JP2010162046A - Production of chitosan and chitin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an alternate method for producing chitin and chitosan without depending on chemicals harmful to the environment or a lot of variations of shellfish animal harvests. <P>SOLUTION: There is provided a method for producing chitin or chitosan by culturing a microorganism, Rhizopus azygosporus or Actinomucor taiwanensis, and isolating chitosan or chitin from the culture medium. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to the production of chitosan and chitin.

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 their derivatives are used in many industrial applications including viscosity modifiers, adhesives, chromatographic carriers, paper enhancers, flocculants, food additives, pharmaceuticals and cosmetic products.
[Problems to be solved by the invention]

Chitin can be produced by demineralization and deproteinization of crab or shrimp shells. Chitosan is then obtained by deacetylation of chitin with a hot alkaline solution. This chitosan production process has many undesirable features. For example, it requires expensive thermal energy and caustic that is potentially harmful to health. This process requires significant disposal costs because it also produces large amounts of waste. In addition, the supply of shrimp or crab shells is highly dependent on seasonal and environmental factors, leading to unpredictable production capacity limitations.
[Means for solving problems]

SUMMARY OF THE INVENTION The present invention is based on the discovery that unexpectedly high yields of chitosan and chitin can be produced from Actinomucor taiwanensis and Rhizopus azygosporus.

  That is, the present invention provides (1) a method for producing chitosan or chitin by culturing Rhizopus azygosporus or Actinomucor taiwanensis in a medium, forming a culture, and (2) isolating chitosan or chitin from cells. It is characterized by. For example, chitosan and chitin can isolate fungal cells from the medium and chitosan or chitin from the separated cells.

  The present invention includes (1) culturing fungi of the family Mucoraceae in a medium useful in the method of the present invention, forming a culture, and (2) separating chitosan or tin from the fungal culture. A method for producing chitosan or chitin is also included.

  Media useful for the methods of the present invention include about 5 to 60 g / L (eg, about 30 g / L) corn steep liquor, about 10-100 g / L (eg, about 50 g / L) glucose, about 0.01 to 30 g / L (eg, about 2.5 g / L) of ammonium sulfate or other suitable component may be included.

  The method of the present invention allows surprisingly high yields of chitosan or chitin production from cultures containing Rhizopus azygosporus or Actinomucor taiwanensis. Furthermore, it has been found that media containing corn steep liquor, glucose, yeast extract and ammonium sulfate can enhance the production of chitin and chitosan from fungal cultures. Thus, the method of the present invention provides an alternative method of producing chitin and chitosan without relying on many changes in environmentally harmful chemicals or crustacean animal yields.

  Other features or advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of chitosan and chitin in high yield from a bacterial culture belonging to the family Mucoraceae.

  Specific fungi useful for the method of the present invention include Rhizopus azygosporus and Actinomucor taiwanensis. Both of these organisms are found in the Food Industry and Research Development Institute, Culture Collection and Research Center (Hinchu 300, Shih-Ping Road, No. 331), Taiwan, Taiwan. CCRC)), available upon request. R. azygosporus is available as catalog number CCRC31558 and A. taiwanensis is available as catalog number CCRC31559.

Procedures for culturing fungi are well known in the art. For example, fungi can be planted on YM agar, and the agar planted with fungus can be cultured at 25 ° C. to 37 ° C. for 3 to 6 days. Spores obtained from the fungus are suspended in the liquid to achieve a 10 ⁴ to 10 ⁷ cfu / ml stock. This stock is planted directly in the fermentation medium.

The fermentation medium has a starting pH of 3-8 and a carbon source of 10-100 g / L (eg glucose, sucrose, corn starch, molasses or soybean oil), a nitrogen source of 5-60 g / L (eg soy flour, peptone) Corn steep liquor), 0.5-20 g / L yeast extract, 0.01-30 g / L (NH ₄ ) ₂ SO ₄ , 0-3 g / L K ₂ HPO ₄ , 0-3 g / L. It can contain L NaCl, 0 to 15 g / L MgSO ₄ .7H ₂ O and / or 0 to 0.3 g / L CaCl ₂ . The fungus can grow in the fermentation medium for an additional 2 to 4 days.

  Chitosan can be isolated and purified from fungal spores by standard methods. For example, chitosan can be isolated by alkali and acid treatment as described in McGahren et al., Process Biochem 19: 88-90, 1984. Additional details and procedures for separating chitosan are described in European Application No. 0531991 A1; Yokoi et al., J Fermen Bioeng 85: 246-249, 1998; US Pat. No. 5,232,842; Rane et al. , Food Biotech 7: 11-33, 1993; and Hang, Biotech Lett 12: 911-912, 1990.

  In general, the cell mass is separated from the fermentation broth and washed with distilled water. The cells are then treated with 0.5-2N NaOH and the alkaline mixture is 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 2% acetic acid solution and incubated at 95 ° C. for 12 hours. The resulting slurry is then separated by centrifugation, resulting in an acid-soluble supernatant (containing chitosan) and an acid-insoluble precipitate (containing chitin).

  Chitosan is precipitated by adjusting the pH of the supernatant to 10 with 2N NaOH. Finally, the chitosan is washed with distilled water and lyophilized. Acid insoluble precipitates are also washed with distilled water and lyophilized. This acid-insoluble and alkali-insoluble fraction is purified chitin.

  Without further elaboration, one of ordinary skill in the art believes that the present invention may be utilized to its fullest extent based on the above disclosure and the following description. The following examples should be construed as merely illustrative of how one skilled in the art can practice the invention, and are not intended to limit the disclosure other than the examples in any way. Should be understood.

  The results of the following examples are summarized in Table 2, which is described after the examples.

Example 1
The spore suspension of Rhizopus azygosporus from the 4-day slant culture was planted directly into a 250 ml shake flask containing 100 ml of fresh fermentation medium. Fermentation was performed at 28 ° C. for 48 hours with shaking at 200 rpm. 10 g nitrogen source (soy flour, peptone or corn steep liquor), 20 g carbon source (glucose or corn starch), 1 g yeast extract, 5 g (NH ₄ ) ₂ SO ₄ , 1 g K ₂ per liter of medium. It contained HPO ₄ , 1 g NaCl, 5 g MgSO ₄ .7H ₂ O and 0.1 g CaCl ₂ .

  Cell mass was recovered from the fermentation broth and treated with 1N NaOH at 121 ° C. for 15 minutes. The alkali insoluble material was suspended in 2% acetic acid and the mixture was incubated at 95 ° C. for 12 hours to solubilize chitosan. Chitosan was precipitated by adjusting the pH of the acid-soluble supernatant to 10. The chitosan was then washed, dried and weighed. Chitin, an acid insoluble material, was also washed, dried and weighed. Yields were calculated in terms of grams of chitin or chitosan per liter of culture and the results are shown in Table 2. The highest yield of chitin and chitosan was achieved using a medium containing corn starch and peptone.

Example 2
Spore suspensions of Actinomucor taiwanensis from 4-day slant cultures were planted directly into 250 ml shake flasks containing 100 ml of fermentation medium. Fermentation and separation of chitosan and chitin were performed as described in Example 1 and the results are shown in Table 2. High yields of chitin and chitosan were achieved by using a medium containing corn steep liquor and glucose.

Example 3
R. azygosporus was cultured and treated as described in Example 1 above. However, all the media contained peptone as the nitrogen source, and the carbon source was changed from glucose, corn starch, sucrose, molasses and soybean oil. In this example, corn starch uptake led to a high yield of 0.9 g / L for both chitosan and chitin, while soy oil uptake led to the highest yield of 1.5 g / L of chitin (Table 2).

Example 4
A. taiwanensis was cultured and treated as described in Example 2 above. However, all media contained corn steep liquor as a nitrogen source, and the carbon source was varied from among glucose, corn starch, sucrose, molasses and soybean oil. In this example, incorporation of glucose as a carbon source led to the best yield for both chitosan and chitin (Table 2).

Example 5
R. azygosporus was cultured and treated as described in Example 1 above. However, 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).

Example 6
A. taiwanensis was cultured and treated as described in Example 2 above. However, 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 the highest combined yield of chitosan (1.7 g / L) and chitin (1.1 g / L) (Table 2).

Example 7
A. taiwanensis was cultured and treated as described in Example 6. However, instead of CaCl ₂ , 0.5 g of K ₂ HPO ₄ was contained per liter of the medium. This culture led to a high yield of 1.4 g / L for chitosan.

  Abbreviations used in Table 2 are as follows: “Ra” means Rhizopus azygosporus; “At” means Actinomucor taiwanensis; “SM” means soy flour “C.S.L.” means corn steep liquor; “C.S.” means corn starch; “S.O.” means soybean oil. All media listed in Table 2 contained yeast extract.

  The results of Examples 1-7 above are: (1) A. taiwanensis and R. azygosporus are superior products of chitin and chitosan, and (2) a medium containing corn steep liquor, glucose and ammonium sulfate This shows that the yield of chitin and chitosan production by fungi can be increased.

Other Embodiments While the invention has been described in connection with a detailed description thereof, the foregoing description is intended to be illustrative and is within the scope of the invention as defined by the appended claims. It should be understood that it is not intended to limit the category. Other aspects, advantages, and modifications are within the scope of the invention.

Claims (20)

  A method of producing chitosan or chitin comprising culturing Rhizopus azygosporus bacteria in a medium, forming a culture, and separating chitosan or chitin from said culture.   2. The method of claim 1, further comprising separating fungal cells from the culture, wherein chitosan or chitin is separated from the separated fungal cells.   The method of claim 2, wherein said medium contains about 5 to 60 g / L corn steep liquor.   4. The method of claim 3, wherein the medium contains about 30 g / L corn steep liquor.   4. The method of claim 3, wherein the medium further comprises about 10-100 g / L glucose and about 0.01-30 g / L ammonium sulfate.   6. The method of claim 5, wherein the medium contains 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 contains about 5 to 60 g / L corn steep liquor, about 10 to 100 g / L glucose, and about 0.01 to 30 g / L ammonium sulfate.   8. 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.   A method for producing chitosan or chitin comprising culturing Actinomucor taiwanensis bacteria in a medium, forming a culture, and separating chitosan or chitin from said culture.   10. The method of claim 9, further comprising separating fungal cells from the culture, wherein chitosan or chitin is separated from the separated fungal cells.   11. The method of claim 10, wherein the medium contains about 5 to 60 g / L corn steep liquor.   12. The method of claim 11, wherein the medium contains about 30 g / L corn steep liquor.   12. The method of claim 11, wherein the medium further comprises about 10-100 g / L glucose and about 0.01-30 g / L ammonium sulfate.   14. The method of claim 13, wherein the medium comprises about 30 g / L corn steep liquor, about 50 g / L glucose, and about 2.5 g / L ammonium sulfate.   10. The method of claim 9, wherein the medium contains about 5 to 60 g / L corn steep liquor, about 10 to 100 g / L glucose, and about 0.01 to 30 g / L ammonium sulfate.   16. The method of claim 15, wherein the medium comprises about 30 g / L corn steep liquor, about 50 g / L glucose, and about 2.5 g / L ammonium sulfate. In a method for producing chitosan or chitin, the method comprises:
Culturing a fungus of the family Mucoraceae in a medium to form a culture, the medium comprising about 5 to 60 g / L corn steep liquor, about 10 to 100 g / L glucose, and about 0.01 A method for producing chitosan or chitin comprising a step of containing 30 g / L of ammonium sulfate and a step of separating chitosan or chitin from the bacterial culture.   18. The method of claim 17, wherein the medium contains about 30 g / L corn steep liquor.   The method of claim 17, wherein the medium contains about 50 g / L glucose.   18. The method of claim 17, wherein the medium contains about 2.5 g / L ammonium sulfate.