JPS59222423A - Method for producing neutralizing substances for cholera toxin and Escherichia coli toxin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a neutralizing substance for cholera toxin and E. coli toxin.

Cholera toxin and Escherichia coli toxin (hereinafter simply referred to as toxins refer to these toxins) are causative agents of diarrhea caused by Vibrio cholerae and Escherichia coli, respectively. Therefore, it should be possible to prevent diarrhea caused by the above-mentioned bacteria by using a substance that can directly act on these toxins and inhibit their toxicity. Methods for preventing or treating the diarrhea that have been developed from this perspective include immunotherapy using the toxin itself, or using GMI, a glycolipid and ganglioside, which is located on the intestinal lining and acts as a mediator between the toxin and the intestinal tract. Antagonism methods are known in which the drug is bound to a carrier such as activated charcoal and then administered. Note 1. D. Mitchell et al. (J. Appl.

Bact, 41.163-174) reported the discovery of a substance that has a toxin neutralizing effect from fermented milk produced by Lactobacillus pluricus or Streptococcus faecalis, but the structure of this substance has a molecular weight of 10. 'We do not know anything other than that it is below, and later reports denied the existence of such substances (J, ^pp1. BacL-45+ 157-160
), and no examples have been found of it being investigated as a drug for the prevention or treatment of the above-mentioned diarrhea.

The present inventors discovered a toxin neutralizing substance in fermented milk that is completely different from these known toxin neutralizing substances, established a method for its purification, and completed the present invention.

That is, the present invention involves fermenting milk with Bifidobacterium or lactic acid bacteria that has the ability to produce toxin neutralizing substances (referred to as Fl in this specification) as described in detail later in milk, and The acid-coagulable proteins released from the microbial cells are removed from the milk, and the two precipitated fractions are collected from the remaining liquid by salting out 50% saturated ammonium sulfate, and the two fractions are fractionated by gel filtration method to obtain a product with a molecular weight of 1.5X10G. This is an invention of a method for producing Fl, which is characterized in that both of the above fractions are separated.

Fl is not a single compound, but mainly glycoproteins (including those in which all or part of the protein portion is a riboprotein).
and glycolipids, and has the following physical and chemical properties.

(1) Molecular weight 1.5x10G or more (by gel filtration method using Bio-Gel A-5m) (2) Carbohydrate content (standard analytical value example) Hexose 8.5% (weight) in terms of galactose %, hereinafter the same) (according to the Anthrone-sulfuric acid method) Hexosamine 0.2% (according to the Elson & Morgan method) in terms of galactosamine (according to the Elson & Morgan method) Sialic acid 1.4% (according to the Warren method) (3) Protein content (standard Analysis value example) 24.8% in terms of bovine serum albumin (by the Rouxeau method) (4) Lipid content (standard analysis value example) 64.8% (by the laser Gottlieb method) (5) Stability ■ Stable at pH 3.5 to 8.4.

■ Can withstand heat treatment at 100°C for 60 minutes.

■ It cannot be inactivated by proteolytic enzymes (pronase, trypsin, etc.).

In this way, Fl is a polymer compound with a molecular weight of 1.5 x 10" or more, which is different from the toxin-neutralizing substance found in fermented milk by Minchell et al. The active ingredients are glycoproteins and glycolipids. This fact is clear from the above analytical values, the infrared absorption spectrum (Fig. 1), and the following facts.

(a) When tested by GMI-ELISA method, it is found that Fl inhibits toxins from being adsorbed to GMI.

(b) Glycoproteins and glycolipids with galactose or/ylamic acid terminals are known to be antagonists to receptors on the intestinal lining for toxins.

(c) Fractionation by RCA, -Seph70- chromatography and examination of the adsorbed and non-adsorbed parts revealed that the former is mainly a glycoprotein with galactose and N-acetylgalactosamine ends, and the latter is mainly a glycoprotein with a galactose end and an N-acetylgalactosamine end. It is known that it is a glycolipid and also contains small amounts of glycoproteins and proteins that do not have galactose ends.

(d) The adsorbing portion and non-adsorbing portion of (c) above each exhibit toxin neutralizing ability even when used alone.

It has been confirmed that Fl as mentioned above is present in many fermented milks, so virtually all Bifidobacteria and lactic acid bacteria extract Fl from milk during the fermentation process.
! It is thought that it will cause t. However, the strength of F1 production ability of these bacteria varies considerably between strains, and F1
It is also true that there are strains that are not practical as bacteria for production. Therefore, the production method of the present invention uses Bifidobacterium or lactic acid bacteria that can produce a certain level or more of Fl in fermented milk, taking economic efficiency into account. "Bacteria that have the ability to produce" means a strain selected based on the above criteria. The presence or absence of F1-producing ability of Bifidobacterium and lactic acid bacteria is determined by measuring the toxin neutralizing ability of the bacteria using the culture supernatant separated from fermented milk by the following method.

First, 400 ng/II+I of commercially available purified cholera toxin
Add 0.2 ml of the above supernatant to 0.1+nl of the solution, and let stand at 37°C for 2 hours. Then, 1.7 ml of HamF 12 culture solution supplemented with 5% calf serum was added, and 0.5 ml of the resulting mixture was added to CHO-cells (preliminarily placed in a chamber slide for 2 to 3 hours at 37°C under 5% carbon dioxide gas).
(previously attached to the surface of the slide) by culturing with After culturing the cells for another 20 hours under the same conditions as above, the cells are stained with Giemsa solution, and the degree of change in cell morphology caused by cholera toxin is examined using a microscope. Based on the cell morphological change rate when a phosphate buffer is used instead of the supernatant, if the morphological change suppression rate is 10% or more, it is judged to be light and have F1 production ability (the morphological change rate is determined to be 10% or more).
Among the 00 cells, those whose cells have elongated due to the toxin are counted and expressed as a percentage. ).

Bifidobacterium or lactic acid bacteria used in the F1 production method of the present invention have a morphological change suppression rate of 20% in the above test among those that have F1 production ability.
The above is desirable in terms of processing efficiency. The morphological change suppression rate in the above test was about 40% or more,
Therefore, specific examples of strains particularly suitable for use in the production method of the present invention include Bifidobacterium breve Y I T-4006 (Huikong iJF strain no. 3906), Bifidobacterium breve ATCC 1569
8. Lactobacillus acidophilus YIT-0154
(Huikou Research Institute No. 7079), Lactobacillus acidophilus YIT-0193 (@1 Research Institute No. 7080)
, Lactobacillus acidophilus ATCC4356,
Examples include Lactobacillus casei YIT-0009 (@1 Laboratory Bacteria Collection No. 7078).

The process of fermenting milk using Bifidobacterium or lactic acid bacteria having F1-producing ability can be carried out in exactly the same manner as in the production of lactic acid bacteria drinks and fermented milk.

In this case, Bifidobacterium and lactic acid bacteria may be used in combination.In addition to whole milk, skimmed milk or reconstituted milk from these milk powders may be used as the milk.
Bacterial growth promoters such as yeast extract, vitamin mixture, etc. may be added to this (1° fermentation is preferably carried out at a pH of 5.0 or less).

After fermentation is complete, the pH of the fermented milk is adjusted to about 6.4 with caustic soda, and then centrifuged (for example, 12,000 rpm, 4
℃ for 30 minutes).

Thereafter, the pH is adjusted to 4.6 with hydrochloric acid to precipitate casein and the like, followed by centrifugation.

Next, from the supernatant remaining after centrifugation, both fractions are collected to be salted out with 50% saturated ammonium sulfate. This step is typically carried out by adding ammonium sulfate to about 50% saturation, but is not limited thereto, and may be carried out by other treatment means with substantially equivalent treatment effects.

The salted-out fraction is subjected to molecular weight fractionation treatment by gel filtration after ammonium sulfate and the like are removed by dialysis or the like using a conventional method. For example, the salting-out fraction is dissolved in a 0.1 M)+7 Solubility-HCl buffer (pH 8.3; hereinafter referred to as Tris-HCl buffer) containing 0.5 M of sodium chloride, and Blo-Ge1 A-5m (
Adsorb onto a column with a molecular weight cutoff range of 10→-1,5×10”, and then elute with Tris-HCl buffer. Fl
appears in both volumes with a molecular weight of 1.5x106 or more (void volume + ne, which elutes first when using the above column), so if this is fractionated, thoroughly dialyzed against distilled water at low temperature, and then freeze-dried, Fl is obtained.

Fl obtained by the production method of the present invention as described above exhibits about 50 times more toxin neutralizing ability than the original culture supernatant.

And this purified F1 has the ability to inhibit the binding of cholera toxin 10 nB to GMI by almost 100% at about 50 to 240 μg (dry weight) when tested by GMl-ELISA method.

Since Fl is produced by simply subjecting fermented milk, which is an old food, to a simple purification process, there is no problem at all in terms of safety. Therefore, according to the present invention, it is possible to provide at low cost a raw material for a highly safe drug that is effective in preventing or treating diarrhea caused by cholera toxin or Escherichia toxin.

The present invention will be explained below with reference to Examples.

Example 1 10% reduced skim milk 2.5C containing 0.5% yeast extract
After sterilization, this was treated with Bifidobacterium breve YIT-4006 (Huikoken Bacteria Collection No. 390), which has F1-producing ability.
No. 6) starter was inoculated and kept at 37°C under anaerobic conditions.
The mixture was cultured for 72 hours to obtain fermented milk with a pH of 4.4. 5N
- After adjusting the pH to 6.4 with NaOH, the fermented milk was centrifuged at 1200 rpm for 30 minutes,
A sterilization operation was performed. Then, the obtained supernatant 1.74E
is ultrafiltered with an ultrafiltration membrane (molecular weight cut off: 13000),
6N-HCI was added to the residual solution to lower the pH to 4.6, and proteins such as casein were removed by centrifugation at 3000 rpm for 15 minutes. Ammonium sulfate was added to the resulting clear supernatant at 50% saturation, resulting in a white precipitate. This precipitate was collected by centrifugation, dissolved in distilled water, thoroughly dialyzed against distilled water at 4 to 5°C, and the dialyzed fluid was freeze-dried. 6. 1 g of the solids of the resulting dialyzed fluid was - B 1o-Gel dissolved in 20 ml of hydrochloric acid buffer
It was supplied to an A-jm column (3.2 cmφX46c+n). Then, the void was eluted with Tris-HCl buffer.
The volume F1 fraction was collected (changes in the absorbance of the eluate in this elution process are shown in Figure 2). Both fractions were dialyzed and lyophilized to obtain Fl. The yield was 500 mg per 2.5 e of reduced skim milk used as the raw material.

The cholera toxin-neutralizing effect of Fl was investigated according to the method for determining the presence or absence of F1-producing ability of Bifidobacterium, Rium bacterium, or lactic acid bacteria described at the end. 60 μg of the above F1 inhibited 100%. Furthermore, in a test using the GMI-ELISA method, 120 μg of the above F1 inhibited the binding of 20 ng of cholera toxin to GMI by almost 100%.

In addition, when the E. coli toxin neutralization effect was investigated in the same manner, it was found that CHO produced by 27 μg (protein equivalent) of E. coli toxin
-60ug of the above F1 caused morphological changes of Kl cells by 10θ%
In a test using the GMl-ELISA method, 135 μg of E. coli toxin bound to GMl was inhibited by 120 μg.
The above F1 was suppressed by 10θ%.

Example 2 100n of 10% reduced skim milk containing 0.5% yeast extract
After sterilizing the Nl, it was inoculated with a starter of Lactobacillus acidophilus Y I T-0154 having F1-producing ability, and cultured under anaerobic conditions at 37°C for 68 hours.
Fermented milk with a pH of 3.9 was obtained.

After adjusting the pH to 7.4 with 5N-NaOH, the fermented milk was centrifuged at 12,000 rpm for 30 minutes to perform a sterilization operation. Then, the obtained supernatant 50I
Dialyze Il, add 6N-FICI to the residual solution, and p
H was lowered to 4.6, and proteins such as casein were removed by centrifugation at 3000 rpm for 15 minutes. Ammonium sulfate was added to the resulting clear supernatant to 50% saturation, resulting in a white precipitate. This precipitate was collected by centrifugation, dissolved in distilled water, thoroughly dialyzed against distilled water at 4 to 5°C, and the dialyzed solution was freeze-dried. Solid matter of the obtained dialysis fluid 125+111? was dissolved in 10+nl of Tris-HCl buffer and applied to a Blo-Ge1 A-5m column (3
, 2ca+φX46c+n). Next, it was eluted with a Tris-HCl buffer, and the voic volume of the F1 fraction was collected. Both fractions were dialyzed and then lyophilized to obtain Fl. The yield is 100ml of reduced skim milk as raw material.
The toxin neutralizing effect of this Fl was investigated in the same manner as in Example 1, and it was found that CH produced by cholera toxin 1008
O-, 45 μg of the above F1 caused cell morphological changes by 61%.
suppressed. In addition, in a test using the GMl-ELISA method,
90 μg of the above F1 inhibited the binding of cholera toxin 10 nH to GMI by almost 100%.

In addition, CHO- produced by 27 μg (protein equivalent) of Escherichia coli heat-labile crude toxin (LT) caused 45% cell morphological change.
μg of the above F1 inhibited 67%, and GMI-ELIS
In a test using Method A, 90 μg of the above F1 inhibited 100% of the binding of 67.5 μg of LT to GMl.

Examples 3 to 6 Fl was produced in the same manner as in Example 1, except that the bacteria used for milk fermentation were variously changed. The yield of Fl on each side and the toxin neutralizing capacity of the resulting Fl were as follows.

Bacteria Strain used Yield [mg/l] pH [μg]*
The amount of Fl required to 100% suppress morphological changes in cells caused by 10 nH of cholera toxin in CHO-

[Brief explanation of the drawing]

Figure 1: Infrared absorption spectrum diagram of toxin neutralizing substance F1. FIG. 2: An explanatory diagram of the elution treatment in Example 1 (graph showing variations in absorbance). Agent Patent Attorney San Itai

Claims (1)

[Claims] Milk is fermented with Bifidobacterium or lactic acid bacteria that have the ability to produce cholera toxin and Escherichia coli toxin neutralizing substance F1 from milk, and acid-coagulable proteins are removed from the centrifuged supernatant of the resulting fermented milk. Cholera characterized by collecting both fractions precipitated from the liquid by salting out 50% saturated ammonium sulfate, and fractionating the fractions by molecular weight using a gel filtration method to separate the fractions having a molecular weight of i, 5xio' or more. Method for producing toxin and Escherichia coli toxin neutralizing substance F1.