Title:  Chitin beads, chitosan beads, process for preparing these beads, carrier comprising said beads, and process for preparing microsporidian spore

United States Patent:  6,156,330

Assignee:  Japan as represented by Director General of National Institute of (Tsukuba, JP)

Filed:  December 30, 1998

PCT NO:  PCT/JP97/03741

102(e) Date:  December 30, 1998

PCT PUB. Date:  November 19, 1998

Foreign Application Priority Data:  May 14, 1997[JP] (9-124255)

The present invention relates to chitin beads having a uniform and fine particle size and comprising microsporidian spores whose principal cell wall substance is chitin; chitosan beads comprising N-deacetylated chitin and having a uniform fine particle size; methods for preparing these beads; carriers comprising these beads; and a method for preparing microsporidian spores. Microsporidian spores are proliferated in insect's bodies or cultured cells to obtain chitin beads having the uniform and fine particle size and whose cell wall substance is mainly composed of chitin. The chitin as the major cell wall substance is subjected to N-deacetylation to give chitosan beads. When the insect is a larva of domesticated silkworm, microsporidian spores are proliferated by orally or percutaneously inoculating the spores into the 2nd instar domesticated silkworm larvae in a concentration of 5x10² to 5x10⁸ spores/ml, then the proliferated microsporidian spores are isolated from the bodies of the grown 5th instar domesticated silkworm larvae, followed by purifying the resulting spores to give chitin beads having the uniform and fine particle size and chitosan beads can be obtained from the chitin beads in the same manner used above. These beads are used as carriers for the immobilization or encapsulation of a variety of substances such as an antibiotic and a microorganism.

DISCLOSURE OF THE INVENTION

In the foregoing conventional method, the particle size and the porosity of the resulting chitosan beads would widely vary depending on, for instance, the rate of desolvation and rates of penetration and diffusion of the coagulating liquid into the resulting beads. For this reason, it is quite difficult to make the particle size of these beads uniform, thus the production of chitosan beads having the uniform particle size requires complicated preparation procedures and skill in the workers and this accordingly makes it difficult to effect mass production thereof. Thus, there has been desired for the development of a method for preparing chitosan beads having the uniform and fine particle size according to production procedures which can easily be carried out and can be excellent in yield, efficiency and economy.

The present invention has solved or eliminated the foregoing problems associated with the conventional techniques by making the most use of the fact that the spores of Microsporozoa having a uniform and fine particle size and whose major cell wall substance comprises chitin can highly efficiently be produced in insect's bodies or within cultured cells. More specifically, the object of the present invention is to provide a method for efficiently and economically preparing chitin beads, chitosan beads and microsporidian spores each having the uniform and fine particle size, while making use of the fact that chitin is the major component of the cell wall substance of particulate microsporidian spores, as well as the beads having the uniform and fine particle size prepared by the method and a carrier which comprises these beads having the uniform and fine particle size.

The inventors of the present invention have conducted intensive investigations to develop a novel technique for utilizing a biopolymer originated from insects. The inventors have taken note of the facts that the microsporidian spores can be produced in the insect's bodies or cultured cells in a high efficiency and each of the resulting microsporidian spores thus produced has the uniform particle size and has a shape of a sphere or an elliptical sphere and that the major cell wall substance of the microsporidian spores is chitin; have found out, for the first time, that if a substance such as an antibiotic or a physiologically active substance is immobilized on the chitin beads or chitosan beads having the uniform particle size, the beads can be used as carriers for sustained release of these substances; and thus have completed the present invention.

When inoculating microsporidian spores into or on domesticated silkworms, wild silkworms or other various insects or cultured cells, there are proliferated a large amount of microsporidian spores whose principal cell wall substance is chitin and having the particle size on the order of several micrometers (.mu.m). The inventors of this invention have also developed a method for purifying/separating the proliferated microsporidian spores thus obtained to give chitin beads, chitosan beads or microsporidian spores each having the uniform particle size and a simple method for preparing carriers, from these beads, for the immobilization of, for instance, an antibiotic or a physiologically active substance.

First of all, the inventors of this invention have clarified optimum conditions for efficiently producing microsporidian spores, such as the time or the stage of an insect to inoculate microsporidian spores into or on insects or cultured cells, the amount thereof to be inoculated and the method for inoculating the same, as well as methods for purifying and/or separating the microsporidian spores proliferated, for instance, in the insect's bodies.

As methods for inoculating microsporidian spores into insects or cultured cells, there have been known, for instance, (i) an oral inoculation method which comprises the step of adding intended microsporidian spores to feeds for insects; and (ii) a direct inoculation method which comprises the step of percutaneously inoculating microsporidian spores into insect's bodies during breeding the same.

Although microsporidian spores may have a variety of shapes, they maintain desired bead shapes depending on the kinds thereof and the major cell wall substance is a complex of chitin and proteins. Thus, these spores may be used in the form of (i) chitin beads, i.e., microsporidian spores per se obtained after the proliferation or (ii) chitosan beads obtained by N-deacetylating the chitin of the microsporidian spores. Alternatively, chitosan beads in which the fine particle's surface is composed of chitosan may likewise be prepared by subjecting the chitin on the surface of microsporidian spores to an N-deacetylation treatment. Moreover, it is also possible to prepare hollow beads which have fine pores formed on the cell walls of the microsporidian spores by activating intracellular substances to thus outwardly release the same through the cell walls.

As has been discussed above, the chitin beads according to the present invention are those having the uniform and fine particle size, composed of microsporidian spores proliferated in insect's bodies or cultured cells and whose major cell wall substance is chitin, while the chitosan beads according to the present invention are the foregoing chitin beads wherein the chitin among the cell wall substances is subjected to N-deacetylation. These chitin beads and chitosan beads may be those from which the proteins have been removed, i.e., non-antigenic ones. The removal of the proteins is carried out by the usual hydrolysis treatment. In addition, the foregoing proliferated microsporidian spores may, if necessary, have pores in their cell walls and the foregoing chitin beads and chitosan beads may be hollow beads. The pores may be formed through a treatment of the beads with hydrogen peroxide or an alkali.

The carrier according to the present invention comprises chitin beads having the uniform and fine particle size which are composed of the aforementioned proliferated microsporidian spores and whose principal cell wall substance is chitin or chitosan beads wherein the chitin as the major cell wall substance is subjected to an N-deacetylation treatment. These chitin beads and chitosan beads may be those from which the proteins have been removed, i.e., non-antigenic ones. The removal of the proteins is carried out by the usual hydrolysis treatment. The foregoing proliferated microsporidian spores may, if necessary, have pores in their cell walls and the foregoing chitin beads and chitosan beads may be hollow beads. The pores may be formed through a treatment of the beads with an alkali or hydroxy peroxide.

The carrier according to the present invention is used for the immobilization or introduction of, for instance, physiologically active substances, antibiotics, biological cells, microorganisms such as bacteria, colorless and colored dyes, medicines, agricultural agents, perfumes, foodstuffs and feedstuffs.

Incidentally, the chitin beads and the chitosan beads according to the present invention are desirably non-antigenic ones when embedding them in, for instance, human bodies.

The method for preparing the uniform and fine particles (the chitin beads and the chitosan beads) according to the present invention comprises the steps of orally or percutaneously inoculating microsporidian spores in a concentration of 5x10² to 5x10⁸ spores/ml, preferably 5x10² to 5x10⁷ spores/ml, into an insect's body to thus proliferate the microsporidian spores, harvesting the proliferated microsporidian spores from the raised or grown insect's body, purifying the spores to thus give uniform and fine particles (chitin beads) and, if necessary, N-deacetylating the chitin of the chitin beads to give uniform and fine particulate chitosan beads. In this regard, if the spore concentration is less than 5x10² spores/ml, the rate of infection of the insect with the spores is low and there is observed a scattering in the rate of infection, while if it exceeds 5x10⁸ spores/ml, the insect is killed prior to the complete formation of spores within the insect's body and therefore, the use thereof in such a concentration is unfavorable in view of profits. Preferably, the time for inoculating microsporozoa in the insect's body is just after the 2nd instar larva and the collection of the spores is started from the 5th instar larva. The insect is preferably larvae of domesticated silkworms.

The method for producing microsporidian spores according to the present invention preferably comprises the steps of adding cultured cells originated from an insect to a cell culture medium containing, on the basis of the weight of the cell culture medium, 5 to 50% by weight and preferably 10 to 40% by weight of the supernatant of hemolymphor or humor of larvae of domesticated silkworms, inoculating microsporidian spores into the culture medium to thus proliferate the cells and then recovering the microsporidian spores from the proliferated cells. In this respect, if the added amount of the supernatant of the hemolymphor of larvae of domesticated silkworms is less than 5% by weight and it exceeds 50% by weight, the spores scarcely undergo any proliferation. In addition, the spores would further efficiently be proliferated if the amount of the supernatant to be added falls within the preferred range defined above.

Claim 1 of 16 Claims

What is claimed is:

1. Chitin beads which are microsporidian spores proliferated in insect bodies or cultured cells, said spores having a uniform and fine particle size and having a cell wall substance mainly composed of chitin.

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