KR20060125995A - Process for preparing collagen from starfish - Google Patents

Abstract

A method for preparing atelocollagen from starfish is provided to mass-produce the atelocollagen without lowering yield by removing non-collagen materials using alkaline materials and adjusting pH of a solution into acidic value. And a method for preparing collagen peptide from starfish using the same is provided to produce collagen peptide from the atelocollagen using a neutral protease without degeneration of the collagen. The method for preparing atelocollagen from starfish comprises the steps of: (a) removing non-collagen materials existing in a starfish body wall using an alkali material selected from the group consisting of NaOH, Ca(OH)2, and Na2CO3 to prepare insoluble collagen fiber; and (b) after adjusting pH using tartaric acid or citric acid into the acidic state, preparing atelocollagen from the collagen fiber using an acidic protease such as 1% pepsin at a temperature of 5-10 deg.C. To prepare collagen peptide from the starfish, the atelocollagen prepared from the above method is hydrolyzed with a neutral protease.

Description

Process for preparing collagen from starfish {Process for preparing collagen from starfish}

Figure 1 shows the collagen content of the starfish enzyme hydrolyzate according to the type of alkaline material.

Figure 2 shows the collagen content of the starfish enzyme hydrolyzate according to the treatment time of calcium hydroxide.

Figure 3 shows the collagen content of the starfish enzyme hydrolyzate according to the treatment temperature of calcium hydroxide.

Figure 4 is a result of measuring the molecular weight of collagen by 5% SDS-polyacrylamide gel electrophoresis (a) is bovine atherocollagen, (b) is starfish atelocollagen.

In the present invention, since the telopeptide is removed from the starfish and the telopeptide is removed, there is no antigen. Thus, the present invention produces atelocollagen, which is highly useful to the human body. It relates to a method of preparation and a method of producing acid-soluble collagen peptides from starfish collagen fibers.

Starfish starfish (Asterina pectinifera) stars as marine benthic belonging to the echinoderms dongmulmun, Amur starfish (Asterias amurensis), prickly leaves Starfish (Astropecten polyacanthus), etc., and that the 1,700 species around the world are looking to academia than 200 in the country offshore This is inhabiting. In particular, the Amur Buddha Starfish and Starfish Starfish (hereinafter referred to as starfish) are carnivorous and have useful shellfish such as shellfish, abalone, clams, and scallops, and are classified as very large pirates in the aquaculture industry. Mass reproduction poses the risk of destroying marine ecosystems. Starfish have been used as a compost in fishing villages since ancient times, and known patent technologies related to this are starfish fertilizers (Korea Patent 10-0088787, 10-0346927, 10-0387936, 10-0420978), calcium supplements for health supplements (Korea) Patent 10-048086), anti-thrombotic composition (Korean Patent No. 10-0408087), anti-cholesterol composition (Korean Patent No. 10-0408089), antimicrobial composition (Korean Patent No. 10-0449910), etc., but to prepare collagen from starfish There is no known patent for the method.

Collagen is a very important fibrous light protein (Albuminoid) that fills cells between cells in humans and animals. Collagen is always present at the site where a large number of cells are collected, and especially collagen is contained in skin, bone, cartilage, blood vessels, teeth, and muscles. Collagen can be classified into soluble collagen and insoluble collagen, and acid-soluble or salt-soluble collagen can be extracted without degeneration from the tissues of immature animals, but the yield is very low and economic efficiency is low. On the other hand, the method of solubilizing and extracting insoluble collagen in tissues is practical. When collagen is present in the tissue, the collagen molecule forms a higher order structure such as fibril and fiver, and the collagen molecule in the structure increases the strength of the tissue by forming crosslinks and insolubilization between molecules. In the crosslinking, non-helix telopeptide exists at both ends of the collagen molecule, and it is possible to solubilize collagen by removing the telopeptide moiety to release the crosslinking. For this solubilization, it is possible to use proteases such as pepsin. Pepsin decomposes only telopeptide and does not act on the helix. Solubilized collagen prepared by this method is called atelocollagen, meaning that telopeptide has been removed. Atelocollagen is preferred for human application because telopeptide and non-collagen proteins are removed by proteolytic enzymes and thus have higher purity than other collagen and have almost the same physical properties as natural collagen. In addition, atelocollagen is not only high purity but also low antigenic protein from which telopeptide, a major antigenic site of collagen, has been removed. This resistance is an important requirement for using collagen as a biomaterial, and atelocollagen is used for artificial skin, hemostatic agents, and cell culture substrates.

Collagen production materials have been mainly supplied from livestock animals such as cows and pigs, but recently, research has been actively conducted on marine organisms that are not related to mad cow disease because of the harmful problem caused by the mad cow disease wave.

The starfish's muscle tissue stretches 1.5 times as large as its own arm and has a variety of physiological functions, including the ability to regenerate tissues in which the damaged arm grows. These characteristics are thought to be closely related to collagen. However, the starfish body wall is composed of bone fragments (calcium carbonate), protein, pigments, odors and so on, there are many differences from the collagen extract of terrestrial animals. Therefore, it is difficult to extract effectively when the non-collagen substance removal method, collagen extraction method, acetic acid extraction method, pepsin extraction method and the like already known directly applied. That is, a large amount (20-30%) of bone fragments (calcium carbonate) is present on the body wall of the starfish, and the condition for removing non-collagen substances needs to be identified.If the collagen is extracted by a known acetic acid extraction method or an acidic protease extraction method, the body wall It is difficult to maintain the optimum extraction condition because calcium carbonate and acetic acid present in the reaction react to cause neutralization reaction.If excess acid is used to adjust the pH, the ions of the solution are caused by the large amount of calcium acetate that is the result of the neutralization reaction. Since the strength is increased, collagen is precipitated and mixed in the residue of the enzyme reaction, so that the loss of collagen is large and economical is low. In addition, while the temperature of denaturation of collagen by heat is about 35-40 ° C. in warm-blooded animals, starfish collagen is relatively low at 25 ° C. and must be treated at low temperature to avoid heat denaturation.

The present invention provides a condition for removing non-collagen substances using an alkaline substance to solve the above problems, and adjusting the pH of the solution to acidic using tartaric acid or citric acid does not affect the ionic strength of the solution collagen The inventors of the present invention have devised a method for preparing atelocollagen without deterioration in yield, and provided a method for preparing collagen peptide from atelocollagen using a neutral protease at low temperature where collagen is not denatured. In addition, the production of acid-soluble collagen peptide from the starfish body wall without a known dialysis process as a technical problem.

The composition of the present invention is largely ① process for producing collagen fiber by removing non-collagen material present in starfish body wall using alkali material, ② process for producing atelocollagen from collagen fiber using acidic protease, ③ neutral protease It comprises a step of producing an unmodified low molecular collagen peptide from atelocollagen, ④ the step of producing an acid-soluble collagen peptide from starfish collagen fibers.

As a starfish material used in the present invention, an amur starfish or a star starfish was used.

① removal of non-collagen substances

The body wall of starfish contains proteins, subcutaneous fat, odors (amines, fatty acids, carbonyl compounds, sulfur compounds) and inorganic substances (calcium carbonate) other than collagen. Therefore, removal of non-collagen substances is essential to increase the purity of collagen. to be. Alkaline, acids, enzymes and the like are used as a method of removing non-collagen substances, but a method generally used in aquatic products such as fish and scales is an alkali treatment method. Therefore, in the present invention, the alkaline treatment method was selected to examine the pretreatment conditions with sodium hydroxide, calcium hydroxide and sodium carbonate, and the optimum treatment temperature and time were identified by using the calcium hydroxide having the best effect. First, the collagen content of an alkali treated sample was measured for 24 hours to find a suitable pretreatment material (FIG. 1).

Collagen content according to alkali type and concentration showed the highest value of 1.5% to 85.3% of calcium hydroxide, 2.5% to 83.6% of sodium hydroxide, and 3.0% to 81.5% of sodium carbonate. Decreased. It is estimated that collagen also elutes and disappears along with non-collagen substance due to high concentration of alkali treatment. In conclusion, 1.5% calcium hydroxide is most suitable as a pretreatment material for extracting collagen from starfish.

In order to determine the proper pretreatment time, the collagen content of the enzyme hydrolyzate was measured after immersion of the starch body wall in 1.5% calcium hydroxide solution (FIG. 2). Collagen content by 1.5% calcium hydroxide treatment time was 73% for 1 hour, 78% for 3 hours, 80% for 5 hours, and 84-85% for 12-48 hours. Therefore, in consideration of degeneration of collagen due to long time treatment, it is appropriate to treat for 12 hours with 1.5% calcium hydroxide.

In order to determine the proper pretreatment temperature, pretreatment was performed for 1.5 hours in 1.5% calcium hydroxide solution at 5, 10, 20, 30 and 40 ° C., respectively, and the collagen content of the enzyme hydrolyzate was measured for each temperature (FIG. 3). Collagen content by treatment temperature was 84-85% did not show a big difference, but considering the thermal denaturation and microbial growth inhibition of starfish collagen, it is preferable to treat at 5 ℃.

 ② Preparation of Atelocollagen

Known methods for producing atelocollagen are insoluble collagen fibers suspended in distilled water, adjusted to pH 3.0 with hydrochloric acid or acetic acid, extracted at 5-20 ° C. with 1-5% acidic protease, followed by salting with NaCl to precipitate collagen. Collagen is produced by dialysis. However, in case of starfish, when hydrochloric acid or acetic acid is used for acid treatment, which is a known method, a large amount of calcium chloride or calcium acetate is produced by reaction with bone fragments (calcium carbonate) present in the tissue (Formula 1). In addition, calcium chloride or calcium acetate produced in this reaction is dissolved in water to increase the ionic strength, causing precipitation of collagen extracted by the enzyme, thereby lowering the yield.

Therefore, in the present invention, an experiment is conducted to find a substance that does not dissolve and reacts with calcium carbonate present in the starfish tissue to maintain acidity (pH 3.0) without affecting the ionic strength of the solution and not harmful to the human body. It was. That is, 10g of starfish bone fragments were added to 200ml of distilled water, and the acid was slowly added until the pH of the solution reached 3.0. Then, the weight of the precipitate was measured as shown in Table 1 below.

 Sediment weight after reaction of starfish fragment with various acids mountain Hydrochloric acid Acetic acid Citric acid Tartaric acid Phosphoric Acid Malic acid Lactic acid Weight of precipitate (g) 0.00 0.00 20.46 41.78 15.38 2.64 0.00

From the above experimental results, calcium salt is produced by the reaction of calcium carbonate contained in the starfish bone fragment with tartaric acid, citric acid, phosphoric acid, and malic acid, and these calcium salts precipitate and do not significantly affect the ionic strength of the solution. In case of tartaric acid and citric acid, the solubility of calcium salt produced by reacting with calcium carbonate in water at 10 ℃ is 0.040g / 100ml and 0.065g / 100ml, respectively. Preferred (Formula 2).

In other words, calcium stannate and calcium citrate are poorly soluble in water, which does not affect the ionic strength of the reaction solution, so that the collagen present in the solution does not precipitate. Focusing on the above principle, the present invention suspended the insoluble collagen fibers in distilled water and adjusted the pH to 3.0 with tartaric acid or citric acid to extract collagen at 5-20 ° C. with 1-5% acidic protease, followed by salting with NaCl to precipitate collagen. A technique for producing atelocollagen was devised by dialysis and then dialysis.

③ Production of Unmodified Collagen Peptides

Atelocollagen extracted by acid protease has a disadvantage in that it is not applicable to cosmetics because it is precipitated and aggregated in the pH range of the cosmetic application (pH5-6) and the molecular weight is not absorbed into the skin with about 100,000. In order to solve this problem, it is necessary to decompose atelocollagen into a low molecular weight peptide having a molecular weight of 3,000 Da or less. Therefore, the method of low molecular weight of atelocollagen using the neutral protease Trypsin, Alcalase, Neutrase, Protamex, Protease NP, and Arazyme under 24 ℃, the heat denaturation temperature of starfish collagen, was investigated. After adding 1% of atelocollagen to each enzyme for 1 hour at the reaction temperature of 20 ° C., the hydrolysis rate was measured for each enzyme (Table 2).

 Collagen Hydrolysis Rate by Enzyme enzyme Trypsin Alcalase Neutrase Protamex Protease NP Arazyme Hydrolysis Rate (%) 67.2 59.0 40.2 37.0 46.5 78.1

The highest hydrolysis rate was 78.1% with Arazyme, a sugar-free spider protease, and Trypsin 67.2%, Alcalase 59.0%, Protease NP 46.5%, Neutrase 40.2%, and Protamex 37.0%. And as an experiment to increase the hydrolysis rate, the hydrolysis rate was measured by reaction time using Arazyme (Table 3).

 Collagen Hydrolysis Rate of Arazyme by Reaction Time Response time (hr) One 3 6 12 18 24 Hydrolysis Rate (%) 78.1 85.8 90.6 93.5 93.6 93.9

The collagen hydrolysis rate of arazyme increased up to 12 hours, but from then on, the increase was minimal and the enzyme reaction time was 12 hours. To summarize the above results, it is preferable that the production conditions of the collagen peptide using starfish-derived atelocollagen are hydrolyzed at 20 ° C. for 12 hours with 1% Arazyme.

④ Production of Acid Soluble Collagen Peptides

This method relates to a technique for easily producing acid-soluble collagen peptides without separate dialysis using the principle of precipitation of calcium stannate from starfish enzyme hydrolyzate. That is, if the starfish body wall is treated with alkali to remove non-collagen material, hydrolyzed with protease, and then the solution from which bone fragments are removed is adjusted to pH 3.0 using tartaric acid, the collagen peptide is dissolved in the solution and the non-collagen component is precipitated. Centrifugation removes the non-collagen components and neutralizes it by adding calcium carbonate. Calcium stannate is produced and precipitated. Therefore, tartaric acid present in the solution can be removed by centrifugation only without separate dialysis process. Production is possible.

The following describes the content of the present invention in more detail using examples and comparative examples, but the present invention does not limit the scope of the claims.

Comparative example

① Cut 100 g of the body wall of the removed starfish into 2-5cm ³ , immerse it in 1 liter of 1.5% calcium hydroxide solution, treat it for 5 hours at 5 ℃, and wash non-collagen components with tap water to remove 90 g of collagen fiber. Manufacture.

② Add 0.9 liter of distilled water to 90 g of collagen fiber produced in ①, crush it with homogenizer (Ultra-Turrax T50), adjust pH to 3.0 using acetic acid, and add 0.9 g of pepsin to extract collagen at 10 ℃. Purification by precipitation of collagen by salting 2-3 times with 5% NaCl was then dialyzed for 12-24 hours and lyophilized to obtain 1.5 g of atelocollagen.

Example 1

① Cut 100 g of the body wall of the removed starfish into 2-5cm ³ , immerse it in 1 liter of 1.5% calcium hydroxide solution, treat it for 5 hours at 5 ℃, and wash non-collagen components with tap water to remove 90 g of collagen fiber. Manufacture.

② Add 0.9 liter of distilled water to 90 g of collagen fiber produced in ①, crush it with homogenizer (Ultra-Turrax T50), adjust pH to 3.0 using tartaric acid, and add 0.9 g of pepsin to extract collagen at 10 ℃. Purification by precipitation of collagen by salting 2-3 times with 5% NaCl was then dialyzed for 12-24 hours and lyophilized to obtain 4.5 g of atelocollagen.

Looking at the obtained amount of atelocollagen from the above results, the comparative example (using acetic acid) was 1.5g, but the example (using dibasic acid) was 4.5g, and the yield was about 3 times higher when the tartaric acid of the present invention was used instead of acetic acid. high.

As a result of measuring the molecular weight of the atelocollagen produced by the above method is as shown in FIG. The atelocollagen isolated from starfish was composed of α1 and α2 chains and had a molecular weight of about 100,000 Da, similar to bovine collagen.

Example 2

10 liters of distilled water was added to 300 g of atelocollagen prepared in Example 1, and hydrolyzed at 20 ° C. with 1 mg of Arazyme for 12 hours using an ultrafiltration membrane to fractionate only a substance having a molecular weight of 1000-3000 Da, and having a molecular weight higher than that. In the same manner as above, 10 liters of low molecular weight collagen peptide having a concentration of 3% was prepared by hydrolysis.

Example 3

① Cut 100 g of the body wall of the removed starfish into 2-5cm ³ , immerse it in 1 liter of 1.5% calcium hydroxide solution, treat it for 5 hours at 5 ℃, and wash non-collagen components with tap water to remove 90 g of collagen fiber. Manufacture.

② After adding 0.9 liter of distilled water to 90 g of the collagen fiber produced in ①, hydrolyzed at 55 ° C by adding 0.9 g of Protease NP, and centrifuging to remove the bone fragments by adjusting the solution to pH 3.0 using tartaric acid and leaving it for 24 hours. After centrifugation, the solution is separated.

Calcium carbonate was slowly added to the solution separated from ② until pH 7.0 and centrifuged to obtain 0.9 liter of 3.6% acid-soluble collagen peptide solution.

The present invention is expected to play a big role in combating starfish by developing demand for starfish, which is a pirate of aquaculture farms, and improving economic value. The water-soluble low molecular collagen peptide, which has been hydrolyzed again, can be used as a cosmetic and food material, and it is expected to contribute greatly to the activation of high value-added marine biotechnology industry.

Claims (8)

A method for producing atelocollagen from the Amur vulgaris or star-stars, including the following steps. ① Process of manufacturing insoluble collagen fiber by removing non-collagen substance on starfish body using alkali material selected from sodium hydroxide, calcium hydroxide and sodium carbonate, ② Adjusting pH to acidity using tartaric acid or citric acid A process for producing atelocollagen from collagen fibers. Method of producing a collagen peptide from the Amur vulgaris or star star including the following steps. ① Process of manufacturing insoluble collagen fiber by removing non-collagen substance present in starfish body using alkali material selected from sodium hydroxide, calcium hydroxide and sodium carbonate, ② Adjusting pH to acidity using tartaric acid or citric acid A process for producing atelocollagen from collagen fibers, ③ process for producing the water-soluble low molecular collagen peptide by hydrolysis of the atelocollagen with a neutral protease. The method according to claim 1 or 2, characterized in that the pH is adjusted to 3.0 using tartaric acid in the step (2). 4. The method according to claim 3, wherein the alkali material in the step (1) is 1.5% calcium hydroxide solution and is treated at 5 ° C to 10 ° C for 10 to 15 hours. The method according to claim 3, wherein the acidic protease in the step 2 is 1% pepsin and the reaction is carried out at 5 ° C to 10 ° C. The method according to claim 2, wherein the neutral protease in the step 3 is arazyme and is reacted at 10 ° C. to 20 ° C. for 10 to 15 hours. 3. The method for producing a water-soluble low molecular collagen peptide according to claim 2, wherein only the substance having a molecular weight of 1000-3000Da is fractionated by using an ultrafiltration membrane, and the one having a molecular weight higher than that is hydrolyzed in step 3). A method for producing an acid-soluble collagen peptide from the Amur vulgaris or star star, including the following steps. ① process to remove insoluble collagen fiber from starfish body wall using alkaline material selected from sodium hydroxide, calcium hydroxide and sodium carbonate, ② ① add distilled water to collagen fiber produced in process and use acidic protease By hydrolysis to remove the non-collagen components by adjusting the pH to 3.0 using tartaric acid in the solution to remove the bone fragments, ③ centrifuged to remove the non-collagen components, neutralize by adding calcium carbonate and centrifuged again Removing acid calcium stannate to produce acid-soluble collagen peptide.

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