KR19990035280A - Lactobacillus ashdophyllus KY 2104 and its uses - Google Patents

Abstract

The present invention relates to a novel Lactobacillus ashdophyllus having excellent acid and bile acid resistance isolated from feces of Koreans, and a new strain Lactobacillus ashdophyllus KY 2104 according to the present invention. Affiliated Biotechnology Research Institute, Date of Access: Sep. 29, 1997, Accession No .: KCTC 8833P) is particularly excellent in acid resistance compared to general Lactobacillus ashdophyllus, and has the ability to inhibit the growth of bile acids and pathogenic bacteria. It is derived from humans and suitable for use on the human body.

This strain comprises (i) primary isolation of acid resistant lactobacillus from Korean feces, (ii) secondary selection of strains resistant to acidic pH from the isolated strains, and (iii) selection of strains. Among them, a bile acid resistant strain having a high resistance to bile acids is selected, and (iv) a strain capable of inhibiting growth against pathogenic bacteria among the selected strains can be finally isolated, and (v) identified.

Lactobacillus ashdophyllus KY 2140 of the present invention can be used as a probiotic, fermentation products, infant foods, functional foods, health foods, such as foods and formal medicines, and other fields, including animal feed additives It can be widely used in.

Description

Lactobacillus ashdophyllus KY 2104 and its uses

The present invention relates to Lactobacillus ashdophilus KY 2104 isolated from Korean feces (Deposit: Biotechnology Research Institute, Korea Institute of Science and Technology, Date of Deposit: September 29, 1997, Accession No .: KCTC 8833P) The present invention relates to a novel Lactobacillus ashidophyllus and its use, which have excellent acid resistance and bile acid resistance and have a growth inhibitory ability against pathogenic bacteria, which can be widely used in food and medicine.

Live microorganisms that promote the health of the host are called probiotics (probiotics), and probiotics are stabilizing the intestinal flora, reducing the production of decay products by inhibiting the establishment of harmful bacteria, preventing disease, anticancer activity due to immune activation, and lowering cholesterol. And intestinal disease prevention effect. Lactic acid bacteria mainly used as probiotics include Lactobacillus, Bifidobacterium, and Enterococcus, among which Lactobacillus is an anaerobic bacillus, which is the first to be used to maintain the equilibrium of the intestinal flora of the animal. Has been. Lactobacillus currently used or available as probiotics includes L. acidophilus, Lactobacillus casi, L. plantarum, L. plantarum, Lactobacillus salivarius (L. salivarius), L. reuteri (L. reuteri) and a variety of normal lactic acid bacteria or abnormal lactic acid bacteria. However, these strains are known to have the following conditions in order to be used as a probiotic for humans. That is, i) must be isolated and safe in the intestine of a non-animal or animal intestine, ii) survive the low pH of the stomach and reach the intestine, iii) be resistant to bile secreted into the small intestine, iv) It should have a beneficial effect in the gut, including inhibition against pathogenic bacteria.

However, most of the lactic acid bacteria that have been developed so far have been separated from livestock and animals such as piglets, but not humans. In addition, some of these strains were difficult to expect a role as probiotics due to the weak acid and bile acid resistance.

Therefore, the present inventors can survive in gastric acid and bile by separating live strains from human feces, and by separating new lactobacillus ashidophilus having excellent acid resistance from Korean feces while studying to obtain safe strains for human body. The invention was completed. Prior to the present invention, the inventors of the present invention, Bifidobacterium bifidem HY 43, which is a new Bifidobacterium derived from human feces, has excellent acid resistance and bile acid resistance, and has a growth inhibitory ability against pathogenic bacteria (Patent Application No. 95-29985). Patent), Bifidobacterium long gum HY 8001 (patent application No. 95-30301), Bifidobacterium breve HY 16 (patent application No. 96-10935), and the like.

The present invention is isolated from the feces of Koreans, Lactobacillus ashdophyllus KY 2104, a new strain with excellent acid resistance, bile acid resistance and growth inhibition ability against pathogenic bacteria (depositing institution: Biotechnology affiliated with Korea Institute of Science and Technology) Institute, Deposit Date: September 29, 1997, Accession No .: KCTC 8833P).

The present invention also provides the use of Lactobacillus ashidophilus KY 2104.

The present invention relates to a novel Lactobacillus ashdophyllus having excellent acid and bile acid resistance isolated from feces of Koreans, and a new strain according to the present invention, Lactobacillus ashdophilus KY 2104 ((Deposit: Korea Science and Technology). Biotechnology Research Institute, Research Institute Date: September 29, 1997, accession number: KCTC 8833P) is particularly acid resistant compared to Lactobacillus ashdophyllus, and has the ability to inhibit the growth of bile acids and pathogenic bacteria. It is derived from humans and suitable for use in humans.Hereinafter, the separation method, bacteriological properties and uses of Lactobacillus ashdophyllus KY 2104 will be described in detail.

[Isolation and Identification of Lactobacillus Ashdophilus KY 2104]

(i). First, the feces of Koreans are collected and mixed in a buffer solution of pH 2.5, the pH is adjusted to 2.3 and shaken for 2 hours, and then plated on LBS agar plate medium and incubated at 37 ° C. for 48 hours. At this time, the grown colonies are separated first, and then separated into Lactobacillus with gram staining, speculum and confirmation of curd formation in milk medium.

(ii). The isolated strains were further left for 2 hours in a buffer solution of pH 2.3, and then the strains resistant to acidic pH were secondarily selected.

(iii). The selected strains are plated in LBS agar plate medium containing 0.3% of Oxal (Bacto oxgall, Difco) and aerobic incubation at 37 ° C. for 48 hours to select bile acid resistant strains highly resistant to bile acids.

(iv). Among the selected strains, strains having growth inhibitory ability against pathogenic bacteria are finally separated.

(v). Identification of the isolated strains through the Burgy's manual and carbohydrate digestion experiments to confirm that the isolated strain is a new strain of Lactobacillus ashidophilus.

[Characteristics of Strains]

(1) form of bacteria

Morphology of bacteria when cultured at 37 ° C for 48 hours in MRS agar plate medium

1) Cell shape: rod type

2) Gram Dyeing: Positive

3) Mobility: None

4) Spore Formability: None

(2) form of colony

Morphology of bacteria when grown at 37 ° C for 48 hours in AMLS agar plate

1) Shape: round or irregular

2) Size: 0.5 ~ 3.0㎜ (diameter)

3) Hue: off-white

4) Surface: Roughness

5) bumps: flat or flat

(3) physiological properties

1) Growth temperature: Growth range: 20 ~ 40 ℃

Optimal temperature: 30 ~ 37 ℃

2) Growth pH: Growth range: pH 2.0 ~ 8.0

Optimum pH: pH 5.0 ~ 7.0

3) Influence of oxygen: breathable anaerobic

4) Milk coagulation: +

5) Catalase:-

6) Beta-Calactose Degrading Enzyme: +

7) alpha-glucose degrading enzyme: +

8) Sugar availability

Erythritol:-

Arabinose:-

Ribose:-

Adonitol:-

Galactose: +

Glucose: +

Fructose: +

Mannose: +

Mannitol:-

Sorbitol:-

N-acetylglucosamine: +

Amigdalin: +

Arbutin:-

Esmullin: +

Raised: +

Cellobiose: +

Maltose: +

Lactose: +

Melibiose: +

Sucrose: +

Trehalose: +

Inulin:-

Raffinose: +

Amidodon:-

Starch:-

Genthiobis: +

Gluconate:-

(4) acid resistance

Lactobacillus ashdophyllus KY 2104 has excellent acid resistance, showing a high survival rate of 50% or more even when incubated in an acidic buffer solution of 2.0 for 60 minutes. In particular, when compared with other Lactobacillus ashidophyllus contained in fermented milk products, lactic acid bacteria, etc., Lactobacillus ashidophilus KY 2104 survives similar to the initial inoculation concentration even after a pH of 3.0 to 2 hours, Even at pH 2.3 and pH 2.0, where the other Lactobacillus ashidophilus strains were almost killed, the survival rates were higher than 10 ⁶ cfu / ml and 10 ⁴ cfu / ml, respectively. From these results, it can be seen that Lactobacillus ashidophilus KY 2104 according to the present invention is a novel strain having excellent acid resistance compared to other Lactobacillus ashidophilus strains.

(5) bile acid resistance

As a result of culturing Lactobacillus ashidophilus KY 2104 in a medium containing bile acid, growth of Lactobacillus ashidophilus KY 2104 was observed even in a medium containing 0.5% (w / v) bile acid. It can be seen from this that Lactobacillus ashidophilus KY 2104 is resistant to bile acids.

(6) Growth inhibition ability against pathogenic bacteria

When the pathogenic bacteria such as Escherichia coli, Salmonella, Shigella and Staphylococcus are mixed with Lactobacillus ashdophyllus and KY 2104, the number of viable cells is greatly reduced compared to the case of single culture.

In other words, by inoculating MLC liquid medium with Escherichia coli and Lactobacillus ashdophilus KY 2104 at the same concentration, and incubating at 37 ° C. for 24 hours, the viable bacteria in single culture The number of viable cells decreased from 10 ⁸ → 10 ⁷ to 10 ⁸ → 10 ² compared to the number, and the growth inhibition rate was 90 ~ 100%. It can be seen that Lactobacillus ashdophilus KY 2104 has excellent growth inhibitory ability against pathogenic bacteria.

(7) proliferative and acid producing ability

Lactobacillus ashdophilus KY 2104 was inoculated 1% in 10% (w / v) reduced skim milk and incubated for 48 hours at 37 ℃ to measure the proliferation and acid production capacity in milk-based culture medium. As a result, the number of viable cells after incubation was about 5.0 × 10 ⁸ cfu / ml and the pH was about 3.65. It can be seen that Lactobacillus ashdophyllus KY 2104 has sufficient proliferation and acid production capacity for use in dairy products such as fermented milk.

(8) other

To test the safety of human Lactobacillus ashidophilus KY 2104, we investigated the activity of beta-glucuronidase, one of carcinogenic enzymes. The enzyme activity was measured using API ZYME kit, which is one of the enzyme activity assays. As a result, Lactobacillus ashidophilus KY 2104 was found to have no activity against beta-glucuronidase. Therefore, it was confirmed that Lactobacillus ashidophilus KY 2104 is a safe strain to human body.

As described above, Lactobacillus ashidophilus KY 2104 is a lactic acid bacterium isolated from feces of Koreans, and has excellent acid resistance compared to conventional Lactobacillus ashdophilus, and has bile acid resistance and pathogenic growth inhibitory ability. It is a new Lactobacillus ashidophilus, isolated from humans and suitable for use in the human body.

[Applications of strains]

Lactobacillus ashdophyllus KY 2104 of the present invention can be used as a fermentation strain of fermented products, including lactic acid bacteria fermented milk, as a probiotic useful to the human body as it is, or dried or concentrated, such as infant food, potential food, health food, etc. It can be added to food. In particular, Lactobacillus ashdophyllus KY 2104 may be usefully used in medicines such as formal preparations, and can be widely used in various fields including animal feed additives.

The present invention will be described in more detail with reference to the following Examples. However, the following examples should not be construed as limiting the scope of the present invention, and of course, ordinary changes may be made by those skilled in the art within the scope of the present invention.

Example 1

Isolation of Lactobacillus ashidophilus KY 2104

Korean feces were collected and mixed well, and then 2.5 to 5.0 g were taken and mixed in a 10-fold volume of pH 2.5 buffer, and the pH was adjusted to 2.3 with HCl stock solution. After standing at 37 ° C. for 2 hours, samples were taken, diluted with dilution buffer solution, plated on LBS agar plate medium, which is a selective medium of Lactobacillus bacteria, and then incubated at 37 ° C. for 48 hours. After colonization, the colonies were Gram-positive, catalase-negative, spore-free, and rod-shaped bacillus.

The isolated strains were further allowed to stand in a buffer solution of pH 2.3 for 2 hours, and then, the strains resistant to acidic pH were secondarily selected, and the extracted strains were LBS agar containing 0.3% of Bacto oxgall (Difco). After plating on the plate medium, aerobic culture was performed at 37 ° C. for 48 hours to select bile acid resistant strains highly resistant to bile acids.

Among the selected strains, the strains capable of inhibiting the growth of pathogenic bacteria were finally isolated and named as Lactobacillus ashdophilus KY 2104 through identification by Burgy's manual and carbohydrate digestion experiments.

Example 2

Acid resistance check

(One). Experiment 1

Lactobacillus ashdophilus KY 2104 was inoculated in a 10ml MRS liquid medium and incubated overnight at 37 ℃, the culture was inoculated at pH 3.0, 2.5, 2.0 buffer solution at an initial concentration of about 10 ⁷ cfu / ml, respectively. After standing at 37 ° C. for 1 hour, 1 ml was taken and diluted in each diluted saline solution. The diluted solution was plated on an MRS agar plate medium and incubated at 37 ° C. for 48 hours, and the number of colonies shown was counted and compared. The results are shown in Table 1 below.

TABLE 1

cfu: colony forming unit

As can be seen from Table 1, Lactobacillus ashidophilus KY 2104 showed excellent survival rate of more than 50% even when incubated for 60 minutes in an acidic solution of pH 2.0 was very excellent acid resistance.

In order to more accurately evaluate the acid resistance of Lactobacillus ashdophilus KY 2104, the following experiment was performed to compare the survival rate of the two-hour culture in acidic solution with other Lactobacillus ashdophilus strains.

(2) Experiment 2 (Comparative Experiment)

The acid resistance of Lactobacillus ashidophilus (hereinafter referred to as A, B, and C) and Lactobacillus ashdophilus KY 2104 of the present invention, which were isolated from fermented milk products and lactic acid bactericidal agents, were compared. KY 2104 and three strains were inoculated into 10 ml MRS liquid medium and incubated overnight at 37 ° C., and then the culture medium was initially treated with pH 7.0, 3.0, 2.5, 2.3 and 2.0 in buffer solution of about 10 ⁷ cfu / ml, respectively. Inoculation was carried out at the inoculation concentration. In this case, the buffer solution was prepared by calibrating 0.05 M sodium phosphate buffer solution with hydrochloric acid (HCL). After stationary incubation at 37 ° C. for 2 hours, 1 ml of each was diluted in physiological saline solution, and the diluted solution was plated on MRS agar plate medium and incubated at 37 ° C. for 48 hours. Resistance to each pH was measured by counting the number of colonies shown in the culture. The results are shown in Table 2 below.

TABLE 2

As a result, KY 2104 showed much better survival rate than A, B, and C strains tested under acidic conditions below pH 3.0. That is, A, B, no death even after 2 hours at pH 3.0 unlike the C strain in the last initial showed similar viability and inoculum concentration, in pH 2.5 that apoptotic action of microorganisms proceeds rapidly than 10 ⁷ cfu / ml High Survival was maintained. In particular, below pH 2.3, B and C strains were almost killed, whereas KY 2104 showed a high survival rate of 10 ⁶ or higher; In the case of strain A, the survival rate of 10 ² was low at pH 2.0, but KY 2104 had a survival rate of 10 ⁴ or more at pH 2.0, and showed a 100 times higher survival rate than the strain A.

The above results indicate that KY 2104 has much higher acid resistance than Lactobacillus ashidophilus strains currently used as probiotics. Therefore, when it is administered to the human body in the form of food or medicine, a large number of people pass through the stomach acid to reach the intestine in a surviving state, it was thought that the usefulness in the future, such as food, medicine.

Example 3

Checking bile acidity

After inoculating a certain amount of Lactobacillus ashidophilus KY 2104 of the present invention into a 10ml MRS liquid medium and incubating at 37 ° C. overnight, the culture solution was oxgal (Bacto oxgall, Difco) of 0.1, 0.3, 0.5% (w / v) inoculated in the containing agar medium. As a result of observing colony formation by incubating at 37 ° C. for 48 hours, it was confirmed that the strain was grown in a medium containing 0.1% and 0.3% as well as 0.5% ox. Therefore, Lactobacillus ashdophilus KY 2104 was found to be resistant to bile acids.

Example 4

Confirmation of pathogenic bacteria growth inhibitory ability

The growth inhibitory ability of Lactobacillus ashdophilus KY 2104 against pathogenic bacteria such as Escherichia coli, Salmonella, Shigella and Staphylococcus, which are known to be the diarrheal agents of infants and adults, was tested. The test method was performed by comparing and measuring the case where the pathogen was cultured by inoculation with Lactobacillus ashidophilus KY 2104 when the pathogen was cultured alone.

Lactobacillus ashdophyllus KY 2104 and pathogenic bacteria were inoculated in 10ml MRS liquid medium, respectively, in a predetermined amount, and then activated by incubating overnight at 37 ° C. After performing the activation process of the above two strains, some were inoculated alone with each of the pathogenic bacteria in 50ml M liquid medium, and the rest was inoculated with Lactobacillus ashdophilus KY 2104, respectively. Single inoculation and mixed inoculation were incubated at 37 ° C. for 24 hours, and the viable cell counts of pathogens were measured and compared. The viable cell count was used for the selection of each pathogenic bacterium. E. coli, Salmonella and Shigella used MacConkey agar plates and Staphylococcus 110 agar plates. The measurement results of viable cells are shown in Table 3.

TABLE 3

As can be seen from Table 3 above, when the pathogenic bacteria were mixed with Lactobacillus ashidophilus KY 2104, the growth of the pathogenic bacteria was greatly suppressed. That is, was the case of E. coli is the number of live cells when single cultures 10 ⁸ inde When mixed culture than the growth inhibition rate of 10 ⁷ appeared as more than 90%, in the case of Salmonella is a decrease in the number of viable cells by 10 ⁸ → 10 ² Growth inhibition is In the case of Shigella and Staphylococcus, the viable cell count decreased to 10 ⁷ → 10 ² and 10 ⁸ → 10 ³ , respectively, and the growth inhibition rate was almost 100%. Therefore, it was found that Lactobacillus ashdophilus KY 2104 of the present invention has excellent growth inhibitory ability against pathogenic bacteria.

Example 5

Culture Example (Proliferation and Productivity Test)

In order to use lactic acid bacteria in dairy products such as fermented milk, it must have excellent acid producing ability in addition to acid resistance to acid, must show high proliferation in milk-based culture medium, and can maintain high viable cell count in dairy products.

1% (w / v) of Lactobacillus ashdophilus KY 2104 of the present invention was inoculated in 1,000 ml of 10% (w / v) reduced skim milk and incubated at 37 ° C. for 48 hours to measure viable cell count and pH. The results are shown in Table 4 below.

TABLE 4

As described above, the viable cell number after completion of the culture was 5.0 × 10 ⁸ cfu / ml, and the pH was 3.65, which was sufficient to prepare the fermented milk.

Example 6

Enzyme Activity Test

The enzyme activity of Lactobacillus ashidophilus KY 2104 was measured. The test enzyme also included beta-glucuronidase (beta-glucuronidase), which is known as a carcinogenic enzyme, and enzyme activity was measured using API ZYME Kit, one of enzyme activity assays. The results are shown in Table 5 below.

TABLE 5 Lactobacillus ashidophilus KY 2104 Enzyme Activity

As described above, it was found that Lactobacillus ashidophilus KY 2104 has no activity at all against beta-glucuronidase, one of carcinogenic enzymes, and was confirmed to be a safe strain that can be used in the human body.

Example 7

Infant clinical trial

Lactobacillus ashdophyllus KY 2104 was incubated and the cells were centrifuged and lyophilized using 10% skim milk powder. The cell powder was taken in four infants (Group I) aged 6 to 17 months for 14 days, and the number of Lactobacillus ashidophilus KY 2104 cells taken by children was about 10 ⁸ to 10 ¹⁰ . Changes in the bacterial flora in feces for 14 days (a), 14 days (b) and 14 days (c) after discontinuation were measured daily.

After repeating the same experiments with four other infants (II), the average values and the deviations of the measurements of group I and group II are shown in Table 6. During the test period (14 days before dosing and 14 days after discontinuation), infants were not fed products containing lactic acid bacteria or oligosaccharides that could affect the intestinal flora.

TABLE 6

As can be seen from Table 6, as a result of taking Lactobacillus ashidophilus KY 2104 cell powder in infants, lactic acid bacteria such as Bifidobacteria, Lactobacillus, Enterococcus, which have beneficial effects in the intestine, increased, and harmful bacteria Staphylococcus is known to have a lower bacterial count than before. The experimental results indicate that the Lactobacillus ashidophilus KY 2104 of the present invention can be used as a probiotic that has a beneficial effect on humans.

As can be seen from the above examples, Lactobacillus ashidophilus KY 2104 according to the present invention is isolated from human feces and has excellent acid resistance, and has the ability to inhibit growth of bile acids and pathogenic bacteria, in particular human It is isolated from and is a suitable strain for use as a probiotic for the human body. Therefore, the strain is a useful new strain, as well as fermented products, foods such as infant foods and functional foods and can be widely used in various fields, such as pharmaceuticals such as dressings, animal feed additives.

Claims (5)

As a strain isolated from feces of Koreans, Sugar-soluble, it is positive for mannose, N-acetylglucosamine, amigdaline, cellobiose, sucrose, trehalose, raffinose and genthiobis; 60 minutes incubation in a pH 2.0 buffer solution is more than 50% excellent acid resistance; Is bile acid resistant and grows in medium containing 0.5% (w / v) oxal; Growth inhibition against E. coli, Shigella, Salmonella and Staphylococcus; Lactobacillus acidophilus KY 2104, characterized by no activity against beta-glucuronidase (β-glucuronidase) (Deposit: Biotechnology Research Institute, Korea Institute of Science and Technology, : September 29, 1997, accession number: KCTC 8833P). Fermentation product characterized by using Lactobacillus ashdophilus KY 2104 (depository: Biotechnology Research Institute, Korea Institute of Science and Technology, Date of Deposit: September 29, 1997, Accession No .: KCTC 8833P) . A food product characterized by containing Lactobacillus ashdophilus KY 2104 (cell deposited: Biotechnology Research Institute, Korea Institute of Science and Technology, Deposit Date: September 29, 1997, Accession No .: KCTC 8833P) Food, including baby food and health food. Lactobacillus ashdophilus KY 2104 (Deposit: Biotechnology Research Institute, Korea Institute of Science and Technology, Date of Deposit: September 29, 1997, Accession No .: KCTC 8833P). An animal feed additive characterized in that it contains the cells of Lactobacillus ashdophilus KY 2104 (depository: Biotechnology Research Institute, Korea Institute of Science and Technology, Date of Deposit: September 29, 1997, Accession No .: KCTC 8833P).