JP2005110566A - Mew lactic acid bacterium, antimicrobial substance produced by the lactic acid bacterium and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an antimicrobial peptide-containing substance that is stable in pH in the neutral range to the alkaline range, especially from the acid to alkalinity ranges, and a new lactic acid bacterium for producing the substance. <P>SOLUTION: The lactic acid bacterium (typically KMR-7 strain) belongs to Lactococcus lactis subsp. lactis capable of producing a peptide-containing substance that is stable at pH 2-9 and exhibits antimicrobial properties against gram-positive bacteria. The antimicrobial substance is obtained by culturing the lactic acid strain and has the following properties of (a) containing antimicrobial peptide or protein, (b) completely being digested by protease, (c) being stable at pH 2-9, especially exhibiting high stability at pH 5-9 and (d) exhibiting heat stability at pH 7.0 at 110°C for 10 minutes. The method for producing the antimicrobial substance comprises culturing the lactic acid strain and collecting the cultured cell. The lactic acid strain is used for producing a food. The food preservative contains the antimicrobial substance or a substance containing it. The fermentation raw material or fermented food is produced by using the lactic acid strain. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel lactic acid strain, a novel antibacterial substance produced by the lactic acid bacterium, and a production method and use thereof, and more specifically, the present invention exhibits antibacterial properties against many gram-positive bacteria including lactic acid bacteria. The present invention relates to a novel lactic acid strain belonging to Lactococcus lactis subspecies lactis producing a substance, a method for producing the antibacterial substance, and its use in foods.

  Lactic acid bacteria are known to have various functions, and among them, antibacterial action has attracted attention in recent years. Antibacterial substances produced by lactic acid bacteria include organic acids such as lactic acid and acetic acid, hydrogen peroxide, diacetyl, reuterin, and bacteriocin.

  Among them, bacteriocin is a proteinaceous or peptidic substance that generally exhibits an antibacterial action against closely related Gram-positive bacteria. The search for bacteriocin of lactic acid bacteria has been vigorously carried out because it is digested and digested by human digestive enzymes, and lactic acid bacteria have been widely used in foods for a long time and are highly safe.

Nisin is a particularly famous bacteriocin. Nisin is currently in practical use as a food preservative in many countries and is added to certain foods such as dairy products and canned foods. Lactococcus lactis subspecies lactis is known to produce nisin (Nature 154 : 551-553 (1944) (reference 1)). Nisin is stable in the acidic range, for example, stable at pH 2.0 and 115.6 ° C., but inactivated at 40% at pH 5.0 and 90% or more at pH 6.8 ( Adv. Appl. Microbiol. 27 : 85-123 (1981) (reference 2)).
Therefore, due to the nature of nisin, the current usage is mainly limited to acidic foods.

On the other hand, using bacteriocin-producing bacteria to try to prevent quality degradation due to contamination with Bacillus during miso fermentation of miso (Biosci. Biotechnol. Biochem., 63 (4), 642-647, 1999) (references) 3)), try applying the culture supernatant or crude product containing bacteriocin to various processed foods (Mentako, Tofu, etc.) (Journal of the Japanese Society for Lactic Acid Bacteria, 10 (1), 2-18, 1999 (Reference 4) Various attempts have been made.
Nature 154: 551-553 (1944) Adv. Appl. Microbiol. 27: 85-123 (1981) Biosci. Biotechnol. Biochem., 63 (4), 642-647, 1999 Japanese Journal of Lactic Acid Bacteria, 10 (1), 2-18, 1999

  Under such circumstances, it is important to search for a bacteriocin-producing bacterium that is useful in place of nisin and that is stable in the neutral range of pH 5 to 7. An object of the present invention is to provide a novel antibacterial substance that is particularly stable in the neutral range and a novel lactic acid bacterium that produces the antibacterial substance.

  In view of the above problems, the present inventors conducted a search for bacteriocin-producing bacteria that are stable not only in the acidic range but also in a wide pH range such as the neutral range. A new lactic acid bacterium KMR-7 belonging to the isolated Lactococcus lactis sub-species lactis is stable in acidic to alkaline regions including neutral, and exhibits antibacterial properties against many gram-positive bacteria including lactic acid bacteria The present inventors have found that a substance is produced, and have completed the present invention based on this knowledge.

That is, the present invention includes the following inventions.
Lactic acid bacteria or lactic acid strains belonging to Lactococcus lactis subspecies lactis, having the ability to produce a peptide (or protein) -containing substance that is stable at pH 2 to 9 and exhibits antibacterial activity against gram-positive bacteria KMR-7 strain.
An antibacterial substance obtained by culturing the above lactic acid strain and having the following properties.
(A) Contains an antibacterial peptide or protein.
(B) Completely digested with protease.
(C) Stable at pH 2-9, particularly high stability at pH 5-9.
(2) Shows thermal stability under treatment conditions of pH 7.0 and 110 ° C. for 10 minutes.
A method for producing an antibacterial substance, comprising culturing a lactic acid bacterium belonging to the aforementioned Lactococcus lactis subspices lactis and collecting the culture.
Use of the above lactic acid bacteria or lactic acid strains for food production. Fermentation raw material or fermented food manufactured using said lactic acid bacteria. A food preservative comprising the above antibacterial substance.

INDUSTRIAL APPLICABILITY According to the present invention, there are provided a novel lactic acid bacterium that produces an antibacterial substance that is stable at acidic to alkaline, particularly near neutral pH, an antibacterial substance thereof, and a method for producing the antibacterial substance.
The lactic acid bacteria of the present invention can be used in various acidic to alkaline foods, particularly foods for fermentation, and can produce foods such as fermented foods while suppressing the propagation of other miscellaneous bacteria. Further, the antibacterial substance according to the present invention is a preservative that is added to various general foods of acidic to alkaline to suppress the propagation of germs in various foods of neutral to alkaline as well as acidic foods. Useful for such as.
Furthermore, due to the property that the antibacterial substance of the present invention is stable at heat and particularly near neutral pH, it is added to foods that cause normal lactic acid bacteria to cause spoilage and deterioration in quality, thereby enhancing its preservability. It is possible.

Novel Lactic Acid Bacteria Lactic acid bacteria or lactic acid bacterial strains belonging to Lactococcus lactis subspecies lactis according to the present invention produce a peptide (or protein) -containing substance that is stable at pH 2-9 and exhibits antibacterial activity against gram-positive bacteria As described above, it has the ability, and a typical example of this lactic acid bacterium is Lactococcus lactis subspecies lactis KMR-7 strain.
The mycological properties of Lactococcus lactis subspecies lactis KMR-7 strain are as follows. These bacteriological properties were based on the method described in Bergey's manual of systematic bacteriology Vol. 2 (1986) and the method described in the lactic acid bacteria experiment manual (Asakura Shoten, published on February 25, 1992) (1992).

(1) Morphology The size of the bacterium is 0.8 to 1.0 μm and is a streptococcus. Gram-positive, non-motile, non-spore-forming bacteria.
(2) Physiological properties
(a) Catalase −
(b) Growth temperature 10 ° C +
45 ° C-
(c) Salt tolerance 4% +
6.5%-
(d) pH 9.2 +
9.6 −
(e) 0.3% methylene blue resistance +
(f) 40% bile resistance +
(g) Arginine hydrolysis +
(h) Presence or absence of citric acid fermentability −
(i) Gas production from glucose −
(j) Presence or absence of sugar fermentability
Glucose +
L-arabinose +
D-ribose +
D-xylose +
Fructose +
Mannose +
Lactose +
Sucrose +
Trehalose +
Raffinose −
Mannitol +
Sorbitol −
Sodium gluconate +
Dextrin +
Rhamnose −
(3) Chemical properties
(a) GC content 35.6%
(b) Peptidoglycan type Lys-Asp

Based on these results, an inquiry was made in Bergey's manual of systematic bacteriology Vol. 2 (1986), and it was identified as Lactococcus lactis subspecies lactis. However, due to differences in the properties of bacteriocin-containing substances to be produced (activity by pH range, antibacterial spectrum, etc.), it is recognized that the bacteria are clearly different from the known bacteria belonging to this species, and Lactococcus lactis subspecies -It was named Lactis KMR-7 strain.
This Lactococcus lactis sub-species lactis KMR-7 strain was deposited by the applicant of the present application on November 14, 2000 at the Institute of Industrial Science and Technology of the Ministry of International Trade and Industry under the accession number: FERM P-18121. Has been.

Lactic acid strains belonging to Lactococcus lactis subspecies lactis as a novel microorganism according to the present invention are not only the KMR-7 strain, which is a representative example, but also the above-mentioned ability (that is, stable at a pH of 2 to 9 and gram). And its variants having the ability to produce peptide-containing substances that exhibit antibacterial activity against positive bacteria.
Mutants have the above-mentioned ability even if they are mutated by natural mutation or normal artificial mutation means (for example, ultraviolet rays, irradiation, transposon, chemicals (for example, sodium nitrite, nitrosoguanidine), etc.) Any present is included in the present invention.

  The strain of the present invention, for example, is a part of a commercially available fermented food material such as Taiwanese menma, which is cultured in a medium for lactic acid bacteria. It can be obtained by screening and isolating strains.

Novel antibacterial substance The antibacterial substance according to the present invention is a peptide-containing substance that is stable at pH 2 to 9 and exhibits antibacterial activity against Gram-positive bacteria. It has properties.
(A) Contains an antibacterial peptide or protein.
(B) Completely digested with protease. (The above peptide or protein is degraded by protease and the activity of the antibacterial substance is lost.)
(C) Stable at pH 2-9, particularly high stability (antibacterial activity) at pH 5-9. Therefore, this antibacterial substance shows a stable and high antibacterial activity even in a neutral range (pH 5-9, especially pH 6-7 or about 6-8) where conventional bacteriocin is unstable.
(2) It exhibits thermal stability (antibacterial activity) under treatment conditions of pH 7.0 and 110 ° C. for 10 minutes.

  Representative examples of the antibacterial substance according to the present invention include an antibacterial peptide or protein having a molecular weight of 3328 consisting of 34 amino acids from instrumental analysis (mass spectrometry, NMR, etc.), gene analysis, etc. relating to various structures. It is a peptide having the same sequence as the amino acid sequence of Z (see Eur. J. Biochem. 201, 581-584 (1991)).

Specifically, the antibacterial substance of the present invention is a culture obtained by culturing the lactic acid bacterium or lactic acid strain of the present invention in a liquid or solid medium, that is, a culture solution (containing microbial cells), a colony, a culture supernatant (excluding microbial cells). ), Roughly purified products (described later), dilutions thereof (with water or the like), etc., but culture supernatants are particularly preferred. The form of the antibacterial substance may be any form such as liquid, paste, and dry. The antibacterial substance according to the present invention is obtained by culturing the lactic acid bacterium of the present invention, and exhibits stability particularly at pH 2 to 9, and becomes unstable in the neutral pH range as described later (antibacterial activity decreases). ) Purified antibacterial peptides (by HPLC, electrophoresis, etc.) are not included in the antibacterial substance of the present invention. In a preferred embodiment of the present invention, the culture supernatant exhibits high stability particularly at pH 5 to 9, and the crude product exhibits high stability particularly at pH 4 to 7 or 5 to 7.
Here, the purified product refers to a peptide having a purity of 70% or more, and the crude product refers to a purity of greater than 0% but less than 70%, such as 60% or less, preferably 40% or less, more preferably It means 20% or less, most preferably 15% or less, and has the above pH stability in the present invention. Here, the degree of purification means purity (antibacterial protein weight / total component weight (other than water)), and the degree of purification of 0% means the culture itself. In this specification, “%” means “% by weight” unless otherwise specified.
The various properties and antibacterial activity of the antibacterial substance according to the present invention are described more specifically in the examples below.

Production of Antibacterial Substance The antibacterial substance according to the present invention is obtained by culturing a lactic acid bacterium or a lactic acid strain (typically KMR-7 strain) belonging to Lactococcus lactis subspecies lactis and a culture (culture medium, culture). (Supernatant or colony) can be obtained. As a medium used for culturing the lactic acid bacteria, a commonly used medium (a liquid medium, a solid medium, etc.) containing a carbon source, a nitrogen source, inorganic salts, vitamins, amino acids, etc. can be used. A liquid medium is used. Examples of the medium for cultivating lactic acid bacteria include GYP, M17, ROGOSA, MRS medium, and the like. Preferably, MRS medium is used.

  The culture conditions may be aerobic conditions or anaerobic conditions. The initial pH of the medium is usually 6-8, preferably 6.8-7.0. The culture temperature is usually 20 to 35 ° C, preferably about 30 ° C. The culture time is usually 5 to 24 hours, preferably 10 to 12 hours. In addition, in culture with a jar fermenter (microorganism culture apparatus or cell culture apparatus) adjusted to have a pH constant between 6 and 8, particularly 6.7 to 6.8, a culture solution with higher activity is used. The culture temperature at this time is preferably 28-30 ° C., and the culture time is preferably about 8-10 hours.

  The culture obtained by the above culture can use any of the cells, a culture solution containing the cells and a culture supernatant not containing the cells, but usually the cells are separated from the culture solution by centrifugation or the like, Preferably, the pH of the culture supernatant is neutralized (about pH 6 to 7) with sodium carbonate, sodium hydroxide, potassium hydroxide or the like, and can be used as an antibacterial substance of the present invention (for example, a food preservative). Moreover, the crudely purified product of the culture supernatant can be used as an antibacterial substance. The roughly purified substance can be obtained, for example, by treating the culture supernatant with a method such as ion exchange, ultrafiltration, reverse osmosis or gel filtration. In addition, about the manufacturing method of the antibacterial substance by this invention, it describes more concretely in the postscript Example.

Uses of the Lactic Acid Bacteria and Antibacterial Substances of the Present Invention Due to the characteristics of the lactic acid bacteria or lactic acid strains of the present invention and antibacterial substances as described above, the present invention is particularly applicable to the food field.
The lactic acid bacteria according to the present invention can be used for food production, particularly for the production of fermentation raw materials or fermented foods. That is, the present invention also includes foods produced using lactic acid bacteria (typically KMR-7 strain) belonging to Lactococcus lactis subspecies lactis according to the present invention, particularly fermented raw materials and fermented foods. .

Fermentation raw materials include fermentation raw materials such as koji, vegetables, fruits and milk, which can be used as seasonings, raw materials and the like added to foods such as pickles, miso, soy sauce and dairy products. For example, in koji, it can be used in combination with koji molds in raw materials such as rice, barley and tofu. Specifically, for example, koji molds and the lactic acid bacterial strain of the present invention are added in an amount of 0.1 to 10% to the cooked ingredients. And it should just be ironed on 20-35 degreeC conditions. In addition, it may be added in an amount of 0.1 to 10% for vegetables, fruits, etc. and fermented at 20 to 35 ° C., and it may be added in an amount of 0.1 to 10% in milk and the like at 20 to 35 ° C. What is necessary is just to ferment (% display is all weight%).
As a fermented food, it can be used for lactic acid fermented foods, such as a pickle and a yoghurt, for example. For example, in pickled food, the lactic acid strain of the present invention can be added to a salted food in an amount of 0.1 to 10% and can be produced at 20 to 35 ° C.

  The lactic acid bacteria of the present invention can be used in combination with other food microorganisms in foods such as fermentation raw materials and fermented foods as necessary. Such microorganisms are those that are not inhibited by this strain, and specific examples include Aspergillus oryzae, Aspergillus sojae, yeast (Saccharomyces cerevisiae, etc.), and the like. .

Use of the antibacterial substance of the present invention The antibacterial substance of the present invention has a particularly strong inhibitory power against gram-positive bacteria such as Lactobacillus, Lactococcus, Enterococcus, and Leuconostoc. In addition, it has inhibitory power against many gram-positive bacteria such as Bacillus, Staphylococcus, and Micrococcus.

  Therefore, the antibacterial substance according to the present invention, for example, the culture of the lactic acid bacterium of the present invention or the lactic acid bacterial strain (typically KMR-7) as described above or the culture supernatant thereof is used for the production of general foods (eg pickles) In particular, it can be used as a preservative, a fermented seasoning and the like. In addition, since the antibacterial substance according to the present invention is stable against heat and pH near neutrality, it is separated from the culture solution in the form of a culture solution or culture supernatant, or is roughly purified (ion exchange, ultrafiltration). After filtration, reverse osmosis, etc., it is possible to add to foods, particularly foods (pickles etc.) that cause normal lactic acid bacteria to cause spoilage or quality deterioration, thereby enhancing their storage stability. The amount of the antibacterial substance to be added to the food may be appropriately set depending on the kind of food. For example, a ratio of 0.001 to 0.1 volume per 1 volume of food (a stationary culture solution of the lactic acid bacteria of the present invention (for example, What is necessary is just to add as the culture solution of Example 1).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by this.

[Example 1]
MRS medium (polypeptone 10.0 g / L, meat extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20 g / L, “Tween 80” 1.0 g / L, dipotassium hydrogen phosphate 2.0 g / L L, diammonium hydrogen citrate 2.0 g / L, sodium acetate trihydrate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L) Lactococcus lactis sub-species lactis KMR7-strain of the present invention was pre-cultured overnight in MRS BROTH (manufactured by OXOID) at 1% in the amount of the medium. Inoculated and cultured at 30 ° C. for 8 hours. After culturing, the cells are removed by centrifugation (7500 × g, 4 ° C., 15 minutes), and the culture supernatant is further diluted with 1.0N or 2.0N sodium hydroxide to remove the influence of organic acids. Was adjusted to 7.0.

[Example 2] Antibacterial spectrum
The antibacterial spectrum was measured using the culture supernatant obtained in Example 1. First, an MRS agar medium (polypeptone 10.0 g / L, meat extract 8.0 g / L, yeast extract 4.0 g / L, glucose 2.0 g / L, with a glucose concentration of 0.2% in a sterilized plastic petri dish. L, “Tween 80” 1.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate trihydrate 5.0 g / L, magnesium sulfate seven water Japanese culture 0.2g / L, manganese sulfate tetrahydrate 0.05g / L, agar 12g / L) MRS soft agar medium (the above composition, agar 7.0 g / L) is overlaid, a hole with a diameter of 3 mm is opened with a cork borer, and 20 μl of the culture supernatant is dispensed at the optimum temperature of each test strain. Incubate around the hole And measuring the diameter of the inhibition circle form (Appl. Environ. Microbiol. 1989 55 (8) 1901-1906). The results of the antibacterial spectrum test are shown in Table 1.

Table 1
Test strain Growth inhibition circle (diameter mm)
Lactobacillus casei ATCC 7469 15.63
(Lactobacillus casei)
Lactobacillus plantarum ATCC 8014 8.59
(Lactobacillus plantarum)
Lactobacillus carbatus ATCC 7469 8.44
(Lactobacillus curvatus)
Lactobacillus halotolerance ATCC 35410 14.96
(Lactobacillus halotolerans)
Lactobacillus hilgardi ATCC 8290 13.65
(Lactobacillus hilgardii)
Lactobacillus brevis IFO 3345 8.66
(Lactobacillus brevis)
Lactococcus lactis
Subspecies Cremolis IFO 3427 11.53
(Lactococcus lactis subsp. Cremoris)
Enterococcus faecalis JCM 5803 4.61
(Enterococcus faecalis)
Enterococcus faecium JCM 5804 5.61
(Enterococcus faecium)
Enterococcus faecium IFO 13131 5.04
(Enterococcus faecium)
Leuconostok Mecenteroides
Subspecies Mecenteroides JCM 6124 6.61
(Leuconostoc mesenteroides subsp.mesenteroides)
Leuconostok Mecenteroides
Subspecies Mecenteroides IFO 3426 8.94
(Leuconostoc mesenteroides subsp.mesenteroides)
Leuconostok Mecenteroides
Subspecies Dextranicum IFO 3349 5.20
(Leuconostoc mesenteroides subsp.dextranicum)
Leuconostok Ractis JCM 6124 5.41
(Leuconostoc lactis)
Leuconostok Paramescenteroides NRIC 1542 12.10
(Leuconostoc paramesenteroides)
Bacillus subtilis IFO 3335 5.33
(Bacillus subtilis)
Bacillus cereus IFO 13494 −
(Bacillus cereus)
Bacillus coagulance IFO 12583 4.70
(Bacillus coagulans)
Bacillus licheniformis IFO 12200 −
(Bacillus licheniformis)
Bacillus Circulation IFO 13262 −
(Bacillus circulans)
Micrococcus luteus IFO 12708 18.28
(Micrococcus luteus)
Staphylococcus aureus IFO 13276 4.93
(Staphylococcus aureus)
Escherichia coli IFO 3301 −
(Escherichia coli)
Pseudomonas florence JCM 5963 −
(Pseudomonas fluorescens)
Acinetobacter crocetics IFO 12552 −
(Acinetobacter cloacetics)
Debariomyces Hanseny IFO 0094 −
(Debaryomyces hansenii)
Saccharomyces cerevisiae IFO 2735 −
(Sacchromyces cerevisiae)

"Example 3" Sensitivity to proteolytic enzymes Various enzymes were added to the culture supernatant obtained in Example 1 to a concentration of 1.0 mg / m1, and reacted at 37 ° C or 25 ° C for 18 hours. Lactobacillus casei ATCC 7469 was used as an indicator bacterium, and the antimicrobial activity was measured to determine the presence or absence of sensitivity. The antibacterial activity was measured by overlaying the MRS soft agar medium described in Example 2 in which Lactobacillus casei ATCC 7469 was inoculated on a plate medium in which the MRS agar medium described in Example 2 was dispensed into a sterilized plastic Petri dish. Then, a hole with a diameter of 3 mm was made with a cork borer, and 20 μl of a preparation of a 2-fold dilution series of the culture supernatant was inoculated into each well and anaerobically cultured at 30 ° C. for 24 hours, after which growth was completely inhibited. The highest dilution factor was examined. For the quantification of the antibacterial activity, the highest dilution factor that completely inhibited the growth was multiplied by 50 to obtain the titer per 1 ml (AU / m1). The results are shown in Table 2.

Table 2
Enzyme Antibacterial activity (AU / ml)
Pepsin 400
Papain 200
Trypsin 400
Protease 0
Untreated 800

[Example 4] Thermal stability
The culture supernatant obtained in Example 1 was heated for 10 minutes at 100, 110, 120, and 130 ° C., respectively, and after cooling, the Lactobacillus casei ATCC 7469 was used as an indicator bacterium by the method of Example 3 to determine the antibacterial activity. The thermal stability was examined. The results are shown in Table 3.

Table 3
Temperature (℃) Antibacterial activity (AU / ml)
100 800
110 800
120 400
130 200
Untreated 800

[Example 5] pH stability
The culture supernatant obtained in Example 1 was adjusted to pH 2-9 with 1.0N or 0.1N NaOH, 1.0N or 0.1N HCl, and treated at 30 ° C. for 60 minutes. The pH was readjusted to 7.0, and Lactobacillus casei ATCC 7469 was used as the indicator bacterium by the method of Example 3, and the pH stability was examined by quantifying the antibacterial activity. The results are shown in Table 4.

Table 4
pH Antibacterial activity (AU / ml)
2 800
3 800
4 800
5 1600
6 1600
7 1600
8 1600
9 1600

[Example 6]
A culture solution obtained by preculturing the Lactococcus lactis sub-species lactis KMR-7 strain of the present invention overnight in MRS BROTH to 5 L of the MRS medium of Example 1 sterilized at 121 ° C. for 30 minutes in an autoclave. The cells were inoculated in an amount of 1% and cultured at 30 ° C. for 8 hours on a jar fermenter while adjusting the pH of the medium during the culture to 6.7 to 6.8. After the cultivation, it was carried out as in Example 1. FIG. 1 shows the relationship between the growth curve and the antibacterial activity when cultured in Example 6 and Example 1, respectively. From Tables 1 to 4, antibacterial substances produced by the Lactococcus lactis subspecies lactis KMR-7 strain of the present invention are gram-positive bacteria including lactic acid bacteria (including food-contaminating bacteria or food spoilage bacteria) It has antibacterial spectrum showing antibacterial activity against, and is sensitive to pepsin, papain, trypsin and protease, and is particularly digested to protease, so it is a protein or peptide. It shows the characteristics. The antibacterial substance of the present invention exhibits high stability at 110 ° C. for 10 minutes, pH 2-9, especially 5-9. Furthermore, when the pH is adjusted (about pH 6.7 to 6.8) and cultured in a jar fermenter, one having an antibacterial activity of 2 times or more can be obtained.

[Example 7] Purification of antibacterial substance
9 L of a culture solution obtained by culturing in a medium excluding Tween 80 from the MRS medium (composition) of Example 1 was centrifuged (9000 × g, 4 ° C., 15 minutes) to obtain a culture supernatant. . Ammonium sulfate was added to the obtained culture supernatant to make a 20% saturated solution, and left overnight at 4 ° C. Thereafter, this was centrifuged (9000 × g, 4 ° C., 15 minutes), the precipitated fraction was collected, and suspended in 25 mM Tris-HCl buffer (pH 7.0).
Next, concentration by centrifugation using ultrafiltration with a fraction size of 3000, purification, concentration, and desalting were performed with a desalter (manufactured by Millipore) (2500 × g, 4 ° C., 30 minutes).
The fraction having a fraction size of 3000 or more obtained here was used as a crude product. This crude product was adsorbed by passing it through a column (column inner diameter 3.5 cm, length 33 cm) packed with CM-Toyopearl 650 (manufactured by Tosoh Corporation) at a flow rate of 0.85 ml / min, and 25 mM Tris containing 1M NaCl. Elution was carried out with a linear gradient of HCl buffer (pH 7.0) (fraction volume 6 ml), and active fractions were obtained at fraction numbers 31 to 36. The result is shown in FIG. The obtained active fraction was concentrated and desalted by a concentration and desalting apparatus by centrifugation using ultrafiltration with a fraction size of 3000 (2500 × g, 4 ° C., 30 minutes). Here, a fraction having a fraction size of 3000 or more was obtained. This was subjected to reversed-phase high-performance liquid chromatography at a flow rate of 1 ml / min using a column (column inner diameter 6.0 mm, length 15 cm) packed with Octadecyl-2PW (Octadecyl-2PW; manufactured by Tosoh Corporation). The active fraction obtained from this was used as a purified bacteriocin.

Example 7-1 A crude product was prepared according to the method described in Example 7. The crude product was adsorbed by passing it through a column packed with CM Toyopearl 650 at a flow rate of 0.85 ml / min, and eluted with a linear gradient of 25 mM Tris-HCl buffer (pH 7.0) containing 1 M NaCl. Got the minute. The ratio of the protein amount of the active fraction to the total protein amount (total fraction) in this method (cation exchange chromatography) was 15% (FIG. 7). Further, when the obtained active fraction was confirmed by high performance liquid chromatography, the protein content ratio of the active fraction was 70% (FIG. 8).
When the obtained purified product was purified twice by high performance liquid chromatography according to the method described in Example 7, the purity became 90% or more.

[Example 8] SDS-polyacrylamide gel electrophoresis
In order to confirm the purity of the purified bacteriocin obtained in Example 7, it was purified by SDS-polyacrylamide gel electrophoresis according to a conventional method. The gel was prepared with a fixed concentration of 25% of acrylamide. As the cross-linking agent, N, N′-methylenebisacrylamide was used. The electrode solution was changed from glycine to tricine. The migrated gel was stained by the silver staining method and detected as a single band.

[Example 9-1] Examination of degree of purification and pH stability Examination of pH stability in each purification stage of Example 7 (culture supernatant, cation exchange chromatography (crude product), HPLC (purified product)) Was performed according to the method of Example 5. The results are shown in Table 5 as the rate of change of antibacterial activity (relative activity%).

Table 5
Antibacterial activity (% change)
(Purification stage) Culture supernatant Cation exchange chromatography HPLC
Matography
pH
2 (100) (100) (100)
3 100 100 200
4 100 200 200
5 200 200 200
6 200 200 200
7 200 200 100
8 200 100 50
9 200 50 50

  From the results shown in Table 5, the culture supernatant and the crudely purified product showed high pH stability in a wide pH range, but the purified product showed reduced stability particularly in the pH neutral to alkaline range (see FIG. 5). .

[Example 9-2] Comparison of pH stability with nisins Culture supernatants of nisin A producing bacteria (Lactococcus lactis subsp. Lactis IFO 12007 strain) and nisin Z producing bacteria (Lactococcus lactis subsp. Lactis JCM 7638 strain) The pH stability was tested in the same manner as for the KMR-7 strain (see Examples 1 and 5).
As a result, as shown in FIG. 6, the nisin A and Z culture supernatants showed stable results in the acidic range, but decreased in the alkaline range. On the other hand, the culture supernatant of KM bacteriocin (according to the KMR-7 strain) showed stability even at low pH to alkali side (particularly pH 5 to 9).

[Example 10] Amino acid sequence The amino acid sequence of the purified bacteriocin obtained in Example 7 was analyzed using a gas phase sequencing apparatus (manufactured by Hewlett-Packard) to obtain sequence information. From the results, it was found that the peptide contained in the antibacterial substance was a peptide having the same sequence as the amino acid sequence of nisin Z (see Eur. J. Biochem. 201, 581-584 (1991)).

[Example 11] Mass spectrometry The ESI-MS spectrum of the purified bacteriocin obtained in Example 7 was measured according to a conventional method. From the measured mass spectrum results, it was revealed that the molecular weight of the purified bacteriocin was 3328, the same as that of Nisin Z (see FIGS. 3 and 4).

[Example 12]
95 ml of commercially available soup for cooking was adjusted to pH 7.0, and after autoclaving, the culture supernatant obtained in Example 1 was added to 1% and 5% to make a total volume of 100 ml. Subsequently, the preculture of Lactobacillus casei ATCC 7469 was inoculated into each test group in an amount of 0.1%, stored at 25 ° C., and the number of bacteria was measured over time. The number of days until the number of general bacteria reached 10 ⁸ / ml was defined as the effective storage day. The results are shown in Table 6.

Table 6
Amount added Effective storage days (25 ° C)
3 days without additive
1% 6 days
5% 8 days

  As is clear from Table 6, the effective storage days are increased by the addition of the antibacterial substance of the present invention.

[Example 13]
Put 100 g of commercially available tofu in a plastic container, add 150 ml of the sterilized preparation solution prepared in Example 1 with adjusted culture supernatant, pH, and salt concentration, store at 25 ° C. and 4 ° C., and store the bacteria over time. A number measurement was made. The number of days until the number of general bacteria reached 10 ⁵ / ml was defined as the effective storage day. The results are shown in Table 7.

Table 7
Test zone Effective preservation days
25 ° C. 1 day 4 ° C. 6 days 4 ° C. + pH 4 7 days 4 ° C. + NaC1 5% 11 days 4 ° C. + culture supernatant 20% 7 days
4 ° C. + pH 4 + NaC1 5% + culture supernatant 20% 16 days

  As is clear from Table 7, the effective storage days are increased by the addition of the antibacterial substance of the present invention, and the effective storage days are further increased by the synergistic effect of pH adjustment and addition of salt.

Explanatory drawing which shows the relationship between the growth curve at the time of culture | cultivating by adjusting pH constant using a jar fermenter in this invention, and the case of carrying out normal culture | cultivation without pH adjustment. Explanatory drawing which shows the elution profile by CM-Toyopearl 650 chromatography of the antimicrobial substance by this invention. Explanatory drawing which shows the ESI-MS spectrum of the antibacterial substance by this invention. Explanatory drawing which shows the ESI-MS spectrum of Nisin Z. The graph which shows the pH stability in the culture supernatant of KMR-7 strain | stump | stock, a crude purified product, and a purified product. The graph which shows the pH stability in the culture supernatant of each bacteriocin producing microbe. Explanatory drawing which shows the elution profile of the crudely purified product by cation exchange chromatography. Explanatory drawing which shows the elution profile of the purified material by HPLC.

Claims (9)

  A lactic acid bacterium belonging to Lactococcus lactis subspecies lactis, characterized in that it has the ability to produce a peptide-containing substance that is stable at pH 2 to 9 and exhibits antibacterial properties against gram-positive bacteria.   The lactic acid bacterium according to claim 1, which is KMR-7 strain (Accession No. FERM P-18121). An antibacterial substance obtained by culturing the lactic acid strain according to claim 1 or 2 and having the following properties.
(A) Contains an antibacterial peptide or protein.
(B) Completely digested with protease.
(C) Stable at pH 2-9, particularly high stability at pH 5-9.
(2) Shows thermal stability under treatment conditions of pH 7.0 and 110 ° C. for 10 minutes.   The antibacterial substance according to claim 3, wherein the antibacterial substance is a culture of a lactic acid strain.   A method for producing an antibacterial substance, comprising culturing the lactic acid strain according to claim 1 or 2 and collecting the culture.   The production method according to claim 5, wherein the culture is performed with a jar fermenter at a pH of 6-8.   Use of the lactic acid strain according to claim 1 or 2 for food production.   Fermentation raw material or fermented food manufactured using the lactic acid strain described in Claim 1 or 2.   A food preservative comprising the antibacterial substance according to claim 3 or 4.

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