JPH0560358B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Fields Lipopolysaccharide has the property of causing antigen-antibody reactions and red blood cell agglutination reactions, so it has been used as a reagent for academic research in fields such as medicine, pharmacy, and biochemistry. It is used.

BACKGROUND ART Lipopolysaccharide is a component of the extracellular membrane of Gram-negative bacteria such as Erwinia bacteria, and it is known that it is produced within the cells of these bacteria.

Regarding the production of lipopolysaccharide, known examples include Escherichia coli, Salmonella
Bacteria, etc. are cultured in a liquid phase in a nutrient medium, and then the bacterial bodies are collected and added to a mixed solution of phenol, water, and petroleum ether (e.g., European Journal of Biochemistry (Eur.J.)).
Biochem.), Vol. 9, p. 245, 1969), or a mixed solution of n-butanol and water (e.g.
Journal. of. Biological. Chemistry (J.Biol.chem.), Volume 250, Page 2911, 1975
(2013) and other methods are used to isolate it from bacterial bodies. However, in all of these methods, the lipopolysaccharide remains inside the microbial cells, and therefore requires an extremely complicated operation of extraction and separation using an organic solvent or the like. Furthermore, there is a limit to the amount that can be accumulated within the bacterial body, making it difficult to produce in large quantities.

Problems to be Solved by the Invention It is an object of the present invention to eliminate the drawbacks of the conventional techniques and to provide a method for easily producing a large amount of lipopolysaccharide outside a bacterial cell. That is, the mechanism by which lipopolysaccharide remains in the bacterial body is released to produce lipopolysaccharide in the culture solution, and the substance, which was conventionally obtained by extraction using an organic solvent, etc., is released into the culture solution. The purpose is to provide a new method.

Means for Solving the Problems The present inventors have conducted research on the culture method of Erwinia bacteria and the substances produced, and as a result, they have found that they can be cultured in two stages using nutrient media each using a different carbon source. We have discovered that the above objective can be achieved by culturing Erwinia bacteria,
Based on this knowledge, we have completed the present invention.
That is, the method of the present invention involves aerobically cultivating Erwinia bacteria using a nutrient medium containing pectin as a carbon source, and culturing the primary growth until the end of the stationary growth phase when almost no pectin is present. The method consists of two stages of secondary growth, in which the bacteria are cultured again using a nutrient medium containing non-pectin as a carbon source.

In the method of the present invention, the bacteria of the genus Erwinia used include bacteria of the Carotobora species group, for example,
Erwinia. Calotobora. Subsp. Carotovora (Erwinia carotovora.subsp.carotovora),
Erwinia. Chrysanthemum (Erwinia)
Chrysanthemi), Erwinia. Preferred examples include Erwinia rhapontici.

The primary growth in the method of the present invention involves using pectic material as a nutrient medium as a carbon source, and nitrogen compounds such as monosodium glutamate and ammonium sulfate, potassium phosphate and potassium phosphate as a nutrient source. A liquid medium to which disodium, magnesium sulfate, etc. are added is used.

On the other hand, for secondary growth, a liquid medium containing a non-pectic substance as a carbon source and the above-mentioned compounds as a nutrient source is used as a nutrient medium. As the non-pectic substance, slowly metabolizing carbon compounds such as glycerin and sorbitol are suitable.

The cultivation of the Erwinia bacteria is carried out aerobically while blowing air, usually at a temperature range of 10 to 60°C, preferably 20 to 40°C, for both primary and secondary growth. Furthermore, it is not necessarily necessary to adjust the pH during either primary or secondary proliferation.

By such a two-step culture method, lipopolysaccharide, which normally remains within the bacterial cells, can be produced even into the culture solution.

The produced lipopolysaccharide is present in the liquid medium, so after removing the bacterial cells by centrifugation or filtration, the filtrate is treated with a commonly used method to obtain crude lipopolysaccharide. will be collected as.
The crude lipopolysaccharide thus obtained can be further purified according to known purification methods, if necessary.

Effects of the Invention In the conventional method for producing lipopolysaccharide, since the substance remains within the cells of Gram-negative bacteria, it was necessary to disintegrate the cells and perform extraction and separation using an organic solvent or the like. In addition, productivity was extremely low due to accumulation within the bacterial body. On the other hand, according to the method of the present invention, lipopolysaccharide is produced in the culture solution, which not only facilitates the separation of lipopolysaccharide, but also significantly increases productivity.

Furthermore, the method of the present invention can be applied to cells other than the above-mentioned bacterial cells.

Examples Next, the present invention will be explained in more detail with reference to Examples.

Example Erwinia. Carotovora FERM P-7576 cells were first cultured in a nutrient medium with the following composition using a 10 jar fermenter under conditions of a temperature of 28°C, an air flow rate of 2.5/min, and a stirring speed of 480 rpm. Ivy.

Composition of primary culture nutrient medium Pectin 25g Monosodium glutamate (monohydrate) 25g Ammonium sulfate 15g Potassium phosphate 12g Disodium phosphate 4g Magnesium sulfate (heptahydrate) 1g Silicone antifoam agent 2.5g Water 5 As a result, the bacteria proliferate until 16 hours after the start of culture, but after that, the cells enter the stationary phase. In this primary culture, a high molecular weight substance is separated from the culture solution from which the bacterial cells have been removed by a known substance separation method. Component analysis of this substance according to a known analytical method revealed that it contained sugar, phosphoric acid, and fatty acid as components. This is equivalent to the constituents of the known substance commonly referred to as lipopolysaccharide. In addition, after electrophoresing the substance according to polyacrylamide gel electrophoresis method, the gel is dyed using a known lipopolysaccharide staining method (for example, Analytical Biochem. Vol. 119, p. 115, 1982). )
When silver staining was carried out according to the method described in 2007, it was found that the material stained clearly. This also confirmed that the substance was lipopolysaccharide.

The amount of lipopolysaccharide produced in the culture solution in the primary culture was 0.1 g/. Next, 300 ml of a nutrient medium consisting of an aqueous solution containing 100 g of glycerin and 100 g of monosodium glutamate (monohydrate) dissolved in the culture solution after 16 hours of primary culture is added to perform secondary culture, and 16 hours after the start of culture. Bacteria proliferate until The bacterial cells are removed by centrifugation from the culture solution that has been subcultured for 16 hours to obtain a sterilized culture solution. Put the solution in a cellophane tube for dialysis, and
Dialyze for 24 hours using 1 part distilled water per 20 ml. When the solution obtained by separating inorganic ions and the like in the nutrient culture by dialysis is freeze-dried to remove water, 3.3 g of powder containing 2 g of lipopolysaccharide is obtained from 1 sterilized culture solution.

Claims (1)

[Scope of Claims] 1. Bacteria of the genus Erwinia are cultured in a nutrient medium containing pectic substances as a carbon source until the end of the stationary growth phase, and then non-pectic substances are added to the culture medium as a carbon source. A method for producing lipopolysaccharide, which comprises adding and culturing a nutrient medium as a source. 2. The method according to claim 1, characterized in that the bacteria are cultured in a nutrient medium using a combination of pectin and a slowly metabolizing carbon compound as a carbon source. 3. The method according to claim 1, wherein the non-pectic substance is a slowly metabolizing carbon compound.