# US 5,705,634 A · High yield preparation of dimeric to decameric chitin oligomers > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,705,634 A - Document ID: US5705634A - Status: Granted - Kind: A - Application number: 08/397,464 - Filed: 1995-03-02 - Granted: 1998-01-06 - Earliest claimed: 1995-03-02 - Assignees: PerImmune Holdings, Inc. - Inventors: Reinhard Bredehorst; Nicholas Pomato; Oliver Scheel; Joachim Thiem - Web page: https://patentgrep.com/patent/US5705634A ## CPC - C08B 37/003: {Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof} - C07H 3/04: Disaccharides - C07H 3/06: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages - C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21) - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) ## Abstract The present invention is directed to a new process for the production of chitin oligomers, including fragmentation, partial hydrolysis and recovery. This process produces high yields, especially of higher (tetra- to deca-) chitin oligomers. ## Claims ### Claim 1 A process for the production of water-soluble chitin oligomers comprising the steps of: (a) fragmenting chitin to particles with a mean diameter of 120 .mu.m or less; (b) mixing the chitin particles with a concentrated hydrohalogen acid solution to achieve partial hydrolysis; (c) neutralizing the reaction mixture to limit the hydrolysis of the chitin particles; (d) desalinating the neutralized chitin hydrolysates from step (c) by a method selected from the group consisting of: (1) electrodialysis, wherein the number of membranes used is proportional to the salt content of the diluate; and (2) flow filtration using a ceramic membrane; and (e) separating the desalted chitin oligomers from the desalted reaction mixture of step (d). ### Claim 2 (depends on 1) The process of claim 1, wherein the chitin oligomers produced have the general formula: ##STR1## wherein m=0-8. ### Claim 3 (depends on 2) The process of claim 2, wherein m=4-8. ### Claim 4 (depends on 1) The process of claim 1, wherein in step (a) the chitin is fragmented to particles having a mean diameter of 120 .mu.m or less by processing the chitin in a mill equipped with one or more beater systems. ### Claim 5 (depends on 1) The process of claim 1, wherein the chitin particles with a diameter of 120 .mu.m or less are fragmented to particles having a mean diameter of about 10 to 50 .mu.m by processing the chitin particles in a fine impact mill or zig zag classifier. ### Claim 6 (depends on 1) The process of claim 1, wherein the chitin particles with a mean diameter of 10-50 .mu.m are fragmented to ultrafine particles having mean diameters of less than 10 .mu.m by processing the chitin particles in an airstress ultrafine classifier. ### Claim 7 (depends on 1) The process of claim 1, wherein in step (b) the chitin particles are dissolved in a concentrated hydrohalogenic acid and subjected to partial hydrolysis at elevated temperatures. ### Claim 8 (depends on 7) The process of claim 7, wherein the concentrated hydrohalogenic acid is 12.5M HCl. ### Claim 9 (depends on 7) The process of claim 7, wherein the chitin particles are dissolved in the acid using a mechanical stirrer at a concentration of 200 to 400 mg chitin particles per ml concentrated hydrohalogenic acid. ### Claim 10 (depends on 7) The process of claim 7, wherein the hydrolysis reaction is carried out at a temperature between 20.degree. C. and 45.degree. C. for a period of 100 to 150 minutes with continuous stirring. ### Claim 11 (depends on 10) The process of claim 10, wherein the temperature is 40.degree. C. ### Claim 12 (depends on 1) The process of claim 1, wherein in step C the neutralization is effected by the addition of ice-cold 50% sodium or potassium hydroxide solution under conditions that keep the temperature of the chitin hydrolysate below 25.degree. C. ### Claim 13 (depends on 1) The process of claim 1, wherein in step (d) the neutralized chitin hydrolysate is filtered to remove insoluble material and is desalinated to a salt concentration of less than 0.1 g/l by electrodialysis. ### Claim 14 (depends on 13) The process of claim 13, wherein desalination is performed by electrodialysis, during which the pH of the desalted fraction is maintained at approximately pH 7. ### Claim 15 (depends on 13) The process of claim 13, wherein desalination is performed by electrodialysis, during which the conductivity of the concentrate is maintained at approximately 100 mS/cm. ### Claim 16 (depends on 13) The process of claim 13, wherein desalination is performed by electrodialysis, during which the temperature is kept at a temperature ranging from about 20.degree. C. to 40.degree. C. ### Claim 17 (depends on 1) The process of claim 1, wherein following step (d) the desalinated hydrolysate is lyophilized. ### Claim 18 (depends on 17) The process of claim 17, wherein the lyophilized chitin hydrolysate is redissolved in water and subjected to the separation process of step (e), which comprises a process that will separate individual chitin oligomers of defined length into individual fractions or groups of chitin oligomers of different lengths within defined ranges into individual fractions. ### Claim 19 (depends on 18) The process of claim 18, wherein the lyophilized chitin hydrolysate is redissolved in water at a concentration of about 2 to 4 g of solid per 40 ml of water. ### Claim 20 (depends on 18) The process of claim 18, wherein the separation of individual chitin oligomers of defined length is accomplished by a chromatographic procedure selected from the group consisting of low pressure gel filtration, high pressure gel filtration and reverse-phase HPLC. ### Claim 21 (depends on 18) The process of claim 18, wherein the separation of groups of chitin oligomers of different lengths is accomplished by salt precipitation of higher chitin oligomers. ### Claim 22 (depends on 1) The process of claim 1, wherein in step (d) the neutralized chitin hydrolysate is filtered to remove insoluble material, and is thereafter desalinated by flow filtration. ### Claim 23 (depends on 22) The process of claim 22, wherein the desalination is effected by tangential flow filtration. ### Claim 24 (depends on 23) The process of claim 23, wherein the tangential flow filtration is performed with membranes having a molecular weight cut-off of 1 KDa or less, whereby salt and dimeric and trimeric chitin oligomers from the chitin hydrolysate are removed and the higher chitin oligomers in the desalinated fraction are enriched. ### Claim 25 (depends on 23) The process of claim 23, wherein the filtration rate ranges from 0.5 L/hour to about 500 L/hour. ### Claim 26 (depends on 22) The process of claim 22, wherein the desalination is effected by flow filtration through a ceramic membrane. ### Claim 27 (depends on 26) The process of claim 26, wherein ceramic membrane filters with pore sizes of about 5 nm to 1 nm are used for desalination and for the removal of lower chitin oligomers from the chitin hydrolysate. ### Claim 28 (depends on 1) The process of claim 1, wherein the chitin hydrolysates, desalinated by flow filtration, are lyophilized. ### Claim 29 (depends on 28) The process of claim 28, wherein the lyophilized chitin hydrolysate is redissolved in water and subjected to the separation process of step (e), which comprises a process that will separate individual chitin oligomers of defined length into individual fractions or groups of chitin oligomers with different lengths within defined ranges into individual fractions. ### Claim 30 (depends on 29) The process of claim 29, wherein the lyophilized chitin hydrolysate is redissolved in water at a concentration of about 2 to 4 g solid per 40 ml of water. ### Claim 31 (depends on 28) The process of claim 28, wherein the separation of individual chitin oligomers of defined length is accomplished by a chromatographic procedure selected from the group consisting of low pressure gel filtration, high pressure gel filtration and reverse-phase HPLC. ### Claim 32 (depends on 28) The process of claim 28, wherein the separation of groups of chitin oligomers of different lengths is accomplished by salt precipitation of higher chitin oligomers. ### Claim 33 (depends on 1) The process of claim 1, wherein after neutralization the chitin hydrolysates are filtered, and are thereafter desalinated by electrodialysis and subsequently subjected to flow filtration without prior lyophilization. ### Claim 34 (depends on 33) The process of claim 33, wherein the neutralized and filtered chitin hydrolysates are desalinated by electrodialysis and subsequently subjected to flow filtration without prior lyophilization, wherein in step (d) the neutralized chitin hydrolysate is desalinated to a salt concentration of less than 0.1 g/l by electrodialysis. ### Claim 35 (depends on 34) The process of claim 34, wherein desalination is performed by electrodialysis, during which the pH of the desalted fraction is maintained at approximately pH 7. ### Claim 36 (depends on 34) The process of claim 34, wherein desalination is performed by electrodialysis, during which the conductivity of the concentrate is maintained at approximately 100 mS/cm. ### Claim 37 (depends on 34) The process of claim 34, wherein desalination is performed by electrodialysis, during which the temperature is kept at a temperature ranging from about 20.degree. C. to 40.degree. C. ### Claim 38 (depends on 33) The process of claim 33, wherein the desalination is effected by tangential flow filtration. ### Claim 39 (depends on 38) The process of claim 38, wherein the tangential flow filtration is performed with membranes having a molecular weight cut-off of 1 KDa or less, whereby salt and dimeric and trimeric chitin oligomers from the chitin hydrolysate are removed and the higher chitin oligomers in the desalinated fraction are enriched. ### Claim 40 (depends on 38) The process of claim 38, wherein the filtration rate ranges from 0.5 L/hour to about 500 L/hour. ### Claim 41 (depends on 38) The process of claim 38, wherein the desalination is effected by flow filtration through a ceramic membrane. ### Claim 42 (depends on 38) The process of claim 38, wherein ceramic membrane filters with pore sizes of about 5 nm to 1 mm are used for the removal of lower chitin oligomers from the chitin hydrolysate. ### Claim 43 (depends on 1) The process of claim 1, wherein the chitin hydrolysate, desalinated by electrodialysis and subsequently subjected to flow filtration, is lyophilized. ### Claim 44 (depends on 43) The process of claim 43, wherein the lyophilized chitin hydrolysate is redissolved in water and subjected to the separation process of step (e), which comprises a process that will separate individual chitin oligomers of defined length into individual fractions or groups of chitin oligomers with different lengths within defined ranges into individual fractions. ### Claim 45 (depends on 44) The process of claim 44, wherein the lyophilized chitin hydrolysate is redissolved in water at a concentration of about 2 to 4 g solid per 40 ml of water. ### Claim 46 (depends on 44) The process of claim 44, wherein the separation of individual chitin oligomers of defined length is accomplished by a chromatographic procedure selected from the group consisting of low pressure gel filtration, high pressure gel filtration and reverse-phase HPLC. ### Claim 47 (depends on 44) The process of claim 44, wherein the separation of groups of chitin oligomers of different lengths is accomplished by salt precipitation of higher chitin oligomers. ### Claim 48 (depends on 1) The process of claim 1, wherein di-N-acetyl-chitobiose is obtained at a yield of 8.6.+-.2.3 g per 100 g chitin. ### Claim 49 (depends on 1) The process of claim 1, wherein tri-N-acetyl-chiotriose is obtained at a yield of about 8.9.+-.2.4 g per 100 chitin. ### Claim 50 (depends on 1) The process of claim 1, wherein tetra-N-acetyl-chitotetraose is obtained at a yield of about 8.7.+-.2.3 g per 100 g chitin. ### Claim 51 (depends on 1) The process of claim 1, wherein penta-N-acetyl-chitopentaose is obtained at a yield of about 8.1.+-.2.1 g per 100 g chitin. ### Claim 52 (depends on 1) The process of claim 1, wherein hexa-N-acetyl-chitohexaose is obtained at a yield of about 3.6.+-.0.7 g per 100 g chitin. ### Claim 53 (depends on 1) The process of claim 1, wherein hepta-N-acetyl-chitoheptaose is obtained at a yield of about 1.0.+-.0.4 g per 100 g chitin. ### Claim 54 (depends on 1) The process of claim 1, wherein octa-N-acetyl-chitooctaose is obtained at a yield of about 0.3.+-.0.2 g per 100 g chitin. ### Claim 55 (depends on 1) The process of claim 1, wherein nona-N-acetyl-chitononaose is obtained at a yield of about 0.2.+-.0.1 g per 100 g chitin. ### Claim 56 (depends on 1) The process of claim 1, wherein deca-N-acetyl-chitodecaose is obtained at a yield of about 0.3.+-.0.2 g per 100 g chitin.