BACKGROUND OF THE INVENTION

The present invention relates to the art of providing purified chitin compositions, and in particular, to deriving highly purified chitin compositions from krill shells.

The biopolymer chitin or poly (β-(1→4)-N-acetyl-d-glucosamine) is a natural polysaccharide of major importance. Chitin is synthesized by myriad living organisms and is one of the most abundant of natural polymers, after cellulose. In its native state, chitin occurs as ordered crystalline microfibrils which form structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast. The main commercial sources of chitin are crab and shrimp shells. In industrial processing, chitin is extracted by acid treatment to dissolve the calcium carbonate followed by alkaline solution to dissolve proteins. Typically, a decolorization step is added. These treatments must be adapted to the chitin source, due to differences in the initial material, to produce a chitin product, and then, after deacetylation, a chitosan product. The lack of solubility of chitin is a major problem in its processing, use and characterization.

Since chitin is a natural polymer, as well as biocompatible and biodegradable in the body, it can be used for biomedical and pharmaceutical applications. Examples include drug release and wound dressing. Additionally, good film forming properties of chitin may be valuable for biofilms, artificial skin and packaging.

In the past methods to perform enzymatic hydrolysis of Antarctic krill has been described. It has been shown that krill shells can be separated from the hydrolyzed matrix of krill. However, the shells are not processed further. Due to the water content, the products are not microbiologically stable and have a low value. Typically, they are discarded after production. There is a need to utilize all the fractions from krill from an economical and environmental perspective.

Krill shells have in the past also been defatted and used for extraction of chitin. The yield of chitin product obtained from defatted krill shell was 27.80±1.48%. This results in a low overall yield and poor commercial potential.

However, up to now, a reproducible quality of chitin has been difficult to obtain. The lack of standardized product has inhibited the full potential of therapeutic applications of chitin, in particular, in the biomedical and/or pharmaceutical fields.

Therefore, there remains a need for improved methods to isolate chitin from krill in a cost-effective, fast and easily controllable industrial process for producing chitin having high purity, high crystallinity index, and high molecular weight.

SUMMARY OF THE INVENTION

The present invention provides production methods by which the properties of chitin compositions derived from krill are reproducible. For example, properties, such as molecular weight (chain length) and degree of acetylation are reproducible. Such reproducible production methods allow for a wide use of the chitin composition end products in pharmaceutical and biomedical applications. The methods also allow for the production of chitin compositions which preserve the native state of chitin.

One aspect of the invention relates to a method of producing a chitin composition wherein deacetylation and depolymerization of krill chitin is minimized, the method including: a) deproteinizing a krill product to provide a deproteinized krill product; and b) isolating the chitin composition, wherein the chitin composition is at least about 30% to about 99% acetylated, and wherein the average molecular weight of the krill chitin is about 300 kDa to about 800 kDa, about 400 kDa to about 700 kDa, or about 400 kDa to about 600 kDa. Preferably, the krill product comprises whole krill. The method may preferably further include demineralizing the krill product to provide a demineralized krill product. The demineralizing step may be performed before the deproteinizing step.

The chitin composition may have a Crystallinity Index (CI) of greater than about 80%, greater than about 85%, or greater than about 90%. The average molecular weight of the krill chitin may be about 400 kDa to about 600 kDa. In one preferred embodiment, the chitin composition has a crystallinity index (CI) between about 85% and about 95%, and the average molecular weight of the krill chitin is about 400 kDa to about 600 kDa. In a preferred embodiment, the chitin composition is at least 90% homogenous.

Another aspect of the invention relates to a bioactive krill-chitin composition prepared by the process including the steps of: a) deproteinizing a krill product to provide a deproteinized krill product; and b) isolating the bioactive krill-chitin composition, wherein the chitin composition is at least about 95% acetylated and wherein the average molecular weight of the chitin composition is about 300 kDa to about 800 kDa, about 400 kDa to about 700 kDa, or about 400 kDa to about 600 kDa.

The bioactive krill-chitin composition may also be prepared with an additional demineralizing step where the krill product is demineralized to provide a demineralized krill product. In a preferred embodiment, the bioactive krill-chitin composition has a Crystallinity Index (CI) of greater than about 80%.

Another aspect of the invention relates to a method of producing a chitin composition, the method including: a) a first enzymatic hydrolysis of a krill product to produce hydrolyzed krill; b) separating the solid portion of the hydrolyzed krill; c) chemical deproteinization and chemical demineralization of the solid portion to produce a chitin composition; and d) providing a chitin composition comprising i) crystallinity index (CI) is greater than about 85%; ii) a purity of greater than about 80%; and iii) average molecular weight of about 300 kDa to about 700 kDa.

In one embodiment, the first enzymatic hydrolysis including contacting with a metalloprotease. In another embodiment, the first enzymatic hydrolysis further includes contacting with a first protease mixture. In a preferred embodiment, the first enzymatic hydrolysis includes contacting with a first protease mixture; and contacting with a second protease mixture. In another preferred embodiment, the method includes a second enzymatic hydrolysis including contacting with a first protease mixture. The first enzymatic hydrolysis may include one or more of acid protease, neutral protease, and alkaline proteases.

The first protease mixture may include at least one alkaline protease; and the second protease mixture may include an acid protease, a neutral protease, and an alkaline protease. The first protease mixture may include 0.3-0.5% of the total weight of the disintegrated crustacean catch; and the second protease mixture may include 0.03-0.05% of the total weight of the disintegrated crustacean catch. In a preferred embodiment, the proteases are fromBacillus licheniformis. The first enzymatic hydrolysis may include contacting with a cell wall degrading enzyme. Preferably, the chemical deproteinization occurs before the demineralization.

Preferably, the solid portion of the hydrolyzed krill includes between about 10% and about 25% lipids, and between about 35% and about 55% protein. Also, preferably, the solid portion of the hydrolyzed krill includes between about 13% and about 18% lipids, and between about 40% and about 50% protein.

Chemical deproteinization may include exposing the solid portion to NaOH. The NaOH may have a molar concentration of about 1M to about 4M. Chemical deproteinization may occur at about 80° C. Chemical deproteinization may last about 1 to about 7 days or about 1 to about 4 days.

Demineralization may include exposing the solid portion to HCl. The HCl may have a molar concentration of about 1M. Demineralization may occur at about 95° C. Demineralization may last about 30 minutes.

In any of the processing steps, it is preferred that the temperature does not exceed 60° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., or 100° C.

The preferred crystallinity index (CI) of the chitin composition is greater than about 85%, greater than about 90%, or greater than about 95%. Preferably, the crystallinity index (CI) of the chitin composition is between about 85% and about 95%.

Preferably, the chitin composition includes a total amount of trace minerals of less than 5 ppm, less than 1 ppm, and less than 0.1 ppm. Preferably, the individual amounts of trace minerals include less than 5 ppm, less than 1 ppm, and less than 0.1 ppm. The trace minerals may include iron, calcium, phosphorus, magnesium, potassium, zinc, nickel, selenium, and copper.

Preferably, the chitin composition is at least 90%, at least 95%, or at least 99% pure. Also, preferably, the chitin composition is at least 90% homogenous.

Another aspect of the invention relates to a chitin composition having a crystallinity index of greater than 80%; a molecular weight of less than 700 kDa; and a purity of greater than 90%. Preferably, the chitin composition is a krill chitin composition.

The chitin composition preferably has a crystallinity index (CI) greater than about 85%, greater than about 90%, or greater than about 95%. Preferably, the chitin composition has a crystallinity index (CI) between about 85% and about 95%.

Also, preferably, the chitin composition is at least 90%, at least 95%, or at least 99% pure. Most preferably, the chitin composition is substantially free of impurities. Impurities may include proteins, lipids, trace minerals, and combinations thereof. Substantially free means containing less than 10%, less than 5%, or less than 1% impurities. In a preferred embodiment, the chitin composition includes a total amount of trace minerals of less than 5 ppm, less than 1 ppm, and less than 0.1 ppm. Preferably, the individual amounts of trace minerals comprise less than 5 ppm, less than 1 ppm, and less than 0.1 ppm. The trace minerals may include iron, calcium, phosphorus, magnesium, potassium, zinc, nickel, selenium, and copper.

In a preferred embodiment, the chitin composition includes less than 10% protein, less than 5% protein, less than 1% protein, or less than 0.1% protein. In another preferred embodiment, the chitin composition includes less than 10% lipid, less than 5% lipid, less than 1% lipid, or less than 0.1% lipid.

Preferably, the chitin composition comprises an average molecular weight of between about 300 kDa to about 800 kDa, about 400 kDa to about 700 kDa, or about 400 kDa to about 600 kDa.

Another aspect of the invention relates to a chitin composition having a crystallinity index of between about 80% and about 90%; an average molecular weight of between about 400 kDa and about 600 kDa; and a purity of between about 90% and about 98%.

