KR100552315B1 - method for producing ?-chitosan using cartilage of cuttlefish - Google Patents

Abstract

The present invention relates to a method for producing β-chitosan using squid cartilage, and more particularly, an immersion step of removing salt by immersing squid cartilage; Hot water extraction process to put the squid cartilage into the reactor for 60 to 120 minutes at a temperature of 115 ~ 140 ℃ and a pressure of 0.5 ~ 1Mpa; An enzyme extraction step of adding water to the reactor remaining only after the residue and then adding alkalase or proteinase of pH 4-12, or mixing alkalase and proteinase and reacting at 20 to 65 ° C; A deacetylation process of filtering out the solid residue and placing it in another reactor and applying 35-50% sodium hydroxide solution for 60-120 minutes at a temperature of 120-140 ° C. and a pressure of 0.1-0.2 Mpa; and washing with running water. By washing with water and drying process to remove protein using hot water extraction and enzyme extraction, thereby providing high purity β-chitosan without destroying the structure of β-chitosan or denatured protein during deproteinization process. By depressurizing at high temperature in the deacetylation process, 90% or more deacetylation degree can be obtained in one step alone, and the high purity and high molecular weight β-chitosan in a short time and the high purity β-chitooligosaccharide having a content of 90-93% Can be provided.

β-chitosan, β-chitooligosaccharide

Description

Method for producing β-chitosan using cuttlefish cartilage

1 is a flow chart schematically showing a method for producing β-chitosan using squid cartilage according to the present invention,

2 is a flowchart schematically showing a process of preparing β-chitooligosaccharides from β-chitosan prepared according to the present invention;

Figure 3 is a Fourier FT-IR (Fourier transfer infrared spectroscopy) chromatogram of β-chitosan prepared according to the method for producing β-chitosan using the squid cartilage of the present invention,

4 is an FT-IR chromatogram of α-chitosan,

5a, 5b and 5c is a figure showing the anticancer activity of β-chitooligosaccharide according to the present invention,

Figure 6 is a figure showing the antimicrobial activity of β-chitooligosaccharide according to the present invention.

The present invention relates to a method for producing beta-chitosan using squid cartilage, and more specifically, to squid cartilage by hydrothermal extraction and enzyme extraction to deproteinized and deacetylated to make beta-chitosan and then to the beta-chitosan. The present invention relates to a method for producing high purity and low molecular weight β-chitooligosaccharides by further enzymatic digestion step.

Chitin is a saccharide that is distributed in large quantities in shells of shellfish such as crabs, shrimps, lobsters, insect shells and fungal hyphae. It contains more than 5,000 residues called N-acetyl-D-glucosamine and has a molecular weight of 100. It is more than 10,000 carbohydrates. Chitin is a very useful substance, but because of its high molecular weight, strong structure, and difficulty in dissolving and dispersing, chitin is used in various states, and chitosan and chitooligosaccharide are examples.

Chitosan is a high-molecular substance obtained through the process of deacetylation of chitin. Unlike chitin, chitosan is known as a positive polymer electrolyte that is well soluble in dilute hydrochloric acid or organic acid solution and insoluble in neutral or alkaline aqueous solution. It is widely used in medicine and industrial goods.

In general, chitosan, a cationic polysaccharide having 5,000 or more glucosamine bound, functions and effects differently depending on the degree of binding. Since chitosan polymer is difficult to be absorbed and used in the body, it is decomposed by an enzyme to bind 2 to glucosamine. It is made of 10 chitosan oligosaccharides. Chitooligosaccharide is a food material having functions such as antibacterial action and liver function improvement, and is widely used in the food and pharmaceutical industries.

Chitin and chitosan are divided into α-type, β-type, and γ-type according to their structure, and α-chitin and α-chitosan are prepared from shells of crabs, shrimps and crayfish, and β- from squid cartilage. Chitin and β-chitosan are prepared, and γ-chitin and γ-chitosan are produced from the bark of longevity sky cow. Until now, research has been mainly focused on α-chitin and α-chitosan. However, since it is known that β-chitin and β-chitosan have excellent physiological functions, studies on this are being actively conducted.

Conventionally, the production of β-chitosan, acid treatment of the shell of crab, shrimp, crayfish, etc. to remove minerals such as calcium, washing with water and then neutralizing and washing with an alkali treatment solution to remove the protein, and then highly concentrated sodium hydroxide solution The conventional method for producing α-chitosan, which is deacetylated by applying, was modified. However, the β-chitosan obtained in this way has a low purity, the structure of β-chitosan is destroyed during the manufacturing process, and the physical and chemical properties peculiar to β-chitosan are changed.

On the other hand, deacetylation of β-chitin produced by deproteinization from squid cartilage has been possible by using a vacuum or nitrogen that has been developed in the past. In other words, the conventional method of reacting with the aqueous base solution under vacuum conditions only needs to be repeated three or more times to obtain a deacetylation degree of only 94%, and the method of reacting with the basic aqueous solution under nitrogen conditions should be repeated two to three times. Only 90% of deacetylation degree was obtained. As the reaction time and the number of reactions were increased, the purity of β-chitosan produced was lowered and a natural polymer could not be obtained. Therefore, a method of reducing the reaction time and the number of reactions was needed.

In addition, 70-75% of α-chito-oligosaccharides have been produced and marketed, but high-purity β-chito-oligosaccharides have not been produced.

The present invention has been made to solve the above-mentioned conventional problems, by removing the protein using hot water extraction and enzyme extraction, β-chitosan structure is not destroyed or protein denaturation during the deproteinization process and high purity β Its purpose is to provide chitosan.

It is still another object of the present invention to obtain a deacetylation degree of 90% or more in a single step by high temperature pressurization in a deacetylation process, thereby providing β-chitosan and high-purity β-chito-oligosaccharides that have been highly purified and polymerized in a short time. will be.

Method for producing β-chitosan using squid cartilage according to the present invention as a means for achieving the above object, an immersion step of removing salt by immersing squid cartilage in fresh water; A hot water extraction step of putting the salt-free squid cartilage into a reactor and reacting for 60 to 120 minutes at a temperature of 115 to 140 ° C. and a pressure of 0.5 to 1 Mpa; After completion of the hot water extraction process, water is added to the reactor remaining only a solid residue, and the enzyme is reacted at 20 to 65 ℃ by mixing alkalase or proteinase of pH 4-12, or alkalase and proteinase Extraction process; After the enzyme extraction process, the solid residue is filtered and put into another reactor to apply a 35 to 50% sodium hydroxide solution and reacted for 60 to 120 minutes at a temperature of 120 to 140 ℃ and a pressure of 0.1 to 0.2 Mpa And a water washing and drying step of washing with water flowing after the deacetylation step and drying.

In addition, in the method for producing β-chitosan using the squid cartilage of the present invention, 20-30% hydrogen chloride solution is added to the above-mentioned solid state β-chitosan and stirred to form a liquid β-chitosan solution, and β- An enzymatic reaction step of adding 1,000 units of chitosanase per 1 kg of chitosan to react at 40 to 50 ° C. for 16 to 20 hours; Ultrafiltration with molecular weight MWCO 10,000 dalton to remove unreacted substances and enzymes in the enzyme reaction; Neutralizing by adding sodium hydroxide to remove hydrogen chloride used for dissolution of the β-chitosan; Desalting and concentrating the neutralized solution by reverse osmosis 2 to 3 times with a molecular weight of MWCO 200 dalton; And further comprising the step of drying the concentrated solution characterized in that the β-chitooligosaccharide is produced.

In addition, in the method for producing β-chitosan using the squid cartilage of the present invention, the pH of alkalase and proteinase used in the enzyme extraction process is characterized in that it is adjusted to electrolyzed water.

Hereinafter, the method for producing β-chitosan using the squid cartilage according to the present invention will be described in detail.

First, squid cartilage is immersed in fresh water to remove salt. Squid cartilage is supplied in a dried state. The dried squid cartilage also contains salt of seawater, so that the salt is removed by immersion in fresh water for about 24 hours.

The squid cartilage from which the salt is removed is subjected to hot water extraction for primary protein removal. When the squid cartilage is put in the hot water, the bone becomes muggy and the porous is formed to facilitate the penetration of enzymes into the squid cartilage. The temperature in the hot water extraction process is 40 to 80% at 115 ° C. or less, and the reaction is better as the temperature and time are reduced, but the protein may be denatured at a temperature above 140 ° C. The pressure varies depending on the temperature, but if the pressure is too high, the reactor itself may be unreasonable. Therefore, the hot water extraction process in the present application is preferably reacted at a temperature of 115 to 140 ° C. for 60 to 120 minutes under a pressure of 0.5 to 1 Mpa. .

The hot water extracted water is removed by a filter, leaving only the residue that is a solid in the reactor.

The squid cartilage from which the protein is first removed by the hydrothermal extraction process is subjected to enzyme extraction to remove the secondary protein. Alkalase and protinase may be used as proteolytic enzymes used in the enzyme extraction process of the present application. The alkalase and proteinase may be used either individually or in combination of two enzymes at a temperature of 20-65 ° C. and a pH of 4-12.

In the case of using the alkalase as a proteolytic enzyme in the enzyme extraction process, it is preferable to react at a temperature of 35-65 ° C. and pH 5-9, and when using proteases, a temperature of 20-60 ° C. It is most preferable to make it react within the range of pH4-12. In addition, when a mixture of alcalase and proteinase is used, it is preferable to adjust the pH to the optimum pH 8 where the activity of both enzymes is best.

Generally, hydrochloric acid or sodium hydroxide is used as a method for adjusting the pH in a chemical reaction. However, if the pH is adjusted using hydrochloric acid or sodium hydroxide, hydrochloric acid or sodium hydroxide remains and is harmful to the human body. Therefore, it is important here to adjust the pH of the enzymes with electrolyzed water. That is, in the present application, since the pH of the alkalase and proteinase is adjusted using acidic electrolytic water and alkaline electrolyzed water obtained by electrolyzing water, it is harmless to the human body.

In the enzyme extraction step, it is preferable to add 100% by weight of water to 30% by weight of squid cartilage and add 0.5 to 2% by weight of enzyme based on the weight of the squid cartilage.

When the protein component is removed through the hot water extraction and enzyme extraction process, a solid residue remains, which is β-chitin.

The solid residue is filtered off and placed in another reactor to deacetylate by applying sodium hydroxide solution. At this time, when the sodium hydroxide solution of 35% or less is applied, the deacetylation reaction does not occur, and when the sodium hydroxide solution is applied at 50% or more, the deacetylation reaction occurs as the concentration of the sodium hydroxide solution increases, but the molecular chain is broken. And the production cost is high. Unlike α-chitosan, in which a deacetylation reaction occurs when the concentration of sodium hydroxide solution is 50% or more, β-chitosan reacts when it is 35% or more. In this application, it is preferable to apply 35-50% of sodium hydroxide solution.

In the deacetylation process of the present application, it is important to increase the purity in a short time. When the temperature condition in the deacetylation process is 80 ° C. or more, the reaction occurs, but it takes a lot of time, and as the reaction time increases, the polymer is decomposed into low molecules rather than pure chitosan in a pure polymer state. Therefore, in the deacetylation process of the present application, it is preferable to react the temperature at 120 to 140 ° C. for 60 to 120 minutes so that the deacetylation reaction can be performed in a pure polymer state in a short time.

In the deacetylation process, the pressure condition is preferably maintained at a pressure of 0.1 to 0.2 Mpa to protect the reactor from being broken and to prevent chitosan from being broken down into low molecules.

In the deacetylation process of the present invention described above, by depressurizing to a high temperature, the deacetylation degree of 90% or more can be obtained by only one process, thereby increasing the purity in a short time and producing polymerized β-chitosan.

The residue from which the acetyl group was removed by the deacetylation process was washed with running water and dried to less than 10% of water content. At this time, the drying method used may be a conventional drying method such as freeze drying, hot air drying.

As described above, β-chitosan is completed.

On the other hand, the method of producing β-chito-oligosaccharide by enzymatic decomposition of the β-chitosan in the solid state is as follows.

Hydrogen chloride solution of 20-30% concentration is added to the generated β-chitosan, followed by stirring to dissolve. 1,000 unit of chitosanase per 1 kg of β-chitosan was added to the dissolved β-chitosan solution, and reacted at a temperature of 40-50 ° C., which is an optimum activity temperature of chitosanase, for 16-20 hours. The enzymatic reaction of chitosanase breaks the intermolecular bond of the polymer, β-chitosan, resulting in a low molecule having an average molecular weight of about 3,000 to 5,000.

Ultrafiltration of the low molecular weight β-chitosan solution by the enzyme reaction to the molecular weight MWCO 10,000 dalton (ultrafiltration) to remove the unreacted reactants and the enzyme having a molecular weight of more than about 30,000 dalton in the enzyme reaction.

Sodium hydroxide is added to the ultrafiltered β-chitosan solution to neutralize the hydrogen chloride used to dissolve the β-chitosan. In other words, Cl ⁻ and Na ⁺ of the hydrogen chloride react with each equivalent to lose their properties as acids and bases and are neutralized.

The neutralized solution is filtered by repeating 2-3 times in reverse osmosis. By repeating the reverse osmosis filtration two to three times, molecules of MWCO 200 dalton or less, that is, water and sodium chloride, are released and removed. Reverse osmosis removes at least 95% of sodium chloride and concentrates to 30 Brix%.

The concentrated solution is dried. At this time, the drying method used may be a conventional drying method such as freeze drying, spray drying.

As described above, β-chito-oligosaccharides having a content of 90 to 93% are completed.

To date, 70-75% of α-chito-oligosaccharides have been produced and are commercially available, but 90-93% of high-purity β-chito-oligosaccharides have not been produced.

Hereinafter, the method for preparing β-chitosan and β-chito-oligosaccharide using the squid cartilage of the present invention as described above will be described in detail with reference to Examples.

Example 1

β - Chitosan

Dried squid cartilage was put in fresh water to soak for 24 hours to remove salt. The salted squid cartilage was placed in a reactor and reacted at 130 ° C. for 90 minutes under a pressure of 0.5 Mpa to remove primary protein components by hot water extraction. The hydrothermal extracted squid cartilage became muggy and formed porous on its surface.

Remove the hot water extracted water and put the water of about three times the mass of the residue back to the reactor remaining only the solid residue, and 2 weight of the alcalase adjusted to pH 8 using electrolysis water based on the residue mass % Was added and the enzyme was reacted at 55 ° C to remove the secondary protein component. The enzyme penetrated through the pores formed on the surface of the squid cartilage, the enzyme reaction was well.

Three reactors were prepared, and each of the deproteinized squid cartilage residue and 35%, 40%, and 45% sodium hydroxide solution were added thereto, and the temperature of 120 ° C, 130 ° C, 140 ° C and 0.1Mpa, 0.15Mpa , 0.2Mpa pressure was set to react for 90 minutes to determine the degree of deacetylation (see Table 1 and Table 2).

Table 1 Deacetylation degree according to temperature and concentration of sodium hydroxide (pressure: 0.15Mpa)

Table 2: Deacetylation degree (NaOH 40%) according to temperature and pressure

Table 1 and Table 2 are the results of one reaction.

It can be seen that the conventional method of reacting with the base aqueous solution under vacuum conditions and the method with the basic aqueous solution under nitrogen conditions shows superior deacetylation effects when compared with the one which shows up to 60% deacetylation degree in one reaction. have.

Through the deacetylation process, the residue of the squid cartilage from which the acetyl group was removed was washed with running water, and then lyophilized to less than 10% of water to prepare β-chitosan according to the present invention.

The β-chitosan prepared according to the present invention was measured by Fourier transform infrared spectroscopy (FT-IR) and the chromatogram was compared with the conventional FT-IR chromatogram of α-chitosan (see FIGS. 3 and 4).

Figure 3 was able to confirm that the material prepared according to the present invention in the above embodiment is β-chitosan, unlike the α-chitosan of Figure 4 where a lot of peaks in the range of 1650 cm ^-1 can be seen that one peak appears there was.

As a result, it can be seen that β-chitosan prepared according to the present invention does not occur intermolecular hydrogen bonds or intramolecular hydrogen bonds due to its structural specificity, and thus its peak appears singly. On the other hand, it can be seen that α-chitosan has various peaks due to intermolecular or intramolecular hydrogen bonding. Eventually, β-chitosan participates in external reactions without the intra-molecular and intramolecular bonds of the functional groups, whereas α-chitosan does not participate in the external reactions due to the hydrogen bonds between the intramolecular and molecular molecules. It is significantly lower than that.

Example 2

β Quitooligosaccharides

Solid β-chitosan and 25% hydrogen chloride solution prepared in Example 1 were added to the reactor and stirred to form a liquid β-chitosan solution.

1,000 unit of chitosanase was added to 1 kg of the β-chitosan solution, followed by enzymatic reaction at 45 ° C. for 18 hours. After enzymatic reaction, the intermolecular bonds of β-chitosan, a polymer, were broken, resulting in low molecular weight and low molecular weight.

The low molecular weight β-chitosan solution obtained by the enzyme reaction was ultrafiltered with a molecular weight of MWCO 10,000 dalton to remove unreacted material and an enzyme having a molecular weight of about 30,000 dalton or more. Sodium hydroxide was added thereto to neutralize the hydrogen chloride used to dissolve the β-chitosan.

The neutralized solution was repeated three times with a molecular weight of MWCO 200 dalton, reverse osmosis to remove sodium chloride and concentrated to 30 Brix%. The concentrated solution was lyophilized to produce 93% β-chitooligosaccharides. By removing the salts contained in the β-chitooligosaccharides, the content was improved to 90% or more and the side reactions that could occur in the human body due to the salts present were minimized.

Experiments on growth inhibition or elimination of gastric cancer cells (SNU-601, SNU-719) and hepatocellular carcinoma cells (Hep G2) of β-chitooligosaccharide according to the present invention made as described above and the results are shown in Figures 5a, 5b and It is shown in Figure 5c.

In addition, the effect of killing or growth inhibition on E. coli, P. aeruginosa, spore-forming bacteria, yeast, and fungi of β-chito-oligosaccharide according to the present invention was tested and the results are shown in FIG.

As can be seen in Figures 5a, 5b, 5c and 6, β-chito-oligosaccharides have an antimicrobial activity of about 2 to 6 times better than α-chito-oligosaccharides, and anticancer activity is 20 to 50% more excellent. As a result of the experiment, it was found that it can be widely used as a functional food material and a pharmaceutical material.

As described above, the method for producing β-chitosan using the squid cartilage of the present invention removes the protein using hot water extraction and enzyme extraction, so that the structure of β-chitosan is not destroyed or protein denaturation occurs during deproteinization. It can provide high β-chitosan.

In addition, the method for producing β-chitosan using the squid cartilage of the present invention can obtain 90% or more of deacetylation degree by only one step by high temperature pressurization in a deacetylation process, and the high purity and high molecular weight of β-chitosan in a short time. It is possible to provide a high purity β-chitooligosaccharide having a content of 90 to 93%.

Claims (3)

An immersion process of removing salt by dipping squid cartilage into fresh water; A hot water extraction step of putting the salt-free squid cartilage into a reactor and reacting for 60 to 120 minutes at a temperature of 115 to 140 ° C. and a pressure of 0.5 to 1 Mpa; After completion of the hot water extraction process, water is added to the reactor remaining only a solid residue, and the enzyme is reacted at 20 to 65 ℃ by mixing alkalase or proteinase of pH 4-12, or alkalase and proteinase Extraction process; After the enzyme extraction process, the solid residue is filtered and put into another reactor to apply a 35 to 50% sodium hydroxide solution and reacted for 60 to 120 minutes at a temperature of 120 to 140 ℃ and a pressure of 0.1 to 0.2 Mpa ;and A method of producing β-chitosan using squid cartilage, comprising: a water washing and drying step of washing with water and drying with running water after the deacetylation process. The method of claim 1, wherein 20 to 30% hydrogen chloride solution is added to the solid-state β-chitosan to form a liquid β-chitosan solution, and 1,000 units of chitosanase per kilogram of β-chitosan. (chitosanase) to put the enzyme reaction step of reacting for 16 to 20 hours at a temperature of 40-50 ℃; Ultrafiltration with molecular weight MWCO 10,000 dalton to remove unreacted substances and enzymes in the enzyme reaction; Neutralizing by adding sodium hydroxide to remove hydrogen chloride used for dissolution of the β-chitosan; Desalting and concentrating the neutralized solution by reverse osmosis 2 to 3 times with a molecular weight of MWCO 200 dalton; And Method for producing β-chitosan using squid cartilage, characterized in that by adding a further step of drying the concentrated solution to produce β-chito-oligosaccharides. According to claim 1, wherein the pH of the alkalase and proteinase used in the enzyme extraction process is a method of producing β-chitosan using squid cartilage, characterized in that the electrolysis water.

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