KR100890462B1 - Pediococcus spissis ＪＮＵ４４ - Google Patents

Abstract

The present invention is isolated from crude oil, fermented milk, kimchi, human feces and livestock feces, and has a function of inhibiting the growth of harmful microorganisms or food-causing microorganisms causing decay pediococcus spissis JNU534 (deposit institution: Korea spawn association The Korea Microorganisms Preservation Center, Accession No .: KCCM-10869P, Accession Date: May 2, 2007), using the Pediococcus spissis JNU534 or its cultures, the natural preservative, food composition, feed composition and It relates to Pediococcus spissis JNU534 that can be widely used in antimicrobial compositions and the like.

Pediococcus spice, antibacterial activity, bacteriocin

Description

Pediococcus spissis ＪＮＪ４４ {PEDIOCOCCUS DAMNOSUS JNU534}

1 is a graph showing the results of fractionation by octyl-sepharose column chromatography of pediococcus spissis JNU534 of the present invention.

Figure 2 is a graph showing the change in activity during the heat treatment of the bacteriocin produced by Pediococcus spissis JNU534 of the present invention.

Figure 3 is a graph showing the change in activity of bacteriocin by the enzyme treatment of bacteriocin produced by Pediococcus spissis JNU534 of the present invention.

The present invention relates to Pediococcus spissis (Pediococcus Damnosus) JNU534, more specifically, it is separated from kimchi, and has the function of inhibiting the growth of harmful microorganisms, such as pathogenic or decaying, natural preservatives, food compositions, feed compositions and antibacterial It relates to a Pediococcus spissis JNU534 that can be widely used in compositions and the like.

Lactic acid bacteria play an important role in improving the production and storage of various fermented foods and inhabiting the intestines of various animals including humans to control microorganisms and improve health.

Especially, among the antimicrobial substances produced by the lactic acid bacteria, bacteriocin is a natural proteinaceous substance, which can be controlled through genetic manipulation and inhibits the growth of pathogenic and decaying bacteria present in various foods. There is an increasing number of studies related to the development of preservatives.

  Recently, consumers are very concerned about food safety and prefer natural foods with little or no artificial preservatives or food additives.

Therefore, there is an urgent need for a method of reducing the use of chemical preservatives or food additives.

The bacteriocin is an antimicrobial protein that is attracting attention as a natural nontoxic preservative produced by microorganisms, and is ingested by the human body and degraded by proteolytic enzymes of the digestive organs, so that it is toxic to humans and has no residual effect. Is increasing.

The bacteriocins are also inert at relatively high temperatures, are characterized by stability at a wide range of pH, and are toxic, colorless, and odorless, and thus are expected to develop a wide range of applications that can replace chemical synthetic preservatives.

In other words, in addition to improving shelf life in fermented milk, fermented alcoholic beverages, canned food, and refrigerated and frozen products, it extends the shelf life of fermented products such as red pepper paste, soybean paste, tofu, and lactic acid bacteria, and prevents rancidity and deterioration of traditional foods such as kimchi, medicinal wine, and takju. It can improve freshness and shelf life of fruits and vegetables such as bean sprouts.

The inventors of the present invention succeeded in obtaining new lactic acid bacteria by separating from kimchi.

Accordingly, the present invention is to solve the above-mentioned disadvantages and problems of the prior art, it is separated from kimchi, pediococcus spissis JNU534 or has the function of inhibiting the growth of harmful microorganisms or microorganisms causing decay It is an object of the present invention to provide a Pediococcus spissis JNU534 that can be widely used in natural preservatives, food compositions, feed compositions and antimicrobial compositions using the culture.

The above object of the present invention has a function of inhibiting the growth of harmful microorganisms or microorganisms that cause deterioration of foods Pediococcus spissis JNU534 (deposit institution: Korea Microbiological Conservation Center affiliated with Korea spawn association, Accession No .: KCCM-10869P, Date of Deposit: May 2, 2007).

In addition, the above object of the present invention is also achieved by the Pediococcus spissis JNU534, characterized in that the Pediococcus spissis JNU534 is separable from kimchi.

In addition, the object of the present invention is that the harmful microorganisms or microorganisms that cause the food to deteriorate Listeria monocytogenes, Listeria monocytogenes KCTC 3569, Staphylococcus aureus, Lactobacillus plantarum KCCM 11322, Lactobacillus plantarum L155, Lactobacillus plantarum L165, Lactobacillus plantarum L167, Lactobacillus ashdophyllus NOCKS, Lactobacillus ashdophilus KCCM 32820, Lactobacillus ashdophyllus CH5, Lactobacillus ashdophilus GP1B, Lactobacillus Bacillus gasser KCTC 3163, Lactobacillus crispatus KCCM 41620, Lactobacillus brevis KCCM 35464, Lactobacillus cascai YIT 9029, Lactobacillus cascai Y1, Lactobacillus rhamnosus GG and Lyukonostock mesenteroides KCCM 35471 It is achieved by pediococcus spice CIS JNU534 characterized by the above .

In addition, the above object of the present invention is also achieved by Pediococcus spissis JNU534, characterized in that the bacteriocin produced from the Pediococcus spisus JNU534 has a molecular weight of 3.4kDa.

In addition, the above object of the present invention is a pedi, characterized in that the first eight amino acid sequence of the bacteriocin produced from the Pediococcus spissis JNU534 is NH ₂ -Ile-Leu-Leu-Glu-Glu-Leu-Asn-Val It is also achieved by Okoccus Spissis JNU534.

In addition, the above object of the present invention is also achieved by the natural preservative, food composition, feed composition and antimicrobial composition comprising the Pediococcus spissis JNU534 or its culture.

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.

1. Selection and Culture Conditions of Lactic Acid Bacteria Producing Antibacterial Substances

Pediococcus spissis JNU534 of the present invention (depositary organization: Korea Microbiological Conservation Center affiliated with the Korean spawn association, Accession No .: KCCM-10869P, date of deposit: May 2, 2007) is the MRS medium from the lactic acid bacteria strains isolated from kimchi (broth) (Difco, USA) and incubated twice at 37 ℃ was used for antimicrobial activity measurement. The lactic acid bacteria used as indicator bacteria were inoculated in Gram (+) and Gram (-) pathogenic bacteria in TS medium (tryptic soy broth) (Difco, USA) and cultured under the same conditions.

Table 1 shows the composition of 1L of the MRS medium, and Table 2 shows the composition of the 1L of TS medium.

Bacto Proteos Pubton 10 g Bakto Beef Extract 10 g Bacto East Extract 5 g Text Ross 20 g Polysorbate BD (or Twwep BD) 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

2. Selection and Identification of Bacteriocin Producing Strains

Selection of the bacteriocin producing strain was performed using the agar-well diffusion method.

That is, the lactic acid bacteria were inoculated in MRS medium, incubated at 37 ° C. for 18 hours, and centrifuged (13,000 rpm, 10 min, 4 ° C.) to recover the supernatant.

This was adjusted to pH4.5 and pH6.5, respectively, and then filtered through a 0.45 μm-pore size membrane filter (Advantec MFS, USA) to be used as a sample for confirming bacteriocin activity.

After placing a sterile metal borer in a petri dish, an aliquot of about 15 ml of MRS solid medium was dispensed and hardened, and TSSA (tryptic) inoculated with 1% concentration of Staphylococcus aureus. soy soft agar) (0.85%) layered and hardened.

The culture supernatant of each strain was dispensed in a well prepared by removing the metal bore, and the culture supernatant of each strain was dispensed and cultured for 18 hours at 37 ° C. Confirmed.

The lactic acid bacteria finally selected by the above method were confirmed morphological characteristics by Gram staining and ATB by examining sugar fermentation using API 50CH carbohydrate test kit (Bio Merieux, France). As a result of the identification on the basis of the results entered into the identification program (Bio Merieux, France), 96.8% homology with the Pediococcus spissis JNU534 of the present invention was shown.

The sugar fermentation test results, the analysis results are summarized in Table 3.

No. Sugar substrate Growth No. Sugar substrate Growth 0 Control - 25 Esulin + One Glycerol - 26 Salicin + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose - 4 L-Arabinose - 29 Lactose - 5 Ribose - 30 Melibiose - 6 D-Xylose - 31 Sucrose - 7 L-Xylose - 32 Trehalose - 8 Adonitol - 33 Inulin - 9 β-Methyl-D-xyloside - 34 Melezitose - 10 Galacotose + 35 Raffinose - 11 Glucose + 36 Starch - 12 Fructose + 37 Glycogen - 13 Manose + 38 Xylitol - 14 Sorbose - 39 Gentiobiose - 15 Rhamnose - 40 D-Turanose - 16 Dulcitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose - 18 Mannitol - 43 D-Fucose - 19 Sorbitol - 44 L-Fucose - 20 α-Methyl-D-mannoside - 45 D-Arabitol - 21 α-Methyl-D-glucoside - 46 L-Arabitol - 22 N-Acetyl glucosamine - 47 Gluconate - 23 Amygdalin - 48 2-keto-gluconate - 24 Arbutin - 49 5-keto-gluconate -

 +: Growing,-: not growing

3. Cell Growth and Bacteriocin Production by Culture Time

The bacteriocin-producing lactic acid bacteria were inoculated in MRS medium at a concentration of 10 ⁶ CFU / ml, followed by incubation at 37 ° C. for 48 hours, and the number of viable cells and pH were measured at 3 hour intervals.

In addition, the change of bacteriocin activity was measured by the critical dilution method (Hoover and Harlander, 1993) using the supernatant obtained by treating the culture solution in each time zone in the same manner as the above method.

That is, a supernatant of two-fold dilution with sterile distilled water was dropped on the MRS medium, and the MRS soft medium inoculated with Lactobacillus plantarum, which is an indicator bacterium, was incubated at 37 ° C. for 18 hours. After the suppression was confirmed.

Table 4 below shows the antimicrobial activity of the selected bacteriocin and shows the results of evaluating the growth inhibitory activity against 11 pathogenic bacteria and 19 lactic acid bacteria using the strains identified above.

Indicator strains Inhibition  Listeria monocytogenes +  Listeria monocytogenes KCTC 3569 +  Listeria ivanovii -  Listeria innocua -  Staphylococcus aureus +  E. coli O0157: H7 ATCC 43889 -  E. coli O0157: H7 ATCC 43894 -  E. coli O0157: H7 ATCC 43895 -  Salmonella typhimurium -  Klebsisella pneumoniase -  Enterobacter aerogenes -  Lactobacillus plantarum L167 +  Lactobacillus acidophilus NOCKS +  Lactobacillus plantarum L165 +  Lactobacillus helveticus CH1 -  Lactobacillus gasseri KCTC 3163 +  Lactobacillus acidophilus KCCM 32820 +  Lactobacillus acidophilus CH5 +  Lactobacillus crispatus KCCM 41620 +  Lactobacillus brevis KCCM 35464 +  Lactobacillus plantarum L155 +  Lactobacillus acidophilus GP1B +  Lactobacillus plantarum KCCM 11322 +  Lactobacillus casei YIT9029 +  Lactobacillus rhamnosus GG +  Lactobacillus paracasei ssp. paracasei P113 -  Lactobacillus casei Y1 +  Lactococcus lactis KCCM 40104 -  Leuconostoc mesenteroides KCCM 35471 +  Pediococcus pentosaceus KCCM 40464 -

+: Suppressed,-: not suppressed.

As shown in Table 4, Listeria monoctogenes, Listeria monoctogenes KCTC 3569, Staphylococcus aureus, Lactobacillus plantarum KCCM 11322, Lactobacillus Lactobacillus plantarum L155, Lactobacillus plantarum L165 Lactobacillus plantarum L167, Lactobacillus acidophilus NOCKS, Lactobacillus ashdophyllus Lactobacillus acidophilus KCCM 32820, Lactobacillus acidophilus CH5, Lactobacillus acidophilus GP1B, Lactobacillus gasseri KCTC 3163, Lactobacillus crispapatus crispatus KCCM 41620, Lactobacillus brevis KCCM 35464, Lactobacillus Lactobacillus casei YIT 9029, Lactobacillus casei Y1, Lactobacillus rhamnosus GG, and Leuconostoc mesenteroides KCCM 35471 show antibacterial activity (E. coli), Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, etc. did not inhibit the growth of gram (-) bacteria.

4. Purification of Bacteriocin

After two passages of Pediococcus spissis JNU534 of the present invention, the supernatant was recovered by centrifugation (8,000rpm, 30min, 4 ° C) after incubating for 18 hours by inoculating 1% concentration in 1,000 ml of MRS medium.

Ammonium sulfate was added to the culture supernatant at a concentration of 30% and stirred slowly at 4 ° C. for at least 12 hours, followed by centrifugation (8,000 rpm, 30 min, 4 ° C.).

A crude bacteriocin solution prepared by dissolving the precipitate obtained in 50 ml of 50 mM MES (2- (4-morpholino) -ethane sulfonic acid) (Sigma, USA) buffer (pH 7.0) was stored at -20 ° C. It was.

Chromatography was performed by injecting a Zobacteriocin solution into an Octyl-Sepharose CL-4B column (Bio-rad, USA) equilibrated to a three bed volume of 1.7 M ammonium sulfate. ) Was performed.

Hydrophobic interaction column chromatography using the crude bacteriocin solution is shown in FIG. 1. Six bacteriocin active fractions were isolated from a total of 78 fractions, and the absorbance at 214 nm was measured. Fractions consistent with the resulting peak and showing the highest bacteriocin activity (400 AU / ml) were used as electrophoretic samples.

The absorbance at 214nm was measured for the fractions obtained, and after confirming the production of antimicrobial substances against indicator bacteria, only the fractions showing the antimicrobial activity were recovered and the respective bacteriocin activities were measured.

Fractions showing the highest bacteriocin activity were frozen at −70 ° C., dried, concentrated, dissolved in 2 × tricine-SDS sample buffer (Novex, USA) and used as electrophoretic samples.

The electrophoresis was performed by preparing a 4% stacking gel, a 10% spacer gel, and a 16% separation gel to produce a mini-PROTEAN 3cell. Tri-SDS-PAGE (Schagger and Von Jagow, 1987) was performed for 60 minutes at a constant voltage of 125 V using (Bio-Rad, USA).

The electrophoresis-completed gel was blotted onto a polyvinylidene difluoride (PVDF) membrane for 3 hours at a constant voltage of 50 V at 4 ° C. using a Mini Trans-Blot Cell (Bio-Rad, USA). blotting).

N-terminal sequencing was performed by Edman degradation of the bloated membrane.

As a result of analyzing the amino acid sequence by Edman digestion, only the first 8 amino acids were read as NH ₂ -Ile-Leu-Leu-Glu-Glu-Leu-Asn-Val, and from the ninth sequence, blocking was not performed. I couldn't.

5. Characteristics of Bacteriocin

The collected supernatant was adjusted to pH 6.5 using 5N NaOH, filtered through a 0.45 μm-pore size membrane filter, and then carried out as follows.

Controlled supernatant using a constant temperature bath and autoclave for 15 minutes at various temperatures (37, 70, 80, 90, 100, 121 ℃) and cooled to room temperature to measure the antimicrobial activity and then not heat treated As a result of investigating changes in bacteriocin activity, as shown in FIG. 2, when heat treated at 37, 70, 80, 90 and 100 ° C. for 15 minutes, the activity of bacteriocin was maintained without being lost, but completely disappeared at 121 ° C.

Thus, the Pediococcus spissis JNU534 of the present invention shows that it does not disappear at temperatures below about 100 ° C.

Trypsin (Sigma, USA), lysozyme (Sigma, USA), and protease (Sigma, USA) are 50 mM Tris-HCl buffers to determine whether the antimicrobial agents are proteinaceous. pH 7.5, pepsin (Sigma, USA) 50 mM citrate buffer (pH 2.0), proteinase K (USB, USA) 10 mM Tris-HCl-50 mM NaCl-5 mM Each was dissolved in EDTA (pH 7.5) to prepare an enzyme solution.

This was added to the bacteriocin solution at a concentration of 4 mg / ml, respectively, and reacted in a 37 ° C. constant temperature water bath for 12 hours, and then the bacteriocin activity was measured. As a control, one that was not treated with enzyme solution under the same conditions was used.

As shown in Figure 3, the bacteriocin activity was maintained by lysozyme, an enzyme that breaks down the polysaccharide chain of the cell wall, but bacteriocin was maintained by proteinases trypsin, pepsin, protease and proteinase K. Since the activity was removed, it was confirmed that it is a proteinaceous material, and thus, the safety as a preservative is considered to be very high.

Thus, the Pediococcus spissis JNU534 of the present invention is Listeria monocytogenes, Listeria monocytogenes KCTC 3569, Staphylococcus oreus, Lactobacillus plantarum KCCM 11322, Lactobacillus plantarum L155, Lactobacillus plantarum L165, Lactobacillus plantarum L167, Lactobacillus ashdophyllus NOCKS, Lactobacillus ashdophyllus KCCM 32820, Lactobacillus ashdophyllus CH5, Lactobacillus ashdophilus GP1B, Lactobacillus gasseri KCTC 3163, Lactobacillus Harmful microorganisms and foods such as Bacillus crispatus KCCM 41620, Lactobacillus brevis KCCM 35464, Lactobacillus cascai YIT 9029, Lactobacillus cascai Y1, Lactobacillus rhamnosus GG, and leukonostock mesenteroides KCCM 35471 Pediococcus spissis with antibacterial ability to restrain microbial growth JNU534 itself or its culture can be used as a natural preservative, food composition, feed composition and antimicrobial composition.

Therefore, the Pediococcus spis JNU534 of the present invention has an antimicrobial ability to inhibit the growth of harmful microorganisms, such as pathogenic microorganisms and decaying microorganisms can be used as natural preservatives, food compositions, feed compositions and antimicrobial 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 (9)

It is separable from kimchi and has the function of inhibiting the growth of harmful microorganisms or microorganisms causing the deterioration of food, and the sequence of the first 8 amino acids is NH ₂ -Ile-Leu-Leu-Glu-Glu-Leu-Asn-Val Pediococcus spissis JNU534, which produces bacteriocin with a molecular weight of 3.4 kDa (depositment institution: Korea Microbiological Conservation Center, affiliated with the Korean spawn association, Accession No .: KCCM-10869P, date of deposit: May 2, 2007). delete The method of claim 1, The harmful microorganisms or microorganisms that cause the food to deteriorate are Listeria monocytogenes, Listeria monocytogenes KCTC 3569, Staphylococcus oreus, Lactobacillus plantarum KCCM 11322, Lactobacillus plantarum L155, Lactobacillus plantarum L165 , Lactobacillus plantarum L167, Lactobacillus ashdophyllus NOCKS, Lactobacillus ashdophilus KCCM 32820, Lactobacillus ashdophyllus CH5, Lactobacillus ashdophilus GP1B, Lactobacillus gaseri KCTC 3163, Lactobacillus Pediococcus, characterized by at least one of Crispaters KCCM 41620, Lactobacillus brevis KCCM 35464, Lactobacillus cassia YIT 9029, Lactobacillus cassia Y1, Lactobacillus ramnosus GG and Leukonostock Mesenteroides KCCM 35471 Spicy JNU534. delete delete Pediococcus spissis JNU534 of claim 1 or a natural preservative comprising the culture thereof. A food composition comprising the pediococcus spissis JNU534 or its culture according to claim 1. Pediococcus spissis JNU534 of claim 1 or a composition for a feed comprising a culture thereof. The antimicrobial composition of Claim 1 containing the pediococcus spisus JNU534 or its culture.

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