KR20040064157A - Lactobacillus fermentum and method of producing culture fluid cotaining conjugated linoleic acid with using this - Google Patents

Abstract

PURPOSE: A microorganism Lactobacillus fermentum and a method for producing conjugated linoleic acid(CLA) using the same microorganism are provided, which microorganism can be used in food because the microorganism is isolated from human, and which conjugated linoleic acid(CLA) has anticancer activity, antioxidative activity, cholesterol reduction, immunity improvement, fat reduction and muscle improvement. CONSTITUTION: The microorganism Lactobacillus fermentum(KACC 91008) is isolated from the feces of human and produces conjugated linoleic acid(CLA), The method for producing conjugated linoleic acid(CLA) comprises the steps of: culturing Lactobacillus fermentum in a medium for 24 hours; adding linoleic acid and tween 80 into the medium; and culturing the medium for 24 hours, thereby the medium contains 0.30 to 0.39 mg of CLA.

Description

LACTOBACILLUS FERMENTUM AND METHOD OF PRODUCING CULTURE FLUID COTAINING CONJUGATED LINOLEIC ACID WITH USING THIS}

The present invention relates to a lactobacillus Lactobacillus fermentum (Lactobacillus fermentum), which produces a large amount of conjugated linoleic acid (CLA), and a method for producing a culture solution containing a large amount of CLA using the same. The present invention relates to a lactobacillus permeum, a lactobacillus bacterium isolated and collected from feces, and a method for preparing a culture solution containing 0.30 to 0.39 mg of CLA per mL of the culture medium using the same.

Linoleic acid is a C18 fatty acid with a double bond in the cis-9, cis-12 configuration, and CLA is a generic term for the position and geometric isomers of this linolenic acid (cis-9, cis-12-octadecadienoic acid). . Among these isomers, cis-9, trans-11 and trans-10 and cis-12 linolenic acid are known to be effective in anticancer, antioxidant, cholesterol lowering, immune activation, body fat reduction and muscle enhancement.

As a result, methods for producing CLA have been researched and developed. Rutin microbe butyrivibrio fibrisolbenz (Butyrivibrio fibrisolvens)It was found that CLA is produced in the process of linolenic acid hydrogenated to stearic acid.

In addition, Propionibacterium Prudenreich, a microorganism that is used as a starting material for the manufacture of dairy products,(Propionibacterium freudenreichiissp.freudenreichii)And Propionibacterium Friedenrich SSP Shermani (Propionibacterium freudenreichiissp.shermanii)And Lactobacillus rutheri piwaial 8 (Lactobacillus reuteriPYR8) and Lactobacillus exidophilus AKYU 1137 (Lactobacillus acidophilusAKU 1137 reported that the CLA was generated.

In particular, the production of CLA by L. reuteri and L. acidophilus was recovered by centrifugation of cultured lactic acid bacteria, followed by tris buffer or phosphoric acid added with linolenic acid. The linolenic acid is converted to CLA anaerobicly in potassium phosphate buffer. Such CLA is accumulated in lipids bound to cells or cells and recovered by centrifugation, or the cells themselves may be used as a source of CLA.

However, the production level of CLA produced by the above-described conventional method is reported differently according to the species, the type and conditions of the medium, and the production amount was also a trace amount of 0.05 mg or less per ml of the culture medium.

Accordingly, the present inventors have succeeded in separating the strain producing large amounts of CLA from the feces of the human body, and have developed a method of producing a large amount of CLA using the same.

Probiotics are living microbial dietary additives that have beneficial effects on the host by intestinal microbial balance. Lactic acid bacilli and Bifidobacteria are most often used as probiotics. By the way, the most important condition to be used as a probiotic should be derived from the human body. This is important because the effect of the probiotic is species specific and the immune relationship between the host and the intestinal microflora.

The present invention is derived from the human body can be used in probiotic and can be useful for fermented milk and health supplements.

An object of the present invention is to provide a strain that can be applied to food and probiotics derived from the human body and produces a large amount of CLA.

Another object of the present invention is to provide a method for producing a culture solution containing 0.30 to 0.39 mg of CLA per mL culture medium using the strain producing a large amount of the above-described CLA.

1 is a graph showing CLA production activity of a strain isolated from feces of infants.

2A is an HPLC analysis diagram of CLA methyl ester as standard.

2B is an HPLC analysis of MRS medium as a control.

Figure 2c is a HPLC analysis of MRS medium containing linolenic acid as a control.

Figure 2d is a HPLC analysis of the strain culture of the present invention.

Figure 3 is a graph showing the time-dependent CLA production amount of the culture medium using the strain of the present invention.

The present invention is characterized by providing a novel strain, Lactobacillus percentage, collected by separating from feces of the human body.

When linolenic acid is added to the culture medium for culturing the Lactobacillus fermentum of the present invention, the culture medium containing 0.30 to 0.39 mg of CLA per mL can be prepared.

Hereinafter, the present invention will be described in more detail in the following examples, and the scope of the present invention is not limited to the following examples.

[Example 1] Selection of Lactobacillus pertumtum, a CLA-producing strain

Fecal samples from healthy infants were taken in MRS liquid medium containing 0.02% sodium azide and incubated at 37 ° C for 24 hours, followed by 0.02% sodium azide using platinum. The resulting colonies were plated on MRS agar plate medium and incubated at 37 ° C. for 48 hours to separate the fungi formed. After culturing for 24 hours in 5 mL of MRS liquid medium (1% tween 80) containing linolenic acid (5 mg / mL), 4 mL of the culture was centrifuged (3,000 g, 10 minutes) to recover the cells. Wash with 0.1M potassium phosphate buffer (pH 7.0). After adding and dispersing 1.3 mL buffer solution, the cells were pulverized by exposure to a sonicator for 3 minutes. The ground cells were centrifuged at 10,000 g for 15 minutes to use the supernatant as a coenzyme solution. 0.01 mL of this enzyme solution was added to 3 mL of reaction solution (1,3-propanediol, 2.7 mL 0.1 M phosphate buffer, 24 μM linolenic acid) and the absorbance was measured at 233 nm.

The absorbance measured indicates the degree of CLA production of each strain isolated from feces of the infant, which is caused by the conversion of linolenic acid to CLA. That is, CLA absorbs light at 233 nm and high absorbance means that a large amount of CLA is generated. The absorbance measured for each strain is shown in Figure 1 and the highest absorbance of the L7-2 strain.

1 mL of the culture solution was pretreated by the method of Jiang et al. (1998) and analyzed by HPLC by the method of Chin (1992). Briefly described as follows.

1 mL of the culture was mixed with 2 mL of isopropanol, 1.5 mL of nucleic acid was added, and the nucleic acid layer was recovered by centrifugation. The extraction process was repeated two more times. 2 mL of 2 mol KOH (in ethanol) was added to the extracted nucleic acid layer and heated at 50 ° C. for 30 minutes to hydrolyze fat to fatty acids, and 2 mL of 3N hydrochloric acid (in methanol) was added to form a methyl ester. 1 mL nucleic acid and 1 mL distilled water were added to the methylated sample, and the organic solvent layer was separated by centrifugation, followed by drying by flushing nitrogen at room temperature. The dried sample was dissolved in 1 mL methanol and analyzed by HPLC. The column was Eclipse XDB-C18 (250 × 4.6, ZORBAX), solvent was 95% acetonitrile: 5% water with a flow rate of 1.2 mL per minute, detected at 245 nm and the results are shown in FIGS. 2A-2D. As a result of analysis by HPLC, only the L7-2 strain of FIG. 1 was able to confirm that CLA was detected.

The characteristics of the strains selected by the above method are as follows.

1) Morphology

Morphology of bacteria when incubated for 2 days at 37 ° C in agar plate (MRS) agar plate

① Cell Type: Bacillus (Figure 1)

Figure 1.Cell Morphology of Selected Lactobacillus Lactobacillus Fermentum

② Mobility: None

③ Spore Formation Capacity: None

④ Gram Dyeing: Positive

2) Form of the flora

Morphology of the Bacillus incubated at 37 ° C for 2 days in MRS agar plate

① Shape: Round

② uplift: convex

③ Surface: Smooth

3) Catalase:-

4) Gas Formation: +

5) Growth at 15 ℃:-

6) Growth at 45 ℃: +

7) Indole Production:-

8) Lactic Acid Production: +

9) sugar fermentation test

Amigdalin:-

D-Arabinose:-

L-Arabinose:-

Cellobiose:-

Esculin:-

Fructose: +

Galactose: +

Glucose: +

Gluconate:-

Lactose:-

Maltose: +

Mannitol:-

Mannose:-

Melegitos:-

Melibiose: +

Rafinos: +

Ribose:-

Rhamnose:-

Salisin:-

Sorbitol:-

Sucrose: +

Trehalose:-

Xylose:-

Glycerol:-

Erythritol:-

Adonitol:-

Beta methyl-xylside:-

Sorboth:-

DoulCitol:-

Inositol:-

Alpha methyl-di-mannoside:-

Alpha Methyl-Di-Glucoside:-

Enacetyl Glucosamine:-

Inulin:-

Amidodon:-

Glycogen:-

Xylitol:-

Beta Genthiobiose:-

Turanos:-

Resources:-

Tagatose:-

Fucose:-

Arabitol:-

2 Keto-Gluconate:-

5 Keto-Gluconate:-

10) Microbial Accession No.: KACC 91008

As described above, the strain was Gram-positive, catalase-negative, and did not grow at 15 ° C. but grew at 45 ° C., and conducted 45 sugar fermentation experiments. This strain was identified as Lactobacillus fermentum.

Example 2 Culture Preparation Using Lactobacillus Percentum

The newly identified strain Lactobacillus fermentum was incubated in a MRS medium (1 liter) for 24 hours using a fermenter, and then 10 mL of Tween 80 and 5 mL of linolenic acid were added. After 2, 4, 6, 24 and 48 hours, 1 mL of the culture was taken and the production of CLA was measured using HPLC.

The yield of CLA over time is shown in FIG. 3. CLA content of culture medium 2 hours after linolenic acid addition was 0.39 mg, which was 0.36 mg after 6 hours, and the amount of CLA decreased after that.

As can be seen from the results of Example 2, the Lactobacillus permeum isolated from the feces of infants incubated for 24 hours in MRS medium, when the strain is sufficiently grown and energy is depleted, if tween 80 is added as an emulsifier and linolenic acid is added The strain transforms linolenic acid to CLA to produce CLA.

The change in the production of CLA over time, the reaction proceeds rapidly due to the nature of the enzyme, the highest production of CLA per mL culture medium 2 hours after linolenic acid addition, and after 6 hours, the production of CLA decreased. This is due to the increase in translocation to and the death of the strain.

As described above, the present invention isolates and selects a novel strain of Lactobacillus fermentum from the feces of the human body, and by using the CLA which is effective in anticancer, antioxidant, cholesterol lowering, immune activation, body fat reduction and muscle enhancement. Can produce large quantities. Since the Lactobacillus permentum of the present invention is derived from the human body, it can be used as a probiotic and applicable to various foods such as fermented milk and health supplement food, thereby being useful for human health.

Claims (5)

Lactobacillus percentage, which produces CLA (conjugated linoleic acid), collected from human feces. A method for producing a culture solution containing 0.30 to 0.39 mg of CLA per mL of culture by adding linolenic acid to a medium for culturing the Lactobacillus fermentum of claim 1. Probiotic comprising a CLA-containing culture prepared in claim 2. Food provided comprising the CLA-containing culture medium prepared in claim 2. The food according to claim 4, wherein the food is one of fermented milk or health supplement.