JP5522857B2 - Method for producing collagen - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a method for producing collagen derived from biological raw materials having high solubility in water in an acidic region, and in particular, an aqueous solution containing collagen obtained by a collagen extraction step and a non-collagenous nitrogen compound. And a method for producing collagen, which is a method for removing non-collagenous nitrogen compounds by contacting activated carbon having a pH of 7 or less.

  Collagen is a protein having a triple helical structure, and when collagen is heated and denatured, it becomes gelatin having no triple helical structure. In the known literature, there are many gelatins called collagen, but in the present invention, only a protein that becomes fibrillated by salting out, that is, a protein having a triple helical structure is called “collagen”. On the other hand, a modified product of collagen by heating or the like is referred to as “gelatin”, and collagen and gelatin are clearly divided and referred to. Further, among nitrogen compounds such as proteins, peptides, amino acids, etc., those other than proteins that become fibrillated by salting out, that is, nitrogen compounds that do not have a triple helical structure are referred to as “non-collagenous nitrogen compounds”.

  Collagen is manufactured through extraction steps using various extraction methods from collagen-containing tissues of biological materials such as mammals and fish shellfish, followed by purification steps that increase the purity of the collagen. The main extraction methods and the names of collagen obtained thereby are: [1] acid-soluble collagen obtained by extraction with dilute acid, [2] enzyme-solubilized collagen obtained by solubilization with enzyme, [3 ] Alkali-solubilized collagen obtained by a method of solubilizing with alkali. Among these, acid-soluble collagen and enzyme-solubilized collagen are highly soluble in water in the acidic region.

  As a method for producing acid-soluble collagen, Patent Document 1 discloses that fish scale is used as a raw material and an extraction step using an acidic solution and then a purification step using a salting-out method are performed under temperature conditions that do not denature the collagen.

  As a method for producing enzyme-solubilized collagen, Patent Document 2 discloses a method for extracting high-purity collagen without denaturing collagen from fish scales. In this method, protease treatment is performed within a temperature range that does not denature collagen in fish scales as much as possible against decalcified fish scales, so that collagen is solubilized to remove residues, and then non-collagenous nitrogen compounds are removed by salting out. High purity collagen is obtained through a purification process.

  The salting-out method is a salting-out process in which collagen is fibrillated by an increase in salt concentration due to sodium chloride, etc., a recovery process in which the fibrillated collagen is recovered by centrifugation, etc. It consists of a dialysis process. The salting-out step may include a step of fibrosis of collagen by making the collagen solution neutral to basic using a basic solution or a buffer solution. In any case, since the addition of inorganic salt is essential in the salting out process, it is necessary to repeat the dialysis process in order to obtain collagen with a low salt concentration as the final product. There was a need.

  On the other hand, in the production of collagen or gelatin, it is widely performed to add activated carbon for the purpose of deodorization and decolorization. For example, in Patent Document 3, activated carbon is added for the purpose of deodorizing and decolorizing the enzymatic degradation product of gelatin.

JP-A-5-93000 JP 2010-193808 A JP 2003-238597 A

  As described above, the salting-out method is used in the collagen purification process to remove non-collagenous nitrogen compounds. However, the salting-out method requires a dialysis process to remove the added salts, which is a complicated process. Time-consuming operation was required. Therefore, there has been a strong demand for the development of a simple method for removing non-collagenous nitrogen compounds in place of the salting-out method.

  As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that activated carbon having a pH of 7 or less among activated carbons commonly used for the purpose of deodorizing and decoloring collagen and gelatin. As a result, it was found that the non-collagenous nitrogen compound can be efficiently removed while maintaining almost the amount of collagen, and the present invention has been completed.

That is, the present invention relates to the following.
(1) In the production of collagen derived from biological raw materials with high solubility in water in the acidic region, the collagen purification method involves contacting an aqueous solution containing collagen and a non-collagenous nitrogen compound with activated carbon having a pH of 7 or less. A method for producing collagen,
(2) The contact method between the aqueous solution and the activated carbon is a method in which the activated carbon is added to the aqueous solution and mixed with stirring. The added amount of the activated carbon is 2 by mass ratio to the non-collagenous nitrogen compound. The method for producing collagen according to the above (1), which is -50 times.
(3) The method for producing collagen according to (1) or (2) above, wherein the activated carbon is in a powder form.

According to the present invention, since the non-collagenous nitrogen compound can be efficiently removed by a simple method using activated carbon having a pH of 7 or less, the time required for the collagen purification step is significantly shorter than the salting-out method. In addition, work labor can be greatly reduced.
Further, since activated carbon is used, it also has a deodorizing effect.

Hereinafter, the method for purifying collagen of the present invention will be described in detail.
In the production of collagen derived from biological raw materials having a high solubility in water in an acidic region, the present invention provides a method for purifying collagen comprising an aqueous solution containing collagen and a non-collagenous nitrogen compound, and activated carbon having a pH of 7 or less. It is related with the manufacturing method of collagen which is a method of removing a non-collagenous nitrogen compound by making it contact.

  As a method for producing collagen derived from biological raw materials having high solubility in water in the acidic region, there is no particular limitation as long as it is a production method for obtaining the collagen, but generally known acid-soluble collagen and enzyme-solubilized collagen are known. It is preferable to use a manufacturing method. In both methods, an aqueous solution containing collagen and a non-collagenous nitrogen compound (hereinafter referred to as “crude collagen solution”) is obtained by an extraction step using an enzyme such as acid or protease. The collagen only needs to have a triple helical structure, and may be atelocollagen from which the terror site has been removed. In order to reduce the work load of the subsequent purification process, a solution obtained by removing coarse residues and protease degradation residues that can be removed by a physical removal method such as centrifugation after the extraction process is used as a crude collagen solution. It is more desirable.

Here, the crude collagen solution will be described.
The collagen concentration in the crude collagen solution is increased as the collagen concentration increases, resulting in poor handling because the collagen concentration decreases. On the other hand, the production efficiency decreases as the collagen concentration decreases, so adjust the concentration to an appropriate concentration range. It is preferable. As an example, when the crude collagen solution obtained from fish scale-derived collagen by the method of Patent Document 2 is used, the collagen concentration is adjusted to 0.7 to 1.5 mass%. At this time, the non-collagenous nitrogen compound concentration is often in the range of about 0.1 to 0.4% by mass.

  Further, the pH of the crude collagen solution is adjusted to be acidic in order to keep it in an aqueous solution state without precipitating collagen, but is preferably 5 or less, more preferably in the range of 1 to 4 in terms of pH value. In the case of collagen derived from fish scales, a pH range of 2 to 3 where the solubility of collagen is highest is preferable.

  The distinction between collagen and non-collagenous nitrogen compounds is made by utilizing the property that collagen is self-organized and fibrillated when the salt concentration in the solution is increased. Specifically, sodium chloride is added to the crude collagen solution to increase the salt concentration in the solution to sufficiently fibrillate the collagen. Next, it is separated into precipitated collagen fibers and a supernatant containing a non-collagenous nitrogen compound by centrifugation. The amount of collagen can be determined according to a quasi-drug raw material standard 2006 component standard: a quantitative method of water-soluble collagen liquid (3). Specifically, it can be obtained by analyzing the nitrogen amount of the precipitate by the Kjeldahl method or the like and then multiplying the nitrogen amount by 5.6 which is a conversion coefficient used for calculating the collagen amount. Further, the amount of non-collagenous nitrogen compound in the present invention is obtained by analyzing the amount of nitrogen in the supernatant by the Kjeldahl method or the like in the same manner as the precipitate, and then multiplying the amount of nitrogen by 5.6.

  The crude collagen solution increases the purity of the collagen by removing non-collagenous nitrogen compounds in the next purification step. As a purification method used in the purification step, a crude collagen solution is brought into contact with activated carbon having a pH of 7 or less.

  As the activated carbon to be brought into contact with the crude collagen solution, one having a pH of 7 or less is used. Here, the pH of the activated carbon is a value measured according to JIS K-1474. Normally, the pH value of activated carbon varies depending on the production lot, so the activated carbon of the commercial product has a range of pH such as 4.5 to 7.5 or 5 to 8, but the median is 7 or less If so, it can be applied as the activated carbon of the present invention.

In the present invention, when the activated carbon has a pH of 7 or less, non-collagenous nitrogen compounds can be removed while avoiding collagen precipitation. In order to more efficiently remove non-collagenous nitrogen compounds, the pH of the crude collagen solution should be set within a pH range where the solubility of collagen is high, and activated carbon having a pH value that matches the pH range should be applied. That's fine. Such activated carbon is preferably in the range of pH 1-5, more preferably in the range of pH 2-4. In addition, about the activated carbon of a commercial item, the median value of pH range should just be in the range of 1-5, More preferably, it is in the range of 2-4.
In the present invention, activated carbon prepared by adjusting the pH of the activated carbon to a desired value with an inorganic acid may be used. For example, commercially available high pH activated carbon is heated in an inorganic acid solution such as hydrochloric acid and sulfuric acid adjusted to an appropriate concentration for a certain period of time, and then washed with water to adjust the pH of the activated carbon.

The shape, raw material, activation method and the like of the activated carbon are not particularly limited. Any shape such as powder, crushed, and granular can be applied. As a particle diameter of powdered activated carbon, a particle diameter of D90 of 5 to 150 μm can be cited as a good example. Here, the particle diameter D90 is an index representing the particle diameter of a general powder, and more specifically, means a particle diameter at which the cumulative weight percentage when sieving activated carbon is 90% by mass. To do.
Examples of the raw material include coconut shell, wood, coal and the like, among which coconut shell and wood are particularly preferable. Examples of the activator include water vapor, carbon dioxide, zinc chloride, and phosphoric acid.

  Since the non-collagenous nitrogen compound is composed of nitrogen compounds having various molecular weights, it is preferable to use a plurality of types of activated carbon having different pore diameters. It is also preferable to use a plurality of types of activated carbon having different adsorption characteristics. In the case of using a plurality of types of activated carbon, the crude collagen solution may be contacted simultaneously with a plurality of types of activated carbon, that is, a mixture of a plurality of types of activated carbon, or may be contacted in multiple stages for each type of activated carbon. .

  The contact method between the crude collagen solution and the activated carbon is not particularly limited as long as the non-collagenous nitrogen compound can be removed by adsorbing the non-collagenous nitrogen compound to the activated carbon. Specific examples include [1] a method of adding activated carbon to a crude collagen solution and mixing with stirring, and [2] a method of passing the crude collagen solution through a single-stage or multistage activated carbon column.

The method [1] will be described.
Since the method [1] requires removal of activated carbon, the purification step consists of two steps: (a) adding activated carbon to the crude collagen solution and mixing with stirring; and (b) removing activated carbon. .
As the method for adding activated carbon in (a) above, a predetermined amount of activated carbon may be added to the crude collagen solution at once, or may be added in portions. When adding in divided portions, the activated carbon added last time may be removed and then added.

  The shape of the activated carbon may be a pulverized form that is pulverized during stirring, but a powder form having a large surface area is particularly preferable in order to increase the adsorption efficiency of the non-collagenous nitrogen compound.

  Regarding stirring, the stirring device, stirring strength, and time are appropriately selected and set so that the non-collagenous nitrogen compound is effectively adsorbed on the activated carbon. A guideline for the stirring time is 1 to 24 hours, and it is desirable that the stirring time is at least 2 hours. It is desirable that the entire solution be suspended with dispersed activated carbon fine particles after a certain time has elapsed since the addition of activated carbon. Such a state is easily obtained when powdered activated carbon is used.

  The addition amount of the activated carbon is preferably 2 to 50 times in mass ratio with respect to the non-collagenous nitrogen compound. In addition, even when activated carbon is added in portions, the total addition amount is preferably within the above range. When the addition amount is less than twice, removal of the non-collagenous nitrogen compound becomes insufficient. On the other hand, adding activated carbon over 50 times is not economical because it is difficult to obtain a removal effect commensurate with the amount of addition, and the labor required for the subsequent removal of activated carbon is also great. The addition amount is preferably 4 to 35 times, more preferably 5 to 25 times, and still more preferably 8 to 20 times.

  Regarding the removal of activated carbon, the removal method is not limited as long as the activated carbon can be appropriately removed. Examples of the removing method include centrifugal filtration, pressure filtration, vacuum filtration, decantation, filter press and the like, and these may be combined.

Next, the method [2] will be described.
In the method [2], the purification step comprises passing the crude collagen solution through a single-stage or multi-stage activated carbon column.
The shape of the activated carbon is not particularly limited as long as it can be applied as an activated carbon column. Also, in order to efficiently remove non-collagenous nitrogen compounds with a wide molecular weight distribution range, multiple types of activated carbon may be stacked in the same column, or as a multi-stage column that changes the type of activated carbon for each column. Good. Conditions such as column diameter and flow rate may be set as appropriate.

  As a purification step, the above methods [1] and [2] may be combined. In this case, in order to reduce clogging of the activated carbon column, the activated carbon is removed after the method [1], and then [ It is preferable to apply the method of 2]. Further, in order to further increase the removal rate of activated carbon, microfiltration with a membrane filter having a pore diameter of 0.1 to 1.0 μm may be performed.

  According to the purification step using the purification method of the present invention, a highly pure collagen solution can be obtained efficiently from a crude collagen solution with a very high yield. The removal rate of the non-collagenous nitrogen compound may be appropriately set according to the purpose of use, such as pharmaceuticals, cosmetics, and health foods, but is preferably at least 30% by mass, more preferably 50% by mass or more.

  Further, the purification step is preferably performed at a temperature lower than the denaturation temperature of collagen as in the extraction step, more preferably at a temperature 5 ° C. or more lower than the denaturation temperature. Further, the purified collagen solution may be concentrated by a moisture removal method not by heating, for example, freeze-drying, evaporation at a denaturation temperature or lower, or the like, if necessary. In addition, what is necessary is just to decolor by a well-known method, when coloring is seen in the collagen solution after refinement | purification.

  In the present invention, the activated carbon having a pH of 7 or less hardly removes collagen, that is, hardly adsorbs, and selectively adsorbs and removes only non-collagenous nitrogen compounds. The reason for this is not clear, but the difference in the three-dimensional structure between collagen and non-collagenous nitrogen compounds, that is, collagen has a cylindrical rigid triple helical structure, whereas gelatin is a random coil that is highly deformable. Since both have different three-dimensional structures so as to have the form, it is assumed that the difference in the structure appears as a difference in the degree of adsorption to the activated carbon.

  EXAMPLES Hereinafter, although an Example is given and the detail of this invention is demonstrated, this invention is not limited by those Examples. In addition, unless otherwise indicated, all% shows the mass%.

[Method for measuring the concentration of collagen and non-collagenous nitrogen compounds]
While stirring the crude collagen solution, sodium chloride was added so that the sodium chloride concentration in the solution was 5.85%, and the collagen was fibrillated. The solution viscosity decreased with the progress of collagen fibrosis, and separated into an agglomerated part and a low-viscosity solution part. After confirming that the separation was sufficiently performed, stirring was continued for about 30 minutes. Next, a fibrillated precipitate and a supernatant were obtained by centrifugation. The precipitate and supernatant were each measured for the nitrogen content by the Kjeldahl method and multiplied by a conversion factor of 5.6 to determine the collagen concentration and the non-collagenous nitrogen compound concentration. The total of the collagen concentration and the non-collagenous nitrogen compound concentration was defined as the total nitrogen compound concentration. The solution after purification of the crude collagen solution was measured in the same manner as described above.

[Preparation of crude collagen solution derived from fish scales]
The tilapia scales were thoroughly washed with water and further washed with a 10% sodium chloride solution to remove impurities such as wrinkles, and then dried at room temperature. The water content was 18.5%. Disperse 1 kg of this tilapia scale in 9 kg of pH 2 hydrochloric acid solution and gently agitate for 2 hours at 25 ° C with the pH kept at 2 while adding 1 M hydrochloric acid solution to dissolve the inorganic components contained in the scale. did. This was put into a colander and washed thoroughly with water, and then a hydrochloric acid solution having a pH of 2 was added so that the total weight was 4 kg. To this, 24 g of pepsin (Wako Pure Chemicals 1: 10000) was added and gently stirred using a stirring blade at 25 ° C. for 72 hours to dissolve the collagen from the scales. To this was added 2 kg of water and stirred to lower the viscosity, and then the mixture was raised to a colander and separated from scale residue (about 3 kg). Furthermore, the supernatant was recovered by centrifugation (10000 G, 60 min) and separated from fine scale residues. To this, 0.5 g of pepsin (Wako Pure Chemicals 1: 10000) was added and kept at 25 ° C. for 24 hours to obtain 2.8 kg of a crude collagen solution. The collagen concentration in the crude collagen solution was 0.79%, and the non-collagenous nitrogen compound concentration was 0.44%.

[Example 1]
350 g of crude collagen solution was provided.
“Bombard 110P-FGZ” (powder, pH 4-7) (Taki Chemical Co., Ltd.) was used as the activated carbon. The amount of activated carbon added was 1, 2, 4, 8, and 16 times the weight of the non-collagenous nitrogen compound amount (1.54 g) in the crude collagen solution.
The above-mentioned added amounts of activated carbon were added to the crude collagen solution with stirring, and then the stirring was continued for 2 hours. After 2 hours, the entire solution was in suspension.
This was centrifuged (11120G, 120 minutes), and the supernatant was filtered with a Nutsche type filtration device coated with diatomaceous earth to obtain a highly transparent solution.
The collagen concentration and non-collagenous nitrogen compound concentration of the obtained collagen solution were measured by the above-described methods.

[Example 2]
As activated carbon, 1 L of 1.8% hydrochloric acid was added to 100 g of “bomb 110P-FGZ”, heated for 5 hours in a water bath, washed with water, and activated carbon prepared to pH 3 was used. Except for the activated carbon, the test was performed in the same manner as in Example 1 to obtain a highly transparent solution. In addition, the whole solution was in a suspended state in the same manner as in Example 1 two hours after addition of the activated carbon.

[Comparative Example 1]
The test was performed in the same manner as in Example 1 except that “bomb 110P-40W” (powder, pH 9 to 11) (Taki Chemical Co., Ltd.) was used as the activated carbon. Two hours after the addition of activated carbon, the whole solution was in a suspended state as in Example 1. However, the solution viscosity increased as the amount of activated carbon added increased, making it difficult to separate and remove the activated carbon. In particular, it was difficult to obtain a highly transparent solution at an addition amount of 4 times or more.

The results are shown in Table 1.
From Table 1, it can be seen that by using activated carbon having a pH of 7 or less, the non-collagenous nitrogen compound can be removed while the amount of collagen is substantially maintained. In addition, the removal rate of the non-collagenous nitrogen compound when activated carbon is added 16 times is 86.4% ((0.44-0.06) ÷ 0.44 × 100 = 86.4%) in Example 1, and 25.0% ((0.44 -0.33) ÷ 0.44 × 100 = 25.0%). Thus, it can be seen that the removal rate of the non-collagenous nitrogen compound by the method of the present invention is extremely excellent.

Claims (2)

In manufacturing solubility of collagen from high biological material in an acidic region to water purification process of collagen, the aqueous solution containing the non-collagenous nitrogen compounds collagen, by a pH contacting the 7 following activated carbon Ri methods der to remove non-collagenous nitrogen compounds, the amount of the activated carbon manufacturing method of the non-collagenous nitrogen compound to Ru 2 to 50 Baidea a weight ratio collagen. The process according to claim 1 Symbol placement of collagen the activated carbon is a powdery.