JP6145630B2 - Protein rich in glycine - Google Patents

Description

  The present invention relates to a water-soluble protein derived from vomeronasal cartilage that is rich in glycine.

  As proteins containing a large amount of glycine, collagen and gelatin obtained from collagen by hot water or alkaline extraction are known. These are characterized by the fact that the primary sequence of amino acids is a repeat of glycine-XY units, and the amount of glycine is very large at a molar ratio of 30% or more. In addition to glycine, there are many prolines and hydroxyprolines. Collagen is widely contained in humans and animals and constitutes skin, bone, tendon, cartilage and the like. In the dermis of human skin, 70% is made of collagen, and when collagen decreases due to aging or the like, the skin peels down and bones and cartilage are disturbed. Collagen has a moisturizing property and is popular as a cosmetic material. In addition, collagen has recently been in demand as a beauty food and drink and health food.

  Since the uproar of mad cow disease, animal-derived collagen and gelatin have been avoided from consumers, and fish-derived collagen and gelatin have been demanded, and collagen has been produced from fish scales (Patent Documents 1, 2, and 3). However, since fish-derived ones have a disadvantage of having a unique fishy odor, there are those that are masked with sucralose (Patent Document 4) and those that are masked with mulberry leaves (Patent Document 5). In addition, since collagen has a helical structure with three polypeptide chains having a molecular weight of about 100,000, it does not dissolve in water, so some peptides are made into peptides with a molecular weight of several thousand by enzymes to improve solubility (patents) Literature 6, 7, 8, 9). However, since bitterness occurs when it is made into a peptide, some have added a linden extract (Patent Document 10).

  Gelatin is obtained by hot water extraction of collagen or the like, but has a limited use because it has the property of gelling and solidifying when cooled. Therefore, it has been carried out to make a peptide by an enzyme (Patent Document 11).

JP 2007-314458 A JP 2006-257014 A JP-A-5-93000 JP 2000-152757 A JP 2004-357484 A Japanese Patent Laid-Open No. 2008-220208 JP 2007-176909 A JP 2006-89 A JP 2006-217876 A JP 2011-211962 A JP 2004-141007 A

  Collagen or gelatin peptide having a molecular weight of several thousand does not gel when dissolved or cooled in water, but has a drawback that water retention is lowered. An object of the present invention is to provide a protein that is derived from fish, has no unpleasant odor, is a water-soluble polymer, does not gel, and is rich in glycine, proline, and hydroxyproline.

  It has been found that the protein contained in vomeronasal cartilage contains 30% or more of glycine, has a molecular weight of 10,000 or more, is water-soluble and does not gel. This protein is obtained by allowing acetic acid to act on vomeronasal cartilage, collecting the obtained acetic acid solution through an ultrafiltration membrane having a fractional molecular weight of 100,000, and further collecting a molecular weight of 10,000 or less from the passing solution having a fractional molecular weight of 100,000. Prepared by removing low molecular weight material.

  The protein of the present invention is a raw material, has a property that it can be obtained in large quantities in Japan, has no unpleasant odor, is a water-soluble polymer, and does not gel. Moreover, it has the characteristic that the obtained protein is not heat-denatured.

It is the figure which measured molecular weight distribution of protein. (Example 1)

    Hereinafter, the present invention will be described in more detail with its preferred modes.

There are various types of salmon (scientific name: Oncorhynchus keta ), mainly in the genus Salmon, such as chum salmon, coho salmon, calaf trout, and sockeye salmon, but the type is not limited in the present invention. The vomeronasal cartilage used in the present invention is a portion of cartilage that constitutes a part of the head of the salmon and is a part eaten as an ice head of a local dish such as Hokkaido or Aomori Prefecture.

  In the method for preparing the protein of the present invention, the head of the shark is selected, the epidermis of the head is peeled off, and the flesh adhering to the head is removed by running water or other methods to obtain the nasal cartilage. The obtained vomeronasal cartilage is soaked in an acetic acid solution to remove solids and obtain an acetic acid solution. The concentration of acetic acid used is 1% or more. The time for soaking the nasal cartilage in acetic acid is preferably one night or longer, and the soaking temperature is not limited. On the way, stirring, shaking, etc. may be performed.

  Next, the acetic acid solution from which vaginal nasal cartilage and other generated solids are removed by filtration or centrifugation is passed through an ultrafiltration membrane having a fractional molecular weight of 100,000, and the passing solution is collected. The ultrafiltration membrane may be a hollow fiber membrane, a flat membrane, or a spiral membrane. Next, a low molecular component having a molecular weight of about 10,000 or less is removed from the passing liquid, and the process is completed. Examples of the removal method include a method using an ultrafiltration membrane having a molecular weight cut-off of 10,000, a dialysis membrane, a cellulose tube, and a visking tube. The removal of low molecules as much as possible by adding water gives a protein with high purity and low ash content. The extracted acetic acid solution and the passing solution having a molecular weight cut off of 100,000 may be concentrated. Finally, this solution may be dry powdered. Moreover, after removing low molecular components, the protein may be recovered by precipitation with an organic solvent.

  The protein thus obtained has water-soluble properties and does not gel. Further, it has a molecular weight of 10,000 or more, contains 30% or more of glycine in a molar ratio among all constituent amino acids, and contains 15% or more of the total of hydroxyproline and proline.

  The present invention will be specifically described below with reference to examples.

  The head was cut from the body of raw chum salmon, the epidermis of the head was peeled off, the meat pieces were removed in water, and the nasal cartilage portion was collected. 4 L of 5% acetic acid (Daicel) was added to 1 kg of vomeronasal cartilage and stirred occasionally at room temperature. After 3 days, the solid content was removed by filtration to obtain a solution. This solution was passed through an ultrafiltration membrane (Millipore) having a molecular weight of 100,000 to obtain 3 L of a passing liquid. This passing solution was put into a cellulose tube for dialysis (Sanko Junyaku Co., Ltd.) and dialyzed against water in a low-temperature room at 4 ° C. for 3 days. The water of the external liquid was changed 3 times a day. It was confirmed that acetic acid in the flow-through was removed, and this solution was lyophilized. The weight of the solid content after lyophilization was measured and found to be 3.3 g. There was no fishy odor.

  The protein content of the lyophilized solid was 95.9% as determined from a calibration curve using a collagen peptide (Wako Pure Chemical Industries, Ltd.) as a standard substance by the Raleigh method which is a colorimetric method.

  The neutral sugar content of the lyophilized solid was 2.5% as determined from a calibration curve using glucose (Tokyo Kasei Kogyo Co., Ltd.) as a standard substance by the phenol-sulfuric acid method, which is a colorimetric method. . The acidic sugar content was measured by the carbazole sulfate method, which is a colorimetric method, but was not detected.

  The freeze-dried solid content was put in a porcelain crucible, heated to a constant weight with a 110 ° C. thermostat to obtain a constant weight, and then heated to a constant weight with a 600 ° C. electric furnace. As a result, the ash content of the lyophilized solid was 1.6%.

The lyophilized solid was dissolved in a 20% hydrochloric acid solution, sealed in a vacuum, and allowed to stand at 110 ° C. for 20 hours for hydrolysis. Hydrochloric acid was removed by evaporation under reduced pressure using an evaporator, placed in a desiccator containing sodium hydroxide, and dried under reduced pressure with a vacuum pump overnight. A buffer solution for amino acid analysis was added to this, redissolved, and the amount of amino acids was measured with a fully automatic amino acid analyzer (JLC500 / V, JEOL Ltd.).

The results are shown in Table 1. Glycine accounted for 33.0% of the total amino acid molar ratio, hydroxyproline 6.1%, and proline 10.3%.

  20 mg of lyophilized solid was subjected to gel filtration chromatography. Sephadex G-75 (GE Healthcare Japan Co., Ltd.) as a gel carrier is packed in a glass column of 2.5 cm in diameter and 113 cm in length, the eluent is 0.1 M NaCl, and the flow rate is 0.5 mL / In minutes, 5 mL of the solution eluted from the gel was collected per fraction. For each fraction, the amount of protein was measured by the Raleigh method (750 nm). The molecular weight markers are Blue Dextran 2000 (molecular weight 2 million or more, GE Healthcare Japan Co., Ltd.), bovine serum-derived albumin (molecular weight 66,000, Sigma-Aldrich Japan), chicken egg white albumin (44,300, Sigma-Aldrich Japan) ), Chymotrypsinogen A (molecular weight 25,000, GE Healthcare Japan), ribonuclease A (molecular weight 13,700, GE Healthcare Japan), glucose (molecular weight 180, Tokyo Chemical Industry Co., Ltd.) ) Was used.

  The result was as shown in FIG. The horizontal axis is the fraction number, and the vertical axis is the absorbance (Abs) of the Raleigh method. The arrow in the figure indicates the elution position of each molecular weight marker. The protein of the present invention was shown to have a molecular weight of 10,000 or more.

  2 g of this lyophilized solid was dissolved in 20 mL of cold water. Then, it heated for 2 hours with a 50 degreeC thermostat, and left to stand in a 4 degreeC refrigerator overnight. As a result, it was in the state of a solution having sufficient fluidity and was not gelled.

  Since the product of the present invention contains a large amount of glycine, is a polymer, is water-soluble and does not gel, it can be used in cosmetics, foods, particularly beverages. In addition, although it is derived from fish, it has no fishy odor and is easily accepted by consumers.

Claims (1)

The nasal cartilage is immersed in an acetic acid solution, the solution and solids are separated, this solution is treated with an ultrafiltration membrane having a fractional molecular weight of 100,000, the passing solution is collected, and then the passing solution is fractionated molecular weight. Glycine is 30% or more in terms of the molar ratio of all amino acids, and the total of hydroxyproline and proline is 15% or more in terms of molar ratio. A method for producing a water-soluble protein that does not have a fishy odor and does not gel.

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