Production of chitosan and chitin

Abstract: The invention relates to a method of producing chitin or chitosan by culturing a Rhizopus azygosporus fungus or an Actinomucor taiwanensis fungus and isolating chitosan or chitin from the culture.

Description: Background of the Invention Chitin is a highly insoluble N-acetylated polymer of β-(1,4)-D-glucosamine. Chitosan is an acid-soluble deacetylated form of chitin. Chitin, chitosan, and derivatives thereof are used in a number of industrial applications, including the production of viscosity control agents, adhesives, chromatography carriers, paper-strengthening agents, flocculent agents, food additives, drugs, and cosmetics. Chitin can be manufactured by the deproteination and decalcification of crab or shrimp shells. Chitosan can then be obtained by deacetylating chitin with a hot alkali solution. This chitosan production process has a number of unfavorable characteristics. For example, the process requires expensive heat energy and caustic alkali, which is a potential health hazard. The process also produces large amounts of waste, thereby necessitating significant disposal costs. In addition, the supply of shrimp or crab shells is highly dependent upon seasonal and environmental factors, leading to unpredictable limitations on production capacity. Summary of the Invention The invention is based on the discovery that unexpectedly high yields of chitosan and chitin can be produced from the fungus Rhizopus azygosporus . Accordingly, the invention features a method of producing chitosan or chitin by (1) culturing a Rhizopus azygosporus fungus in a medium to form a culture, and (2) isolating chitosan or chitin from the cells. For example, chitosan and chitin can be isolated from the culture by separating the fungal cells from the culture and isolating the chitosan or chitin from the separated cells. A medium useful for the methods of the invention can include about 5 to 60 g/L (e.g., about 30 g/L) corn steep liquor, about 10-100 g/L (e.g., about 50 g/L) glucose, about 0.01 to 30 g/L (e.g., about 2.5 g/L) ammonium sulfate, or other suitable ingredients. The methods of the invention allow surprisingly high-yield production of chitosan or chitin from a culture containing a Rhizopus azygosporus fungus. Further, it has been discovered that a medium containing corn steep liquor, glucose, yeast extract, and ammonium sulfate is capable of increasing the output of chitin and chitosan from a fungal culture. The methods of the invention, therefore, provide an alternative to producing chitin and chitosan without reliance on environmentally harmful chemicals or the variable abundance of the crustacean crop. Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims. Detailed Description The invention relates to high yield production of chitosan or chitin from fungal cultures belonging to the family Mucoraceae, namely Rhizopus azygosporus . This organism is available upon request from the Culture Collection and Research Center (CCRC), Food Industry and Research Development Institute, No. 331, Shih-Ping Road, Hsinchu 300, Taiwan, Republic of China. R. azygosporus is available as Catalog No. CCRC31558. Procedures for culturing fungi are well known in the art. For example, YM agar can be inoculated with a fungus, and the inoculated agar incubated at 25°C to 37°C for 3 to 6 days. Spores obtained from the fungus are suspended in liquid to achieve a 10 4 to 10 7 cfu/ml stock. This stock is directly inoculated into a fermentation medium. The fermentation medium can have an initial pH ranging from 3 to 8 and can contain 10 to 100 g/L of a carbon source (e.g., glucose, sucrose, corn starch, molasses, or soybean oil), 5 to 60 g/L of a nitrogen source (e.g., soybean meal, peptone, or corn steep liquor), 0.5 to 20 g/L of yeast extract, 0.01 to 30 g/L (NH 4 ) 2 SO 4 , 0 to 3 g/L K 2 HPO 4 , 0 to 3 g/L NaCl, 0 to 15 g/L MgSO 4 ·7H 2 O, and/or 0 to 0.3 g/L CaCl 2 . The fungus is grown in the fermentation medium for an additional two to four days. Chitosan can be isolated and purified from fungal mycelia by standard methods. For example, alkaline and acid treatment can be used to isolate chitosan as described in McGahren et al., Process Biochem 19:88-90, 1984. Additional details and procedures for isolating chitosan can be found in European Application No. 0531991 A2; Yokoi et al., J Fermen Bioeng 85:246-249, 1998; U.S. Patent No. 5,232,842,; Rane et al., Food Biotech 7:11-33, 1993; and Hang, Biotech Lett 12:911-912, 1990. S.C. Tan et al (1996) Carbohydr. Polym. 30, 239-242 describe the extraction of chitosan from the Mycelia of fungi of the genus Rhizopus . JP-A-8 013 250 (Abstract, AN 1996-113 203) describe the preparation of a chitosan-chitin based hollow fiber comprising hot alkali treating a Rhizopus genus biomass which is cultured at a pH of 4.0-6.0. In general, the cell mass is separated from the fermentation broth and washed with distilled water. The cells are then treated with 0.5 to 2 N NaOH, and the alkaline mixture incubated at 121°C for 15 minutes. The solid material is then pelleted by centrifugation and washed with distilled water and ethanol. The washed material is treated with a 2% acetic acid solution and incubated at 95°C for 12 hours. The resulting slurry is then isolated by centrifugation, yielding an acid-soluble supernatant (containing chitosan) and an acid-insoluble precipitate (containing chitin). The pH of the supernatant is adjusted to 10 with 2 N NaOH, thereby precipitating out the chitosan. The chitosan is finally washed with distilled water and freeze-dried. The acid-insoluble precipitate is also washed with distilled water and freeze-dried. This acid-insoluble and alkali-insoluble fraction is purified chitin. Without further elaboration, it is believed that one skilled in the art can, based on the above disclosure and the description below, utilize the present invention to its fullest extent. The following examples are to be construed as merely illustrative of how one skilled in the art can practice the invention and are not limitative of the remainder of the disclosure in any way. The results for the following examples are summarized in Table 2, which appears after the Examples. Example 1 Spore suspension of Rhizopus azygosporus from 4-day slant cultures was inoculated directly into 250 ml shaker flasks containing 100 ml of fresh fermentation medium. Fermentation was carried out at 28°C for 48 hours, while shaking at 200 rpm. Each liter of medium contained 10 g of a nitrogen source (soybean meal, peptone, or corn steep liquor), 20 g of a carbon source (glucose or corn starch), 1 g yeast extract, 5 g (NH 4 ) 2 SO 4 , 1 g K 2 HPO 4 , 1 g NaCl, 5 g MgSO 4 ·7H 2 O, and 0.1 g CaCl 2 . The cell mass was recovered from the fermented broth and treated with 1 N NaOH at 121°C for 15 minutes. The alkali-insoluble material was suspended in 2% acetic acid, and the mixture incubated at 95°C for 12 hours to solubilize the chitosan. Chitosan was precipitated by adjusting pH of the acid-soluble supernatant to 10. The chitosan was then washed, dried, and weighed. The acid-insoluble material, chitin, also was washed, dried, and weighed. Yields were calculated in terms of grams chitin or chitosan per liter of culture, and the results are presented in Table 2. The highest yields of chitin and chitosan were achieved using the medium containing corn starch and peptone. Example 2 R. azygosporus was cultured and processed as described in Example 1 above, except that all media contained peptone as the nitrogen source, and the source of carbon was varied among glucose, corn starch, sucrose, molasses, and soybean oil. Inclusion of corn starch led to a high yield of 0.9 g/L for both chitosan and chitin, while inclusion of soybean oil led to the highest yield of 1.5 g/L for chitin for this Example (Table 2). Example 3 R. azygosporus was cultured and processed as described in Example 1, except that each liter of medium contained 30 g corn steep liquor, 50 g glucose, 2 g yeast extract, 2.5 g (NH 4 ) 2 SO 4 , and 0.05 g CaCl 2 . This culture led to a high yield of 1.1 g/L for chitosan (Table 2). The abbreviations used in Table 2 are as follows: "R.a." stands for Rhizopus azygosporus ; "S.M." stands for soybean meal; "C.S.L." stands for corn steep liquor; "C.S." stands for corn starch; and "S.O." stands for soybean oil. All cultures described in Table 2 contained yeast extract. The results in Examples 1-3 above indicated that (1) R. azygosporus is superior producers of chitin and chitosan, and (2) media containing corn steep liquor, glucose, and ammonium sulfate can increase the yield of chitin and chitosan produced by fungi.

Claims: A method of producing chitosan or chitin, the method comprising culturing a Rhizopus azygosporus fungus in a medium to form a culture, and isolating chitosan or chitin from the culture. The method of claim 1, further comprising separating fungal cells from the culture, and wherein the chitosan or chitin is isolated from the separated fungal cells. The method of claim 2, wherein the medium comprises about 5 to 60 g/L corn steep liquor. The method of claim 3, wherein the medium comprises about 30 g/L corn steep liquor. The method of claim 3, wherein the medium further comprises about 10-100 g/L glucose and about 0.01 to 30 g/L ammonium sulfate. The method of claim 5, wherein the medium comprises about 30 g/L corn steep liquor, about 50 g/L glucose, and about 2.5 g/L ammonium sulfate. The method of claim 1, wherein the medium comprises about 5 to 60 g/L corn steep liquor, about 10-100 g/L glucose, and about 0.01 to 30 g/L ammonium sulfate. The method of claim 7, wherein the medium comprises about 30 g/L corn steep liquor, about 50 g/L glucose, and about 2.5 g/L ammonium sulfate.

Inventors: CHEN HING-YUEN, CHEN MEI-HUEI, CHEN YEN-LI, CHUANG SU-HUI, HWANG ING-ER, WU CHIH-LU, CHEN, HING-YUEN, CHEN, MEI-HUEI, CHEN, YEN-LI, CHUANG, SU-HUI, HWANG, ING-ER, WU, CHIH-LU
Applicants: FOOD IND RES & DEV INST, FOOD INDUSTRY RESEARCH AND DEVELOPMENT INSTITUTE
Application year: 2000
Main classification: C12P19
IPC codes: C12P19, C12N1