KR920001375B1 - New microorganism lacto bacillius s.p. - Google Patents

Abstract

New strains of lactobacillus acidophilus SB603 KFCC 10686 (I), lactobacillus plantarum SB602 KFCC 10685 (I), lactobacillus plantarum SB602 KFCC 10685 (II) and lactobacillus salivarius P-24 KFCC 10684 (III) are sped. by (a) crushing the intestines of pig and cattle, and suspending in the saline, (b) spreading the suspension on the BCP agar medium and culturing at 37 deg.C for 24 hrs. in the CO2 incubator, and (C) selecting the (I), (II) and (III) having acid tolerance, bile acid tolerance and bactericidal activity against pathogenic bacteria. Each of the obtd. strains can be used for correcting the inbalance in instestinal microflora.

Description

New Lactobacillus

1 is a graph showing a comparison of the sensitivity to the pH of the lactic acid bacteria and known lactic acid bacteria of the present invention.

Figure 2 is a graph showing the comparison of the inhibitory effect of E. coli growth in vitro of the lactic acid bacteria and known lactic acid bacteria of the present invention.

The present invention relates to a novel lactic acid bacterium which is stable against acidic pH and bile acids and is strong in bactericidal power. More specifically, the present invention relates to novel lactic acid bacteria isolated from microorganisms inhabiting the intestines of mammals.

Lactobacillus is a final metabolite, commonly referred to as lactic acid bacteria, which refers to bacteria that produce significant amounts of lactic acid. Lactic acid bacteria are a kind of microorganisms that humans have been using for a long time, and are widely used in kimchi in Korea and dairy fermentation in the West.

In recent years, research on lactic acid bacteria has been actively conducted, and its application range has been widened, such as being used as health food and medicine as well as general foods. Etc.

These bacteria are Gram-positive bacteria that are anaerobic or unilaterally anaerobic. They decompose nutrients and fiber ingested by the animals and use them as energy sources to produce lactic acid and antibiotics to inhibit the growth of harmful bacteria in the intestines. Therefore, it is known to play an important role in maintaining the gut health.

However, young animals require lactic acid bacteria, which are beneficial strains for normal intestinal flora, because the intestinal flora is not fully formed, and diarrhea caused by harmful strains such as Escherichia coli is destroyed by intestinal flora in stress or by absorption of harmful substances, travel and drinking. In order to suppress the occurrence of diseases such as lactic acid bacteria are required.

In addition, lactic acid bacteria are used for promoting animal growth and improving feed utilization, increasing resistance to disease, inhibiting growth of harmful bacteria, reducing mortality, inhibiting the production of toxic toxins, and producing various vitamins.

In order to exert such effects, lactic acid bacteria introduced from the outside should reach the intestine without any disorder and adhere to the intestinal mucosa, and then exhibit its function. To this end, it must be a strain that can be directly attached to the intestine, have little destruction by gastric acid during oral administration, have a strong resistance to bile acids and have a strong bactericidal ability.

However, until now, isolated lactic acid bacteria are known to have very low stability against acids and bile acids. Accordingly, the present inventors have led to the present invention by separating lactic acid bacteria having a stronger resistance to acidic pH and bile acids than the known lactic acid bacteria and having strong growth inhibitory and bactericidal power.

Hereinafter, the present invention will be described in detail.

The present invention is Lactobacillus acidophilus SB603, Lactobacillus salivarius (Lactobacillus salivarius) P-24, Lactobacillus plantarta, which are stable to acidic pH and bile acids, and are new lactic acid bacteria that are resistant to pathogenic growth and strong bactericidal properties. Lactobacillus plantarum SB602, Streptococcus faecium L-20.

The lactic acid bacteria according to the present invention were identified by the Burgy's manual as a strain directly isolated from the intestine of cattle or pigs after slaughter.

The method of obtaining strains of the present invention is to crush the intestine of cattle or pigs immediately after slaughtering and then suspended in physiological saline and plated in prepared BP agar plate medium and cultured for 24 hours at 37 ℃, carbon dioxide incubator to show a yellow strain First, the isolate was separated first by gram staining and speculum. The isolated strains were classified into Lactobacillus and Streptococcus bacteria by temperature sensitivity, pH sensitivity, and fermentation form, and thus, resistant to acidic pH, high resistance to bile acids, excellent growth inhibition and bactericidal activity of pathogens were obtained.

The strains of the present invention were deposited with the Korean spawn association on December 1, 1989. Specifically, Lactobacillus esidophilus SB603 is deposited with the KFCC-10686, Lactobacillus salivarius P-24 is deposited with the KFCC-10684, Lactobacillus plantarum SB602 has been deposited under accession number KFCC-10685 and Streptococcus fascini L-20 with accession number KFCC-10683.

Hereinafter, Lactobacillus esidophilus SB603, Lactobacillus salivarius P-24, Lactobacillus plantarum SB602, and Streptococcus fascitus L-20 according to the present invention have the following characteristics.

[Bacterial Properties]

Lactobacillus esidophilus SB603, Lactobacillus salivarius P-24, Lactobacillus plantarum SB602, Streptococcus fasciparum L-20 of the present invention have bacteriological properties as shown in Table 1.

TABLE 1

* Composition of B.C.P.Agar Medium:

5g yeast extract

5 g peptone

1 g of glucose

Agar 15g

Bromocresol Purple 0.06 ~ 0.04g

1ℓ of distilled water

[Resistant to acidic pH]

In general, lactic acid bacteria lose significantly their resistance to acidic pH during culture or storage. However, the strains according to the present invention show high resistance to acidic pH or hardly lose such resistance compared to conventional lactic acid bacteria.

Experiments with resistance to acidic pH are described in the journal Food Science Vol. 17, No.3 (1985) pp192-196] by the pH resistance test method of "Identification of Lactic Acid Bacteria Isolated from Domestic Liquid Fermented Milk and Investigation of Digestive Fluids Resistance" and the method described in Yuhan Colax Technical Data Book. Was carried out.

Each of the strains is streaked on an AM plate to obtain pure colonies and single colonies are incubated in a carbon dioxide incubator for 12-14 hours in an AM liquid medium. Subsequently, 100 ml of 1 / 15M KH ₂ PO ₄ -HCl buffer solution at pH 2.5 and pH 3.0 was inoculated 100 ml each, stored in a carbon dioxide incubator at 20 ° C or 37 ° C, sampled every 30 to 60 minutes, and serially diluted with physiological saline. Then, smear it on an AML plate and incubate it again in a 37 ° C. carbon dioxide incubator for 24 hours, and then measure the degree of resistance by comparing the number of colonies before storage and the number of colonies after storage.

The composition of the AM medium is used as follows.

10 g peptone

Meat Extract 10g

5g yeast extract

20 g of glucose

Twin 80 1 g

2 g ammonium citrate

5 g sodium acetate

0.1 g magnesium sulfate

0.05 g manganese sulfate

Dipotassium phosphate 2g

1ℓ of distilled water

In this experiment, the resistance of the conventional bacteria was significantly reduced to 5-40% after 60 minutes at pH 2.5, whereas the resistance of the bacteria according to the present invention was 68-98%, and the resistance to acidic pH was maintained intact. In addition, in the 1 / 15M KH ₂ PO ₄ -HCl buffer solution of pH 3.0, the resistance was significantly high (71-99%) (see FIG. 1).

[Resistance to bile acids]

Conventional lactic acid bacteria ingested from the outside are inhibited by bile acids as well as low pH. However, the fungi according to the present invention grows well in the medium plate containing bile acid, which shows that the resistance to the bile acid is superior to the conventional strain. The test for resistance to bile acid was inoculated with a single colony of the above strains in a medium medium and incubated at 37 ° C. in a carbon dioxide incubator for 12 to 14 hours, and then diluted with physiological saline to give a bile acid containing 0.05% to 0.3%. This was done by comparing the number of colonies shown by smearing on S reputation medium [Reference: Korean J. Food Sci. Technol. Vol 17, No. 3 (1985) pp 192-196].

In this test, the conventional lactic acid bacteria showed resistance of 40% or less while the lactic acid bacteria of the present invention showed high resistance of 85 to 93%.

[Antibacterial and bactericidal activity against pathogens]

Lactic acid bacteria should have properties that can inhibit the growth of harmful bacteria. Existing strains inhibit the growth of 100-fold viable cell counts for E. coli after 20 to 24 hours (Ref. 83-31).

On the other hand, as shown in Figure 2, the strains according to the present invention exhibited a bactericidal power of 10 ² ~ 10 ³ times than the culture of E. coli 15 hours after the culture alone and 10 ⁷ ~ 10 ⁸ times more than 18 hours It can be seen that the growth inhibitory and bactericidal power of pathogens is stronger than existing strains showing bactericidal activity.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1

[Separation of strains]

After slaughter, cattle and pigs were purchased and ground in a blender and used as a source of lactic acid bacteria. The prepared isolate was suspended in physiological saline and plated on a plate of AMLS, a lactic acid bacteria culture medium, and incubated in a 37 ° C. carbon dioxide incubator for 24 hours. Single colonies were isolated from colonies resulting from the culture, and each was plated on a BP plate containing bromocresol purple and cultured in the same manner as above. As a result of lactic acid production, colonies were changed from purple to yellow. . The composition of the BP plate used in this is mentioned in Table 1. In order to separate the lactic acid bacteria from the isolated lactic acid bacteria were carried out according to the strain characteristics of Lactobacillus and Streptococcus buggy manual (Bergey's manual). First, strains Gram stained (+) and catalase (-) were selected and divided into lactic acid bacteria of cocci and bacillus through a microscope, and then strains grown on KF medium were isolated to selectively isolate strains of S. aureus. The composition of KF medium used for this is as follows.

10 g peptone

10g yeast extract

5 g sodium chloride

10 g sodium phosphate

Maltose 20g

1 g of lactose

Sodium azide 0.4g

Bromocresol Pain 0.015g

Agar 20g

1ℓ of distilled water

Sterilization 121 ℃, 15 minutes

In order to identify the isolated Lactobacillus and Streptococcus strains, it was cultured in an incubator at 15 ° C and 45 ° C according to the Burgy's manual to measure the temperature sensitivity and to determine whether growth in medium of pH 6.5 and pH 9.6 It was measured and the magnetization of the carbohydrate was measured.

The measurement of carbohydrate magnetization was carried out by adding 1-2% of carbohydrates except for hexaextract and glucose in the above-mentioned AMS medium, and the magnetization was generated by adding 0.05% of chlorophenol red. It was confirmed that the color of the indicator changed from purple to yellowish brown by lactic acid. The above results are shown in Table 1. The species name of each strain was confirmed by comparing the characteristics of each strain of the bugis manual and the characteristics of the newly isolated strain.

Example 2

[Resistant to acidic pH]

Measurement of the pH resistance of the lactic acid bacteria of the present invention was carried out in two ways. The first method merely suspended lactic acid bacteria incubated in AMLS solution for 12-14 hours in 1/15 molar sodium phosphate-hydrochloride buffer at pH 2.5 and pH 3.0 to determine resistance to acidic pH. The viable cell count was measured by.

The second method was applied to artificial gastric fluid containing pepsin at pH 3.0 for measurement in an environment similar to the conditions of the digestive tract in vivo according to Kobayashi's method (Ref .: Japan. J. Microbiol., Vol 24, pp691, 1974). Lactobacillus cultured in AMLS solution for 14 hours was suspended and the number of viable cells was measured at 30-60 minute intervals. The composition of the artificial gastric juice used is as follows.

For the determination of viable cell count, the suspension of lactic acid bacteria in physiological saline was continuously diluted and smeared on an AML plate medium, and then measured by colony number generated by incubating in a 37 ° C. carbon dioxide incubator for 24 hours. The results are as shown in Table 2 and FIG.

TABLE 2

Example 3

[Resistance to bile acids]

Resistance of the lactic acid bacteria to bile acids of the present invention is described in the Journal of the Korean Society for Industrial Microbiology Vol. 17, No.3 (1985) pp192-196], lactic acid bacteria were incubated for 12-14 hours in an AM liquid medium, diluted with physiological saline, and plated in a plate medium containing 0% -0.3% bile acid. Was measured. The viable cell count was measured in the same manner as in Example 2. Resistance to bile acids is shown in Table 3, wherein the bile acid addition medium used was as follows.

0-3 g bile acids (depending on treatment concentration)

10 g peptone

Meat Extract 10g

5g yeast extract

20 g of glucose

Twin 80 1 g

2 g ammonium citrate

5 g sodium acetate

Magnesium Sulfate 0.1g

Manganese Slicate 0.05g

Dipotassium phosphate 2g

Agar 20g

1ℓ of distilled water

Sterilization 121 ℃, 15 minutes

TABLE 3

Example 4

[E. coli growth inhibition]

In order to measure the growth inhibitory ability and bactericidal power of Escherichia coli, which is a pathogenic bacterium of the present invention, mixed inoculation of Lactobacillus and Escherichia coli in an AM liquid medium capable of growing both Lactobacillus and Escherichia coli and single inoculation of E. coli alone at 37 ° C. Incubated. The culture samples were taken at intervals of 1-3 hours and the growth inhibition was measured by measuring the number of Escherichia coli. The measurement of the number of Escherichia coli was carried out in the same manner as in the method for measuring the number of live bacteria of lactic acid bacteria in Example 2 and the results are shown in Table 4 and FIG. In the above results, when the mixed culture for 15 hours, the number of E. coli was reduced by 10 ² -10 ³ times than that of E. coli alone, and after more than 18 hours it was reduced to 10 ⁷ -10 ⁸ times, showing a strong bactericidal power.

At this time, the spawn of lactic acid bacteria was cultured in the above-mentioned AMLS medium, and E. coli was cultured in the following Elby medium.

Tryptone 10g

5g yeast extract

5 g sodium chloride

1ℓ of distilled water

Sterilization 121 ℃, 15 minutes

In summary, the above is as follows.

TABLE 4

Claims (3)

Lactobacillus esidophilus SB603 (Accession No. KFCC-10686), a lactic acid bacterium with enhanced resistance to acidic pH and bile acids and bactericidal activity. Lactobacillus silivarius P-24 (Accession No. KFCC-10684), a lactic acid bacterium with enhanced resistance to acidic pH and bile acids and bactericidal activity. Lactobacillus plantarum SB602 (Accession No. KFCC-10685), a lactic acid bacterium with enhanced resistance to acidic pH and bile acids and bactericidal activity.

Images