KR101206855B1 - Method For Obtaining Chitin And Chitosan - Google Patents

Abstract

The present invention relates to a method for obtaining chitin, and more specifically, (a) grinding the shell of shellfish; (b) a decolorizing step of extracting astaxanthin contained in the ground crustacean shell with alcohol; (c) a deliming step of extracting and separating ash by putting the decolored crustacean shell in (b) into an extraction tank containing lactic acid and boiling; (d) a method of obtaining chitin comprising a deproteinization step of obtaining the chitin by injecting boiled crustacean shells into an extraction tank containing potassium carbonate (K ₂ CO ₃ ) and boiling to extract and separate proteins. . In addition, the present invention relates to a method for obtaining chitosan obtained by chitin obtained in the method for obtaining chitin, washed with distilled water and neutralized, and then put into a fermentation tank, and inoculating microorganisms in the fermentation tank to deacetylate to obtain chitosan. will be.

Chitin, Chitosan, Decolorization, Deliming, Deproteinization, Deacetylation

Description

Method for obtaining chitin and chitosan

The present invention relates to a method for obtaining chitin and chitosan, which is environmentally friendly, simple and convenient and can utilize by-product resources.

Chitin is a natural macromolecular substance present in the shells of shellfish such as crabs, crayfish, shrimp, and higher plant cells such as fungi and algae. Chitosan is a substance produced by deacetylation of chitin, and it is known as a substance that does not dissolve well in water depending on the degree of deacetylation, but dissolves in an acidic solution of low concentration. Chitin and chitosan are widely used in various medical fields due to their excellent non-toxicity, environmental friendliness and biocompatibility.In recent years, chitin and chitosan are eco-friendly materials for producing agricultural products. Is emerging as a new gun. As a result, its use in food and pharmaceuticals has been greatly expanded. In addition, it is considered to be a new material that is widely used in new industrial products including eco-friendly films, and is widely used in various fields such as polymer protein or heavy metals to reduce contamination.

Conventionally, the shells of crustaceans could be produced at relatively low cost by using strong acid (hydrochloric acid) and strong base (caustic soda solution), but the facility itself is treated with hydrochloric acid or caustic soda, which are environmentally harmful chemicals. There is a problem that inevitable moisture absorption occurs when neutralizing it to remove the salt. Now, it is considered that it is a step that should apply the process (the technique) that does not cause the environment. However, due to the cost, the eco-friendly alternative method is not widely applied at present, and it has not escaped the application of the existing method.

Reflecting this, the contents of the Ganggu Agricultural Cooperative Dalsan Branch homepage (09-0822) still indicate that it is still using strong acid and strong base. In addition, the use of the red pigment astaxanthin contained in red crab (shell) is not considered at all, and it has been described as oxidizing with a decolorizing agent such as sodium hypochlorite to be decolorized to produce chitin. Neutralization is not described, and the use of salts or strong base caustic soda is not described as neutralizing or desalting, so it is considered to cause environmental pollution. In particular, in the deacetylation process using strong alkali (caustic soda 40-50% aqueous solution), contamination is a significant problem. However, the treatment of enzymes or microorganisms is mentioned, but high concentration caustic soda (NaOH) treatment method is common. It is clear from the description that environmentally friendly treatment methods are not being applied.

Therefore, more environmentally friendly, strong acid or strong base treatment, and a simple and convenient process should be developed. If possible, the red pigment which can be obtained from the crab shell according to the treatment step and the antioxidant astaxanthin and the human body are necessary. There is a need for a technique to increase the profits of utilizing minerals, including calcium, and the proteins that make up the shell. At the same time chitin and chitosan also need a process to obtain the desired quality.

Conventional techniques that use strong acids and strong bases to produce (produce) chitin and chitosan from crustacean shells such as crabs, crayfish or shrimp not only cause environmental pollution, but also have very limited use of by-products other than chitin and chitosan among the extracted materials. It is observed that there is no utilization because of difficulty. Therefore, by using the extracted materials using organic acids and alkalis and microorganisms that can be used for edible use, it can reduce the environmental pollution and use as a resource to generate secondary profits, and obtain higher quality chitin and chitosan, At the same time, it aims to provide a way to use by-products as a resource for the production of food and various industrial products.

One aspect of the invention relates to a method for obtaining chitin, and more specifically, (a) washing, drying and grinding a crustacean shell; (b) a decolorizing step of extracting astaxanthin contained in the shellfish crust crushed in (a) as a spirit; (c) a deliming step of extracting and separating ash by putting the decolored crustacean shell in (b) into an extraction tank containing lactic acid and boiling; (d) a method of obtaining chitin comprising the deproteinization step of obtaining the chitin by injecting a shellfish shell subjected to the deliming step into an extraction tank containing potassium carbonate (K ₂ CO ₃ ) and boiling to extract and separate proteins. .

In the step (a), the shell of crustacean is placed in an extraction tank, and the drinking water is administered so that the contents are submerged. Then, the stirrer is rotated. The mill is then crushed into a preparatory stage to increase efficiency in subsequent operations. The crustacean is not particularly limited, but is mainly intended for shells such as crabs, crabs such as crabs, crawfish, and shrimps.

 In the bleaching step (b), the antioxidant astaxanthin (red pigment) contained in the crustacean shell is first repeated twice with alcohol (80% to 85%, preferably 85%) for 3 to 4 hours, preferably 3 The crustacean shell is decolorized by extraction and separation by time. The alcohol used in the decolorizing operation can be evaporated and recycled under low vacuum, the pigment astaxanthin is concentrated, and a dry powder is prepared as necessary, using red pigment or antioxidant.

In the deliming step of (c), the crustacean shell decolorized in the step (b) is placed in an extraction tank containing lactic acid of 1.5 to 3 times, preferably 2 times of 1.5N to 2.5N, preferably 2 N. Charge and boil it for 12 to 24 hours (18 hours is optimal) to extract and separate ash to demineralize the discolored crustacean shell. The extracted and separated ash can be concentrated to produce calcium lactate, which can be used as a resource.

In the deproteinization step of (d), the shellfish shell demineralized in step (c) is 1.5 to 3 times, preferably 2 times of 1N to 1.5N, preferably 1N of potassium carbonate (K ₂ CO ₃ ). It is put in this extraction tank and boiled for 18 to 36 hours (30 hours is optimal) to extract and separate proteins and the like to obtain chitin. The extracted and separated proteins are concentrated, neutralized with calcium lactate obtained in step (c), and used for preparing crab & shrimp sponge cake or crab meat and shrimp flavor as calcium and protein sources. It can be used as a protein source or by neutralizing with vinegar.

Another embodiment of the present invention relates to a method for obtaining chitosan, and more particularly, the chitin obtained in the method for obtaining chitin, comprising the steps (a) to (d), is washed with distilled water, neutralized and then introduced into a fermentation tank. And inoculating the microorganisms in the fermentation tank to deacetylate to obtain chitosan, and more specifically, the chitin obtained in the method for obtaining chitin, comprising the steps (a) to (d). Washed with distilled water, neutralized and put into a fermentation tank, and then sterilized and microbial culture and deacetylated medium listed therein, and cooled to obtain the microorganisms obtained by the method shown in Example 1 (hereinafter referred to as 'group 1A'). Fermentation at room temperature (25 ° C.) for 4 to 6 days, preferably 6 days, to deacetylate to obtain chitosan.

The liquid medium composition for microbial culture and deacetylation of chitin is as follows.

1) Yeast extract 10.0g

2) Ammonium phosphate 3.0g

3) Magnesium sulfate 2.5g

4) Powder Chitin 5.0g

5) Drinking water 1,000ml

According to the present invention, astaxanthin (red pigment) is extracted and enriched with alcohol (85%) from crustacean shells such as crab, crayfish or shrimp to obtain antioxidant astaxanthin, which can be utilized as a health functional food and food coloring. This allows you to generate new revenue. When this red pigment is mixed into noodle production, yellow noodle is produced, and at high concentration, it can be used as a pigment for crab or shrimp flavor.

In addition, crustacean shells such as crabs, crawfish or shrimp can be demineralized with lactic acid and converted to calcium lactate to utilize it as a calcium source. That is, it can be used as a raw material of calcium-enriched noodles or health functional foods, which can be an additional source of income.

In addition, decolorized and demineralized crustacean shells are treated with potassium carbonate to separate and remove proteins, produce chitin, and neutralize concomitantly obtained proteins to produce egg whites for crab and shrimp sponge cakes and crab meat. It can be used as a substitute to generate new revenue.

In addition, by culturing and deacetylating microorganisms, it eliminates environmental pollution caused by deacetylation using a strong base (caustic soda 40-50%), and incidentally, substances produced by microorganisms, such as single cell proteins, etc. If it finds research, development and its use as a new material, it can be used for food or feed.

1. Preparation

Since it may be a problem to collect red crab and shrimp shells and collect them from a crab or shrimp processing facility to use them as a resource, and to extract the ingredients contained in them, use them as food. Put the drinking water in the shell and heat it to boil (100 ℃) to wash and remove the dirt.Reduce the number of microorganisms, put it in the dryer, dry it at 60 ℃ with hot air, and grind (100 mesh) to extract the contents. Be prepared.

2. Astaxanthin Extract Step-Decolorization Step

30 kg of pulverized powder is placed in the extraction tank (for 500 liter capacity), twice the volume of alcohol (85%) is added and agitated while stirring astaxanthin twice at room temperature for 3 hours. Case), and in a concentration tank, remove all the alcohol by evaporation under low pressure (vacuum) at 40 ° C, continue to concentrate, and mix with astaxanthin (antioxidant) to prepare yellow noodle, or use as red pigment. It can be used to make crabmeat or shrimp flavored products by adjusting and drying it to a suitable concentration.

3. Demineralization Step

30 kg of bleached red crab or shrimp skin is put into an extraction tank containing 2 times lactic acid, and stirred for 18 hours while stirring to extract, isolate and concentrate the ash to produce calcium lactate. The calcium lactate is initially in a liquid state, but gradually solidifies at room temperature to become a solid, so when divided into a clean glass bottle (2L) in the initial liquid state, it can be conveniently used by dissolving it in boiling water and dissolving it when necessary. Do.

The demineralized red crab or shrimp skin is washed with distilled water or water passed through reverse osmosis (R / O) and neutralized. By reducing the weight of the degassing unit, it is possible to increase the efficiency by incorporating 2 ~ 3 times of the process into the deproteinizing operation once.

4. Deproteinization step-obtaining chitin

Collect depigmented and demineralized red crab or shrimp skin (3 times is best) into the extraction tank, add 2 times of 1N potassium carbonate, and stir to extract protein by heating and boiling (100 ℃). Although there is a difference depending on the size, the concentration and amount of the solvent, the rotation speed of the stirrer, etc., most proteins are extracted after 30 hours extraction.

In order to reduce the water content contained in the extracted protein, vacuum concentration at 50 ~ 60 ℃ and, if necessary, can be used in the manufacture of sponge cake replacing the egg white using a bubble generated by neutralizing by reacting with calcium lactate, organic acid For example, the protein can be used as precipitated collagen by neutralizing with vinegar and administering the same amount (or twice) of alcohol (90%).

The red crab or shrimp shell that has been subjected to the above process is born as chitin and washed until it is neutralized with distilled water or water passed through reverse osmosis (R / O). It can be dried and supplied to chitin or used for subsequent deacetylation.

5. Deacetylation Step-Obtaining Chitosan

Conventional methods of using high concentration (40-50%) alkaline solutions to make chitin into chitosan are now unwelcome for environmental pollution. Therefore, in this fifth step, the cultivation of microorganisms (terminal 1A) in a chitin-based medium is responsible for the deacetylation operation. A suitable liquid medium (for a 300 liter, 500 liter extractor) is prepared, sterilized, cooled and inoculated using sterile platinum from a strain tube stored in the first 100 ml medium (500 ml flask). .

The flask is shaken in an incubator and incubated at room temperature (25 ° C.) for 24 hours to increase the turbidity considerably to increase microorganisms. This platinum is inoculated and cultured in a flask containing 200 ml of medium, and at the same time prepared Petri dish (Plate) Inoculation Check whether the microorganisms are pure. In the next step, the inoculation amount is increased, and finally, the inoculation culture is carried out in a 1.000ml culture solution, and all of them are added to the fermenter to promote the culture. Deacetylation is performed simultaneously with incubation while agitating with the valve open to ensure sufficient sterile air. As described above, it was observed that the deacetylation operation simultaneously with the cultivation of microorganisms resulted in 50-75% deacetylation in 5 to 6 days. Continue fermentation when more deacetylation is required, or continue to deacetylate by adding new sterile media nutrients and continuing to use chitin and chitosan in the fermentation tank to achieve the desired deacetylation. It becomes possible.

To check the progress of deacetylation, collect 10 ml of the sample once a day and centrifuge for 12,000 g for 15 minutes to remove the supernatant, and mix well with 10 ml of 0.1 N caustic soda solution in a tube containing precipitated microorganism, chitin and chitosan, and mix well. Heat-treated (autoclave) for 15 minutes and centrifuged again for 15 minutes to remove supernatant (such as cytosol dissolved in alkaline solution) and 10 ml of 0.2% acetic acid solution in a tube containing precipitated chitin, chitosan and microbial polymer. Put it in the shake incubator to dissolve chitosan overnight and shake it completely to dissolve chitosan. This was again centrifuged for 15 minutes and 12,000 g of the supernatant in which the chitosan was dissolved, and the drop was neutralized by dropping 1N caustic soda solution to form a suspension. The increased production of chitosan leaves the white precipitate to stand still. This confirms that chitosan is produced, but in order to confirm more precisely, prepare the same medium composition as a control, but add 100ml of the culture medium without chitin into a 250ml flask, sterilize, cool, inoculate and incubate the microorganisms, and contain the chitin when sampling. When chitin is administered in the same ratio as the culture tank and treated under the same conditions as described above, chitosan production can be more accurately confirmed.

When deacetylation of the culture tank confirms that the progress of the process is more than 75%, the culture solution is separated from the precipitates (microorganisms, chitin, and chitosan) by centrifugation using a continuous centrifuge. As the above-mentioned chitosan identification procedure, the precipitate was poured into 0.1N caustic soda solution (double volume is optimal) and stirred for 15 minutes while stirring (121 ° C) to dissolve and cool the other materials except chitin and chitosan. Remove the supernatant by centrifugation again. In addition, the obtained precipitate was added twice the amount of vinegar or 5% acetic acid solution to the precipitate chitin and chitosan, and stirred for 24 hours to dissolve the chitosan and centrifuge again to separate and remove the precipitated chitin, and neutralize and aggregate the dissolved chitosan with alkaline solution. do. The centrifuged culture solution is sterilized and concentrated to dry the spray dried lumps in a hot plate dryer to use as a single cell protein.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely examples for describing the present invention, whereby the scope of the present invention is not limited.

<Examples>

Example 1-Method of Obtaining Microbial Spawn A

1.5% (15g / 1000ml) of agar was added to the medium containing chitin and sterilized to prepare a solid medium Petri dish. Diluted 10 times and 100 times of salted fish paste (fish sauce), 0.1 ml of each was applied to the plate and inoculated well. Then, 5 kinds of strains were grown in the incubator at 25 ° C. for 2 days, and then streaked in different Petri dishes, and preliminary experiments were performed to select spawn A capable of deacetylating chitin.

Example 2

Put 20g red crab shell powder (80 mesh) and 200 ml alcohol (85%) in a 500 ml Erlenmeyer flask, shake it for 3 hours, shake it for 3 hours, extract the red crab pigment twice, combine it, and decompress it at 40 ℃ with a vacuum rotary concentrator. When concentrated and mixed with 250g of wheat flour, yellow (carotenoid color) resulted, and the noodle containing it was prepared and sampled, which was evaluated as crab flavored crab and confirmed that it could be used for making noodles.

Example 3

In a 2-liter round flask, 50 g of bleached red crab shell and 1.500 ml of 2N lactic acid were added to a heating mantle, and heated and boiled to extract 1,000 ml of ash containing calcium carbonate contained in the crab shell for 18 hours. It was concentrated to a rotary vacuum concentrator 500ml and left overnight at room temperature to give crude calcium lactate as a white solid.

The demineralized crab shell was washed 4 times with 3 liters of purified water with a reverse osmosis (R / O) water purifier, and the pH was neutralized. The dried crab was dried at 60 ° C for 24 hours, and then weighed at 13.25 g (26.5%). 73% of the shell was found to be ash.

Example 4

Into a 2-liter flask, 13.25 g of decolorized and demineralized crab shell (obtained in Example 3) and 1,500 ml of potassium 1N potassium carbonate were added and boiled with an electric heater to extract and remove the protein contained in the crab shell for 30 hours. 12.24 g was obtained by washing, neutralizing and drying four times with 2-3 liters of purified water with a reverse osmosis (R / O) water purifier. Thus, the crab shell contained about 2% (1.0 / 50X100 = 2%) of protein.

The potassium carbonate extract obtained in Example 4 was concentrated with a rotary vacuum concentrator to obtain 500 ml, and a portion of the 20 ml was reacted with 20 g of crude calcium lactate to form a foam and a sponge cake mix containing no egg white (containing 200 g of flour). Mixed well and baked to create a sponge cake that tastes good. The hydrogen gas produced by the acid-base reaction was captured by the protein collagen in the shell of the crab and was interpreted as acting as a white egg protein.

Example 5

The medium as shown in Table 1 was prepared in order to perform the task of deacetylating chitin to chitosan by using a microorganism (selected seed 1A).

Table 1 Composition of microbial medium used to deacetylate chitin

Yeast extract 10g Ammonium phosphate 3.0 g Magnesium Sulfate 2.5 g Powder Chitin  5.0 g drinking water  1,000ml

The culture solution was divided into four 500 ml Erlenmeyer flasks (100 ml each for fermentation without the addition of chitin) and three 200 ml each (for fermentation without the addition of chitin). After sterilization at 121 ° C. for 15 minutes, and then cooled, the first seedlings were inoculated using the sterilized platinum from the strain tube stored in two 500 ml flask medium containing 100 ml first (two experiments at a time). The flask was shaken and placed in an incubator at room temperature (24 hours at 25 ° C. for turbidity to be noticeable, and then grown inoculated in a flask containing 200 ml medium of platinum each, inoculated in a prepared plate at the same time. [To confirm the purity of the culture microorganism] and placed in the incubator and tested at 25 ℃ to confirm that each of the pure spawn.

10 ml each of the microorganism 200 ml of the flask incubated for 2 days was taken, and at this time, chitin was added to the incubated flask without any chitin, mixed well, and 10 ml was taken. 10 ml of 0.1 N caustic soda solution was added to the tube containing precipitated precipitates (cells, chitin and chitosan), mixed well, and then sterilized (autoclave). Organics except chitin and chitosan were dissolved, cooled and centrifuged under the same conditions to remove the precipitated chitin and chitosan free supernatant. 10 ml of 0.2% acetic acid was added to each tube, and the resulting chitosan was shaken for 1 day. The four tubes thus treated were centrifuged again under the above-mentioned conditions, and the supernatant containing chitosan was transferred to four new tubes and neutralized with 1N caustic soda solution. Suspension was remarkable in the tube in which the spawn was incubated and it was confirmed that the white polymer precipitated (agglomerated) after 1 day was chitosan. This was reconfirmed by centrifuging the tube under the conditions described above to dissolve the precipitate in 0.2% acetic acid.

As described above, it will be understood by those skilled in the art that the present invention may be implemented in other specific forms without changing the technical spirit or essential features thereof. The scope of the present invention is shown by the claims to be described later rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. Should be.

Claims (5)

(a) washing, drying, and grinding the crustacean shell; (b) a decolorizing step of extracting astaxanthin contained in the shellfish crust crushed in step (a) as a spirit; (c) the crustacean shell decolorized in step (b) is put into an extraction tank containing 1.5 to 2.5 N lactic acid having a volume of 1.5 to 3 times, boiled for 12 to 24 hours, made into a liquid state, and cooled to room temperature to solid ( Demineralizing the resulting crustacean shell by separating the resulting solid after the formation of ash); And (d) The shellfish shell after the deliming step is put into an extraction tank containing 1.5 to 3 times the volume of 1N to 1.5N potassium carbonate (K ₂ CO ₃ ) and boiled for 18 to 36 hours to make a liquid state to room temperature A process for obtaining chitin, comprising a deproteination step of cooling to form a solid (protein) and then separating and removing the resulting solid to obtain chitin. The method of claim 1, The crustacean is a method for obtaining chitin, characterized in that the crab, crayfish or shrimp. delete delete delete