Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-10-27
2001-12-25
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S017200, C536S101000, C536S127000, C536S128000, C536S123000, C536S123120, C536S123100, C435S101000, C424S184100, C424S185100
Reexamination Certificate
active
06333399
ABSTRACT:
The present invention pertains to a process for the preparation of chitosan-glucan complexes from biological sources, a material essentially consisting of a chitosan-glucan complex obtainable by the process according to the invention, materials shaped therefrom, a wound healing means, and uses of the materials according to the invention.
Cell walls of microorganisms containing chitin have already been used for many applications in the prior art. Thus, it is possible to bind and accumulate metal ions from water by means of microorganisms. The sorptive power of the microorganisms is attributed to the cell walls. Especially cell walls of microscopic fungi, actinomycetes and yeasts contain various complexes of polysaccharides. Thus, for instance, the inner layer of the cell wall of
Neurospora crassa
, Penicillium sp., Aspergillus sp. consists of chitin microfibers incorporated in a 1,3-polyglucan-matrix. The cell walls form a barrier against deleterious environmental influences and possess a protective function for the corresponding microorganisms. Thus, the chitin content in fungi can reach up to 45% of the cell components (e.g. in
Penicillium digitatum
). An important aminopolysaccharid, chitosan, can be obtained from chitin. The primary amino groups of chitosan or its complexes can selectively form particular bonds with metal ions. Chitosan has been used as a sorbent in the heavy metal elimination and as a flocculant for negatively charged colloids. In addition, polymers based on chitin are known to be capable of causing a decrease in cholesterol content. Chitosan could achieve much importance for the application on many technical fields. To date, however, no satisfactory process has been provided for obtaining chitosan derivatives which may be employed, for example, in medicine, food industry or environmental and disposal engineering. Thus, to date, the mycelium, for example, from
Rhizopus arhisus
, has been treated with 4% sodium dodecylsulfate solution, which is an aggressive detergent, mechanically disrupted, centrifuged and washed with water. However, a total disruption of the native cell wall structure is unavoidable thereby. This has a negative effect on sorptivity. In addition, it has been known to process cells, for instance, from
Saccharomyces cerevisiae
, with denaturing agents, such as 8 M urea solution, 1 M aqueous potassium hydroxide and 33 mM acetic acid solution. This yields substances having a low sorption capacity for heavy metal ions. These methods are described in Biotechnol. & Bioeng., Vol. 24, N2, p. 385-401, 1982, The Mechanism of Uranium Biosorption by Rhizopus arhisus, The Fungi, Eds. Ainsworth G. C., Sussman, A. V., Academic Press 1965, p. 46-76, and in GB-2,188,135 A1. GB-2,102,506 pertains to the recovery of chitin-containing materials from the molds Mucor mucedo and
Risomucor miecheli
. This involves incubation with 8% H
2
O
2
solution at 100° C. for one hour followed by treatment with sodium silicate solution. A material is obtained which has high protein and ash contents. This may have an adverse effect on the flexibility of the corresponding materials. The recovery of chitin-containing materials for use in wound healing is described in U.S. Pat. No. 3,632,575. This involves first extraction with chloroform, treatment with 1.0 N aqueous sodium hydroxide at room temperature for 18 hours, followed by the addition of low-concentrated hydrochloric acid and dialysis with distilled water. Said chloroform extraction is a problematic step in industrial scale since the use of large amounts of chloroform is not recommendable for ecological and industrial hygiene reasons.
DE 29 23 802 A1 pertains to a process for treating mycelia of the mold
Mucor rouxii
with 40% aqueous sodium hydroxide at 128° C. for four hours. In a similar way, mycelia of
Aspergillus niger
, for example, are treated with 40% aqueous sodium hydroxide for six hours. A chitosan-glucan complex is obtained in an amount of from 32 to 52% of the total weight of the fungal mycelia, the chitosan-glucan complex obtained being capable of binding heavy metal ions, e.g., copper. However, this process has an advantage in that it is impossible to ensure the required degree of purity with respect to proteins, lipids and carbonates in this complex. A high carbonate content has also an adverse effect on the flexibility and applicability of the materials obtained. In addition, it is a drawback that the native structure of the mold cell walls is completely disrupted, and that control of the degree of deacetylation of the chitin is not possible, either.
It has been the object of the invention to provide a process which, in the first place, generally avoids the disadvantages of the prior art, i.e. is capable, in particular, of processing the fungal mycelia in such a way that as extensive as possible a use thereof in different technical fields becomes possible, and, in particular, of controlling the degree of deacetylation. In addition, the process is to be environmentally safe and still ecological.
The object of the invention is surprisingly achieved by a process according to the features and to preferred embodiments in the following description of the process according to the invention.
The process according to the invention for the preparation of chitosan-glucan complexes proceeds from biological sources which are essentially chitin-containing microorganisms. The latter are treated with solutions, the process according to the invention being characterized in that said chitin-containing microorganisms are treated at least once with a first alkaline solution. Then, the product obtained from this step a) is treated with diluted mineral acid. This step b) is followed by a further treatment with a second alkaline solution, the alkalinity of said second alkaline solution being higher than that of said first alkaline solution. The alkalinity of the second alkaline solution is adjusted such that deacetylation of the product obtained from the preceding step does not proceed to completion. This is followed by separating the solid components from this treatment step c), and then discarding the supernatant, neutralizing the residue and washing the residue with water.
The process according to the invention has the advantage that it modifies the fungal mycelia mildly so that the native structure of the cell walls is essentially retained and the degree of deacetylation becomes controllable. This is especially advantageous for adjusting the properties of the materials according to the invention obtainable by the process according to the invention in order to direct them to a wide variety of applications. Thus, for example, a higher acetylation level is desirable if the materials according to the invention are employed in wound healing, for example. However, if the product obtainable by the process according to the invention is to be employed as a sorbent, for example, it may be advantageous to increase the degree of deacetylation to obtain more complexing sites or sorption sites through free amino groups.
In the process according to the invention, there are preferably used chitin-containing microorganisms, such as zygomycetes, ascomycetes, plectomycetes, streptomycetes, pyrenomycetes, discomycetes and/or yeasts. These include, in particular, Aspergillus sp., Penicillium sp., Mucor sp., Phycomyces sp., Choanephora sp., Zygorrhynchus sp., Blakeslia sp., Actinomyces sp. and Saccharomyces sp. Preferably, the microorganisms mentioned are employed in the stage of highest chitin content and/or in the final condition of their application for industrial purposes. Basically, however, the stage of development is not critical. Said fungi or yeasts are characterized by having a chitin content in their cell walls.
According to the invention, it is preferred to perform the treatment with said first alkaline solution according to step a) twice to four times.
The alkalinity of the first alkaline solution is preferably from 1 to 15%, in particular from 5 to 10%. The temperatures are preferably in the range of from 50 to 110° C., i
Nikolaewna Woewodina Irina
Teslenko Alexander
Holman PLLC Jacobson
Minnifield Nita
Teslenko Alexander
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