bergey's Manual of Systematic Bacteriology and Bergey's Manual of Determinative Bacteriology.

Therefore, the characteristics of Lactobacillus carvitus HY7601 according to the present invention are as follows.

1) Types of bacteria

The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days

① Cell shape: Bacillus

② Mobility: None

③ Spore forming ability: None

④ Gram staining: positive

2) Morphology

When cultured on MRS agar plate medium at 37 ° C for 2 days,

① Shape: Circular

② Bump: convex

③ Surface: Smooth

3) Physiological properties

① Growth temperature: Growth possible Growth temperature 15 ~ 40 ℃

            Optimum growth temperature 36 ~ 38 ℃

② Growth pH: Growable growth pH 5.0 ~ 7.5

           Optimum pH 6.0 to 6.5

③ Effect on oxygen: Tumor anaerobic

4) Catalase: -

5) Whether gas is formed: -

6) Growth at 15 ° C: +

7) Growth at 45 ℃:-

8) Indole production: -

9) Lactic acid production: +

10) Fermentation experiment and identification

Table 1 shows the results of sugar fermentation experiments using api 50 CH kit from Biomerieux.

0-24 per Whether or not to use Per 25-49 Whether or not to use 0 Control - 25 esculin - 1 glycerol - 26 Living + 2 Erythritol - 27 Cellobios - 3 D arabinose - 28 Maltose + 4 L arabinose - 29 lactose - 5 ribos + 30 Melibiose - 6 D xylose - 31 sucrose + 7 L xylose - 32 Trehalose - 8 Adonitor - 33 inulin - 9 beta methyl-D-xyloside - 34 Melitos - 10 galactose + 35 raffinos - 11 glucose + 36 starches - 12 fructose + 37 glycogen - 13 Manno + 38 xylitol - 14 sorbos - 39 Gentiobios - 15 Lambos - 40 D Toranos - 16 Dorsitol - 41 License Source - 17 Synoshitol - 42 D Tagatos - 18 Mannitol - 43 D Fuchos - 19 sorbitol - 44 L Fuchos - 20 [alpha] -methyl-D-mannoside - 45 D arabitol - 21? -Methyl-D-glucoside + 46 L arabitol - 22 N-acetyl-glucosamine + 47 Gluconate - 23 Amigalline - 48 2-keto-gluconate - 24 Arbutin - 49 5-keto-gluconate -

Based on the morphological, physiological and growth characteristics of the above bacteria, the strain of the present invention was identified as Lactobacillus curvatus , and the present inventors named it Lactobacillus curvatus HY7601, (Deposited with KCTC 11456BP, Korea Research Institute of Bioscience and Biotechnology as of Jan. 22, 2009), which is a Korean Patent Registration No. 0996577 entitled "Lactobacillus lucerus Vetus HY7601 and products containing it as active ingredients).

The characteristics of Lactobacillus plantarum KY1032 according to the present invention are as follows.

1) Form of bacteria: Bacillus

2) Colony color: Milk light circle shape

3) Optimum growth temperature: 37 ℃

4) Motility: None

5) Gram staining: positive

6) Catalase: negative

7) Oxygen effect: Beta anaerobic

8) Fermentation experiment and identification

Table 2 shows the results of sugar fermentation experiments using API 50CHL kit from Biomerieux.

0-24 per Whether or not to use Per 25-49 Whether or not to use 0 Control - 25 esculin + 1 glycerol - 26 Living + 2 Erythritol - 27 Cellobios + 3 D arabinose - 28 Maltose + 4 L arabinose + 29 lactose + 5 ribos + 30 Melibiose + 6 D xylose - 31 sucrose + 7 L xylose - 32 Trehalose + 8 Adonitor - 33 inulin - 9 beta methyl-D-xyloside - 34 Melitos + 10 galactose + 35 raffinos + 11 glucose + 36 starches - 12 fructose + 37 glycogen - 13 Manno + 38 xylitol - 14 sorbos - 39 Gentiobios + 15 Lambos + 40 D Toranos + 16 Dorsitol - 41 License Source - 17 Synoshitol - 42 D Tagatos - 18 Mannitol + 43 D Fuchos - 19 sorbitol + 44 L Fuchos - 20 [alpha] -methyl-D-mannoside + 45 D arabitol + 21? -Methyl-D-glucoside + 46 L arabitol - 22 N-acetyl-glucosamine + 47 Gluconate + 23 Amigalline + 48 2-keto-gluconate - 24 Arbutin + 49 5-keto-gluconate -

The strain of the present invention was named Lactobacillus plantarum KY1032 based on the morphological, physiological and growth characteristics of the above bacteria, and was deposited on October 2, 2002 at the Korea Microorganism Conservation Center (Accession No .: KCCM- 10430). This strain has been already patented in Korea Patent No. 0464642 (entitled Lactobacillus planta kewaii 1032).

On the other hand, Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, which have the effect of improving liver function and improving the hyperinsulinemia, hyperglycemia and hypertriglyceridemia of the present invention, The pharmaceutical composition containing the pharmaceutical composition may be used alone or in combination with pharmaceutically acceptable excipients in the form of tablets, capsules or the like according to a method commonly used in pharmaceutical practice.

In the case of humans, a typical daily dose may range from 1 to 30 mg / kg body weight, and may be administered in single or divided doses. However, the actual dosage should be determined in light of various relevant factors such as route of administration, age, sex, weight, health status and severity of the disease.

Of course, since the pharmaceutical composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long time.

In addition, fermented milk containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, which have an effect of improving liver function and improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia, as active ingredients Lactobacillus plantarum KY1032, Lactobacillus carvitus HY7601 and mixed juice syrup are mixed at a constant ratio of 150 bar, cooled to below 10 ° C and packaged in a container.

In addition, beverages containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, which have the effect of improving liver function and improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia, as active ingredients Lactobacillus plantarum KY1032, Lactobacillus carvitus HY7601 and water were mixed at a constant ratio and homogenized at 150 bar, cooled to below 10 ° C, and packaged in glass bottles, plastic bottles, etc. .

In addition, the present invention relates to a pharmaceutical composition containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032, which have an effect of improving liver function, and improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia, The functional food contains not only the above-mentioned Lactobacillus plantarum KY1032 and Lactobacillus cabutus HY7601 but also vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide, May be added and other food additives may be added.

The Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 according to the present invention reduce liver weight gain caused by obesity and improve the symptoms of fatty liver in which neutral lipids and cholesterol accumulate in the liver , Decreases the expression of the genes FAS, ME, ACC1 and genes related to cholesterol synthesis, HMGCR, ACAT1, and ACAT2, which are related to fatty acid synthesis, decreases hepatic AST and ALT and decreases insulin, ricinsein, glucose, C-peptide and triglyceride ( Lactobacillus curvatus HY7601 ) and Lactobacillus plantarum (KY1032) to improve liver function and to improve hyperinsulinemia, hyperglycemia and hypertriglyceridemia Functional food and pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of measurement of liver tissue and body weight reduction effects of obesity test animals by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 2 is a graph showing the inhibitory effects of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 on the fat accumulation in liver tissues of obese animals by hematoxylin and eosin (H & E) staining method Fig.
FIG. 3 is a graph showing the results of measuring the AST and ALT in the blood of the test animals by the ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 4 shows the expression levels of FAS, ME and ACC1 mRNA expressions related to the fatty acid synthesis in liver tissues of obese animals by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 And a real-time PCR method.
FIG. 5 shows the mRNA expression levels of HMGCR, ACAT1 and ACAT2 genes in cholesterol synthesis-related genes in the liver of obese test animals by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 Time PCR method.
FIG. 6 is a graph showing the results of measuring the insulin concentration-decreasing activity of insulin in the high-fat diet-fed mice by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 7 is a graph showing the results of measurement of blood ridicidin concentration-decreasing activity of high-fat diet-fed mice by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 8 is a graph showing the results of measuring the insulin concentration-decreasing activity of high-fructose-fed rats by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 9 is a graph showing the results of measurement of blood glucose-lowering activity of high-fructose-fed rats by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 10 is a graph showing the results of measuring the C-peptide concentration-decreasing activity of a high-fat diet-fed rat by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.
FIG. 11 is a graph showing the results of measurement of blood triglyceride concentration-decreasing activity of high-fat diet-fed rats by ingestion of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art within the scope of the technical idea of the present invention are possible.

≪ Example 1 >

Manufacture of freeze-dried powder

Lactobacillus charvetus ( Lactobacillus curvatus ) Lyophilized powder containing HY7601

The Lactobacillus curvatus HY7601 of the present invention was prepared by cultivating a liquid medium supplemented with Proteose peptone # 3, Yeast Extract, Beef Extract and glucose for food for about 16 hours at 37 ° C, The cells were separated and washed with sterilized physiological saline and lyophilized to obtain about 10 ¹¹ cfu bacterium per gram of freeze-dried powder (g).

Meanwhile, Lactobacillus curvatus HY7601 of the present invention may be provided in the form of a lyophilized powder or a culture as described above.

Lactobacillus plantar room Lactobacillus plantarum ) Freeze-dried powder containing KY1032

The Lactobacillus plantarum KY1032 of the present invention was prepared in the same manner as the preparation of the lyophilized powder containing the above-mentioned Lactobacillus curvatus HY7601.

<Example 2>

Lactobacillus charvetus ( Lactobacillus curvatus ) HY7601 and Lactobacillus plantarum ( Lactobacillus plantarum ) Preparation of a pharmaceutical composition containing KY1032 as an active ingredient

The preparation examples of the pharmaceutical composition containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 of the present invention as an active ingredient will be described, but the present invention is not limited thereto, This is specifically intended to explain.

Manufacture of tablets

A lyophilized powder 100 obtained by mixing the lyophilized powder containing Lactobacillus curvatus HY7601 of Example 1 and the lyophilized powder containing Lactobacillus plantarum KY1032 in a weight ratio of 1: 1 100 mg of corn starch, 100 mg of lactose, and 97 mg of polyvinylpyrrolidone were homogeneously mixed and granulated by a wet granulation method, and 2 mg of magnesium stearate was added and mixed.

Preparation of capsules

A lyophilized powder 100 obtained by mixing the lyophilized powder containing Lactobacillus curvatus HY7601 of Example 1 and the lyophilized powder containing Lactobacillus plantarum KY1032 in a weight ratio of 1: 1 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were thoroughly mixed and filled in hard gelatin capsules according to a conventional preparation method of capsules to prepare capsules.

<Example 3>

Lactobacillus charvetus ( Lactobacillus curvatus ) HY7601 and Lactobacillus plantarum ( Lactobacillus plantarum Preparation of fermented milk containing KY1032 as active ingredient

A method for producing a fermented milk composed of a culture medium of lactic acid bacteria and Lactobacillus curvatus HY7601, Lactobacillus plantarum KY1032 and a mixed juice syrup of the present invention is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix ^o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 × 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.

Next, the mixed fruit juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix ^o , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 캜 Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.

Then, the lyophilized powder containing 69.5% by weight of the culture liquid of the above-mentioned lactic acid bacteria and the Lactobacillus curvatus HY7601 of Example 1 and the lyophilized powder containing Lactobacillus plantarum KY1032 were mixed at a ratio of 1: 1 0.1% by weight of the lyophilized powder mixed with 30.4% by weight of the mixed juice syrup was homogenized at 150 bar and then cooled to 10 캜 or lower to obtain Lactobacillus curvatus HY7601 and Lactobacillus planta Fermented milk containing Lactobacillus plantarum KY1032 as an active ingredient was prepared.

<Example 4>

Lactobacillus charvetus ( Lactobacillus curvatus ) HY7601 and Lactobacillus plantarum ( Lactobacillus plantarum ) Preparation of functional beverage containing KY1032 as an active ingredient

A method for producing a functional beverage composed of Lactobacillus curvatus HY7601, Lactobacillus plantarum KY1032 and a mixed juice syrup of the present invention is as follows.

First, a mixed juice syrup was 13% by weight of liquid fructose, white sugar, 2.5 wt%, brown sugar 2.5% by weight, mixed juice concentrate 56Brix ^o 10.9% by weight of pectin, 1.0% by weight, fresh 0.1% by weight fruit mix essence and purified water 70% The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.

The lyophilized powder containing 30.4% by weight of the mixed fruit juice syrup prepared by the above method and Lactobacillus curvatus HY7601 of Example 1 and the lyophilized powder containing Lactobacillus plantarum KY1032 0.1% by weight of the lyophilized powder mixed at a weight ratio of 1: 1 and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar. The mixture was cooled to 10 ° C or lower and packed in a glass bottle, plastic bottles, A functional beverage containing Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 as an active ingredient was prepared.

<Example 5>

Lactobacillus charvetus ( Lactobacillus curvatus ) HY7601 and Lactobacillus plantarum ( Lactobacillus plantarum Production of Health Functional Foods Containing KY1032 as Active Ingredient

The lyophilized powder containing Lactobacillus curvatus HY7601 of Example 1 and lyophilized powder containing Lactobacillus plantarum KY1032 in a weight ratio of 1: 1 were mixed to prepare a lyophilized powder (Vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide) and oligosaccharide were added to 0.1 wt% of the lyophilized powder mixed in a ratio of ₁ : Dried powder containing Lactobacillus curvatus HY7601 and lyophilized powder containing Lactobacillus plantarum KY1032 in a weight ratio of 1: 1 was added in an amount of 10 parts by weight per 100 parts by weight of the lyophilized powder, And mixed in a high-speed rotary mixer. To 100 parts by weight of the mixture, 10 parts by weight of sterilized purified water was added and mixed to form granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

&Lt; Test Example 1 >

Liver weight, liver lipid accumulation, liver level, enzymatic activity relating to hepatic lipid synthesis, gene related to hepatic fatty acid synthesis, and cholesterol synthesis-related gene mRNA expression in high fat-fed mice by Lactobacillus carbetus HV7601 and Lactobacillus plantarum KY1032, Decrease in blood insulin concentration, decrease in blood ritestine concentration

1-1. Experimental animal breeding and sacrifice

C57BL / 6J 4 week old male mice were purchased from Jackson Labratories (Bar Harbor, ME) and used for high fat diet for 1 week. All mice in the experimental group were randomly assigned to 10 mice. The experimental groups were divided into three groups: normal diet group, high fat diet group, lactic acid bacteria supplement group (high fat diet + Lactobacillus carvitus HY7601 + Lactobacillus plantarum KY1032), and composition of general diet and high fat diet were as shown in Table 3 below .

The lyophilized powder of Lactobacillus carvitus HY7601 of Example 1 and the lyophilized powder of Lactobacillus plantarum KY1032 were mixed with the diet to give a concentration of 5 x 10 ⁹ CFU per g of the dietary weight, and then administered to the mice. The total body weight and feed intake were measured twice a week by feeding the experimental diet for a total of 9 weeks. After 9 weeks of feeding, the animals were fasted for 12 hours, sacrificed after being anesthetized with diethyl ether. Blood was collected from the lower vena cava to analyze the blood of the experimental animals.

Liver and adipose tissue were collected, washed with PBS and weighed and stored in nitrogen for enzymatic activity and gene expression analysis.

Ingredient (g) A normal diet Highland method Casein 200.00 200.00 D, L-methionine 3.00 3.00 Corn starch 150.00 111.00 Sucrose 500.00 370.00 Cellulose powder 50.00 50.00 Corn oil 50.00 30.00 Lard - 170.00 mineral mixture ^a 35.00 42.00 vitamin mixture ^b 10.00 12.00 Choline bitartrate 2.00 2.00 Cholesterol - 10.00 tert-Butylhydroquinone 0.01 0.04 Total (g) 1000.0 1000.0

^a AIN-76 mineral mixture contained in (g / kg of mixture): calcium phosphate dibasic, 500.0; sodium chloride, 74.00; potassium citrate H ₂ O, 222.00; potassium sulfate, 52.00; magnesium oxide, 24.00; manganous carbonate, 3.50; ferric citrate USP, 6.00; zinc carbonate, 1.60; cupric carbonate, 0.30; potassium lodate, 0.01; sodium selenite, 0.01; chromium potassium sulfate 12H ₂ O, 0.55; sucrose, finely powdered, 118.03.

^b AIN-76 vitamin mixture contained in (g / kg of mixture): thiamine HCl, 0.60; riboflavin, 0.60; pyridoxine HCl, 0.70; niacin, 3.00; calcium pantothenate, 1.60; folic acid, 0.20; biotin, 0.02; vitamin B12 (0.1%), 1.00; vitamin A palmitate (500,000 IU / g), 0.80; vitatin D3 (400,000 IU / g), 0.25; vitamin E acetate (500 IU / g); 10.00; menadione sodium bisulfate, 0.08; sucrose, finely powdered, 981.15.

1-2. Liver tissue and body fat weight reduction

The results of weighing liver and body fat weighed are shown in Fig.

As can be seen in FIG. 1, liver and body fat weights increased in the high fat diet group compared to the general diet group were inhibited by the ingestion of Lactobacillus cabutus HY7601 and Lactobacillus plantarum KY1032. The significant difference between the high fat diet group and the lactobacillus supplement group was shown as * p <0.05, ** p <0.01.

1-3. Suppress fat accumulation in liver tissue

Liver was washed with PBS and fixed in 10% (v / v) formalin / PBS for hematoxylin and eosin (H & E) staining. The sections were stained with hematoxylin and eosin (H & E) and examined under a microscope.

The results are shown in Fig.

As can be seen from FIG. 2, it was confirmed that the high fat diet inhibited the accumulation of fat in the liver tissue and the increase in fat pore size by the ingestion of Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 .

1-4. Suppress neutral lipid and cholesterol accumulation in liver tissue

Lipids in liver tissues were extracted using modified Folch's method, and dried lipids were dissolved in 1 ml of isopropanol. The amount of neutral lipids and cholesterol in the liver tissues were measured by Kit (Asan Co., Seoul, Korea) . One enzyme enzyme reagent and one buffer solution were mixed to make an enzyme solution, and the reaction was compared by mixing 1.5 ml of the enzyme solution and 10 μl of the serum or 10 μl of the standard solution. After reacting at 37 ° C for 5 minutes, absorbance was measured at 500 nm. The cholesterol and triglyceride concentrations of the samples were calculated as follows.

Concentration of sample (mg / dl) = absorbance of sample / absorbance of standard solution x concentration of standard solution

The results are shown in Table 4.

As shown in Table 4, in the group (lactobacillus supplement group) fed with Lactobacillus cabutus HY7601 and Lactobacillus plantarum KY1032, the amount of neutral lipids and cholesterol in the liver increased Respectively. ** p <0.01 and *** p <0.001 were significant differences between high fat diet group and lactobacillus supplement group.

General diet It's a high-fat diet. Lactic acid bacteria supplement group Triglyceride (mg / g liver) 9.25 + 0.28 14.75 ± 0.37 ^* 6.64 + - 0.51 *** Cholesterol (mg / g liver) 2.80 ± 0.15 12.46 + 0.74 ^* 9.66 + 1.07 **

1-5. Reduced AST and ALT in blood

AST and ALT in the blood of the experimental animals were measured using Kit (Asan Co., Seoul, Korea).

The results are shown in Fig.

As can be seen from FIG. 3, in the group (lactobacillus supplement group) fed with Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032, the decrease of hepatic AST and ALT was observed, which suppressed the increase of hepatic secretion due to high fat dietary intake Respectively. The significant difference between the high fat diet group and the lactobacillus supplement group was shown as * p <0.05.

1-6. Reduced enzyme activity related to lipid synthesis in liver tissue

Fatty acid synthase (FAS) activity was measured by the method of Nepokroeff, Nepokroeff, CM, Lakshmanan, MR, Poter, JW, Fatty acid synthase from rat liver method Enzymol. 1975, 35, 37-44 Oxidized NADPH (nmol / min / mg protein).

The activity of malic enzyme (ME) was evaluated by Ochoa (Ochoa, S., in: Colowick, SP, Kaplan, NO (Ed), Malic enzyme: malic enzymes from pigeon and wheat germ, Methods in Enzymol. Academic 1995, pp. NADPH (nmol / min / mg protein), which was measured by the method of Nippon Kayaku Co., Ltd., 323-326).

The results are shown in Table 5.

As can be seen in Table 5, the activities of the enzymes involved in the fatty acid synthesis (FAS, ME) in the Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032-supplemented groups (lactic acid bacteria supplement group) were decreased. Therefore, it was confirmed that Lactobacillus carbetus HY7601 and Lactobacillus plantarum KY1032 inhibit lipid accumulation in liver tissue by reducing lipid synthesis in liver tissue by high fat diet.

** p <0.05, ** p <0.01, *** p <0.001 were significant differences between high fat dietary group and lactic acid supplement group.

General diet It's a high-fat diet. Lactic acid bacteria supplement group FAS 16.29 ± 1.10 17.97 ± 1.20 11.13 + - 0.95 ** ME 166.66 + 7.66 ^* 68.69 ± 3.14 48.19 ± 3.83 ***

1-7. Reduction of mRNA expression of genes related to fatty acid synthesis and cholesterol synthesis in liver tissues

Expression of genes related to fatty acid synthesis (FAS, ME, ACC1) in liver tissues and expression of genes related to cholesterol synthesis (HMGCR, ACAT1, ACAT2) were analyzed. RNA was isolated from the liver using TRIZOL reagent (Invitrogen, Carsbad, CA, USA). CDNA was synthesized from the separated RNA using QuantiTect reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantified by real-time PCR using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) and the CFX384 real-time RT-PCR detection system (Bio-Rad, Hercules, CA). Mouse primer sequences are shown in Table 6. GAPDH was used as an internal control.

gene primer sequence Fatty acid synthase (FAS) forward; 5'-CGCTCCTCGCTTGTCGTCTG-3 '
reverse; 5'-AGCCTTCCATCTCCTGTCATCATC-3 ' Malic enzyme (ME) forward; 5'-CAGCAGTACAGTTTGGCATTCCG-3 '
reverse; 5'-AATAGCCTTGACGACATCCTCTGG-3 ' Acetyl-CoA carboxylase 1
(ACC1) forward; 5'-gcctcttcctgacaaacgag-3 '
reverse; 5'-tgactgccgaaacatctctg-3 ' 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) forward; 5'-TTCACGCTCATAGTCGCTGGATAG-3 '
reverse; 5'-GGTTCAATTCTCTTGGACACATCTTC-3 ' Acetyl-CoA acetyltransferase 1
(ACAT1) forward; 5'-CTCACGGCAGGAACAGGATACG-3 '
reverse; 5'-TTCTTCATCTTCTTTCACCACCACATC-3 ' Acetyl-CoA acetyltransferase 2
(ACAT2) forward; 5'-GAGACCAGAGCGACAAGATGAATG-3 '
reverse; 5'-GACAGGCACGGTGGACAGG-3 '

The results are shown in Fig. 4 and Fig.

As can be seen from FIG. 4, the expression of genes involved in fatty acid synthesis (FAS, ME, ACC1) in liver tissues was significantly higher in the groups fed lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 Respectively.

As can be seen from FIG. 5, the expression of genes related to cholesterol synthesis (HMGCR, ACAT1, ACAT2) was significantly increased in Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 Respectively.

1-8. Decrease blood insulin levels

The insulin concentration in the blood was measured using a mouse insulin ELISA kit purchased from Millipore Co. The results are shown in FIG.

As can be seen in FIG. 6, the blood insulin levels increased in the high-fat diet group compared with the normal diet group, and the blood insulin levels were significantly decreased in the lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032-supplemented group (lactic acid bacteria supplement group) . The significant difference between the high fat diet group and the lactobacillus supplement group was shown as * p <0.05.

1-9. Decrease blood ritestine concentration

The concentration of ridicutin in the blood was measured using a mouse insulin ELISA kit purchased from Millipore Co. The results are shown in FIG.

As can be seen from FIG. 7, the blood ryestine concentration increased in the high fat diet group compared to the general diet group was significantly decreased in the lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 (lactic acid bacteria supplement group) Respectively. The significant difference between the high fat diet group and the lactobacillus supplement group was *** p <0.001.

&Lt; Test Example 2 &

Reduction of insulin, glucose and C-peptide levels in the blood of rats with high fructose intake by Lactobacillus carbetus HY7601 and Lactobacillus plantarum KY1032

2-1. Experimental animal breeding and sacrifice

Experimental animals were purchased from Wistar 4 week old male rats in Jackson Labratories (Bar Harbor, Me.) And adapted for feeding for 1 week. Rats in all experimental groups were randomly assigned to 8 rats. The experimental groups were divided into three groups: normal diet group, high fructose diet group, and lactobacillus supplement group (high fructose diet + Lactobacillus cabutus HY7601 + Lactobacillus plantarum KY1032). The lyophilized powder of Lactobacillus carvitus HY7601 of Example 1 and the lyophilized powder of Lactobacillus plantarum KY1032 were mixed with the diet to give a concentration of 5 x 10 ⁹ CFU per gram of food weight, and then administered to rats. The body weight and feed intake were measured at intervals of two times a week for a total of three weeks. After 3 weeks of feeding, the animals were fasted for 12 hours, sacrificed after being anesthetized with diethyl ether. Blood was collected from the lower vena cava to analyze the blood of the experimental animals.

Ingredient (g) High fructose diet Casein 200.00 L-Cystine 3.00 Fructose 700.00 Cellulose powder 50.00 Corn oil 25.00 Lard 20.00 Mineral Mix S10026 10.00 DiCalcium Phosphate 13.00 Calcium Carbonate 5.50 Potassium citrate 16.50 Vitamin Mix V10001 10.00 Choline bitartrate 2.00 Total (g) 1000.0

2-2. Decrease blood insulin levels

The insulin concentration in the blood was measured using a rat insulin ELISA kit purchased from Millipore Co. The results are shown in FIG.

As can be seen from FIG. 8, the blood insulin level increased in the high fructose diet group compared to the normal diet group was significantly decreased in the Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 (lactic acid bacteria supplement group) Respectively. The significant difference between the high fructose diet group and the lactobacillus supplement group was shown as * p <0.05.

2-3. Decrease in blood glucose concentration

The blood glucose concentration was measured using a glucose assay kit purchased from Cayman Co. The results are shown in FIG.

As can be seen from FIG. 9, the increase in blood glucose level in the high-fructose diet group compared to the normal diet group was significantly decreased in the lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032-fed group (lactic acid bacteria supplement group) Respectively. The significant difference between the high fructose diet group and the lactobacillus supplement group was shown as * p <0.05.

2-4. Decrease in blood C-peptide concentration

The C-peptide concentration in the blood was measured using a rat C-peptide ELISA kit purchased from Biovendor, and the results are shown in FIG.

As can be seen from FIG. 10, the serum C-peptide concentration increased in the high-fructose diet group compared to the normal diet group was significantly higher in the Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 (lactic acid bacteria supplement group) Respectively. The significant difference between the high fructose diet group and the lactobacillus supplement group was shown as * p <0.05.

2-5. Decrease blood triglyceride level

The triglyceride assay kit purchased from Asan Pharmaceutical Co., Ltd. was used to measure the blood triglyceride concentration and the results are shown in Fig.

As can be seen from FIG. 11, the blood triglyceride levels increased in the high fructose diet group compared to the general diet group, and the blood triglyceride concentration in the high fat fructose diet group significantly increased in the Lactobacillus carvettus HY7601 and Lactobacillus plantarum KY1032 Respectively. The significant difference between the high fructose diet group and the lactobacillus supplement group was shown as * p <0.05.

Korea Biotechnology Research Institute KCTC11456BP 20090122 Korea Microorganism Conservation Center (overseas) KCCM10430 20021008

Claims (8)

Reduce liver weight gain due to obesity,
Improves the symptoms of fatty liver that accumulates in the liver,
Lower the amount of triglycerides and cholesterol in the liver,
Fatty Acid Synthase (FAS), Malic Enzyme (ME), Acetyl-CoA Carboxylase 1 (ACC1), and Genes Related to Cholesterol Synthesis HMGCR (3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase), ACAT1 ( Decreases the expression of Acetyl-CoA Acetyltransferase 1), ACAT2 (Acetyl-CoA Acetyltransferase 2),
Gansuchi AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase), characterized in that for having an effect of reducing Lactobacillus greater Better bus (Lactobacillus curvatus) HY7601 (Accession No: KCTC 11456BP) and Lactobacillus Planta room (Lactobacillus plantarum ) A pharmaceutical composition for improving liver function containing KY1032 (Accession Number: KCCM10430) as an active ingredient.
Reduce liver weight gain due to obesity,
Improves the symptoms of fatty liver that accumulates in the liver,
Lower the amount of triglycerides and cholesterol in the liver,
Fatty Acid Synthase (FAS), Malic Enzyme (ME), Acetyl-CoA Carboxylase 1 (ACC1), and Genes Related to Cholesterol Synthesis HMGCR (3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase), ACAT1 ( Decreases the expression of Acetyl-CoA Acetyltransferase 1), ACAT2 (Acetyl-CoA Acetyltransferase 2),
Gansuchi AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase), characterized in that for having an effect of reducing Lactobacillus greater Better bus (Lactobacillus curvatus) HY7601 (Accession No: KCTC 11456BP) and Lactobacillus Planta room (Lactobacillus plantarum ) Fermented milk for improving liver function containing KY1032 (Accession No .: KCCM10430) as an active ingredient.
Reduce liver weight gain due to obesity,
Improves the symptoms of fatty liver that accumulates in the liver,
Lower the amount of triglycerides and cholesterol in the liver,
Fatty Acid Synthase (FAS), Malic Enzyme (ME), Acetyl-CoA Carboxylase 1 (ACC1), and Genes Related to Cholesterol Synthesis HMGCR (3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase), ACAT1 ( Decreases the expression of Acetyl-CoA Acetyltransferase 1), ACAT2 (Acetyl-CoA Acetyltransferase 2),
Gansuchi AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase), characterized in that for having an effect of reducing Lactobacillus greater Better bus (Lactobacillus curvatus) HY7601 (Accession No: KCTC 11456BP) and Lactobacillus Planta room (Lactobacillus plantarum ) Drink for improving liver function containing KY1032 (Accession Number: KCCM10430) as an active ingredient.
Reduce liver weight gain due to obesity,
Improves the symptoms of fatty liver that accumulates in the liver,
Lower the amount of triglycerides and cholesterol in the liver,
Fatty Acid Synthase (FAS), Malic Enzyme (ME), Acetyl-CoA Carboxylase 1 (ACC1), and Genes Related to Cholesterol Synthesis HMGCR (3-Hydroxy-3-Methyl-Glutaryl-CoA Reductase), ACAT1 ( Decreases the expression of Acetyl-CoA Acetyltransferase 1), ACAT2 (Acetyl-CoA Acetyltransferase 2),
Gansuchi AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase), characterized in that for having an effect of reducing Lactobacillus greater Better bus (Lactobacillus curvatus) HY7601 (Accession No: KCTC 11456BP) and Lactobacillus Planta room (Lactobacillus plantarum ) Health functional food for improving liver function containing KY1032 (Accession Number: KCCM10430) as an active ingredient.
( Lactobacillus &lt; RTI ID = 0.0 &gt; ( Lactobacillus ) &lt; / RTI &gt; ( Lactobacillus ), which has an effect of reducing the levels of blood insulin, resistin, glucose, C-peptide and triglyceride Curvatus HY7601 (Accession No .: KCTC 11456BP) and Lactobacillus plantarum ) A pharmaceutical composition for improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia containing KY1032 (Accession Number: KCCM10430) as an active ingredient.
( Lactobacillus &lt; RTI ID = 0.0 &gt; ( Lactobacillus ) &lt; / RTI &gt; ( Lactobacillus ), which has an effect of reducing the levels of blood insulin, resistin, glucose, C-peptide and triglyceride Curvatus HY7601 (Accession No .: KCTC 11456BP) and Lactobacillus plantarum ) KY1032 (Accession No .: KCCM10430) as an active ingredient for improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia.
( Lactobacillus &lt; RTI ID = 0.0 &gt; ( Lactobacillus ) &lt; / RTI &gt; ( Lactobacillus ), which has an effect of reducing the levels of blood insulin, resistin, glucose, C-peptide and triglyceride Curvatus HY7601 (Accession No .: KCTC 11456BP) and Lactobacillus plantarum ) KY1032 (Accession No .: KCCM10430) as an active ingredient for improving hyperinsulinemia, hyperglycemia and hypertriglyceridemia.
( Lactobacillus &lt; RTI ID = 0.0 &gt; ( Lactobacillus ) &lt; / RTI &gt; ( Lactobacillus ), which has an effect of reducing the levels of blood insulin, resistin, glucose, C-peptide and triglyceride Curvatus HY7601 (Accession No .: KCTC 11456BP) and Lactobacillus plantarum ) KY1032 (Accession No .: KCCM10430) as an active ingredient for the treatment of hyperinsulinemia, hyperglycemia and hypertriglyceridemia.

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