KR100725012B1 - The new lactobacillus acidophilus HY 7036 isolated from korean adult, having high constipation improvement effect, high relieving intestinal disorders and high organic acid producibility, and products containing it - Google Patents

Abstract

The present invention relates to a novel L. acidophilus HY 7036 strain and a product containing the same, which have excellent organic acid generating ability from feces of healthy Korean adults and have excellent constipation improvement ability and intestinal action.

Since the Lactobacillus ashidophyllus HY7036 strain according to the present invention is isolated from the living body of a healthy Korean adult, it can be improved in constipation by promoting intestinal motility in the intestine of Koreans. It is excellent and has a fast growth rate in milk, so it can be used in various fermented foods.

Description

Lactobacillus acidophilus HY 7036 isolated from korean adult, having high constipation improvement effect with excellent intestinal separation and organic acid generation ability , high relieving intestinal disorders and high organic acid producibility, and products containing it}

1 is a graph showing the acid resistance test results of L. acidophilus HY 7036.

Figure 2 is a graph showing the results of the bile resistance test of L. acidophilus HY 7036.

Figure 3 is a graph showing the growth test results in the medium (milk components) of L. acidophilus HY 7036.

The present invention relates to a novel Lactobacillus ashdophyllus strain, and more particularly, excellent in the production of organic acids, excellent viability in the digestive organs of the human body, excellent inhibitory ability against intestinal harmful bacteria, fast growth rate Lactobacillus Ashdophilus HY 7036 and products containing the same.

Lactic acid bacteria are distributed in the intestinal tract of animals, have the effect of inhibiting the growth of harmful microorganisms, treatment of abnormal fermentation, intestinal motility through stimulation of the intestinal wall of the intestinal tract, enhancement of immune function, and also has anti-cancer activity.

Fermented dairy products have been commercialized and widely consumed or edible so that humans can consume such lactic acid bacteria and promote their health. Representative strains used in the fermented dairy products are Lactobacillus ashdophyllus ( L. acidophilus ), Lactobacillus casei ( L. casei ), Lactobacillus delburactki subspecies Bulgaricus ( L. delbruckii subspp. Bulgaricus ), Streptococcus Thermophilus and the like.

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, lactic acid bacteria having this property can be settled because of their excellent organic acid production ability, they can settle in the intestine by counteracting toxins and metabolites produced by various harmful bacteria in the intestine and inhibiting their growth. When used to produce fermented products will be able to function substantially useful to the human body.

In addition, the generated organic acid stimulates the intestinal cell wall and affects the intestinal activity, and the increase in the intestinal wall activity has a positive effect on bowel movements. Efforts to isolate and develop excellent strains have been attempted in various ways.

On the other hand, in order for the lactic acid bacteria to continue to have a useful function, in addition to the ease of bowel movement through the intestinal wall stimulation by the organic acid described above, the intestinal fixation rate should be high. However, because 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 the desired effect.

In addition, lactic acid bacteria are the most common among the digestive disorders of modern people, but because of the effect of improving the constipation that many people have, it is required to develop lactic acid bacteria excellent in these characteristics.

In particular, 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 inventors of the present invention, while separating and testing lactic acid from Korean adults to isolate strains suitable for producing fermentation products suitable for the physiology of Koreans, have excellent organic acid production ability, inhibit intestinal harmful bacteria growth, and stimulate intestinal wall irritation. The present invention has been successfully achieved by separating new lactic acid bacteria having fast bowel movement function and fast growth rate.

Therefore, an object of the present invention is a new Lactobacillus ashophilus HY 7036 ( Lactobacillus acidophilus HY) having a high organic acid production, rapid growth rate, excellent growth inhibitory effect on human harmful bacteria, excellent ability to increase the intestinal motility of the human body 7036).

Another object of the present invention is to provide a new Lactobacillus ashidophilus HY 7036 suitable for Koreans.

It is another object of the present invention to provide a fermentation product prepared using Lactobacillus ashidophilus HY 7036.

In order to achieve the above object, according to the present invention, suitable for Korean adults, organic acid generation is vigorous, the growth rate is fast, the growth inhibitory effect on human harmful bacteria is excellent, and the new lactobacillus excellent in the ability to increase the intestinal motility of the human body Ashdophilus HY 7036 strain is provided.

Hereinafter will be described in detail the isolation, identification, mycological properties and uses of the new Lactobacillus ashidophyllus HY 7036 strain according to the present invention.

Isolation of Lactobacillus ashidophilus HY 7036 Strain

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 collected using platinum, and then plated 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 acid resistance and bile acid resistance. The lactic acid bacteria of the present invention were deposited with the Agricultural Biotechnology Research Institute on August 1, 2005 (accession number: KACC 91167P).

Identification of Lactobacillus ashidophilus HY 7036 Strain

The characteristics of the new strain 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: 4.0 ~ 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

Reactor result Reactor result Glycerol - Salicin - Erythritol - Celliobiose + D-Arabinose - Maltose + L-Arabinose - Lactose + Ribose - Melibiose - D-Xylose - Sucrose + L-Xylose - Trehalose + Adonitol - Inulin - β-Methyl-D-Xyloside - Melezitose - Galactose + Raffinose + Glucose + Starch + Fructose + Glycogen - Mannose + Xylitol - Sorbose - Genitobiose + Rhamnose - D-Turanose - Dulcitol - D, L-Xylose - Inositol - D-Tagatose + Mannitol - D-Fucose - Sorbitol - L-Fucose - α-Methyl-D-Mannoside - D-Arabitol - α-Methyl-D-Glucoside - L-Arabitol - N-Acetyl-Glucosamine + Gluconate - Amygdalin - 2-keto-Gluconate - Esulin + 5-keto-Gluconate -

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 HY 7036.

Strains of the invention can be provided in the form of cultures or lyophilized powders. As such, the cultures containing the strains of the present invention obtained by fermenting sugars or proteins using the strains of the present invention or the strains of the present invention can be used as a dressing agent because of the expected form effect. 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. These examples are only for illustrating the present invention, and the scope of the present invention is not limited to these 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

0.2% sodium thioglycollate was added to the MRS liquid medium to prepare a MRS Thio medium. The medium was inoculated with Lactobacillus ashidophilus HY 7036 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. In order to compare the acid resistance of the Lactobacillus ashidophyllus HY 7036 of the present invention, Lactobacillus ashdophyllus CH-5 (CHR. Hansen, Denmark), Lactobacillus NCFM, which was frequently used as a commercial spawn, was used. The acid resistance results of the lactic acid bacteria tested in Comparative Example with Lactobacillus ashidophilus HY 7036 of the present invention are shown in FIG. 1.

  As can be seen in Figure 1, the Lactobacillus ashdophyllus HY 7036 of the present invention was found to be superior in acid resistance than conventional Lactobacillus ashdophilus CH-5 or Lactobacillus NCFM.

Example 2: Bile Acid Resistance Test

Oxal (oxgall, manufactured by Difco) was added to the MRS liquid medium so that the final concentration was 0%, 0.3%, and 0.5%, and then inoculated with 1% of Lactobacillus ashidophilus HY 7036 and incubated at 37 ° C. Absorbance was measured at 620 nm using UV-VISIBLE SPECTROPHOTOMETER (UV-1601, SHIMADZU, Co. Japan). Lactobacillus ashdophilus CH-5 (CHR. Hansen, Denmark) and Lactobacillus NCFM were used to compare the bile acid resistance of the strain Lactobacillus ashidophilus HY 7036 of the present invention. The results of the bile resistance of lactic acid bacteria tested in comparison with Lactobacillus ashidophilus HY 7036 of the present invention are shown in FIG. 2.

  As can be seen in Figure 2 Lactobacillus ashdophyllus HY 7036 of the present invention was found to be superior to the bile acid resistance than conventional Lactobacillus ashdophilus CH-5 or Lactobacillus NCFM.

Example 3 Growth Test in Medium (Milk)

Inoculated in 12% sterile reduced skim milk at 1% concentration of Lactobacillus ashidophilus HY 7036 of the present invention, inoculated in MRS agar plate medium while incubated at 37 ° C. for 24 hours and cultured at 37 ° C. for 2 days. Counting viable cells was measured. Lactobacillus ashdophyllus CH-5 (CHR. Hansen, Denmark) and Lactobacillus NCFM were used to compare the growth test in milk of strain Lactobacillus ashidophilus HY 7036 of the present invention. A comparison of the milk growth test of lactic acid bacteria tested with Lactobacillus ashidophilus HY 7036 of the present invention as a comparative example is shown in FIG. 3.

  As can be seen in Figure 3, the Lactobacillus ashidophyllus HY 7036 of the present invention was found to be superior to the existing Lactobacillus ashdophilus CH-5 or Lactobacillus NCFM.

Example 4 Preparation of Fermented Milk Using Lactobacillus Ashdophilus HY 7036

Milk prepared with 8.5% milk solid content was sterilized at 135 ° C. for 3-5 seconds using an ultrahigh temperature sterilizer and then cooled to 37 ° C. Lactobacillus Lactobacillus ashidophilus HY 7036 of the present invention was inoculated at 5.0 × 10 ⁶ cfu / ml level and incubated at 37 ° C. for 24 hours. The culture was stopped when the final acidity and pH of the culture reached 0.65%, 4.8. After the incubation was terminated, the mixture was cooled to 8 ° C. or less and mixed well, and then homogenized to 130-150 mBar using a homogenizer (AlphaLaval, Netherlands). The product was prepared by mixing the prepared syrup so as to contain 5% by weight of apple juice, 3% by weight of sugar, and 5% by weight of high fructose in the final product in a homogenized culture medium. Physicochemical characteristics of the finished product were pH 4.8, TA 0.68, Brix 18, lactic acid bacteria 4.0 × 10 ⁸ cfu / ml or more. The fermented milk prepared was filled in clean bottles prepared for use in clinical experiments and used 150ml each. Lactobacillus ashdophyllus CH-5 (CH. Hansen, Denmark), Lactobacillus NCFM to prepare the same lactobacillus fermented milk in order to compare the constipation improvement effect of lactobacillus fermented milk prepared with Lactobacillus ashdophyllus HY 7036 It was.

Example 5: Constipation Improvement Efficacy Experiment

Selected 160 subjects complaining of constipation symptoms of the lactic acid bacterium fermented milk composition prepared in Example 4 randomly divided into four groups, and then confirmed the experimental results through a survey while supplying two compositions each day for two weeks. It was. In the constipation improvement function of the product, the change of the number of bowel movements, the improvement of constipation symptoms and the change of the residual stool after stool according to the drinking of fermented milk were checked through questionnaire. Each experimental group confirmed the results are shown in Table 3, Table 4, Table 5.

Changes in the frequency of bowel movements after drinking fermented milk (Unit: person) division collection L. acidophilus HY 7036 Lactobacillus ashdophyllus CH-5 Lactobacillus NCFM Item term Bowel movements Before Intake 1 time or less 32 32 32 2-3 times 8 8 8 4-5 times 0 0 0 6 or more times 0 0 0 1 week after ingestion 1 time or less 22 30 33 2-3 times 18 One 7 4-5 times 0 0 0 6 or more times 0 0 0 2 weeks after ingestion 1 time or less 11 26 31 2-3 times 18 11 9 4-5 times 8 3 0 6 or more times 3 0 0

Improvement of Constipation Symptoms by Drinking Fermented Milk (Unit: Persons) division collection L. acidophilus HY 7036 Lactobacillus ashdophyllus CH-5 Lactobacillus NCFM Item term Bowel movements 1 week after ingestion same 12 30 31 A little improvement 22 10 9 Generally improved 6 0 0 Full improvement 0 0 0 2 weeks after ingestion same 6 25 31 A little improvement 12 12 9 Generally improved 16 3 0 Full improvement 6 0 0

Changes in Residual Sensitivity due to Drinking Fermented Milk (Unit: Persons) division collection L. acidophilus HY 7036 Lactobacillus ashdophyllus CH-5 Lactobacillus NCFM Item term Bowel movements 1 week after ingestion none One 0 0 Sometimes 17 0 0 often 12 12 8 always 10 28 32 2 weeks after ingestion none 8 0 0 Sometimes 12 8 0 often 16 21 10 always 4 11 30

As can be seen in Tables 3, 4, and 5, the Lactobacillus ashidophyllus HY 7036 of the present invention was found to have superior constipation improvement effect than the existing Lactobacillus ashidophilus CH-5 or Lactobacillus NCFM.

Example 6 Experiment of Intestinal Harmful Microorganism Growth Capacity

When a fermented milk composition prepared using Lactobacillus ashidophilus HY 7036 of the present invention was supplied to a subject complaining of constipation, changes in the level of harmful microorganisms present in the feces were observed. Lactobacillus fermented milk (150ml) prepared using Lactobacillus ashdophyllus HY7036 was supplied two times a day for two weeks, and feces were collected from the subjects to observe the number of harmful indicator microorganisms in the feces. The harmful microorganisms were designated as E. coli , Staphylococcus, and Clostridium perfrigens , and were observed before ingestion, 1 week of ingestion, 2 weeks of ingestion, 1 week of ingestion, and 2 stops of ingestion. The main distinction was to observe changes in microbial levels in feces. The observed experimental results are shown in Table 6.

Changes in Microbiological Characteristics of Stool According to Fermented Milk Drinking with Lactobacillus Ashdophilus HY 7036 term Before Intake Ingestion 1 week Ingestion 2 weeks 1 week of suspension 2 weeks pH 6.9-7.6 6.4-6.7 5.4-5.7 6.6-6.8 6.9-7.1 Lactic acid bacteria count (cfu / ml) 6.5 × 10 ⁶ 1.2 × 10 ⁸ 4.2 × 10 ⁸ 3.0 × 10 ⁷ 8.3 × 10 ⁶ Harmful bacteria count (cfu / ml) E. coli 6.5 × 10 ⁷ 8.2 × 10 ⁶ 1.3 × 10 ⁶ 5.2 × 10 ⁶ 3.3 × 10 ⁷ Staphylococcus 4.8 × 10 ⁵ 1.0 × 10 ⁵ 4.4 × 10 ⁴ 6.0 × 10 ⁴ 1.3 × 10 ⁵ C. perfrigens 3.3 × 10 ⁵ 2.1 × 10 ³ 1.2 × 10 ³ 4.4 × 10 ⁴ 1.6 × 10 ⁵

As can be seen in Table 6, the Lactobacillus ashidophilus HY 7036 of the present invention was found to be excellent in the ability to inhibit the growth of harmful microorganisms in the intestine.

As described in detail above, the Lactobacillus ashidophilus HY7036 strain according to the present invention is isolated from the living body of a healthy Korean adult settled in the intestine of the Korean can promote the mobility of the intestine to improve constipation and acid resistance It has excellent bile acidity, harmful bacterium growth inhibition ability, and fast growth rate in milk. Therefore, the strain of the present invention can be used in a variety of fermented foods and the like as a useful new strain.

Claims (2)

Lactobacillus ashdophyllus HY 7036 (Excellence date and place of deposit: August 1, 2005), excellent in acid resistance, bile acid resistance, intestinal harmful bacteria growth inhibition and constipation improvement and fast growth rate isolated from Korean feces Institute of Engineering Accession No .: KACC 91167P). Fermented food fermented using Lactobacillus ashidophilus HY 7036 according to claim 1.

Images