JP5084093B2 - Method for producing gasericin A, antibacterial agent and therapeutic agent for inflammation - Google Patents

Description

The present invention relates to a method for producing gassericin A which is a bacteriocin produced by Lactobacillus gasseri ( Lactobacillus gasseri ) LA39 strain of Lactobacillus acidophilus group lactic acid bacteria, and an antibacterial agent and inflammation containing gassericin A produced by this production method Relates to a therapeutic agent for

Lactobacillus acidophilus ( Lactobacillus acidophilus , Lactobacillus acidophilus ) group lactic acid bacteria isolated from human intestinal tract and feces with high frequency have found many strains that produce antibacterial peptides (bacteriocin). It is attracting attention as a probiotic that brings about an intestinal regulating action.

Among the acidophilus group lactic acid bacteria considered to be particularly safe for the host, Lactobacillus gasseri ( Lactobacillus gasseri ), a bacteriocin produced by LA39 strain, is a bacteriocin produced by Listeria monocytogenes. ) And Staphylococcus aureus are known to exhibit a high antibacterial effect (see Non-Patent Document 1). Focusing on this high antibacterial effect, it is expected to be used in the food and pharmaceutical fields.

  However, until now, gasericin A could not be used in the food and pharmaceutical fields. In general, ingredients used in foods and pharmaceuticals are required to have a high degree of safety for humans and animals. Gasericin A is produced using a commercially available synthetic medium (for example, MRS medium manufactured by Difco). This synthetic medium contains oleic acid, which is essential for the construction of the cell wall of Lactobacillus, as a component. However, this surfactant cannot be used from the viewpoint of safety in the field of food and medicine and pharmacy. No matter how much the purification level of gasericin A is increased, this surfactant and medium components will be contained in foods and pharmaceuticals, etc., and conventionally gaselin A cannot be used in the food and pharmaceutical fields. It was.

  In particular, in the case of a pharmaceutical that directly injects gassericin A into the body, a very high level of safety is required for humans and animals due to problems of immune response and host damage. It was impossible to use A as a medicine for direct injection into the body.

Therefore, it has been studied to produce gassericin A using a skim milk medium that is safe as a food instead of a synthetic medium, but useful koji molds derived from the human intestine, including gasseri bacteria, are extremely viable on skim milk medium. Therefore, it was very difficult to efficiently produce gasericin A using skim milk medium.
Itoh, T., Y. Fujimoto, Y. Kawai, T. Toba and T. Saito, Inhibition of food-borne pathogenic bacteria by bacteriocins from Lactobacillus gasseri, Letters in Applied Microbiology, 21, 137-141, 1995

  It is an object of the present invention to provide a method for efficiently producing gasericin A using a skim milk medium that is safe for humans and animals. It is another object of the present invention to provide an antibacterial agent and a therapeutic agent for inflammation containing gassericin A produced using a skim milk medium that is safe for humans and animals as an active ingredient.

In the present invention, Lactobacillus gasseri ( Lactobacillus gasseri ) LA39 strain (hereinafter referred to as Gaseri bacteria LA39 strain) is cultured in a skim milk medium containing proteose peptone (hereinafter referred to as pp). A method for producing gasericin A is provided. The skim milk medium preferably contains pp of 0.1% (w / v) or more.

  The present invention also provides an antibacterial agent containing gassericin A produced by the above method as an active ingredient.

  Furthermore, the present invention provides a therapeutic agent for inflammation comprising gassericin A produced by the above method as an active ingredient.

  The inflammation may be inflammation caused by Staphylococcus aureus or mastitis.

  According to the present invention, it is possible to obtain a large amount of gassericin A that is safe for humans and animals at low cost. In addition, according to the present invention, it is possible to provide an antibacterial agent containing gericin A as an active ingredient and a therapeutic agent for inflammation that are safe for humans and animals. Furthermore, according to the present invention, it is possible to treat inflammation caused by Staphylococcus aureus using gasericin A. Furthermore, even when the therapeutic agent for inflammation according to the present invention is directly injected into bovine breast, it is possible to treat mastitis without problems of immune response and host damage.

  In the present invention, gasserin A strain 39 is cultured in a skim milk medium containing pp to produce gassericin A.

  pp, or proteose peptone, refers to a protein that is strongly hydrophilic and has lost its coagulation property upon heating, and is usually a mixture of a plurality of peptides rather than a single component. Examples of available pp include product number No. 211693 manufactured by Difco and product number LP0085 manufactured by Oxoid, and product number No. 211693 manufactured by Difco is preferable.

  The skim milk medium is not particularly limited as long as it is bovine milk, and commercially available skim milk or milk can be used. When producing gassericin A using skim milk medium, the impurities contained in the purified gassericin A are components derived from bovine milk. Therefore, when this gasericin A is used as an antibacterial agent and an anti-inflammatory agent, safety is ensured for humans and animals. In addition, when this gasericin A is used as a therapeutic agent for bovine mastitis, even if it is directly injected into the bovine mammary gland, no foreign substance is injected, so the safety for the bovine breast is very high.

  This skim milk medium contains the above-mentioned pp, preferably 0.1% (w / v), more preferably 0.5% (w / v).

The gasseri LA39 strain is the gasseri LA39 strain belonging to the B1 subgroup among the Lactobacillus acidophilus group lactic acid bacteria. Gaselicin A, a bacteriocin produced outside of the cells by the gasseri strain LA39, is known to exhibit an antibacterial effect, particularly against Listeria monocytogenes and Staphylococcus aureus. Gasericin A is a cyclic bacteriocin having a molecular weight of 5,652 daltons, a total number of amino acid residues of 58, and does not contain modified amino acids such as lanthionine, and has N- and C-termini bound in the molecule. This strain was isolated from infant stool by the present inventors and deposited in 2001 at RIKEN (Tokyo, Wako City). The registration number of this strain is Lb. gasseri JCM11657 strain, and anyone can easily obtain it.

  As for the production method of gasseri bacteria LA39 strain, pp is added to skim milk medium, and when the gasseri bacteria LA39 strain is inoculated and cultured, it is produced in the culture supernatant. The culture time is preferably 8 hours or more, more preferably 12 hours. Since the number of molecules of the gasseri LA39 strain and the number of molecules produced by the gassericin A are in a parallel relationship, the gasseri LA39 depends on the pH and titration acidity. By measuring the amount of lactic acid produced from the strain, the viability of gasseri strain LA39 can be estimated, whereby the viability of gassericin A can be known. The culture temperature is preferably 35 to 40 ° C, more preferably around 37 ° C.

  In order to extract gasericin A from the culture, gel filtration, ion exchange chromatography, hydrophobic chromatography and the like can be combined as necessary. For example, an ion exchange method using a DEAE resin or a pH adjustment method may be used.

  Gasericin A obtained by the production method of the present invention can be used for foods, beverages, cosmetics, pharmaceuticals and the like. When used in these applications, gasericin A may be used as a whole without being extracted from the culture, or may be extracted from the culture and used alone, depending on the properties of the target article. Can be extracted. Even if the whole culture is used, since this skimming milk medium is used in this production method, it is highly safe for humans and animals. Moreover, even if extracted from the culture and used alone, the impurities contained in gasericin A after extraction are components derived from skim milk medium, and thus are extremely safe for humans and animals.

  Gasericin A has an excellent antibacterial action, and gasericin A may be used as an antibacterial agent or anti-inflammatory agent. In particular, since gassericin A has an excellent antibacterial action against S. aureus, it may be used as a therapeutic agent for inflammation caused by S. aureus or as a therapeutic agent for mastitis.

  When gasericin A is used as an antibacterial agent and a therapeutic agent for inflammation, it may be dissolved or dispersed in a liquid carrier or mixed with a powder carrier. Examples of additives include surfactants, pigments, dyes, emulsifiers, dispersants, suspending agents, wetting agents, and stabilizers. Examples of the form include emulsions, aqueous agents, gels, claims, ointments, aerosols, capsule powders and tablets.

  In particular, when gassericin A is used as a therapeutic agent for mastitis, it may be mixed with a sample orally and administered orally, and the teat may be dipped in the form of an aqueous preparation containing gassericin A in a solvent. In order to obtain the maximum therapeutic effect, it may be administered into the breast from the nipple in the form of an injection preparation. A mastitis therapeutic agent containing gasericin A can also be administered during lactation and dry periods of dairy cows.

  Test examples are shown below. Unless otherwise specified in the test examples, the reagent used was a special grade reagent or a first grade reagent manufactured by Wako Pure Chemical Industries, Ltd. (Osaka).

  For the gasseri strain LA39, a bacteriocin-producing strain isolated from human infant feces in the Animal Resource Chemistry Laboratory, Graduate School of Agriculture, Tohoku University was used.

(Test Example 1 Changes in the productivity of gasericin A when various components are added to skim milk medium)
Gasseri strain LA39 was inoculated at 1% (w / v) in MRS medium (Difco, Detroit, MI, USA) and subcultured 3 times (37 ° C, 18-24) before the test. Time).

  Next, 10% (w / v) skim milk medium (Morinaga Milk Industry) supplemented with various components shown in Table 1 (addition amounts were set based on the amounts commonly used in artificial synthetic media for lactic acid bacteria). In order to know the viability of gasericin A, pH / ion meter (Horiba, Model F-24) Was used to measure the pH.

表１に示される通り、pp添加脱脂乳培地ではガセリシンAの生産性は「＋」、すなわち、良好であった。これに対して、その他の添加成分を脱脂乳培地に加えた場合のガセリシンAの生産性は「−」であり、また、添加成分がない場合と比べて特に生育性に大きな変化が
なかった。 There is a parallel relationship between the production of gassericin A and the viability of the gasseri strain LA39. Therefore, the productivity of gastricin A was determined by knowing the viability of the lactic acid bacteria LA39 strain by pH measurement. When the pH is 5.0 or less, the productivity of gasericin A is good, and is shown as “+” in Table 1. If a higher pH is measured, it is shown as “-” in Table 1.

As shown in Table 1, the productivity of gasericin A was “+” in the pp-added skim milk medium, that is, good. In contrast, the productivity of gasericin A when other additive components were added to the skim milk medium was “−”, and there was no significant change in growth particularly compared to the case where there was no additive component.

表２より、ｐｐ添加量が０．１%(w/v)では、１〜５回目の脱脂乳培地のｐHが全て５以下で好ましい値になった。また、ｐｐ添加量が０．５%(w/v)では、ｐHが全て４以下で、より好ましい値となった。ｐｐは一般に非常に高価であるが、わずかな添加量でガセリシンAの生育性が良好となった。 (Test Example 2 Change in pH accompanying change in pp concentration added to skim milk medium)
10% (w / v) skim milk medium (manufactured by Morinaga Milk Industry Co., Ltd., product name: skim milk) pp (Difco, Bacto proteose peptone No. 3) is 0.001, 0.01, 0.1, 0.5 and 1% (w / v), respectively. After adding and cultivating the gasseri LA39 strain subcultured in the MRS medium in the same manner as in Test Example 1, the pH was measured for 5 days. By measuring the pH, it is possible to know the viability of the Lactobacillus gasseri strain LA39, that is, the productivity of gassericin A. If the pH is 5 or less, the productivity of gassericin A is good.

From Table 2, when the addition amount of pp was 0.1% (w / v), the pH values of the skim milk media for the first to fifth times were all 5 or less, which was a preferable value. Further, when the addition amount of pp was 0.5% (w / v), the pH was all 4 or less, which was a more preferable value. Although pp is generally very expensive, the growth of gasericin A was improved with a small addition amount.

(Test example 3 Viable cell count and pH change over time of gasseri LA39 strain)
In order to investigate the relationship between productivity and pH and time of gassericin A, using 10% skim milk medium (manufactured by Morinaga Milk Industry Co., Ltd., trade name skim milk) with a pp addition amount of 0.5% (w / v), The viable cell count and pH value of gasseri strain LA39 in parallel with productivity were measured.

  The number of viable bacteria was diluted serially with 10 mmol phosphate buffer (pH 6.8), smeared on 10% (w / v) skim milk plate medium containing 0.5% (w / v) pp, Anaerobic culture was performed at 37 ° C. for 48 hours, and the number of bacteria was indicated by “colony forming unit (CFU) / ml”. The pH value was measured in the same manner as in Test Example 2. These results are shown in FIG.

According to FIG. 1, good growth of gastricis LA39 strain, that is, about 10 ⁹ CFU / ml in 8 hours of culture and about 10 ^9.5 CFU / ml in 12 hours of culture, that is, gasericin A was produced well. Is understood. In addition, a value of about pH 5 was obtained after 8 hours of culture, and a pH of 4.2 was obtained after 12 hours of culture. From this result, it is understood that gasericin A was produced satisfactorily.

(Test Example 4 Purification of produced gasericin A using pp-added skim milk medium)
Passage 3 times of 1% (v / v) inoculum of gasseri strain LA39 in 10% (w / v) skim milk medium with 0.5% (w / v) pp and culture (18 hours at 37 ° C) After that, the final culture for preparation was performed at 37 ° C. for 24 hours. The culture supernatant obtained by centrifugation (5,000 × g, 20 minutes, 4 ° C.) from the main culture solution was adjusted to pH 7 with 2N sodium hydroxide solution (fraction P1). Fraction P1 was then subjected to reverse phase chromatography using C4 resin (C4-Butyl Toyopearl 650M, Tosoh, Tokyo) equilibrated with water. Elution was performed in a stepwise manner with water, 40% (v / v) and 90% methanol. Fractions containing bacteriocin activity (fractions P2, C4 pass-through fraction) were subjected to reverse phase chromatography with C8 resin (Licloprep RP-8, Merck, Darmstadt, Germany) equilibrated with water. Elution was performed in a stepwise manner with water, 30%, 60%, 90% acetonitrile, 20%, 40%, 60% and 90% isopropanol. Fractions showing bacteriocin activity eluted with 60% isopropanol were stored at −20 ° C. (fraction P3, purified gericin A fraction).

(Test Example 5 SDS-polyacrylamide gel electrophoresis and detection of bacteriocin activity on the gel)
SDS-polyacrylamide gel electrophoresis was performed according to the Laemmli method using a rapidus mini slab electrophoresis apparatus AE-6440 type (W90 × H80 mm, Ato, Tokyo). The polyacrylamide concentration was 4.5% (w / v) for the concentrated gel and 15% (w / v) for the separation gel. The gel after electrophoresis was stained and decolored using Coomassie Brilliant Blue staining kit: Rapid CBB KANTO (Kanto Chemical, Tokyo).

  Detection of bacteriocin activity on the electrophoresed gel (gel activity detection method) was performed as follows according to the method of Daba et al. The gel immediately after electrophoresis was lightly washed with distilled water and then fixed for 30 minutes while shaking with a fixing solution (20% isopropanol, 10% acetic acid). Subsequently, the gel was sufficiently washed (30 minutes × 3 times) while shaking with distilled water, and then excess water was removed as much as possible and placed in a sterilized petri dish. MRS soft agar medium (20 ml) containing indicator bacteria diluted 400-fold on this gel was poured and solidified, then cultured at 37 ° C for 18 hours, where bacteriocin was located (the indicator bacteria did not grow and became transparent. The part observed in the state was confirmed.

In addition, Lactobacillus is the indicator bacteria Bacillus del Burekki subsp. Bulgaricus JCM1002 ^T Corporation ^{(Lactobacillus delbrueckii subsp. Bulgaricus JCM1002 T} , hereinafter abbreviated simply 1002 shares), RIKEN microorganism strain preservation facility (JCM, Purchased from Wako City), inoculated with MRS medium (Difco, Detroit, MI, USA) at 1% (w / v) volume and subcultured 3 times (37 ° C, 18- 24 hours).

  In addition, 1.5% (w / v) amount of agar medium and 0.75% (w / v) amount of agar (Agar Bacteriological, Agar No.1, Unipath Ltd., Hampshire, UK) for soft agar medium Added and prepared.

  Sterilization is performed by autoclaving (SS-305 type, Tommy Seiko, Tokyo) at 115 ° C for skim milk medium and 121 ° C for other medium for 15 minutes, and dissolution of the soft agar medium for layering is performed at 100 ° C for 1 minute. It was.

この結果によると、わずか２回段階の逆相クロマトグラフィーの実施により、ガセリシンAは300倍に精製できたことになる。 These results are shown in Table 3 and FIG. FIG. 2 shows the results of SDS-polyacrylamide electrophoresis (I) and bacteriocin activity detection (II) of gassericin A using pp skim milk medium, lane 1 is a molecular weight marker, and lane 2 is fraction P1. (Culture supernatant), lane 2 is fraction P2 (C4 reverse phase chromatography) and lane 3 is fraction P3 (C8 reverse phase chromatography).

According to this result, gasericin A could be purified 300 times by performing reverse phase chromatography in only two steps.

  In addition, purification with the conventional MRS medium requires 7 days including medium preparation and culture (36 hours), but in this study, high purity gassericin A can be prepared in 2 days with pp skim milk medium. It has become possible. From the above results, it was considered that the usefulness of this medium is extremely high because gasericin A can be easily prepared from pp skim milk medium in a short time.

(Test Example 6 Measurement of protein content)
The protein content in the sample was measured using a Biciconic acid quantification kit: Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA) applying the method of Smith et al. As the standard protein, insulin or bovine serum albumin (for biochemistry) was used.

  The results are also shown in Table 3.

  Although the total protein amount was reduced to about 17 mg, 0.08%, the inactivity increased 1800 times, indicating that it was purified to high purity.

(Test Example 7 molecular weight measurement)
Gaselicin A purified fraction purified from pp-added skim milk medium (Fraction P3) was measured using a fast atom bombardment mass spectrometer: FAB (Fast Atom Bombardment) MASS JMS-700 (JEOL, Tokyo). It was measured. The result is shown in FIG.

According to this result, in the molecular weight measurement of fraction P3 by FAB-MASS, a peak of 5,653 (m / z) (molecular ion peak M ⁺ + H) peculiar to gasericin A was detected, and gaslicin obtained in a conventional MRS medium It was confirmed that it had the same molecular weight as A.

(Test Example 8 administration of gasericin A to bovine quarters)
In order to examine the immune response of bovine organisms in the administration of gasericin A obtained in pp-added skim milk medium, using healthy Holstein quarters, milk was collected over time after the introduction of dry milk, and the bacteria test of milk Further, the α1 acidic glycoprotein concentration serving as an inflammation marker and the number of cells (SCC, CD11b, CD4, and CD8) were measured. In the test quarter, antibiotics (cephazone) were administered for 3 days to remove bacteria in the milk tank, and on the third day after antibiotic administration, the activity was 300 AU / quarter with PBS (phosphate buffered saline). After injecting 2.5 ml of gasericin A solution (culture supernatant of skim milk medium supplemented with pp) or PBS (control) into the milk tank, measuring the number of bacteria in milk and measuring the effect of inflammation markers over time did.

  The results are shown in FIGS.

  From these results, as a result of the administration test of gasericin A in the normal bovine breast, there was a great effect in reducing the number of Staphylococcus aureus (Fig. 4), and the inflammatory marker α1AG concentration did not increase compared to the control (Fig. 5). ), The number of somatic cells did not increase (FIG. 6), and the ratio of inflammatory lymphocytes did not increase (FIG. 7). Overall, there was no adverse effect on the body. That is, it is possible to treat bovine mastitis using highly safe gasericin A.

  According to the method for producing gassericin A according to the present invention, gassericin A having antibacterial activity can be produced efficiently and at low cost using a skim milk medium, which is useful in the fields of foods, beverages and pharmaceuticals using the same.

It is a figure which shows the time-dependent change of viable count and pH of gasseri bacteria LA39 strain. It is a figure which shows SDS-polyacrylamide electrophoresis (I) and the activity detection (II) result of bacteriocin. It is a figure which shows the FAB-MS spectrum of gasericin A (fraction). It is a figure which shows the time-dependent change of staphylococci in the ghasselicin A administration and PBS administration quarter. It is a figure which shows the time-dependent change of the alpha 1 acidic glycoprotein density | concentration in a gasserin A administration and PBS administration quarter. It is a figure which shows the time-dependent change of the cell number in a gaslicin A administration and PBS administration quarter. It is a figure which shows CD4 / CD8 ratio in a gassericin A administration and PBS administration quarter.

Claims (1)

A method for producing gasericin A, comprising culturing Lactobacillus gasseri LA39 strain in skim milk medium containing 0.1% (w / v) or more of proteose peptone to produce Lactobacillus, gasseri LA39 strain .

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