KR100299306B1 - Lactic acid bacteria with high acid resistance and growth rate and excellent blood cholesterol lowering ability - Google Patents

Abstract

The present invention relates to Lactobacillus ashdophilus SNUL 01 strain, which is excellent in acid resistance, bile acid resistance, blood cholesterol lowering ability and fast growth rate isolated from feces of healthy Korean adults, and the strain of the present invention is acid resistant and bile acid. Because of excellent properties related to resistance and lowering blood cholesterol and fast growth rate, the strain of the present invention has a high survival rate in the digestive organs, and in particular, the strain of the present invention has been isolated from living organisms of healthy Korean adults and thus can be established in Korean intestines. Most likely. Therefore, when the culture or fermented food cultured strain or strain of the present invention can be eaten, it can be expected to have a formal effect and cholesterol lowering effect.

Description

Lactic acid bacteria with high acid resistance and growth rate and excellent blood cholesterol lowering ability

The present invention relates to a new lactic acid bacterium having excellent acid resistance and bile acid resistance, excellent viability in the digestive organs of the human body, excellent blood cholesterol lowering ability, and fast growth rate, and more particularly, lactose exhibiting the above-described excellent characteristics. It relates to Bacillus ashdophyllus.

Lactic acid bacteria are distributed in the intestines of animals, have the effect of inhibiting the growth of harmful microorganisms, treatment of abnormal fermentation, lowering blood cholesterol, enhancing immune function, and also have anti-cancer effects.

Fermented dairy products are commercialized and widely consumed or edible so that humans can ingest these lactic acid bacteria to promote their health. Representative strains used in fermented dairy products are L. acidophilus, Lactobacillus casei, L. bulgaricus, Streptococcus thermophilus Etc.

For lactic acid bacteria to have a beneficial effect on the human body, it must be able to survive the passage through the digestive tract and settle in the small intestine. Therefore, the higher resistance to acid secreted from the stomach and bile acids secreted from the small intestine increases the number of viable bacteria that survive in the harsh environment of these digestive tracts and reach the small intestine. When manufactured, it can function substantially useful to the human body.

Therefore, acid resistance and bile acid resistance have become important issues as properties required for lactic acid bacteria used in fermented dairy products, and various efforts have been made to isolate and develop strains having excellent characteristics.

On the other hand, in order for the lactic acid bacteria to have a continuous useful function, in addition to the acid resistance and bile acid resistance described above, the intestinal fixation rate should be high. However, since lactic acid bacteria exhibit host specificity, for fermentation products for humans, lactic acid bacteria isolated from humans should actually settle in the human body to obtain desired effects.

In addition, since lactic acid bacteria also have an effect of lowering blood cholesterol, which is known to be harmful to adult diseases of humans, in particular, diseases of the circulatory system, it is required to develop lactic acid bacteria having excellent characteristics.

Accordingly, an object of the present invention is to provide a new lactic acid bacterium having high acid resistance and bile acid resistance, fast growth rate, and excellent ability to lower cholesterol in the blood of the human body.

Another object of the present invention is to provide Lactobacillus ashidophilus (Lactobacillus ashidophyllus SNUL 01), which exhibits useful properties when ingested in the human body.

Another object of the present invention is to provide a new lactic acid bacteria suitable for Koreans.

Another object of the present invention is to provide a fermentation product prepared using Lactobacillus ashidophilus SNUL 01.

Another object of the present invention is to provide a formal preparation prepared using Lactobacillus ashidophilus SNUL 01.

1 is a graph showing the results of the acid resistance test (1) of Example 1 and Comparative Examples 1 and 2,

2 is a graph showing the results of the acid resistance test (2) of Example 2 and Comparative Examples 3 to 5,

3A to 3D are graphs showing the results of the bile acid resistance test of Example 3 and Comparative Examples 6 to 8,

Figure 4 is a graph showing the results of the growth test of Example 5 and Comparative Examples 12 to 14,

Figure 5 is a graph showing the results of the growth test in skimmed milk of Example 6 is Comparative Example 15.

Lactic acid bacteria are widely distributed in human oral cavity, intestine, vagina and feces. Therefore, a method of obtaining lactic acid bacteria having desired properties by separating the lactic acid bacteria from these and testing their properties has been performed, and in particular, a method of separating the lactic acid bacteria from feces has been usefully used.

In the present invention, in consideration of the host specificity, by separating the lactic acid bacteria from the human body of the Korean as a lactic acid bacteria used in fermentation products for Koreans, it is possible to maximize the useful effect of lactic acid bacteria in the relationship of the host specificity.

In view of this, the present inventors have tested lactic acid from Korean adults to isolate a strain suitable for producing fermentation products suitable for Korean physiology, and have excellent acid resistance, bile acid resistance, blood cholesterol lowering ability and rapid growth rate. Successful isolation of new lactic acid bacteria has reached the present invention.

In order to isolate the strains according to the present invention, feces of healthy Korean adults were placed in MRS liquid medium containing 0.02% sodium azide and incubated at 37 ° C for 24 hours. After incubation, the culture medium was taken using 10 μl platinum, and then plated again on agar plate agar containing 0.02% sodium azide and incubated at 37 ° C. for 48 hours. Among the fungi formed, the strains of the present invention were isolated by testing for acid resistance and bile acid resistance. Lactobacillus of the present invention was deposited on July 3, 1998 by the Korean spawn association (Repair number: KFCC-11041).

The characteristics of the lactic acid bacteria according to the present invention 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

② Mobility: None

③ Spore Formation Capacity: None

④ Gram staining: 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: rough

3) physiological properties

① Growth temperature: Possible growth temperature: 25 ~ 45 ℃

Optimum growth temperature: 37-40 ℃

② Growth pH: Growth possible Growth pH: 5.0 ~ 7.5

Optimum pH: 6.0 to 6.5

③ Effect on oxygen: anaerobic anaerobic

4) Catalase:-

5) Gas formation:-

6) Growth at 15 ℃:-

7) Growth at 45 ℃: +

8) Indole Production:-

9) Lactic Acid Production: +

10) Sugar fermentation test of Bergey's manual

Amigdalin:-

Arabian:-

Cellobiose: +

Esculin: +

Fructose: +

Galactose: +

Glucose: +

Gluconate:-

Lactose: +

Maltose: +

Mannitol:-

Mannose: +

Melegitos:-

Melibiose:-

Rafinos: +

Ribose:-

Rhamnose:-

Raised:-

Sorbitol:-

Sucrose: +

Trehalose: +

Xylose:-

Based on the morphological, physiological and growth characteristics of such bacteria, the strain of the present invention was identified as Lactobacillus ashdophyllus, and the inventors named it Lactobacillus ashdophyllus SNUL 01.

Lactic acid bacteria of the present invention may be provided in the form of a culture or lyophilized powder. Thus, the culture containing the strain of the present invention obtained by fermenting the sugar or protein using the lactic acid bacteria of the present invention or the strain of the present invention, excellent survival rate and growth in the intestines of Korean adults, inhibiting the growth of harmful microorganisms in the intestine Since it is confirmed that the effect is high, it can be used as a formal agent to obtain formal effects such as maintenance of normal intestinal flora or suppression of harmful microorganisms. Particularly widely fermented foods such as yogurt and cheese can be prepared using the strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. The composition of the MRS medium and MRS agar plate medium used in these Examples and Comparative Examples are shown in Tables 1 and 2, respectively.

Composition of the MRS Badge Proteose Chengton No. 3 10 g Hex extract 10 g Yeast extract 5 g glucose 20 g Tween 80 1 g Ammonium citrate 2 g Sodium acetate 5 g Magnesium sulfate (MgSO ₄ .7H ₂ O) 0.1 g Manganese Sulfate (MnSO ₄ .4H ₂ O) 0.05 g Potassium phosphate (dibasic) 2 g Distilled water 1000 ml

MRS agar plate MRS Badge 100 ml Agar 1.5 g

Example 1: Acid Resistance Test (1)

0.2% sodium thioglycollate was added to the MRS liquid medium to prepare a MRS Thio medium. The medium was inoculated with the Lactobacillus ashidophilus SNUL 01 strain of the present invention and incubated at 37 ° C for 24 hours. Sterilized 1.25 N hydrochloric acid solution was added to the culture to adjust the medium to pH 2. Samples were taken at 30 minute intervals while the medium was subsequently incubated at 37 ° C.

The sample taken with a mixed solution of 8.5 ml of sterilized 0.1% peptone solution and 0.5 ml of 0.5 N NaOH solution was neutralized and diluted, followed by serial dilution with 9 ml of 0.1% peptone solution. Diluted samples were inoculated into MRS agar plate medium and incubated at 37 ° C. for 2 days to count the number of cells formed to measure viable cells.

Comparative Examples 1 and 2: Acid Resistance Test (1)

In the same manner and conditions as in the above Example, Lactobacillus ashdophilus 145 (WIESBY, Germany) and Lactobacillus ashdophilus NCFM (MARSCHALL, USA) strains of the test strains were tested for acid resistance to make Comparative Examples 1 and 2. . The result of Example 1, the comparative example 1, and the comparative example 2 is shown in FIG.

Example 2: Acid Resistance Test (2)

The Lactobacillus ashidophilus SNUL 01 of the present invention was inoculated in a MRS liquid medium and incubated at 37 ° C. for 18 hours, and then the cells were recovered by centrifugation (3,000 × g) for 20 minutes using a centrifuge. The suspension obtained by suspending the recovered cells in physiological saline was diluted with physiological saline.

0.05 M sterile phosphate buffer with pH adjusted to 2 by addition of 10% hydrochloric acid was added to the suspension to 10 cfu / ml. While culturing again at 37 ° C., the bacterial solution was collected and diluted every hour, inoculated in MRS agar plate medium, and cultured at 37 ° C. for 2 days to count viable cells.

Comparative Examples 3 to 5: acid resistance test (2)

Lactobacillus ashdophyllus CH-5 (CHR. HANSEN'S LAB., A / S DENMARK), Lactobacillus ashdophilus 145 and Lactobacillus ashdophilus NCFM, which are the disclosed strains in the same manner and conditions as in Example 2 Acid resistance was tested for the strains to make Comparative Examples 3 to 5. The result of Example 2 and Comparative Examples 3-5 is shown in FIG.

Example 3: Bile Acid Resistance Test

Oxal (oxgall, manufactured by Difco) was added to the MRS broth so that the final concentration was 0%, 0.3%, and 0.5%, and then inoculated with 1% of Lactobacillus ashidophilus SNUL 01 and incubated at 37 ° C. Absorbance was measured at 620 nm using a UV-VISIBLE spectrophotometer (UV-1601, SHIMADZU, Co. Japan).

Comparative Examples 6 to 8: Bile Acid Resistance Test

By using the same methods and conditions as in Example 3, Lactobacillus ashdophilus CH-5, Lactobacillus ashdophilus 145 and Lactobacillus ashdophilus NCFM strains were tested for bile acid resistance to Comparative Examples 6 to 8 was set. The results of Example 3 and Comparative Examples 6 to 8 are shown in Figs. 3A to 3D.

Example 4: Cholesterol Removal Ability Test

Lactobacillus ash of the present invention in MRS liquid medium containing 0.3% oxgall (Difco), 0.2% thiocholic acid and cholesterol (Water soluble cholesterol, Sigma, Co., 100 ㎍ / ㎖) Dofilus SNUL 01 was inoculated 1%. After 24 hours of incubation at 37 ℃, the content of cholesterol remaining in the medium was measured by the method of Rudel and Morris (Rudel & Morris, J. Lipid Research, 1973. 14: 364-366).

Comparative Examples 9 to 11

In the same conditions and methods as in Example 4, the test of cholesterol removal ability was measured for the tested strains Lactobacillus ashidophilus ATCC 43121, Lactobacillus ashidophilus CH-5 and Lactobacillus ashidophilus 145 strains. .

Results of Example 4 and Comparative Examples 9 to 11 are shown in Table 3 below.

Cholesterol removal Strain name Cholesterol removal after incubation (%) Example 4 SNUL 01 41.17 Comparative Example 9 ATCC 43121 24.85 Comparative Example 10 CH-5 28.4 Comparative Example 11 145 24.85

Example 5: Growth Test in MRS Liquid Medium

The sterilized MRS liquid medium was inoculated with about 10 ⁶ cfu / mL of Lactobacillus ashidophilus SNUL 01 according to the present invention and incubated at 37 ° C for 24 hours. The change of viable cell number over the cultivation time was measured by inoculating the diluted sample into MRS agar plate medium and incubating at 37 ° C. for 2 days to count the viable cell number.

Comparative Examples 12 to 14

The number of viable cells was measured after incubating the strains Lactobacillus ashidophilus CH-5, Lactobacillus ashidophilus 145, and Lactobacillus ashidophilus NCFM in the same manner and in the same manner as in Example 5. The result of Example 5 and Comparative Examples 12-14 is shown in FIG.

Example 6: Growth Test in Milk

Inoculated with 12% sterile reduced skim milk at 1% concentration of Lactobacillus ashidophilus SNUL 01 of the present invention and incubated at 37 ° C. for 24 hours while inoculating MRS agar plate medium and incubating at 37 ° C. for 2 days Counting viable cells was measured.

Comparative Examples 15 and 16

By virtue of the same methods and conditions as in Example 6, Lactobacillus ashidophilus CH-5 and Lactobacillus ashdophilus NCFM were cultured, and the number of viable cells was measured. The result of Example 6 and Comparative Examples 15 and 16 is shown in FIG.

As is apparent from the above description and the examples, the Lactobacillus ashidophilus SNUL 01 according to the present invention has high acid resistance, bile acid resistance, cholesterol lowering ability, and rapid growth rate. Therefore, the survival rate of lactic acid bacteria reaching the small intestine when ingesting the food containing the strain of the present invention is higher than the conventional strain, in particular, because the strain of the present invention is isolated from the living body of healthy Korean adults, the possibility of intestinal settlement of Koreans This is high.

Therefore, when the culture or fermented food cultured strain or strain of the present invention can be eaten, it can be expected to have a formal effect and cholesterol lowering effect.

Claims (3)

Lactobacillus ashdophyllus SNUL 01 with excellent acid resistance, bile acid resistance, blood cholesterol lowering ability and fast growth rate isolated from Korean feces (Repair No. KFCC-11041). Fermented food fermented using the strain of claim 1. A formal agent containing the strain of claim 1.