JP2016113382A - Proteoglycan production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a convenient and efficient production method for proteoglycan with reduced unpleasant odors.SOLUTION: The present invention provides a proteoglycan production method comprising (a) extracting proteoglycan from fish cartilage with plum vinegar.SELECTED DRAWING: None

Description

  The present invention relates to a proteoglycan production method using fish cartilage as a material.

  Proteoglycans are a type of polymer that forms the extracellular matrix of animals. Proteoglycans have high water retention and are known to have various physiological functions such as wound healing, anti-inflammatory, and cell growth promotion. In addition to pharmaceuticals and experimental reagents, cosmetics, food and drinks, etc. Can be used in a wide range of areas. For this reason, development of the method of manufacturing the proteoglycan which can be utilized for these simply and efficiently is calculated | required.

  Conventionally, as a method for producing proteoglycan, a method of extracting from fish cartilage such as salmon with a guanidine hydrochloride solution has been reported (Patent Document 1). However, this method requires a degreasing process using chloroform, methanol, etc. in order to remove the peculiar unpleasant odor (animal odor, fish odor) of fish, and completely removes the unpleasant odor even if the degreasing process is performed. It was impossible. In addition, since guanidine hydrochloride, chloroform, methanol and the like used in this method are considered harmful to the human body, the use of proteoglycans obtained by this method in cosmetics, foods and drinks, etc. has been restricted.

  As a method for solving such a problem, a method using acetic acid as an extraction solvent has been reported (Patent Document 2). However, the proteoglycan obtained by this method has a problem of generating an unpleasant odor, which is considered to be caused by acetic acid (Patent Document 3).

  On the other hand, ume vinegar has long been used as a long-term preservation solution for livestock processed meats, raw seafood, vegetables, etc., and has not been reported to be harmful to the human body. Moreover, since ume vinegar is normally discarded when manufacturing an umeboshi, it can be utilized cheaply.

JP 2001-172296 A JP 2002-069097 JP 2009-173702

  An object of the present invention is to provide a simpler and more efficient production method of proteoglycans with reduced unpleasant odor.

  As a result of diligent research, the present inventor has found that proteoglycan can be extracted from fish cartilage with ume vinegar, and further has found that the above problems can be solved. That is, the present invention includes the following aspects.

  Item 1. (A) A method for producing proteoglycan comprising extracting proteoglycan from fish cartilage with plum vinegar.

  Item 2. Furthermore, (b) The manufacturing method of claim | item 1 including removing an insoluble matter from the extract obtained at the process a.

  Item 3. Furthermore, (c) The manufacturing method of claim | item 2 including refine | purifying proteoglycan by alcohol precipitation after process b.

  Item 4. Item 4. The production method according to Item 3, wherein the alcohol precipitation is ethanol precipitation.

  Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the extraction temperature is 10 to 40 ° C.

  Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the pH of ume vinegar is 1.5 to 3.0.

  According to the present invention, proteoglycan can be more easily and efficiently produced by extracting proteoglycan from fish cartilage using ume vinegar as an extraction solvent. The resulting proteoglycan has almost no unpleasant odor (animal odor, fish odor, acetic acid odor) derived from raw materials and extraction solvents, and has no odor or good plum scent.

  In addition, according to the present invention, proteoglycan can be produced with ume vinegar, which has been used as a food preservation solution for a long time, without using substances that are harmful to the human body such as guanidine hydrochloride, chloroform, and methanol. Proteoglycan is considered safer for the human body.

  Conventionally, when a proteoglycan was extracted from fish cartilage, a preservative was used. However, in the production method of the present invention, ume vinegar, which is known to have antiseptic action, is used as an extraction solvent. It is not necessary to add an agent.

The result of having carried out the cellulose acetate film | membrane electrophoresis of the proteoglycan obtained in Example 1 is shown (Test Example 1). The HPLC result of the proteoglycan obtained in Example 1 is shown (Test Example 2).

  The present invention relates to a proteoglycan production method comprising (a) extracting proteoglycan from fish cartilage with ume vinegar (hereinafter also referred to as “step a”).

  Fish cartilage is fish cartilage. The fish is not particularly limited, and examples thereof include teleost fish and cartilaginous fish. Examples of teleost fish include cod, tuna, salmon, trout and the like, and examples of cartilaginous fish include shark and ray. The cartilage is not particularly limited, but head cartilage, particularly nasal cartilage is preferable. In addition, since the head is usually discarded when fish is processed into food products or the like, there is also an advantage that the cost of obtaining the head cartilage is low and can be stably supplied in large quantities.

  It is preferable that the fish cartilage is removed if there is an extra attached solid fat. The fish cartilage is preferably crushed from the viewpoint of extraction efficiency. It does not specifically limit as a method to crush, A well-known crushing method is employable.

  One type of fish cartilage may be used alone, or two or more types may be used in combination.

  The plum vinegar is not particularly limited, and examples thereof include white plum vinegar and red plum vinegar. Among these, white plum vinegar is preferable from the viewpoint of suppressing coloring to proteoglycan. White plum vinegar is typically obtained by mixing plum and salt, and then placing the weight on the plate for several days (1 to 5 days) when producing plum dried. The pH of ume vinegar is not particularly limited, but may be, for example, 1.0 to 4.0, preferably 1.5 to 3.0, and more preferably 1.5 to 2.5.

  Plum vinegar may be used alone or in combination of two or more.

  Proteoglycan can be extracted according to a known extraction method. For example, it can be carried out by mixing fish cartilage and ume vinegar and then leaving it (preferably with stirring).

  The weight ratio of fish cartilage and ume vinegar is not particularly limited, but is preferably such that the entire fish cartilage is immersed in ume vinegar. Specifically, the weight ratio (fish cartilage: plum vinegar) can be, for example, 1: 1 to 1:50, preferably 1: 5 to 1:35, and more preferably 1:10 to 1:25.

  The extraction time is not particularly limited as long as proteoglycan can be extracted. The extraction time can be, for example, 3 to 72 hours, preferably 8 to 48 hours, more preferably 12 to 36 hours, and still more preferably 18 to 30 hours. If the upper limit of extraction time is the said time, the ratio of the proteoglycan in an extraction component can be raised more.

  The extraction temperature is not particularly limited as long as proteoglycan can be extracted. Extraction temperature can be 10-40 degreeC, for example, Preferably it can be 15-30 degreeC.

  A proteoglycan-containing extract is obtained by the step a. This proteoglycan-containing extract can be used as it is as “proteoglycan”, or a product obtained through the following steps can be used as “proteoglycan”.

  In the production method of the present invention, (b) it is preferable to remove insoluble matter (extraction residue) from the extract obtained in step a (hereinafter also referred to as “step b”). A method for removing insoluble matters is not particularly limited, and a known method such as filtration or centrifugation can be employed.

  In the production method of the present invention, in order to further increase the purity of the proteoglycan obtained, purification may be performed after the step a or the step b. Examples of the purification method include alcohol precipitation, dialysis, column (preferably anion exchange column) chromatography, and the like. Among these, alcohol precipitation is preferable from the viewpoint of simplicity. According to the production method of the present invention, even if the purification is only alcohol precipitation, it is of high purity (for example, the purity of the degree other than the proteoglycan and the GAG band derived from it as a result of cellulose acetate membrane electrophoresis), Moreover, proteoglycans with reduced unpleasant odor can be produced. Therefore, in the production method of the present invention, (c) after step b, it is preferable to purify proteoglycan by alcohol precipitation (hereinafter also referred to as “step c”).

  Alcohol precipitation can be performed according to a known method. Typically, the proteoglycan-containing extract obtained through step b is mixed with 1 to 5 times (preferably 2 to 4 times) the amount of alcohol, and then allowed to stand for a certain period of time to form a precipitate. Thereafter, the pellet obtained by centrifugation is collected.

  The alcohol is not particularly limited as long as the proteoglycan can be precipitated. Examples of the alcohol include ethanol and isopropanol. Among these, ethanol is preferably used from the viewpoint of lower toxicity. One kind of alcohol may be used alone, or two or more kinds of alcohols may be used in combination.

  It is preferable that the alcohol contains a salt. The salt is not particularly limited, and examples include ordinary salts used for alcohol precipitation, such as sodium chloride, sodium acetate, ammonium acetate, lithium chloride, magnesium chloride and the like. One type of salt may be used alone, or a combination of two or more types may be used. The concentration of the salt in the alcohol is not particularly limited, and may be a normal salt concentration employed in alcohol precipitation, for example, a saturated concentration with respect to the alcohol in the case of sodium chloride.

  The temperature at which the precipitate is left to form a precipitate is not particularly limited as long as the proteoglycan can be precipitated. The temperature can be, for example, about −80 ° C. to room temperature, preferably about 0 to 10 ° C.

  The time for leaving the precipitate to form is not particularly limited as long as the proteoglycan can be precipitated, and is appropriately set according to the temperature. If the temperature is 0 to 10 ° C., the time can be, for example, 4 to 24 hours, preferably 8 to 16 hours.

  The centrifugal force is not particularly limited as long as a pellet can be formed. The centrifugal force can be, for example, 700-2500 g.

  In the manufacturing method of this invention, unless the effect of this invention is impaired, you may give processes other than an above-described process. The proteoglycan obtained by the production method of the present invention can be preferably used as a raw material for foods, cosmetics, pharmaceuticals and the like.

  The use aspect of the proteoglycan obtained by the production method of the present invention is not particularly limited. Since proteoglycan has the effects described above, for example, it is preferable to use it as an external composition or an oral composition. That is, it is preferably used as an external composition or an oral composition containing proteoglycan obtained by the production method of the present invention. Moreover, the composition for external use or oral composition can be used as a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, a food composition, and the like. These can be produced by a conventional method using the proteoglycan obtained by the production method of the present invention. In particular, these compositions can be preferably used in the cosmetics field and the food and drink field.

  The cosmetic composition containing the proteoglycan obtained by the production method of the present invention used in the cosmetic field (hereinafter sometimes referred to as “the cosmetic composition according to the present invention”) may be the proteoglycan itself. The proteoglycan and a medium acceptable for cosmetics, bases, carriers, additives, and other components and materials acceptable for cosmetics may be appropriately blended and produced according to a conventional method. Specific examples include emulsions, lotions, creams, serums, foundations, packs, sunscreens, and the like that are produced containing the proteoglycans. Such a cosmetic composition according to the present invention can be preferably used for inflammation improvement or anti-aging, and more specifically, for example, for sunburn prevention, suntan care, moisturizing and anti-skin aging ( For example, it can be preferably used for the prevention or improvement of dry skin, rough skin, wrinkles and sagging of the skin).

  A food / beverage product composition (hereinafter sometimes referred to as “a food / beverage product composition according to the present invention”) containing a proteoglycan obtained by the production method of the present invention used in the field of food / beverage products (beverages and foods) The proteoglycan itself may be appropriately blended with the proteoglycan and a food hygienically acceptable base, carrier, additive, and other ingredients and materials that can be used as food. . For example, processed foods, beverages and health foods (nutrient functional foods, foods for specified health use) for moisturizing and anti-skin aging (for prevention or improvement of dry skin, rough skin, wrinkles and sagging skin etc.) containing the proteoglycan Etc.), supplements, beauty foods, foods for the sick, and the like. Furthermore, the humectant and the anti-skin aging agent comprising such a food or drink composition according to the present invention are also included in the present invention. The moisturizer and anti-skin aging agent are drinks, tablets, tablets, capsules, granules for beauty and anti-skin aging (for example, prevention or improvement of dry skin, rough skin, wrinkles and sagging skin). , Jelly, troche and the like.

  In the pharmaceutical composition, quasi-drug composition, cosmetic composition, or food and beverage composition according to the present invention, the amount of the proteoglycan-containing extract contained in these compositions is not particularly limited. For example, it is 0.001-100 mass% normally with respect to the whole composition, Preferably it is 0.01-95 mass%.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Reference Example 1: Preparation of Ume Vinegar Ripe Minami Takame plum (3 kg) and salt (360 g) from Wakayama Prefecture were mixed and kneaded well. This was put into a jar, a weight was placed on it, and allowed to stand in a cold place for 3 days. After standing, the solution obtained after removing plums was filtered to obtain 630 mL of plum vinegar. The pH of this ume vinegar was 2.1.

Example 1: Extracting and crushing fish cartilage (1 g) of proteoglycan using ume vinegar and ume vinegar (20 mL) obtained in Reference Example 1 were mixed and allowed to stand at room temperature for 24 hours with gentle stirring. The obtained mixed liquid was filtered with a filter paper to remove insoluble components. Ethanol (salt saturated ethanol) (60 mL) in which a saturated amount of sodium chloride was dissolved was added to the obtained filtrate and mixed, and the mixture was allowed to stand at 4 ° C. for 12 hours. After centrifugation (1300 g × 10 minutes), the pellet (white) was recovered. The white precipitate was washed with ethanol (20 mL) and air-dried to obtain 8.7 mg of proteoglycan.

Comparative Example 1: Extraction of proteoglycan using acetic acid Proteoglycan was obtained in the same manner as in Example 1 except that 4% acetic acid aqueous solution was used instead of ume vinegar.

Test Example 1: Confirmation of proteoglycan and identification of glycosaminoglycan The proteoglycan obtained in Example 1 was dissolved in water to prepare a 0.1% proteoglycan aqueous solution, among which (5 μL) and a separately prepared standard solution (hyaluronic acid , Heparan sulfate, heparin, or chondroitin sulfate aqueous solution) was spotted on a cellulose acetate membrane previously impregnated with a migration buffer solution (0.1 M formic acid-pyridine buffer solution (pH 3.0)). Next, the cellulose acetate membrane was placed on the electrophoresis layer and subjected to electrophoresis at 1 mA / cm for 15 minutes. After electrophoresis, the cellulose acetate membrane was subjected to Alcian blue staining, decolorized with 70% ethanol, and the stained band was visually observed.

  The results are shown in FIG. In FIG. 1, the outline of the stained band is surrounded by a black line. In FIG. 1, HP is a band indicating heparin, ChS is a band indicating chondroitin sulfate, HS is a band indicating heparan sulfate, and HA is a band indicating hyaluronic acid. As shown in FIG. 1, the detection of proteoglycan was confirmed as the darkest band (*) at the bottom. Moreover, since the band (**) of the same mobility as chondroitin sulfate was confirmed above the proteoglycan, it was speculated that the glycosaminoglycan in the proteoglycan obtained in Example 1 was chondroitin sulfate.

Test Example 2: Identification of molecular weight of proteoglycan The proteoglycan obtained in Example 1 was dissolved in water to prepare a 0.1% proteoglycan aqueous solution, 50 μL of which was subjected to high performance liquid chromatography analysis. The analysis conditions are as shown below.
Detector: Differential refractometer (RI) YRD-882midget (Shimmura Keiki Seisakusho)
Column: TOSOH TSK-gel G5000PWXL
Eluent: 0.2M-NaCl
Flow rate: 0.5ml / min
Column temp .: 30 ° C
The results are shown in FIG. The numerical values in FIG. 2 indicate the holding time. As shown in FIG. 2, a proteoglycan peak was observed at a retention time of 17.668 minutes, and the average molecular weight was calculated to be 170,000. The average molecular weight was calculated using pullulan standard (manufactured by Shodex, P-82). In addition, a chondroitin sulfate peak was observed at a retention time of 20 minutes.

Test Example 3: Proteoglycan Odor Test Regarding the proteoglycan obtained in Example 1 and the proteoglycan obtained in Comparative Example 1, eight evaluators evaluated the odor in five stages (5: smell of plum, 4: It had a faint plum scent, 3: no odor, 2: a faint unpleasant odor, 1: a strong unpleasant odor. The results are shown in Table 1.

  As shown in Table 1, the proteoglycan obtained in Comparative Example 1 was evaluated as having an unpleasant odor by nearly half of the evaluators (3/8), whereas the proteoglycan obtained in Example 1 was There were no people who evaluated it as having an unpleasant odor, and nearly half (3/8) of the evaluators rated it as having a good aroma of plum.

Claims (6)

(A) A method for producing proteoglycan comprising extracting proteoglycan from fish cartilage with plum vinegar. Furthermore, (b) The manufacturing method of Claim 1 including removing an insoluble matter from the extract obtained at the process a. Furthermore, (c) The manufacturing method of Claim 2 including refine | purifying proteoglycan by alcohol precipitation after the process b. The production method according to claim 3, wherein the alcohol precipitation is ethanol precipitation. The manufacturing method in any one of Claims 1-4 whose extraction temperature is 10-40 degreeC. The manufacturing method in any one of Claims 1-5 whose pH of ume vinegar is 1.5-3.0.