JP4413133B2 - Method for measuring fucoidan content and food containing fucoidan - Google Patents

Description

  The present invention relates to a method for measuring the fucoidan content, and further relates to a fucoidan-containing food that is measured by the measurement method.

Fucoidan is known as a bioactive substance derived from aquatic organisms. Fucoidan is a sulfated fucose-containing polysaccharide contained in algae, echinoderms, etc. (hereinafter referred to as “fucoidan raw material”), and contains sulfated fucose as a constituent sugar.

  Fucoidan has effects such as anticoagulant activity, antitumor activity, blood cholesterol lowering activity, antiviral activity, anti-HIV activity, antihypertensive activity, antiallergic activity, etc., as a health-oriented food and / or material Development of applications is expected.

Various fucoidan-containing health foods aiming at these effects are manufactured and sold, and many seaweed-based foods and food materials that promote the health image of fucoidan are commercially available (using seaweed of Patent Document 1 as a raw material) Refer to the method for producing beverage foods).
JP 2003-259844 A

  For foods that use seaweed containing fucoidan, such as mozuku, if the raw seaweed is subjected to salting, freezing, heating, etc., the tissue is likely to be damaged, and the damaged raw materials are washed with water. In this case, fucoidan flows out because it is highly water-soluble. For this reason, the amount of fucoidan contained in the final product is considerably smaller than seaweed as a raw material. In addition, the degree of reduction varies depending on the washing conditions, and the actual condition is not constant.

  For this reason, in order to know the amount of fucoidan in the food, it was necessary to measure the content of fucoidan in the food. However, a method for quantifying fucoidan in food has not been established. Fucoidan is a general term for polysaccharides containing fucose. When the type of fucoidan raw material is different, the constituent ratio of fucose (fucoidan conversion coefficient) is also different.

  In other words, since the fucoidan conversion coefficient is specific for each fucoidan raw material, when the fucoidan raw material type is known, the fucoidan content can be converted from the fucose content in the food and measured.

  On the other hand, when the fucoidan raw material species used in the food was unknown, the fucoidan content could not be measured. Also, when multiple types of fucoidan are used and the mixing ratio of each fucoidan is unknown, or even if the mixing ratio of multiple types of fucoidan is known, fucoidan conversion of any fucoidan raw material used When the coefficient was unknown, the fucoidan content in food or food material could not be measured.

  Under these circumstances, even when trying to ingest fucoidan for maintaining health, it was not possible to know how much fucoidan was actually contained in the food to be eaten, so it was not possible to take it effectively. In order to solve these problems, it was necessary to establish a method for measuring the fucoidan content, and to use this measurement method to produce a food whose fucoidan content was controlled and measured.

  An object of the present invention is to solve the above-mentioned conventional problems, and an object of the present invention is to realize a method for measuring the fucoidan content in foods or food materials.

  In order to solve the above-mentioned problems, the present invention specifies fucoidan raw material species and fucose amount, and measures fucoidan content based on fucoidan content rate (fucoidan conversion coefficient) relative to fucoidan specific to fucoidan raw material species and the fucose amount. A method for measuring fucoidan content is provided.

  It is preferable to identify the fucoidan raw material species by identifying the type and ratio of sugars constituting the fucoidan.

  The fucoidan raw material species is preferably identified from the molecular weight distribution of fucoidan.

  The fucoidan raw material species is preferably specified using a substance that specifically identifies the fucoidan raw material species.

  The substance that specifically identifies is preferably an antibody.

  According to the method for measuring the fucoidan content according to the present invention, it is possible to specify the type of fucoidan raw material and the fucoidan content ratio in the food or the food material that could not be achieved conventionally, and as a result, in the food or the food material. The fucoidan content can be measured easily and accurately, and the fucoidan can be applied to products such as various processed foods.

  Furthermore, as fucoidan raw materials, not only the above-mentioned seaweeds and echinoderms, but also purified fucoidans themselves can be included, and the types and fucoidan content ratios of fucoidan raw materials in foods or food materials containing these raw materials are specified. As a result, the fucoidan content in the food or in the food material can be measured easily and accurately.

The best mode for carrying out the fucoidan content measuring method according to the present invention will be described below, and further examples will be described. Note that the following ratio-related terms are defined as follows for clarity of explanation.
(1) “Content rate” is used for “Fucoidan content in each fucoidan raw material”.
(2) “Content ratio” is used for “content ratio of plural types of fucoidan raw materials per se in processed foods”, in other words, “content ratio of different types of fucoidan raw materials per se in foods”.
(3) “Content ratio” is used for “content ratio of different fucoidans derived from different fucoidan raw materials”.

(Overview)
The basic principle of the fucoidan content measuring method according to the present invention will be described. About seaweed etc. which are fucoidan raw materials containing fucoidan, the content of fucose in fucoidan is known for each type.

  Now, when the content percentage (%) of fucose in fucoidan is K%, if k = 100 / K, fucose content × k = fucoidan content. In the present specification, this k is referred to as “fucoidan conversion coefficient”.

  Therefore, if the fucoidan raw material species is identified and specified, the fucoidan content can be calculated by measuring the fucose amount of the food or food material and multiplying this by the fucoidan conversion coefficient.

  However, if the fucoidan raw material is a composite material composed of a plurality of types of raw materials, the content ratio of fucoidan derived from each fucoidan raw material must be taken into consideration. This fucoidan content ratio should be specified by analyzing fucoidan contained in the food, or the content ratio of the plurality of fucoidan raw materials constituting the food should be specified and specified from the content of fucoidan in each fucoidan raw material. Can do. Below, the measuring method of the fucoidan content in a foodstuff is demonstrated.

(Method for measuring fucoidan content)
Measuring method 1:
This measuring method 1 is a fucoidan content measuring method when the fucoidan raw material in the food is one kind (single raw material). In this measuring method 1, the fucose content in the food is measured.

  Then, the fucoidan content in the food can be obtained by multiplying the fucoidan conversion coefficient k known for each fucoidan raw material type such as seaweed and the fucose content in the food. In addition, when it is unknown whether there is a single fucoidan raw material species such as seaweed, it may be identified and identified by the fucoidan raw material species identification method described later.

Measurement method 2:
This measuring method 2 is a fucoidan content measuring method in the case where there are a plurality of fucoidan raw materials (composite raw materials) in food. In this measurement method 2, as in measurement method 1, first, the fucose content in the food is measured.

  And it is necessary to specify the content ratio of a plurality of fucoidan raw material species in the food and fucoidan derived from the fucoidan raw material species. The measurement of the content ratio of a plurality of fucoidan raw material species and fucoidan derived from the fucoidan raw material species will be described later.

  Here, if the fucoidan content (Xn) is calculated for each fucoidan raw material type (n = 1, 2,..., N) and summed (ΣXn), the fucoidan content in food (F = ΣXn) ). The fucoidan content of each fucoidan raw material type is calculated by the following formula.

Here, Xn, An, kn, and R are
[Amount of fucoidan derived from the fucoidan raw material species in food]: Xn
[Content ratio of fucoidan derived from the fucoidan raw material species in food]: An
[Fucoidan conversion coefficient for each fucoidan raw material]: kn
[Fucose content in food]: R
When defined as
If Kn = 1 / kn,
X1 = A1 / (K1 × A1 + K2 × A2 + K3 × A3 +... + Kn × An) × R
X2 = A2 / (K1 × A1 + K2 × A2 + K3 × A3 +... + Kn × An) × R
X3 = A3 / (K1 × A1 + K2 × A2 + K3 × A3 +... + Kn × An) × R
・
・
・
Xn = An / (K1 × A1 + K2 × A2 + K3 × A3 +... + Kn × An) × R
Therefore,
F (fucoidan content) = ΣXn = (X1 + X2 + X3 +... + Xn)
= (A1 + A2 + A3 +... + An) / (K1 × A1 + K2 × A2 + K3 × A3 +... + Kn × An) × R

In other words,
[Fucoidan content derived from the fucoidan raw material in food] = [content ratio of fucoidan derived from the fucoidan raw material based on the fucoidan content in food] / [fucoidan raw material in food processed fucoidan raw material in processed food Sum of numerical values obtained by multiplying the content ratio of fucoidan derived from each fucoidan raw material based on the reciprocal of the conversion coefficient and the fucoidan content in the processed food] x [fucose content in the processed food]
And
[Fucoidan content in food] = [Total content of fucoidan derived from all fucoidan raw materials based on fucoidan content in food] / [Conversion coefficient of fucoidan raw material in processed food] Sum of numerical values of the fucoidan content ratio derived from each fucoidan raw material based on the fucoidan content in the processed food] x [fucose content in food]
It becomes.

This calculation formula is obtained as follows. In other words, from the above definition,
X1, X2,..., Xn is the amount of fucoidan for each fucoidan raw material in the food, and the amount of fucose in the food determined by measurement is R, and the content of fucoidan in the food by fucoidan raw material The ratio is X1: X2: ...: Xn = A1: A2: ...: An, and the fucoidan conversion coefficient of each fucoidan raw material type in the food is k1, k2, ..., kn, respectively. When
If Kn = 1 / kn,
R = K1X1 + K2X2 + ... + KnXn From the content ratio, X1 = A1b, X2 = A2b,..., Xn = Anb (where b is a value other than zero), so R = K1X1 + K2X2 + ... + KnXn
= K1A1b + K2A2b + ... + KnAnb
= (K1A1 + K2A2 + ... + KnAn) b
Therefore, b = R / (K1A1 + K2A2 +... + KnAn)
Therefore, Xn = Anb = An / (K1A1 + K2A2 +... + KnAn) × R
It was requested from becoming.

  In the above formula, the fucoidan content of each fucoidan raw material type was obtained and calculated by the sum, but the total fucoidan conversion coefficient of the food (this is defined as kT) is obtained and this is the fucose in the processed food. The total fucoidan content in the food or food material can be calculated by multiplying the content.

  That is, a plurality of fucoidan raw material species used in foods are identified, the content ratio of each fucoidan species derived from the fucoidan raw material is measured, the content ratio of fucoidan derived from the fucoidan raw material species is obtained, and each ratio is determined as the fucoidan raw material species. The value obtained by multiplying the fucoidan conversion coefficient of the species (conversion coefficient that can calculate the amount of fucoidan from the amount of fucose: K), and summing them (the sum of the values calculated for multiple fucoidan raw materials) By dividing the sum of the content ratios of fucoidan derived from seeds, the overall conversion coefficient (kT) that can calculate the amount of fucoidan from the amount of fucose of food is determined.

By multiplying the total conversion coefficient (kT) by the fucose amount of the processed food, the fucoidan content (F) is obtained. According to the above definition,
kT = (A1 + A2 +... + An) / (K1A1 + K2A2 +... + KnAn)
It is represented by

  For example, if the fucoidan raw material species used as a raw material for food are two types, A and B, and the ratio of both is A type content: B type content = α: β, the fucoidan conversion coefficient is A type a When the B type is b, the overall conversion coefficient (kT) is kT = (α + β) / (α × 1 / a + β × 1 / b), where the processed food has a fucose content of Z. The fucoidan content Y to be obtained is Y = Z × K = Z × [(α + β) / (α × 1 / a + β × 1 / b)].

Measurement method 3:
This measuring method 3 is a method for measuring the fucoidan content in a food when there are a plurality of fucoidan raw material species in the food and the fucoidan content of each of the plurality of fucoidan raw material species is known. In this measurement method 3, as in measurement method 1, first, the fucose content in the processed food is measured.

  Then, a plurality of fucoidan species in the food is specified, and the content ratio of each fucoidan raw material in the food is specified. The identification of the plurality of fucoidan raw material species and the measurement of the content ratio thereof will be described later. In the measurement method 2, a plurality of fucoidans derived from a plurality of fucoidan raw material species in foods are specified, and the content ratio of a plurality of fucoidan derived from fucoidan raw materials is specified. In this measurement method 3, the fucoidan raw material species itself is specified. It is different in that the content ratio is specified.

  If the fucoidan content is calculated for each fucoidan raw material type and summed, the fucoidan content in the food can be determined. The fucoidan content for each fucoidan species is calculated by the following formula.

  [Fucoidan content derived from the fucoidan raw material in the processed food] = [Fucoidan content in the fucoidan raw material] × [Content ratio of the fucoidan raw material itself in the processed food] / [All processed fucoidan raw materials in the processed food Of the conversion coefficient of the fucoidan raw material, the sum of the values of the fucoidan content in the fucoidan raw material and the content of the fucoidan raw material itself in the processed food] × [fucose content in the processed food]

In order to carry out the fucoidan content measuring method according to the present invention, the following identification or measurement process is required.
(1) Measure the amount of fucose in the processed food.
(2) Identify the types of fucoidan raw materials in processed foods.
(3) When the processed food is composed of a plurality of types of fucoidan raw materials (measurement methods 2 and 3), the content ratio of the fucoidan derived from the plurality of types of raw materials in the food sample is specified. In this case, measurement method 2 identifies the content ratio of multiple types of fucoidan by analyzing fucoidan contained in the processed food, and measurement method 3 identifies the content ratio of the plurality of fucoidan raw materials themselves that constitute the processed food. And it specifies from the content rate of the fucoidan in each fucoidan raw material.

(Identification of type and content ratio of seaweed or fucoidan)
In the following, with regard to the above (2) and (3), the type and content ratio of fucoidan contained in the fucoidan raw material constituting the processed food invented by the present inventors, or the specific type of fucoidan raw material and the content ratio A method of identifying will be described.

First identification method:
The first identification method is that the molecular weight distribution of fucoidan before hydrolysis of the fucoidan raw material is specific to the type of fucoidan raw material such as seaweed (for example, fucoidan having a molecular weight of 240,000 is a molecular weight specific to Ito Mozuku, molecular weight 42,000 is Focusing on the specific molecular weight of Hijiki fucoidan, the molecular weight of 500,000 is the molecular weight specific to Okinawa mozuku fucoidan), and by examining this unique molecular weight distribution by gel filtration method, fucoidan raw material species in foods And the fucoidan content ratio.

  This method is as follows in detail. Crude fucoidan is extracted from food samples by hydrochloric acid extraction, filtration, and ethanol precipitation. 1 g of this crude fucoidan is dissolved in 100 ml of distilled water. After overnight dialysis, 2% cetylpyridinium chloride is added, and the resulting precipitate is recovered by centrifugation, washed with distilled water and dissolved in 100 ml of 4M calcium chloride. After filtration, dialysis was performed to obtain a purified fucoidan solution.

  The molecular weight distribution of this solution is measured by gel filtration, and the content ratio of the fucoidan raw material species and fucoidan in the food can be determined by comparing with the composition ratio of the standard product.

Second identification method:
The second identification method pays attention to the fact that the ratio of the constituent sugar of the fucoidan raw material is also unique depending on the type of fucoidan raw material. Analysis using HPLC (High Performance Liquid Chromatography) or TLC (Thin-Layer Chromatgraphy) Is a method for determining the fucoidan raw material species and the fucoidan content ratio in foods by analysis using thin layer chromatography).

  The details of this method are as follows. Crude fucoidan is extracted from seaweed powder, a food sample, by hydrochloric acid extraction, filtration, and ethanol precipitation. 1 g of this crude fucoidan is dissolved in 100 ml of distilled water. After overnight dialysis, 2% cetylpyridinium chloride is added, and the resulting precipitate is recovered by centrifugation, washed with distilled water and dissolved in 100 ml of 4M calcium chloride. After filtration, dialysis was performed to obtain a purified fucoidan solution. This was subjected to hydrolysis in 2.0 N sulfuric acid at 100 ° C. for 2 hours.

  Here, when HPLC is used, the purified fucoidan subjected to hydrolysis is quantitatively analyzed by HPLC, and the composition ratio of sugars such as L-fucose and D-xylose is compared with the composition ratio of the standard product. As a result, the content ratio of raw material species such as seaweed and fucoidan could be clarified. In this HPLC, Shimak ISA-07 (4.0 x 250 mm) was used for the column (column hot bath), the eluent was 0.1 to 0.4 M borate buffer, and the flow rate of the eluent was 0. The temperature was 6 ml / min and the temperature was 65 ° C.

  When using TLC, the purified fucoidan subjected to hydrolysis is quantitatively analyzed by TLC, and the composition ratio of sugars such as L-fucose and D-xylose is compared with the TLC pattern of the standard product. Fucoidan raw material species and fucoidan content ratio can be clarified.

Third identification method:
In the third identification method, when a plurality of fucoidan raw material-derived fucoidans are mixed, first, an immunological measurement method such as an ELISA analysis method using a specific antibody against fucoidan derived from each fucoidan raw material. Alternatively, the fucoidan raw material species and the fucoidan content ratio are measured by a measurement method using a lectin specific to fucoidan derived from the fucoidan raw material.

Fourth identification method:
The fourth identification method is the fucoidan content (% of raw fucoidan raw material actually used after storage water washing) used in the product for the case where fucoidan derived from multiple seaweeds is mixed. ) Is known in advance, measure the content of fucoidan raw materials and the content of each fucoidan raw material by the measurement method using specific antibodies obtained by immunizing specific antigens for fucoidan raw materials such as seaweeds other than fucoidan. By multiplying this by the fucoidan content (%) of the individual fucoidan raw material, it is possible to obtain the individual fucoidan content ratio.

Fifth identification method:
The fifth identification method is for a case where fucoidans derived from a plurality of fucoidan raw materials are mixed, and when the fucoidan content (%) of each fucoidan raw material used in the product is known in advance, each fucoidan raw material Specific fucoidan raw materials used by various DNA quantification methods such as PCR were measured, and the fucoidan content (%) of each fucoidan raw material was measured. ) Can also be used to determine individual fucoidan content ratios.

(Measurement of fucose content in processed foods)
Hereinafter, the measurement of the fucose content (R) in the processed food multiplied by the conversion coefficient (k) listed in (1) above will be described. Conventionally, it has been difficult to measure fucose in processed foods containing various additives, particularly when other sugars or substances having sulfate groups are mixed.

For example, the rosin acid method (KSDodgson, RGPrice, Biochem. J., 84, 106 (1962)), which has been used to measure the purity of fucoidan in the past, hydrolyzes sugar with hydrochloric acid, removes hydrochloric acid, and removes ethanol. After adding and removing the precipitate by centrifugation, BaCl ₂ and a sodium rosinate solution are added and mixed, placed in a dark place at room temperature, and measured at an absorbance of 520 nm.

  However, in this method, in order to measure the amount of sulfate groups, many other food materials containing sulfate groups, such as animal-derived chondroitin, seaweed-derived carrageenan, and inorganic compound alum, are also included in processed foods. There was a problem that it could not be used to measure fucoidan. In the fucoidan content measuring method according to the present invention, fucose in a processed food, which has been difficult in the past, can be measured by performing the following method.

  That is, in the method for measuring fucoidan content according to the present invention, food is treated with hydrochloric acid and then treated with ethanol. Further, in the next color development process, in the next color development process, oligosaccharides and monosaccharides that are obstructive for color development similar to fucose, etc. The low molecular fraction containing is filtered off by ultrafiltration and then hydrolyzed.

  When the obtained fucoidan is decomposed into fucose, in the phenol / sulfuric acid method used in the conventional measurement of fucoidan, the mixing of sucrose and glucose is affected by color development. do not use. In the fucoidan content measurement method according to the present invention, sucrose and glucose do not develop color, and at this stage, only the amount of fucose that is methyl pentose can be measured (Gibbons method: Gibbons, MN (1955) Analyst, 80, 268.).

  This Gibbons method is a kind of cysteine-sulfuric acid method. The exact basic reaction of the principle of this cysteine-sulfuric acid method is unknown, but roughly, the substance produced by the reaction of sugar and sulfuric acid reacts with an SH reagent such as cysteine and changes color. The principle of this method is to quantitate the sugar from the coloration degree.

  In particular, the Gibbons method is one of the methods for quantifying methylpentose as described above, but using thioglycolic acid as the SH reagent. The absorbance at 400 nm and 430 nm of the absorption spectrum when colored with thioglycolic acid was measured, the difference was calculated, and the amount of fucose was measured. This made it possible to grasp the amount of fucose in the processed food. When there was no problem with mixing of sucrose and glucose, it was possible to use the phenol / sulfuric acid method.

  As described above, the fucoidan content measurement method according to the present invention has been described mainly with respect to the case where the fucoidan raw material is seaweed, but the fucoidan raw material can include not only seagrass and echinoderms, but also purified fucoidan itself. The type and fucoidan content ratio of the fucoidan raw material in the food containing the raw material or in the food material can be specified. As a result, the fucoidan content in the food or the food raw material can be measured easily and accurately.

  The object of the method for measuring the fucoidan content according to the present invention is fucoidan raw materials such as seaweed, but as foods containing fucoidan made from such fucoidan raw materials, there are many foods. .

  Examples include side dishes such as vinegar, soups and salads, konjac and tofu, dressings, kneaded products such as fried tempura and tempura, various beverages such as seaweed-containing beverages, seaweed-containing ham and sausages, hamburgers and meatballs. Processed meat products such as bread, rice crackers, rice cakes, cakes, cookies, candy and gum, sprinkled seaweed powder and teas, jams, dairy products such as butter, cheese and yogurt, various canned goods, retort Various foods such as foods, frozen foods, and dry matter can be listed. Furthermore, this method is applicable not only to foods but also to cosmetics and pharmaceuticals.

(Identification by sugar composition)
As an embodiment 1, an embodiment characterized by the identification method 2 will be described below. As a test specimen, seaweed powder consisting only of Okinawa mozuku was prepared. Crude fucoidan was extracted from this seaweed powder by hydrochloric acid extraction, filtration, and ethanol precipitation. 1 g of this crude fucoidan is dissolved in 100 ml of distilled water. After overnight dialysis, 2% cetylpyridinium chloride is added, and the resulting precipitate is recovered by centrifugation, washed with distilled water and dissolved in 100 ml of 4M calcium chloride. After filtration, dialysis was performed to obtain a purified fucoidan solution. This was subjected to hydrolysis in 2.0 N sulfuric acid at 100 ° C. for 2 hours.

  This was quantitatively analyzed by HPLC (column, Shimpak ISA-07 (4.0 × 250 mm); eluent, 0.1 to 0.4 M borate buffer; flow rate, 0.6 ml / min; temperature 65 ° C.). However, it was revealed that the composition ratio of L-fucose and D-xylose is 97: 3 and shows a sugar composition specific to Okinawa Mozuku that other sugars are hardly contained. From this result, it was confirmed that the seaweed powder was made only from Okinawa mozuku.

(Identification by sugar composition)
As a second embodiment, another embodiment characterized by the identification method 2 will be described below. When the sugar composition of fucoidan contained in seaweed-filled fish cakes with only hijiki added as seaweed was examined in the same manner as in Example 1, L-fucose: D-galactose: D-glucose, a sugar composition specific to hijiki : D-mannose: D-xylose: D-glucuronic acid was found to be 5: 1: 1: 0.5: 0.5: 2. From this result, it was confirmed that the seaweed used was only hijiki.

(Identification of raw seaweed by antibody)
As a third embodiment, an embodiment characterized by the identification method 3 will be described below. A white rabbit is dosed intradermally with 1.0 mg of Okinawa Mozuku-derived purified fucoidan (as an emulsion with Freund's complete adjuvant), and one week later, an equivalent amount of Okinawa Mozuku-derived purified fucoidan is further administered ( Whole blood was collected 49 days after repeated administration (as an emulsion with Freund's incomplete adjuvant) four times to obtain antiserum. This antiserum was purified by affinity chromatography to obtain a purified fucoidan polyclonal antibody derived from anti-Okinawa mozuku.

  The same treatment was also applied to the kombu to obtain an anti-comb-derived purified fucoidan polyclonal antibody. Here, a seaweed drink was prepared by mixing Okinawa mozuku with a fucoidan content of 20% (6% by weight in a beverage) and a kombu with a fucoidan content of 10% (4% by weight in a beverage), and using this antibody in a seaweed beverage The amount of fucoidan derived from Okinawa Mozuku was measured by ELISA.

  Similarly, the amount of fucoidan derived from kombu in seaweed drink was measured by ELISA using this antibody. As a result, the ratio of Okinawa mozuku-derived fucoidan content: kombu-derived fucoidan content = 3: 1 could be obtained as the fucoidan content ratio of Okinawa mozuku and kombu.

  As Example 4, an example in which the fucoidan content in processed food is measured by the process of identification by molecular weight distribution (discrimination method 1) and measurement of fucose content in processed food will be described below.

First process (identification by molecular weight distribution):
First, as a test sample, seaweed soup was prepared using seaweed, hijiki and Okinawa mozuku as raw materials. By purifying fucoidan in these raw materials in advance and measuring the fucoidan content in each seaweed, this seaweed soup has a fucoidan content ratio derived from each seaweed: fucoidan derived from hijiki: fucoidan derived from Okinawa mozuku = At 20:10, it was confirmed that the fucoidan content was 53 mg.

  Crude fucoidan was extracted from this seaweed soup by hydrochloric acid extraction, filtration, and ethanol precipitation. 1 g of this crude fucoidan is dissolved in 100 ml of distilled water. After overnight dialysis, 2% cetylpyridinium chloride is added, and the resulting precipitate is recovered by centrifugation, washed with distilled water and dissolved in 100 ml of 4M calcium chloride. After filtration, dialysis was performed to obtain a purified fucoidan solution.

  Here, the molecular weight of 42,000 is a molecular weight specific to Hijiki Fucoidan, and the molecular weight of 500,000 is a molecular weight specific to Okinawa Mozuku's fucoidan. Therefore, the molecular weight distribution of this solution was measured by gel filtration. The amount of fucoidan showing 42,000: the amount of fucoidan showing a molecular weight of 500,000 was 20:10.

  From this, it was confirmed that the fucoidan contained in each fucoidan raw material was contained in the sample soup in a ratio of fucoidan content of hijiki: fucoidan content of Okinawa mozuku = 20: 10.

Second process (measuring fucose content in processed food):
Hydrochloric acid was added to 50 ml of the seaweed soup to a concentration of 0.2N HCl (final concentration), and extracted overnight with stirring at room temperature. Solid matter was removed at 2,000 rpm for 10 min, suction filtration (ADVANTEC 5A), neutralized with NaOH (pH 6-7), 30 ml EtOH was added, and the precipitate was collected by centrifugation (5,000 rpm x 15 min). Subsequently, it was dissolved in 150 ml of pure water, and the molecular weight of 200,000 or less was eliminated by ultrafiltration (4 ° C.), and washed once.

  2.7 ml concentrated sulfuric acid was added to make up to 50 ml, hydrolyzed (100 ° C. for 2 hours), and fucose quantification was performed. The fucose was quantified by the Gibbons method. In other words, serially dilute a fucose standard solution (1% solution (10 mg / ml)), dilute the sample, put 1 ml of the standard solution or sample in a capped tube (samples prepared in triplicate), and cool under ice 4.5 ml sulfuric acid (pure water: concentrated sulfuric acid = 1: 6) was added.

  The lid was returned to room temperature, boiled for 10 minutes, and then cooled on ice. 0.1 ml of thioglycolic acid (1 ml of thioglycolic acid diluted to 30 ml with pure water) was added. One set of samples was prepared by adding water instead of thioglycolic acid and used to measure the background value. It was left for 3-24 hours in the dark. The absorbance at 400 nm and 430 nm was measured, and the difference was calculated. As a measurement result, 20 mg was obtained as the fucose content.

Third process (calculation of fucoidan content in processed food):
Here, since the fucose content of fucoidan in hijiki is 31.4%, the fucoidan conversion coefficient is 100 / 31.4 = 3.18. In addition, the fucoidan fucose content in Okinawa mozuku is 50.5%, so the fucoidan conversion coefficient is 100 / 50.5 = 1.98.

  And since the fucoidan content of hijiki: the fucoidan content of Okinawa Mozuku = 20:10, the overall conversion factor is 30 / (1 / 3.18 × 20 + 1 / 1.98 × 10) = 2.65. Therefore, the fucoidan content in 50 ml of this seaweed soup was 20 mg × 2.65 = 53 mg.

  The best mode for carrying out the fucoidan content measuring method according to the present invention has been described based on the embodiments. However, the present invention is not limited to such embodiments, and the scope of the claims is as follows. It goes without saying that there are various embodiments within the technical scope described.

  As described above, since the present invention can measure the amount of fucoidan in food and accurately evaluate the amount, it can accurately contain the amount of fucoidan according to the purpose of the food using fucoidan, The added value of each processed food can be increased by inclusion in various foods such as foods, processed meat products, side dishes and frozen foods. The present invention can also be applied to the measurement of fucoidan content in health foods and cosmetics.

Claims (5)

  A method for measuring the content of fucoidan, characterized in that a fucoidan raw material species and fucose amount are specified, and the fucoidan content is measured based on the fucoidan conversion coefficient specific to the fucoidan raw material species and the fucose amount. Content measuring method.   2. The method for measuring fucoidan content according to claim 1, wherein the identification of the raw material species is performed by identifying the type and ratio of sugars constituting the fucoidan.   2. The fucoidan content measuring method according to claim 1, wherein the raw material species is identified by identifying it from the molecular weight of fucoidan.   2. The fucoidan content measuring method according to claim 1, wherein the material species is specified using a substance that specifically identifies the material species.   5. The method for measuring fucoidan content according to claim 4, wherein the substance specifically identified is an antibody.