JP5252623B2 - Extraction method of proteoglycan - Google Patents

Description

  The present invention relates to a method for extracting and recovering proteoglycans from aquatic animal tissues, especially fish heads that are discarded in large quantities.

  In the past, proteoglycans were extracted and separated from those contained in mammalian cartilage. However, since the occurrence of BSE in cattle has been reported, extraction and separation of proteoglycans from aquatic animal tissues has been performed. Extraction of proteoglycans contained in whale and shark cartilage has already been carried out, but the amount of animals that can be used as raw materials is limited, and the production of proteoglycans is limited. Furthermore, the extraction and separation operation is complicated, and the solvent used for the extraction is often unsuitable for food, and the use of the product is limited and the price is high.

  There are various methods for extracting proteoglycans contained in animal tissues, but many methods in which the proteoglycans prepared are decomposed and reduced in molecular weight in the methods in which a heating operation is performed during extraction. In the extraction method in which a protease inhibitor is added to prevent hydrolysis, the recovery rate is poor, and various operations contained in the tissue are mixed and complicated operations are required to remove it, which greatly affects the extraction cost. The products that have been marketed so far have complicated manufacturing methods and are very expensive. In addition, because a special extraction solvent such as acetic acid or alkali is used, the odor of the solvent remaining in the product remains, limiting the range of use such as medical use, and the use of the proteoglycan's health functions is closed. It is.

Moreover, each animal has a peculiar body odor, and whenever a tissue is taken out from the animal, a body odor or a modified odor is attached to the tissue. Sometimes the odor may be desirable, but in many cases it is not desirable. The animal's body odor must be completely removed to feel in trace amounts. In addition, many body odors are easily dissolved in oil, and even if the tissue is taken out and deodorized during the purification process, complete deodorization is very difficult due to the residual trace amount of fat.
In particular, in order to remove the off-flavor attached when separated and extracted from fish, it is necessary to use a large amount of various polar and non-polar solvents or to use a special apparatus such as supercritical extraction. When these solvents are used, many of them cannot be used for foods. Solvents that can be used for foods have low efficacy, and a sufficient effect cannot be obtained when the use conditions are set strictly and are not used.

  WO 2004/083275 discloses a method for producing a proteoglycan by pulverizing cartilage derived from cartilaginous fish, adding water to extract a water-soluble component, separating an aqueous layer, adding an alcohol to obtain a precipitate. Yes. However, this method has a problem that it is difficult to completely separate proteoglycan and fat from a cartilage raw material containing a large amount of lipid, and a fishy odor remains.

WO2004 / 083257

  An object of the present invention is to provide a method for efficiently separating, extracting and purifying fish that are caught in large quantities, particularly from cartilage tissues of salmon and carp heads that are discarded as residues after being caught and processed in the North Sea. is there. Another object of the present invention is to provide a method for obtaining a proteoglycan composition having almost no off-flavor and having a wide range of uses.

As a result of various studies on the extraction conditions for proteoglycans in order to achieve the above object, the present inventor has reached the present invention. That is, the present invention is as follows.
1. A method for extracting a proteoglycan composition comprising the following steps A to E.
A. Crushing frozen aquatic animal tissue, adding water thereto, and treating at a temperature of 0 to 20 ° C. and a pH of 4.8 to 7,
B. Centrifuging the solid-liquid mixture of A, removing the uppermost lipid layer and the intermediate aqueous layer, and collecting the precipitate;
C. Drying the precipitate and pulverizing it,
D. A step of adding hexane, acetone or ethanol as a solvent to the resulting dry fine powder, and extracting and removing residual lipids;
E. Removing the solvent.
2. 2. The extraction method according to 1 above, wherein the organic solvent is ethanol.
3. The method according to claim 1 or 2, wherein the volume ratio of the dried precipitate to the organic solvent is 1: 1 to 10.
4). A proteoglycan composition obtained by the method of 1, 2 or 3.

  According to the method of the present invention, a composition mainly used for proteoglycan can be separated and extracted from aquatic animal tissues to provide a composition for use in foods. By carrying out the treatment operation of the present invention, the composition can be made into a state where it can be used as a food and other materials by removing the off-flavor by using a solvent permitted for the food. Furthermore, the composition obtained in the present invention can be further purified to provide a composition that can be used for food and various other uses.

  In the extraction method of the present invention, the delicate temperature and pH of water used for extraction from aquatic animals are set appropriately, and the lipid is levitated to the upper part to perform extraction, thereby preventing unnecessary lipid degradation while preventing proteoglycan degradation. And the second stage of processes D to E that removes a bad odor coexisting in the lipid remaining in a trace amount using an organic solvent such as ethanol.

  Since the lipids contained in living tissue and other compositions are completely integrated to form a tissue body, a powerful operation method such as heat extraction is used to separate the two, so usually the composition Often cause denaturation. In the present invention, as a first step operation for separating the lipid and the water-soluble composition, as a result of earnest research, 75% of the lipid is removed by separating the lipid contained in the ground cartilage tissue by the difference in specific gravity. It is possible.

  Examples of the aquatic animal in the present invention include sharks, whales, sharks, and sharks, and preferably sharks and sharks. The aquatic animal tissue may be any tissue, but salmon that has been caught and processed in the North Sea and discarded as a residue is preferred from the viewpoint of effective use of resources. These cartilage tissues need to be frozen in order to suppress degradation of proteoglycans contained in the cartilage tissues by hydrolase. For reference, the composition of salmon nasal cartilage is shown in Table 1.

  The crushing in the step A may be performed by a known crusher, for example, a chopper (freezing / bone meat grinder). The water added to the aquatic animal tissue can be tap water, deionized water or distilled water. The amount of water is preferably 100 to 200 parts by weight with respect to 100 parts by weight of aquatic animal tissue on a wet basis. In the present invention, in Step A, it is important that the pH is 4.8 to 7 and the temperature is sufficiently mixed at 0 to 20 ° C., and then preferably centrifuged at 0 to 15 ° C. Under acidic conditions where the pH is less than 4.8, acetic acid or the like is used as a solvent, and it becomes difficult to completely remove the solvent from the precipitate, so that the range of use is limited. Under alkaline conditions exceeding pH 7, the dissolution rate of fat increases, but the glycosaminoglycan sugar chain and core protein of proteoglycan are also degraded. If the temperature is lower than 0 ° C., it becomes ice and cannot be used as an extraction solvent. If the temperature is higher than 20 ° C., the function of proteoglycan hydrolase contained in aquatic animal tissues is promoted, and the proteoglycan is reduced in molecular weight. Particularly preferably, the pH is 5.0 to 6.5 and the temperature is 10 to 15 ° C. When water is added and the pH and temperature are adjusted to the desired ranges, they are preferably mixed well with stirring. Lipids float near the top and the proteoglycan composition precipitates.

  Next, as the B stage, the lipid and the proteoglycan composition are immediately separated by centrifugation. Although the rotation speed of centrifugation and the separation time have a correlation, 3,000 to 12,000 rpm and 10 to 40 minutes are preferable, and 5,000 to 10,000 rpm and 20 to 30 minutes are more preferable. The centrifugal separation layer is divided into three layers, and the uppermost lipid layer and the intermediate aqueous layer are removed to collect the precipitate. The temperature at the time of centrifugation is preferably 0 to 15 ° C. in order to form clear three layers.

  Next, in step C, the precipitate is dried, preferably freeze-dried, and then made into a fine powder by a pulverizer. The pulverization may be performed by a known pulverizer such as a centrifugal type or impact type pulverizer. The degree of grinding is approximately 100 to 300 mesh. The resulting dry fine powder has about 10% lipid. Therefore, about 75% of lipid was removed in the A to C steps.

  The steps A to C, which are the first stage treatment in the present invention, can remove most of the lipids that are not preferred to coexist, but are about the same as the compositions marketed in proteoglycan compositions. However, there was still a strange odor and there was a limit to the food used. Therefore, in the present invention, intensive studies were conducted with the primary purpose of further expanding the use of proteoglycans having various functions. In this sense, the remaining off-flavor must be removed.

  There have been many studies on odor so far, and most odors are rarely composed of a single component, and specific odors are constituted by specific collections of many chemical substances. There are many research results on the market regarding particularly favorable odors or “scents”. The object of the present invention is to remove the odor that is the object of objection, but it is necessary to know its overall properties, and the present invention conducts intensive research to find out that it coexists with lipids. It was.

Although the amount of the proteoglycan composition obtained in the steps A to C is very small, lipids still remain. In order to remove the remaining lipid, it is necessary to use a solvent that dissolves the lipid. Since the proteoglycan composition in the present invention is intended for a wide range of uses including foods, restrictions are imposed on the solvent that dissolves lipids. From this point, hexane, acetone or ethanol permitted by the Food Sanitation Law, which is safe for workers and product users, is used as the solvent. Ethanol is preferable.
When extracting a lipid by adding a solvent, proteins such as collagen coexisting with the lipid often show an emulsified state, and a method has been developed that simplifies the operation as much as possible and removes lipids containing off-flavors at low cost.

That is, in the present invention, in order to prevent emulsification of the solvent in which the lipid is dissolved and the proteoglycan composition, as the step D, a specific solvent is added to the dry fine powder obtained in the step C, and the residual lipid is extracted and removed. Thus, the amount of the solvent to be used can be reduced by adding the solvent while reducing the moisture and volume. As for the quantity of a solvent, 1-10 are preferable for an organic solvent with respect to the dry precipitate 1 by volume ratio, More preferably, it is 5-10.
Finally, as the step E, the target solvent can be removed without decomposing proteoglycans in the composition by air drying or low-temperature vacuum drying of the solvent used. Solvent extraction can be performed once to multiple times as necessary.

Example 1
100 g of frozen salmon nasal cartilage was crushed, and the same volume of tap water at 15 ° C. was added thereto, gently stirred and mixed thoroughly, and the mixture maintained at around 5 ° C. was immediately centrifuged at 9,000 rpm for 30 minutes. Centrifugation was performed at 4 ° C. to separate lipids and proteoglycan and other compositions. The centrifugal separation layer was divided into three layers, the uppermost lipid layer and the intermediate aqueous layer were removed, and the precipitate was collected. The precipitate was freeze-dried and then pulverized with a centrifugal pulverizer to obtain a water-defatted fine powder. At this stage, lipids were partially extracted by ether extraction, and as a result, 8.8% of the lipids remained. The removal rate when the lipids before degreasing was 100% was 75.0%, with a weak odor. Was included.

  Subsequently, 10-fold volume of ethanol was added to the water-degreased fine powder to dissolve and extract the lipid containing off-flavor. This operation was repeated twice to remove the ethanol solution by filtration and evaporate the solvent to obtain a slightly yellowish brown odorless proteoglycan composition powder. The yield based on salmon nasal cartilage was 58.7% (dry base), and the proteoglycan content was 77.7%. The off-flavor of the proteoglycan composition powder disappeared at all.

  The composition of the proteoglycan composition of the sample, water degreased powder and water-ethanol degreased powder is shown in Table 2, and the molecular weight constitution of proteoglycan contained in the water degreased fine powder is shown in FIG. For comparison, Table 3 shows the composition of the proteoglycan composition obtained after organic solvent degreasing using chloroform-methanol, which is a general degreasing method, and FIG. 2 shows the molecular weight structure.

  Since the proteoglycan composition obtained by the method of the present invention has no off-flavor, it is particularly suitable for use in the food field.

2 is a diagram showing the molecular weight structure of proteoglycan contained in the water-degreased powder obtained in the intermediate stage of Example 1. FIG. It is a figure showing the molecular weight structure of the proteoglycan contained in the powder obtained by organic solvent degreasing (chloroform-methanol).

Claims (3)

A method for extracting a proteoglycan composition comprising the following steps A to E.
A. Crushing frozen aquatic animal tissue, adding water thereto, and treating at a temperature of 0 to 20 ° C. and a pH of 4.8 to 7,
B. Centrifuging the solid-liquid mixture of A, removing the uppermost lipid layer and the intermediate aqueous layer, and collecting the precipitate;
C. Drying the precipitate and pulverizing it,
D. A step of adding hexane, acetone or ethanol as a solvent to the resulting dry fine powder, and extracting and removing residual lipids;
E. Removing the solvent.   The extraction method according to claim 1, wherein the solvent is ethanol. The method according to claim 1 or 2, wherein the volume ratio of the dried precipitate to the organic solvent is 1: 1 to 10.

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