KR100871904B1 - Lactobacillus acidophillus kcnu - Google Patents

Abstract

The present invention is isolated from the small intestine of pigs, and has the function of inhibiting the growth of pathogenic microorganisms and decaying microorganisms Lactobacillus ashdo that can be widely used in food compositions, feed compositions, antimicrobial compositions and household goods, such as pharmaceutical compositions, beverages, etc. It relates to Philus KCNU and its culture.

Lactobacillus, Ashdophilus, Antibacterial activity, Bacteriocin

Description

Lactobacillus ACIDOPHILLUS KCNU}

1 is a graph evaluating acid resistance of Lactobacillus ashidophilus KCNU according to an embodiment of the present invention.

Figure 2 is a graph evaluating the bile acid resistance of Lactobacillus ashirophilus KCNU according to an embodiment of the present invention.

Figure 3 shows the transmission electron microscope observation of the indicator after the bacteriocin treatment produced by Lactobacillus ashdophilus KCNU according to an embodiment of the present invention.

Figure 4 shows the low molecular weight electrophoresis of bacteriocin produced by Lactobacillus ashidophilus KCNU according to an embodiment of the present invention.

The present invention relates to Lactobacillus ashophilus KCNU, more specifically, it is separated from the small intestine of pigs, and has the function of inhibiting the growth of harmful microorganisms, such as pathogenic or rot, food composition, such as pharmaceutical compositions, beverages, etc. It relates to Lactobacillus ashidophilus KCNU which can be widely used in compositions, feed compositions, antimicrobial compositions and household goods.

There are microorganisms that inhabit the intestine of humans or animals and have various beneficial effects. These microorganisms are called probiotics, and among them, Ashidophillus probiotics is one of the representative probiotic microorganisms.

Lactobacillus is a bacterium that decomposes and utilizes carbohydrates such as glucose or lactose to make lactic acid. Up to now, lactic acid bacteria have been known as 300 to 400 kinds. Lactic acid bacteria are widely distributed in the natural world such as human and animal digestive tracts, milk, yogurt, cheese, kimchi, plants and soil, and these lactic acid bacteria are beneficial commensal bodies that are directly or indirectly related to human life. The lactic acid bacteria used in the fermented milk production and fermentation business mainly include Lactobacillus, Lactococcus, Streptococcus, and Leuconostoc and Bifidobacterium. .

In this case, in order to use the lactic acid bacteria in food or medicine, there are various selection criteria, and the cholesterol lowering ability and antimicrobial ability should be confirmed. Only those lactic acid bacteria having this ability normalize the intestinal flora and reduce lactose intolerance. In addition, it can be expected to lower blood cholesterol levels, anti-cancer effects and strengthen the immune system.

The inventors of the present invention have long been studying the functional problem of lactic acid bacteria by separating live strains from the small intestine of pigs, which is safer to humans than conventional Lactobacillus ashdophyllus bacteria, and has excellent physiological efficacy and antibacterial substances. Successfully obtained strains to produce.

Therefore, the present invention is to solve all the disadvantages and problems of the prior art as described above, is separated from the small intestine of the pig from the small intestine of the pig, and has the function of inhibiting the growth of harmful microorganisms of pathogenicity and decay, pharmaceutical composition, beverage It is an object of the present invention to provide Lactobacillus ashdophyllus KCNU which can be widely used in food compositions, feed compositions, antimicrobial compositions and household goods such as and the like.

The object of the present invention is acid-resistant, bile acid resistance and cholesterol adsorption capacity, Lactobacillus ashdophyllus KCNU (depositing institution: Korea Microbiological Conservation Center, Accession No .: KCCM) having the function of inhibiting the growth of harmful microorganisms -10753P, entrusted date: May 22, 2006).

In addition, the object of the present invention is that the harmful microorganisms Bacillus cereus, Enterococcus aerogenes, Escherichia coli K21052, Escherichia coli O157: H7 ATCC 43889, Listeria innocua, Listeria monocytogenes, Listeria Ivanobi, Shigella sonei, Star Lactobacillus ashdophilus KCNU, characterized by at least one of: Philococcus oreus, Staphylococcus epidermidis KCCM 35494, Staphylococcus intermedius KCCM 40149, Pseudomonas aeruginosa KCCM 11363, Elcinia enterocholicica It is also achieved by

In addition, the above object of the present invention is also achieved by the Lactobacillus ashidophilus KCNU is characterized in that it is collected from the small intestine of pigs.

In addition, the above object of the present invention is also achieved by the pharmaceutical composition characterized in that it comprises the Lactobacillus ashidophilus KCNU or its culture.

In addition, the object of the present invention is also achieved by a food composition comprising the Lactobacillus ashidophilus KCNU or its culture.

In addition, the above object of the present invention is also achieved by a feed composition comprising a Lactobacillus ashidophilus KCNU or a culture thereof.

In addition, the above object of the present invention is also achieved by an antimicrobial composition comprising the Lactobacillus ashidophilus KCNU of claim 1 or a culture thereof.

Details of the above object, technical configuration and effects according to the present invention will be more clearly understood by the following detailed description with reference to the drawings showing preferred embodiments of the present invention. In addition, in the drawings, the length, thickness, etc. of layers and regions may be exaggerated for convenience. Like numbers refer to like elements throughout.

Lactobacillus ashdo philus KCNU of the present invention (depositary organization: Korea Microbiological Conservation Center affiliated with the Korean spawn association, Accession No .: KCCM-10753P, date of deposit: May 22, 2006) is a saline collected by collecting the small intestine of pigs After serial dilution with, smeared on MRS solid medium containing bromocresol purple and incubated in a 37 ° C. incubator for 24 hours to obtain a yellow colony.

The characteristics of the Lactobacillus ashidophilus KCNU strain obtained by the above method are shown in Table 1 below, and the sugar fermentation properties are shown in Table 2 below.

Morphology Stick Colony color milk white Growth temperature 37 ℃ motility none Gram Dyeing positivity Catalase voice Oxygen effect Combination anaerobic

Sugar fermentation test Party kind Usability Glycerol - Erythritol - D-Arabinose - L-Arabinose - Ribose - D-Xylose - L-Xylose - Adonitol - βMethyl-xyloside - Galactose + D-Glucose + D-Frucose + D-Mannose + L-Sorbose - Rhamnose - Dulcitol - Inositol + Mannitol - Sorbitol - αMethyl-D-mannoside - αMethyl-D-glucosie - N acetyl glucosamine + Amygdaline + Arbutine - Esulin + Salicine - Cellobiose - Maltose + Lactose + Melibiose + Saccharose + Trehalose - Inuline - Melezitose + D-Raffinose + Amidon + Glycogen - Xylitol - βGentiobiose + D-Turanose + D-Lyxose - D-Tagatose - D-Fucose - L-Fucose + D-Arabitol - L-Arabitol - Gluconate - 2 ceto-gluconate - 5 ceto-gluconate -

Example 1 Acid Resistance Evaluation

First, pH (hydrogen ion concentration) was adjusted to 2.5, and the sterilized MRS liquid medium was prepared by adding a pepsin sterilized with a 0.22 micron filter to 1000 units per milliliter of the acid resistance measurement artificial gastric juice was prepared.

The Lactobacillus ashidophilus KCNU of the present invention was incubated in a constant temperature water bath at 18 ° C. for 18 hours, and then the cells were precipitated using a centrifuge, followed by washing the precipitated cells with physiological saline twice.

After the cells obtained by the above method were incubated for 1, 2 and 3 hours in artificial gastric juice as described above, the viable cell count was measured. In this case, when measuring the viable cell number was used MRS medium containing 2 parts by weight of beta-glycero phosphate.

1 is a graph evaluating acid resistance of Lactobacillus ashidophilus KCNU according to an embodiment of the present invention. 1 is Lactobacillus ashidophilus CH5 (commercial strain), B is Lactobacillus ashidophilus NCFM (commercial strain), and C is Lactobacillus ashidophilus NOCKS (commercial strain). D is a Lactobacillus casei (commercial strain) and E is a Lactobacillus ashidophilus KCNU strain.

Referring to Figure 1, Lactobacillus ashdophilus KCNU of the present invention and four representative Lactobacillus genus among the lactic acid bacteria used in the production of conventional fermented milk, that is, Ashdophilus CH5, Ashdophilus NCFM , Ashdophyllus NOCK, Casey and Ashdophyllus KCNU strains used as probiotic lactobacillus is shown a graph comparing the results of the acid resistance. At this time, the acid resistance test is the result of treatment for 3 hours at pH 2.5.

As a result, it can be seen that the Lactobacillus ashidophilus KCNU strain of the present invention does not die when treated at pH 2.5 for 3 hours.

In addition, the acid resistance of the Lactobacillus ashidophilus KCNU strain of the present invention is different from that of other lactic acid bacteria currently used in fermented milk production, namely, Ashdophilus CH5, Ashdophilus NCFM, Ashdophilus NOCK, and Kasei strains. It can be seen that the comparison is similar or superior.

Therefore, the Lactobacillus ashidophilus KCNU and its culture of the present invention is a pharmaceutical composition, a food composition, which can be ingested by humans, even if ingested by humans or animals is not killed by gastric acid of humans or animals. And antimicrobial compositions and the like.

Example 2 Bile Acid Resistance Evaluation

Figure 2 is a graph evaluating the bile acid resistance of Lactobacillus ashirophilus KCNU according to an embodiment of the present invention. 2 is Lactobacillus ashidophilus CH5 (commercial strain), B is Lactobacillus ashidophilus NCFM (commercial strain), and C is Lactobacillus ashidophilus NOCKS (commercial strain). D is a Lactobacillus casei (commercial strain) and E is a Lactobacillus ashidophilus KCNU strain.

Referring to Figure 2, the resistance to the bile acid of the Lactobacillus ashirophilus KCNU of the present invention can be seen by examining the state of growth in MRS medium containing 0.5% bile acid (Oxgall).

That is, the Lactobacillus ashirophilus KCNU of the present invention was incubated in a constant temperature water bath at 18 ° C. for 18 hours, followed by precipitating the cells using a centrifuge, followed by washing the cells precipitated with physiological saline twice and containing bile acids. After growth for 12 and 24 hours in each medium was observed.

As a result, the Lactobacillus ashirophilus KCNU of the present invention did not die in the medium containing 0.5% bile acid, as shown in the graph shown in Figure 2 showed excellent bile acid resistance, lactic acid bacteria used in conventional fermented milk production Among the representative four types of Lactobacillus, namely Ashdophilus CH5, Ashdophilus NCFM, Ashdophilus NOCK, it can be seen that the similar or superior growth compared to the strains.

Therefore, the Lactobacillus ashidophyllus KCNU and its culture of the present invention is a pharmaceutical composition, a food composition that can be ingested by humans because it is not killed by human or animal bile acids even if ingested by humans or animals. It can be used as a composition and antimicrobial composition.

Example 3 Cholesterol Degradation Capacity Evaluation

First, Thiolacolate and Oxal or Taurocholic acid were added to MRS liquid medium at 0.2 to 0.3% by weight, respectively, and sterilized at 121 ° C. for 15 minutes, and then 9 ml of 1 ml cholesterol micelle was prepared. Was added to the prepared medium so that the cholesterol level in the final medium was 100 μg / ml.

In addition, Lactobacillus ashidophilus KCNU of the present invention was cultured in the treated medium, and the cholesterol micelle content remaining in the supernatant after culture was measured as follows.

That is, 9.1 mg cholesterol and 0.2 ml L-α-lecithin were dissolved in 1 ml of chloroform, dried with nitrogen gas, and ultrasonic crushing was performed for 15 minutes by adding 0.4 M and 10 ml sucrose.

At this time, the analysis of residual cholesterol was added to 3ml 97% ethanol and 2ml 50% by weight potassium hydroxide in 0.5ml supernatant and heated at 60 ℃ for 10 minutes and mixed well with 5ml hexane in a solution cooled at room temperature for 30 seconds to extract cholesterol It was.

And, 2.5 ml of the extracted hexane was dispensed into a test tube and dried with nitrogen gas, but drying was promoted by heating at 60 ° C.

4 ml O-phthalaldehyde (50 mg / 100 ml acetic acid, glacial) solution was added to the dried test tube and allowed to stand for 10 minutes for smooth color development. After standing, 2 ml concentrated sulfuric acid was added to induce color development. Cholesterol was quantified by measuring absorbance at 550 nm after 10 minutes of concentrated sulfuric acid.

As shown in Table 3 below, the Lactobacillus ashidophilus KCNU strain of the present invention was shown to adsorb 45.05% of cholesterol and has moderate adsorption.

In contrast, the ashdophilus ATCC 43121 strain showed the highest adsorption at 57% and the lactis ATCC 4797 strain had the lowest at 16.26%.

Therefore, the Lactobacillus ashidophilus KCNU and its culture of the present invention shows that it can be used as a pharmaceutical composition and food composition or animal feed composition which can be ingested by humans because of its cholesterol-degrading effect.

Fungus % Decomposition Lactobacillus ashdophyllus KCNU 45.05 Lactobacillus ashdophyllus ATCC 4356 33.24 Lactobacillus ashdophyllus ATCC 43121 57.50 Lactobacillus ashdophyllus ATCC 4962 26.14 Lactobacillus delbruekki Subspicis Lactis ATCC 4797 16.26 Lactobacillus helvetica 33.43 Lactobacillus casei 911 24.75 Lactobacillus rhamnosus 7469 25.13

Example 4 Antibacterial Activity Measurement

Evaluation of the antimicrobial activity of Lactobacillus ashidophilus KCNU of the present invention was performed as follows. The Lactobacillus ashidophilus KCNU was incubated in MRS liquid medium in a 37 ° C. constant temperature water bath for 18 hours, followed by centrifugation (6000 g, 30 minutes, 4 ° C.) to remove the cells, and then the pH was adjusted to 7.0.

The supernatant obtained by filtration using a 0.45 micron filter was dispensed into 20 microliters of MRS solid medium and TS solid medium, respectively, to layer the supporting bacteria.

After culturing for 18 to 24 hours at the optimum growth temperature of each indicator, the antimicrobial activity was measured by inhibition of production.

At this time, lactic acid bacteria were used for MRS medium and other microorganisms for TS medium, respectively, and the composition of 1 L of MRS medium is shown in Table 4 below, and the composition of 1 L of TS medium is shown in Table 5 below.

Bacterium Proteases Peptone 10 g Bakto Beef Extract 10 g Bacto East Extract 5 g Text Ross 20 g Polysorbate 80 (or Tween 80) 1 g Ammonium citrate 2 g Sodium acetate 5 g Magnesium sulfate 0.1g Manganese Sulfate 0.05g Dipotassium Phosphate 2 g

Bacto trypton 17 g Bakto Soyton 3 g Text Ross 2.5g Sodium chloride 5 g Dipotassium Phosphate 2.5g

At this time, Table 6 and Table 7 show the growth inhibitory activity against 34 kinds of lactic acid bacteria and 24 pathogenic microorganisms of the Lactobacillus ashidophilus KCNU of the present invention and different spectrum from the bacteriocin produced by the existing lactic acid bacteria Bacillus cereus, Enterococcus aerogenes, Escherichia coli K21052, Escherichia coli O157: H7 ATCC 43889 It has been shown to effectively inhibit the growth of Epidermidis KCCM 35494, Staphylococcus intermedius KCCM 40149, Pseudomonas aeruginosa KCCM 11363, and Elcinia enterocholicica.

Therefore, the Lactobacillus ashidophilus KCNU and its culture of the present invention has an antimicrobial action against pathogenic microorganisms and can be used as a pharmaceutical composition, a food composition, or a feed composition for animals. give.

Indicator Inhibition L. acidophilus 4962 + L. acidophilus GP1B - L. acidophilus 606 + L. acidophilus KU107A + L. acidophilus 30SC + L. acidophilus GP2A + L. acidophilus GP4A + L. acidophilus A4 - L. acidophilus ATCC 4356 + L. acidophilus ATCC 43121 - L. acidophilus NCFM + L. casei 9018 + L. casei 393 - L. casei sp. + L. delbrueckii subsp. lactis ATCC 4797 + L.helveticus 1042 - L. helveticus KU116 + L. paracasei subsp. paracasei 910 - L. paracasei subsp. paracasei 911 - L. plantarum EH2 + L. plantarum CS1 - L. plantarum K 4 - L. plantarum M 2 - L. plantarum B 1 + L. plantarum F7 1 + L. rhamnosus ATCC 7469 - L. lactis subsp. lactis ATCC 11454 + Lactococcus sp. 449 - Lactococcus sp. LN518 + Lactococcus sp. LO518 + Lactococcus sp. HT61 - Lactococcus sp. HT51 + Leuconostoc sp. K2 - Leuconostoc sp. K1 +

(+) Inhibited, (-) not inhibited

Pathogens Strains GP1B Acinetobacter baumannii - Bacillus cereus + Bacillus subtilis - Enterococcus aerogenes + Escherichia coli K11234 - Escherichia coli K21052 + Escherichia coli O15: H7 ATCC 43894 - Escherichia coli O15: H7 ATCC 43895 - Escherichia coli O15: H7 ATCC 43889 + Escherichia coli O15: H7 ATCC 43893 - Fecal streptococcus - Klebstella pneumoniae - Listeria inoccua + Listeria monocytogene + Listeria ivanovii + Salmonella typhimurium K11806 - Shigella sonnei + Staphylococcus aureus + Staphylococcus epidermidis KCCM35494 + Staphylococcus intermedius KCCM40149 + Pseudomonas aeruginosa KCCM11321 + Pseudomonas chororaphis K11363 - Pseudomonas fluorescens - Yersinia enterocolitica +

(+) Inhibited, (-) not inhibited

Example 5 Observation of Antimicrobial Activity

In order to observe the antimicrobial activity by scanning electron microscope (Scanning Electron Microscope), the sample was first cut into a suitable size and attached to the sample table, and then the sample powder to be observed was lightly dropped on it. The prepared specimens were then coated under vacuum for 60 seconds with a gold-palladium layer using an ion sputter coater for electron microscopy. In addition, the shape of the specimen was observed at an accelerating voltage of 15 kV.

Figure 3 shows the transmission electron microscope observation of the indicator after the bacteriocin treatment produced by Lactobacillus ashdophilus KCNU according to an embodiment of the present invention. 3 (A) is a photograph immediately after the bacteriocin treatment, (B) 30 minutes after the bacteriocin treatment, and (C) 90 minutes after the bacteriocin treatment.

As a result, as shown in the photographs shown in Figure 3 immediately after the treatment of the bacteriocin (A in Fig. 3) there is no change in the cytoplasm no damage to the cell wall, 30 minutes after the treatment of the bacteriocin (B in Fig. 3 ) Shows that the cell wall is destroyed, and after 90 minutes have elapsed (FIG. 3C), the cell wall is destroyed and the internal cytoplasmic component is lost.

Therefore, after treatment with the selection bacteriocin and observed by electron microscopy, cell wall collapse was observed, it was estimated that the selection bacteriocin has a cell lysis action.

Example 6 Purification of Antibacterial Substances

Bacteriocin produced by the Lactobacillus ashidophilus KCNU of the present invention was the highest yield (yeild) to precipitate in 40% ammonium sulfate (Sepharose) G-50, octyl-sepharo When fractionated with an OSC CL4B column (Octyl-sepharose CL4B column) it can be seen that it is purified as a single peak in the final high performance liquid chromatography (HPLC). When precipitated with 40% ammonium sulfate, 67.9% of bacteriocin could be recovered and the specific activity was 9.37 times higher (see Table 8).

Figure 4 shows the low molecular weight electrophoresis of bacteriocin produced by Lactobacillus ashidophilus KCNU according to an embodiment of the present invention. At this time, Figure 4 (A) is a gel after silver staining (Lane 1: polypeptide standard sample, Lane 2: bacteriocin sample), (B) is a layer of 0.8% MRS solid medium inoculated with the indicator bacteria on the same gel Antibacterial appearance.

As shown in FIG. 4, low molecular weight electrophoresis was carried out after the final purification, and the molecular weight was measured to be about 3.6 kDs.

N-terminal amino acid analysis showed that the amino acid sequence of NH ₂ -Pro-Ala-X-Leu-Met-Tyr-Arg was read and was identified as a novel bacteriocin different from the existing bacteriocin sequence in the NCBI-BLAST program. .

Purification Characteristics of Bacteriocin According to Ammonium Sulfate Concentration Purification stage Total protein (mg) Total acivity (AU / ml) Specific acivity (AU / mg) Purification fold Recovery (%) Culture solution (895 ml) 203.2 2,864,000 14,094 One 100 Ammonium Sulfate Concentration 30% 1.05 35,840 34,133 2.42 1.25 30-40% 14.73 1,945,600 132,084 9.37 67.9 40-50% 11.94 53,760 4,502 0.32 1.88

Therefore, the Lactobacillus ashidophilus KCNU and its culture of the present invention has superior antimicrobial ability to inhibit the growth of harmful microorganisms such as pathogenic microorganisms and perishable microorganisms, and thus can be utilized as a probiotic of the human body. It is provided as a useful strain for fermented products, lactic acid bacteria food, and can be used as an additive as an animal feed, and can be used as a food composition, a feed composition, an antimicrobial composition, and a daily necessity such as a pharmaceutical composition and a beverage. There is an effect that can be used very widely.

Although the present invention has been shown and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

Claims (8)

delete Lactobacillus ashdophilus KCNU (deposited institution: Korea Microbiological Conservation Center, Accessory No .: KCCM-10753P, Accession Date) : May 22, 2006). The method of claim 2, The harmful microorganisms are Bacillus cereus, Enterococcus aerogenes, Escherichia coli K21052, Escherichia coli O157: H7 ATCC 43889, Listeria innocua, Listeria monocytogenes, Listeria ivanobi, Shigella sonei, Staphylococcus oreus, Staphylococcus Lactobacillus ashdophilus KCNU, characterized in that any one or more of Epidermidis KCCM 35494, Staphylococcus intermedius KCCM 40149, Pseudomonas aeruginosa KCCM 11363, Elcinia enterocholicica. The method of claim 2, The Lactobacillus ashdophyllus KCNU is Lactobacillus ashdophilus KCNU, characterized in that obtained from the small intestine of pigs. A pharmaceutical composition comprising the Lactobacillus ashidophilus KCNU of claim 2 or a culture thereof. A food composition comprising the Lactobacillus ashidophilus KCNU of claim 2 or a culture thereof. Feed composition comprising the Lactobacillus ashidophilus KCNU of claim 2 or its culture. The antimicrobial composition of claim 2 comprising the Lactobacillus ashidophilus KCNU or its culture.

Images