KR920000115B1 - New microorganism streptococcus sp - Google Patents

Abstract

The strain streptococcus faecium L-20, KFCC 10683 is sepd. 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, (c) selecting the streptococcus faecium L-20. The obtd. strain has acid- and bile acid tolerance, and bactericidal activity against pathogenic bacteria. The obtd. strain can be used for collecting the unbalance in intestinal 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.

FIG. 2 is a graph showing the effect of inhibiting the growth of E. coli in the test tube of 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.

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

Recently, research on lactic acid bacteria has been actively conducted, and its range of application has been widened, including not only general food but also health food and medicine. Among these bacteria, Streptococcus, Pediococcus, Leuconostoc, Lactobacillus, Sporolactobacilus and Bifidobacterium Etc.

These bacteria are Gram-positive bacteria that are anaerobic or ectopic, and decompose the nutrients and fibrin 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 intestinal health.

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

In addition, lactic acid bacteria are used to promote the growth of animals and improve feed utilization, increase resistance to disease, inhibit the growth of harmful bacteria, reduce mortality, inhibit the production of toxic toxins and produce various vitamins.

In order to exert the above effects, lactic acid bacteria introduced from the outside should reach the intestine without any disorder, 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, the destruction by gastric acid during oral administration, the resistance to bile acids must be strong and the bactericidal ability of the pathogen should be strong.

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 which are much more resistant to acidic pH and bile acids than the known lactic acid bacteria, and which have strong inhibition of pathogen growth and sterilization.

Hereinafter, the present invention will be described in detail.

In the present invention, Lactobacillus acidophilus SB 603, Lactobacillus salivarius (Lactobacillus salivarius) P-24, Lactobacillus plantar, even in Lactobacillus, a novel lactic acid bacterium that is stable against acidic pH and bile acids and is strong in inhibiting the growth and pathogenicity of pathogens. Lactobacillus plantarum SB602, Streptococcus faecium L-20.

Other lactic acid bacteria in 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 the 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 a prepared BP agar plate medium and cultured in a carbon dioxide incubator for 24 hours at 37 ℃, yellow strain First, the isolate was separated first by gram staining and speculum. The isolated strains were classified into Lactobacillus bacteria and Streptococcus bacteria by temperature sensitivity, pH sensitivity and fermentation form, so that they were resistant to acidic pH, high resistance to bile acids, excellent growth inhibition and bactericidal activity of pathogens.

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 accession number KFCC-10686, Lactobacillus salivarius P-24 is deposited with the KFCC-10684, Lactobacillus plantarum SB 602 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 SB 602, and Streptococcus fasciparum L-20 according to the present invention have the following characteristics.

[Bacterial Properties]

Lactobacillus esidophilus SB603, Lactobacillus salivarius P-24, Lactobacillus platarum SB 602, Streptococcus fascita L L-20 of the present invention have bacteriologic properties as shown in Table 1.

TABLE 1

[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 method described in the article "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 in an AM plate medium to obtain pure colonies, and a single colony is incubated in a carbon dioxide incubator for 12 to 14 hours in an AM liquid medium. Subsequently, inoculate 100 μl into 10 ml of 1 / 15M KH ₂ PO ₄ -HCl buffer solution at pH2.5 and pH3.0, respectively, and store it in a carbon dioxide incubator at 20 ° C or 37 ° C. After serial dilution, smear it on an AM plate and incubate it for 24 hours in a 37 ℃ carbon dioxide incubator, 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.

In this experiment, the conventional bacteria were markedly degraded at 5 to 40% after 60 minutes at pH 2.5, whereas the resistance of the bacteria according to the present invention was 68 to 98%. In addition, the 1 / 15M KH ₂ PO ₄ -HCl buffer solution at pH 3.0 showed a significantly higher resistance of 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 bacterium according to the present invention grows well even in the batch of AMLS plate containing bile acid, which shows that the resistance to the bile acid is superior to the conventional strain. To test the resistance to bile acids, inoculate a single colony of the above strains into an AMS medium, incubate at 37 ° C. in a carbon dioxide incubator for 12 to 14 hours, and then dilute it with physiological saline to obtain a bile acid containing 0.05% to 0.3%. This was done by comparing the number of colonies shown by smearing on S plate medium [Reference: Korea 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 times the number of viable cells in E. coli after 20 to 24 hours (Ref. RDA 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 to 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 bactericidal activity of pathogens is stronger than that of existing strains.

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

Example 1

[Separation of strains]

The cattle and pig intestines immediately after slaughter were purchased and ground in a blender and used as a source of lactic acid bacteria. The prepared isolates were suspended in physiological saline and plated on MLC plate medium, which is 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 medium used for this is mentioned in Table 1. In order to separate the lactic acid bacteria from the isolated lactic acid bacteria, it was carried out according to the strain characteristics of Streptococcus genus of Lactobacillus of the Burgy's manual. First, gram stain (+), catalase (-) strains were selected and divided into lactic acid bacteria of cocci and bacillus through a microscope and then strains grown in KF medium to selectively isolate the strains of Streptococcus. KF medium composition was used as follows.

In order to identify the Lactobacillus and Streptococcus strains isolated in this way, it was cultured in a incubator at 15 ° C and 45 ° C according to the buggy's manual (Bergey's nanual) to measure the temperature sensitivity and to determine whether growth was achieved in the 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 only suspended the lactic acid bacteria incubated in the AMLS solution for 12-14 hours in 1/15 mol sodium phosphate-hydrochloric acid buffer solution at pH 2.5 and pH 3.0 to measure resistance to acidic pH at 30-60 minute intervals. The viable cell count was measured by.

The second method was applied to a 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 counts, the suspension of lactic acid bacteria in physiological saline was serially diluted and plated in an AML plate medium, followed by colony counts generated in a 24-hour culture in a carbon dioxide incubator. The results are shown in Table 2 and FIG.

TABLE 2

Example 3

[Resistance to bile acids]

The resistance of the lactic acid bacteria to the bile acid of the present invention was incubated for 12-14 hours in a liquid medium of AMLS according to the Korean Industrial Microbiological Society Vo, 17, No. 3 (1985) pp192-196, followed by physiological saline. Diluted and plated in a plate medium containing 0% -0.3% bile acid was measured for the viable count. 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.

TABLE 3

Example 4

[E. coli growth inhibition]

In order to measure the growth inhibitory ability and bactericidal power of Escherichia coli, which are pathogenic bacteria of the present invention, a mixed inoculation of Lactobacillus and Escherichia coli and a single inoculation of Escherichia coli in a liquid medium in which both Lactobacillus and E. coli can grow are respectively inoculated at 37 ° C. Incubated at. The culture samples were taken at intervals of 1 to 3 hours to measure the number of Escherichia coli and growth inhibition. 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 mixed culture for 15 hours, the number of Escherichia coli was reduced by 10 ² -10 ³ times than that of Escherichia coli alone, and after more than 18 hours, it was reduced to 10 ⁷ -10 ⁸ times and showed strong bactericidal power.

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

In summary, the above is as follows.

TABLE 4

Claims (1)

Lactobacillus Fetacean L-20 (Accession No. KFCC-10683), a lactic acid bacterium with enhanced resistance to acidic pH and bile acids and bactericidal activity.