JP7563757B2 - Method for quantifying proteoglycan in a composition - Google Patents

Description

The present invention relates to a method for quantifying proteoglycans contained in a composition containing a protein used in protein drinks, etc.

Proteoglycan is a major biopolymer that forms the substrate in the extracellular matrix together with collagen and hyaluronic acid. It is found in the cartilage of mammals such as humans and cows, and fish such as salmon and sharks, and many foods such as supplements containing proteoglycan and methods for producing proteoglycan have been reported. In addition, many useful properties of proteoglycan itself are known, and for example, it has been reported that proteoglycan has the effect of promoting cartilage differentiation, improving knee joints, and inhibiting skin pigmentation (see Patent Document 1).

As mentioned above, proteoglycan is a complex, and it is difficult to accurately measure the amount of proteoglycan contained in a composition such as a food or beverage supplement. Therefore, the reality is that no technology has yet been established to measure the amount of proteoglycan contained in a composition. For example, a method for quantifying proteoglycan has been reported in which pretreatment is performed in a hollow container with a molecular weight cutoff membrane to remove free chondroitin sulfate and hyaluronic acid, and this is combined with a reagent for colorimetric quantification of glycosaminoglycan (see Patent Document 2).

JP 2016-138060 A JP 2018-151206 A

The problem that the present invention aims to solve is to find a new method for quantifying proteoglycans contained in compositions such as functional foods that contain proteins (such as milk proteins).

As a result of intensive research conducted by the present inventors to solve the above problems, the inventors have found that, for example, by optimizing the solvent for extracting a composition containing proteoglycan and protein, and measuring the proteoglycan concentration in the extract using a color reagent containing methylene blue or a derivative thereof, the proteoglycan content in the composition can be quantified. That is, the present invention includes the following embodiments.

(1) A method for quantifying proteoglycan in a composition, comprising the steps of: extracting a composition containing proteoglycan and a protein (such as a milk protein) with water or a PIPES buffer solution; recovering a water-soluble fraction after extraction; measuring the proteoglycan concentration in the water-soluble fraction using a color reagent containing methylene blue or a derivative thereof; and calculating the proteoglycan content in the composition from the proteoglycan concentration in the water-soluble fraction.
(2) A method for analyzing proteoglycan in a composition, comprising the steps of: extracting a composition containing proteoglycan and protein with water or a PIPES buffer solution; recovering a water-soluble fraction after extraction; adsorbing the recovered water-soluble fraction onto an ion exchange resin; eluting the adsorbed proteoglycan from the ion exchange resin with an elution solution containing a PIPES buffer solution; and measuring the molecular weight of the eluted proteoglycan by HPLC.
(3) The method according to (1) or (2), wherein the molecular weight of the proteoglycan is 100,000 to 2,000,000.
(4) The method according to any one of (1) to (3), wherein the extraction step is carried out multiple times and the proteoglycan concentration in a mixture of multiple water-soluble fractions is measured.
(5) A quantitative method according to any one of (1) to (4), in which a portion of the water-soluble fraction is used to analyze proteoglycan by the method described in (2), and the proteoglycan content in the composition is calculated in relation to the molecular weight of the proteoglycan.

The method of the present invention makes it possible to easily and precisely quantify the proteoglycan content in a composition containing proteoglycan and a protein (such as milk protein).

FIG. 1 is a process diagram showing the flow of a method for quantitatively determining and qualitatively analyzing proteoglycan in one embodiment of the present invention. FIG. 2 is a process diagram showing the flow of the qualitative analysis method for the proteoglycan recovered in the water-soluble fraction. FIG. 3 shows an example of a calibration curve prepared using a proteoglycan standard in one embodiment of the present invention. FIG. 4 is an example of an HPLC analysis chart obtained by the qualitative analysis of proteoglycan. 5(A) shows the results of qualitative analysis (arrows indicate peaks) for Sample 3-1 (containing Proteoglycan F) in Example 3. FIG. 5(B) shows the results of quantitative analysis for Sample 3-2 (not containing Proteoglycan F) in Example 3. FIG. 6(A) shows the results of quantitative analysis (arrows indicate peaks) for sample solution 4-1-1 (containing proteoglycan) in Example 4. FIG. 6(B) shows the results of quantitative analysis for sample solution 4-1-2 (not containing proteoglycan) in Example 4. FIG. 6(C) shows the results (peaks) of quantitative analysis for sample solution 4-2-1 (containing proteoglycan) in Example 4. FIG. 6(D) shows the results of quantitative analysis for sample solution 4-2-2 (not containing proteoglycan) in Example 4.

First, a brief explanation of the terms used in this specification will be provided.
(Composition)
The composition to be measured by the method of the present invention may be, for example, a composition containing milk protein and proteoglycan used in protein drinks, etc., and may be in the form of a liquid, solid, or semi-solid, but is preferably in a solid state, and more preferably in a powdered form. The lower limit of the proteoglycan content contained in this composition is 0 μg/mg or more, preferably 0.1 μg/mg, and more preferably 1 μg/mg. The proteoglycan content in the composition can be measured if it is equal to or greater than the detection limit, and if it cannot be detected, it is found that the proteoglycan content is equal to or less than the detection limit. There is no particular limit to the upper limit of the proteoglycan content contained in this composition, but it is preferably equal to or less than 100 μg/mg in terms of the cost of producing the composition.

(protein)
The proteins contained in the composition include, for example, milk proteins, plant-derived proteins (e.g., soy proteins, pea-derived proteins), and seafood-derived proteins. Milk proteins include, for example, skim milk powder, whole milk powder, total milk protein, whey protein, casein protein, sodium caseinate, whey protein, buttermilk powder, cow's milk, whole fat concentrated milk, skim milk concentrated milk, and natural cheese. For example, W80 Instant sold by Wheyco is a whey protein derived from cheese whey, and is composed of the main components β-lactoglobulin (a dimer with a molecular weight of 36,600) and α-lactalbumin (a molecular weight of 14,200), as well as BSA and lactoferrin.

(Proteoglycan)
Proteoglycans are glycoproteins in which sugar chains called glycosaminoglycans (GAGs), such as chondroitin sulfate and dermatan sulfate, are covalently bonded to a core protein. Proteoglycans are one of the main components of the extracellular matrix and are widely distributed throughout the body, including in skin and cartilage. GAG chains have a long linear structure with no branches. They are negatively charged due to the presence of many sulfate and carboxyl groups, and the GAG chains take on an extended shape due to their electrical repulsive force. Proteoglycans can also retain a large amount of water due to the water affinity of sugars. The large number of GAG chains contained in proteoglycans retain water like a sponge, providing cartilage with functions unique to cartilage, such as elasticity and resistance to impact.

The type of proteoglycan that can be measured by the method of the present invention is not particularly limited, and may be classified as any of chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, or keratan sulfate proteoglycans extracted from salmon nasal cartilage, squid head cartilage, and pig cartilage. Specific examples include aggrecan, versican, neurocan, brevican, decorin, biglycan, serglycin, perlecan, syndecan, glypican, lumican, and keratocan. Among these, the method of the present invention is preferably suitable for measuring the content of chondroitin sulfate proteoglycans, and more preferably aggrecan. The molecular weight of the proteoglycan used in the present invention is not particularly limited, but is preferably 100,000 to 2,000,000.

(Extract)
The extraction liquid used in the method of the present invention is water or a PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution. The PIPES buffer solution can be used at a concentration of about 1 mM or more, preferably about 5 mM or more, and more preferably about 10 mM or more. The upper limit of the PIPES buffer solution concentration is about 100 mM, preferably about 50 mM or less, and more preferably about 20 mM or less. The pH of the extraction liquid is about 6.0 to about 8.0 (e.g., about 7.0). A specific extraction liquid used in the examples described below contains purified water or 10 mM PIPES buffer solution and has a pH of about 7.0. The buffer solution used in the method of the present invention may also contain a salt (e.g., NaCl, KCl, or NaOAc) at the same time.

(Ion exchange resin)
The term ion exchange resin refers to a negatively charged (i.e., a cation exchange resin) or positively charged (i.e., an anion exchange resin) solid phase. The charge is provided by attaching (e.g., by covalent bonding) one or more charged ionic groups to the solid phase. Alternatively, the charge can be an intrinsic property of the solid phase, such as chitin or chitosan, which have an overall positive charge.

By solid phase is meant a non-aqueous matrix to which one or more charged ionic groups can adhere. The solid phase can be a purification column, a discontinuous phase of discrete particles, a membrane, or a filter, etc. Examples of materials for forming the solid phase include polysaccharides (e.g., agarose and cellulose) and other mechanically stable matrices (e.g., silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above).

Anion exchange resin refers to a solid phase that is positively charged (e.g., has one or more positively charged ionic groups, such as quaternary amino groups, attached to the solid phase). Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX ^™ , and FAST Q SEPHAROSE ^™ (Pharmacia). Anion exchange resins are classified as strong or weak basic depending on the dissociation of their ionic groups.

A weakly basic anion exchange resin refers to an anion exchange resin that exhibits weak basicity, such as a resin that has primary, secondary, or tertiary amino groups as functional groups. Since weakly basic functional groups do not dissociate in alkaline solutions and do not exhibit ion exchange capacity, it is preferable to use them in the neutral region. In addition, although they will exchange with relatively strong acidic groups such as sulfo groups in proteoglycans, they are thought to have difficulty exchanging with weakly acidic groups such as carboxy groups in impurity proteins.

(Color Reagent)
The color reagent used in this embodiment includes methylene blue or a derivative thereof. Methylene blue (MB) is a basic dye of the thiazine family that is positively charged and is used as a staining reagent because it binds to acidic substances such as nucleic acids and mucopolysaccharides to exhibit a blue color tone. MB is known to exist as a monomer and a dimer in an aqueous solution, and can be confirmed by absorbance measurement using a spectrophotometer (maximum absorption wavelength, monomer: 664 nm, dimer: 616 nm). Examples of methylene blue derivatives include, but are not limited to, 1-methylmethylene blue, 1,9-dimethylmethylene blue, methylene violet, bromomethylene violet, 4-iodomethylene violet, 1,9-dimethyl-3-dimethyl-amino-7-diethyl-amino-phenothiazine, and 1,9-dimethyl-3-dimethylamino-7-dibutyl-amino-phenothiazine.

Among these reagents, those capable of selectively quantifying glycosaminoglycans colorimetrically are preferred, and commercially available kits include the BLYSCAN GLYCOSAMINOGLYCAN ASSAY KIT (manufactured by ACCURAT CHEMICAL & SCIENTIFIC CORPORATION), the Rheumera kit (manufactured by Astarte Biologics), and the Blyscan Glycosaminoglycan Assay Kit (manufactured by Biocolor).

The quantitative method and qualitative analysis method of the present invention will be described below with reference to the drawings. FIG. 1 is a process diagram showing the flow of the quantitative method and qualitative analysis method of proteoglycan in one embodiment. As shown in FIG. 1, the method of this embodiment includes a step (S1) of collecting a measurement sample from a proteoglycan-containing composition to be measured, an extraction step (S2) of dissolving the sample in water or a PIPES buffer to extract proteoglycan, a recovery step (S3) of recovering the water-soluble fraction after extraction, and a colorimetric determination step (S4) of measuring the proteoglycan concentration of the water-soluble fraction using a color reagent containing methylene blue or a derivative thereof. In addition, a qualitative analysis step (S5) of measuring the molecular weight of proteoglycan using a part of the water-soluble fraction obtained in the recovery step (S3) may be provided. This allows quantitative analysis and qualitative analysis of proteoglycan in the composition to be performed simultaneously.

Here, the measurement sample extraction step S2 is the most important step in the method of the present invention. As described above, proteoglycan is a glycoprotein with an extremely high molecular weight, and therefore it is necessary to prevent aggregation in the solution. For this reason, the composition to be measured is dissolved in water or a buffer suitable for dissociating the functional groups of proteoglycan. Examples of such buffers include PIPES sodium buffer and/or PIPES potassium buffer. A PIPES buffer containing sodium ions and/or potassium ions and not containing calcium ions is preferable. The concentrations of sodium ions and potassium ions are not particularly limited, but are preferably 5 mM or more, and more preferably 10 mM or more.

The method for extracting proteoglycan from the measurement sample using the above extraction solution is not particularly limited, but may be agitation using a vortex mixer or ultrasonic treatment, either alone or in combination. After extraction, the water-soluble fraction is recovered (S3), for example, by centrifugation at 25°C and 3000 rpm for 10 minutes. It is preferable to perform this extraction step (S2) multiple times, mix multiple water-soluble fractions, and measure the proteoglycan concentration in the mixed solution.

The subsequent colorimetric determination step (S4) includes a step of measuring the proteoglycan concentration of the water-soluble fraction using a color reagent containing methylene blue or a derivative thereof, and a step of calculating the proteoglycan content in the composition from the obtained proteoglycan concentration. This color reaction is a quantitative dye-binding method for analyzing sulfated proteoglycans. When 1,9-dimethylmethylene blue, a typical dye label, is used, it specifically binds to the sulfated polysaccharide component of proteoglycan and shows a blue color. In this case, a linear graph is obtained by plotting the absorbance measured using a proteoglycan standard against the known concentration. An example of a calibration curve created in this manner is shown in Figure 3. The concentration of the test substance can be read from the graph or calculated from the degree of slope. The absorption spectrum of 1,9-dimethylmethylene blue has a maximum peak at 656 nm, but the absorbance peak is broad, so it is sufficient to measure the absorbance between 625 and 675 nm.

Figure 2 is a more detailed explanation of the qualitative analysis step (S5). The qualitative analysis step (S5) includes a step (S51) of first adsorbing the water-soluble fraction recovered above to an ion exchange resin equilibrated with an equilibration buffer, a step (S52) of washing the ion exchange resin with the adsorbed proteoglycan with a washing buffer containing the above buffer, and then eluting and/or desorbing the proteoglycan from the ion exchange resin with an elution buffer, and an HPLC analysis step (S53) of measuring the molecular weight of the eluted proteoglycan by HPLC.

The proteoglycan in the sample loaded in the adsorption step (S51) is dissolved in a buffer suitable for dissociation of functional groups, and is therefore efficiently adsorbed to the ion exchange resin. In order to promote adsorption of proteoglycan, it is preferable to charge the measurement sample at room temperature or low temperature (4°C to 10°C) and at a low flow rate. For washing the ion exchange resin, it is preferable to use the same buffer as the buffer used in preparing the measurement sample and/or the equilibration buffer. The washing buffer may contain a salt to increase the ionic strength of the solution in order to remove impurities from the column. For example, when NaCl is added, the concentration is 0 to less than 0.4 M, and preferably a buffer containing no NaCl and a buffer containing about 0.2 M NaCl may be used sequentially.

The elution buffer used in the elution step (S52) may or may not contain the buffer used in preparing the measurement sample, but it is preferable to use the same buffer as the equilibration buffer in order to prevent aggregation of the eluted proteoglycan. The ionic strength of the elution solution is preferably such that it weakens the adsorption force of the proteoglycan to the ion exchange resin. For example, when NaCl is added, the concentration is 0.4 M or more, and a buffer containing about 0.5 M NaCl can be used.

In the HPLC analysis step (S53), it is preferable to first desalt the eluate. This is to reduce the salt concentration in the eluate, which facilitates detection by HPLC. As the column for size exclusion gel chromatography in the HPLC analysis step (S53), a column that can analyze molecular weights of 100,000 to 2,000,000 may be used, but it is preferable to use a column containing a packing material with a particle size of 10 μm or more or a column for protein analysis, as this improves separation ability, and it is particularly preferable to use a column whose base material is a methacrylate polymer.

Detection in the HPLC method can be performed using a refractive index detector that uses the refractive index of the analyte compared to the solvent, or a method that uses a UV detector to detect absorbance at 200 to 280 nm, with a UV detector with a UV wavelength of 200 to 220 nm being particularly preferred. In addition, proteoglycan peaks can be detected by preparing a standard proteoglycan derived from salmon nasal cartilage (manufactured by Fujifilm Wako Pure Chemical Industries), overlaying the chromatogram with the sample to be analyzed, and if there is a peak in approximately the same elution range, it can be determined that this peak is the peak where proteoglycan has been detected. In addition, proteoglycan in a sample can be quantified by creating a calibration curve in advance using a proteoglycan standard.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % of the numerical values showing the amount of each component added means mass %.

Example 1
To approximately 454.5 mg of sample powder containing 0.44% by mass of proteoglycan and a predetermined amount of a predetermined milk protein, 15 mL of each of 10 mM PIPES-NaOH (pH 7.0), phosphate buffer (pH 6.8), citrate buffer (pH 4.6) or acetate buffer (pH 6.0) was added. Next, these aqueous solutions were extracted by ultrasonic treatment, and the supernatant was collected by centrifugation. The supernatant was filtered through filter paper to prepare a 50 mL solution. This preparation is shown in "PG content (mg/50 mL)" in Table 1 below. This was diluted 2-fold with purified water to prepare a measurement sample.

The above buffer solutions were prepared as follows.
10 mM PIPES-NaOH (pH 7.0) buffer solution: About 800 mL of purified water was added to 3.05 g of PIPES (DOJINDO), stirred at room temperature, and adjusted to pH 7.0 with 5N NaOH. Purified water was added to this to make the total volume 1000 mL, and a 10 mM PIPES solution was prepared.
Phosphate buffer solution (pH 6.8): Prepared based on the "Food Additives Standards, General Testing Methods, Reagents and Test Solutions."
Acetic acid buffer solution (pH 6.0): 0.3 g of sodium acetate trihydrate was dissolved in 80 mL of water, the pH was adjusted to 6.0 with acetic acid, and water was added to make 100 mL.

Separately, about 2 mg of a standard sample of proteoglycan was precisely weighed, and phosphate buffer (pH 6.8) was added to make the volume exactly 10 mL. This solution was diluted 4/10, 2/10, 1/10, 1/20, and 1/40 to obtain standard solutions, and phosphate buffer (pH 6.8) was used as the zero concentration, and the amount of proteoglycan in the sample was determined from the obtained calibration curve by the DMMB colorimetric method. The DMMB method (Dimethylmethylene Blue Assay) was performed using the Blyscan ^TM GLYCOSAMINOGLYCAN ASSAY kit (Biocolor Ltd.) according to the following procedure.

Blyscan ^™ dye reagent (1.0 ml) was added to 100 μl of the measurement sample. The mixture was mixed in a vortex mixer and stirred in a shaker at room temperature at 1000 rpm for 30 minutes. After centrifugation, the supernatant was removed and dissociation reagent (0.5 ml) was added thereto. The measurement sample was prepared by mixing well in a vortex mixer to dissolve the precipitate. 200 μl of each sample was transferred to each well of a 96-microwell plate, and the absorbance at a wavelength of 656 nm was measured using a microplate reader. The absorbance of the standard and test samples was measured, and the proteoglycan concentration of each sample was determined from the standard curve. The results are shown in Table 1 below. "PG" in Table 1 refers to proteoglycan.

As shown in Table 1, proteoglycan was most quantitatively recovered when PIPES buffer was used.

(Example 2) Measurement of proteoglycan in the composition 1. Preparation of powder Two types of powder were prepared.
To prepare a sample for the measurement shown in FIG. 4(A), about 454.5 mg of sample powder containing 0.44% by mass of proteoglycan and a given amount of a given milk protein was prepared.
To prepare a sample (blank sample) for the measurement shown in FIG. 4(B), about 454.5 mg of sample powder containing no proteoglycan but a given amount of a given milk protein was prepared.

Specific examples of the milk proteins contained in the powder include skim milk powder, whole milk powder, total milk protein, sodium caseinate, whey protein, buttermilk powder, milk, whole fat concentrated milk, skim milk concentrated milk, and natural cheese.

2. Preparation of solution The powder was precisely weighed and 15 mL of 10 mM PIPES-NaOH buffer (pH 7.0) was added to prepare a solution. This solution was then subjected to ultrasonic treatment under specified conditions, and after ultrasonic treatment, the supernatant was collected by centrifugation. The supernatant was filtered through filter paper to prepare 50 mL of solution.

3. Ion exchange treatment After 10 mL of purified water was poured into a column of TOYOPAK DEAE M (gel volume 2.0 mL, manufactured by Tosoh Corporation), which is a weakly basic anion exchange gel, 10 mL of the 10 mM PIPES-NaOH buffer (pH 7.0) prepared above was poured to equilibrate the ion exchange resin. 10 mL of the measurement sample dissolved in the same buffer was loaded onto this column. Next, the column was washed with 5 mL of the same buffer, and then further washed with 10 mL of the same buffer containing 0.35 M NaCl. After washing, 10 mL of the same buffer containing 0.5 M NaCl was poured into the column to desorb the proteoglycan from the column.

4. Desalting Treatment 10 mL of the desorption fraction recovered in the above ion exchange treatment was desalted by first washing once with the desorption solution and then with purified water using an Amicon Ultra centrifugal filter unit (molecular weight cutoff 100K, Merck Ltd.). The concentrated solution was recovered from the filter cup after centrifugation, and phosphate buffer (pH 6.8) was added to make 5 mL of solution (measurement sample).

Separately, approximately 1 mg of a proteoglycan standard (manufactured by Fujifilm Wako Pure Chemical Industries) was precisely weighed and phosphate buffer (pH 6.8) was added to make 10 mL (standard solution).

5. HPLC Analysis Using the measurement samples and standard solutions prepared above, HPLC analysis was carried out under the following operating conditions to determine whether the measurement samples contained proteoglycan.
Detector: UV-VIS detector SPD-20A (manufactured by Shimadzu Corporation)
Measurement wavelength: 215nm
Column: TSKgel G5000-PWXL φ7.8 mm × 300 mm (Tosoh Corporation)
Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5mL/min Injection volume: 100μL

The results of this measurement are shown in Figure 4. The arrow in Figure 4(A) indicates the elution position of proteoglycan (12.733 min). Although not shown, in HPLC analysis using the standard solution, the elution position of proteoglycan F was around 12.7 min.
On the other hand, Fig. 4(B) shows the results of an analysis of a blank sample that does not contain proteoglycan. The elution position of proteoglycan could not be confirmed.

(Example 3) Quantitative analysis of proteoglycan in the composition 1. Preparation of powder (sample 3-1 and sample 3-2)
In order to prepare samples used in the measurements shown in FIG. 5, the following two types of powder (sample 3-1 and sample 3-2) were prepared.

Sample 3-1: Approximately 231.7 mg of sample powder containing 6.4% by mass of Proteoglycan F (Ichimaru Falcos) and a specified amount of a specified pectin was prepared.
Sample 3-2: Approximately 231.7 mg of sample powder was prepared that did not contain Proteoglycan F (Ichimaru Falcos) but contained a specified amount of a specified pectin.

2. Preparation of standard solution A standard solution was prepared in parallel with 3. below. Approximately 2 mg of a proteoglycan standard sample (Fujifilm Wako Pure Chemical Industries) was precisely weighed, and approximately 2 mg of powder was prepared. Phosphate buffer (pH 6.8) was added to dissolve the powder, and phosphate buffer (pH 6.8) was added again to prepare a standard solution.

The phosphate buffer solution (pH 6.8) used in this Example 3 is in the "Food Additives Official Standards, General Testing Methods, Reagents and Test Solutions."

3. Preparation of sample solutions (sample 3-1 and sample 3-2)
The powders (sample 3-1, sample 3-2) were precisely weighed and prepared at about 231.7 mg each. The powders were dissolved in 15 mL of purified water and sonicated for 15 minutes to prepare a solution. To this solution, 20 μL of pectinase (Pectinase from Aspergillus aculeatus, SIGMA, P2611) was added and subjected to enzyme treatment at 50° C. for 60 minutes. In order to inactivate the pectinase (enzyme), the treated solution was treated at 80° C. for 30 minutes.

The solution after the 30-minute treatment was centrifuged at about 1800G for 10 minutes, and the supernatant after the centrifugation was collected. The supernatant was centrifuged at about 2400G for 30 minutes using an Amicon Ultra Centrifugal Filter Unit (molecular weight cutoff 100K, Merck Ltd.). The proteoglycan fraction (fraction containing concentrated proteoglycan) that does not permeate the membrane was purified. The proteoglycan fraction was washed five times with water. Phosphate buffer (pH 6.8) was added to the resulting proteoglycan fraction to make it exactly 20 mL (a 20 mL solution was prepared). The prepared solution was filtered through a 0.45 μm membrane filter. The filtered filtrate was used as the sample solution. Note that other methods can be used to prepare the sample solution as long as they provide an equivalent recovery rate.

3. Measurement by liquid chromatography The standard solution and the sample solution were tested by liquid chromatography under the following operating conditions, the proteoglycan concentration in the test solution was calculated from the calibration curve, and the proteoglycan content in the standard solution and the sample solutions (sample 3-1 and sample 3-2) was calculated by the following formula.

Calculation method:
Proteoglycan content (%) = (A x V x N x 1/W) x 100
A: Proteoglycan concentration in the test solution (mg/mL)
V: constant volume (mL)
N: Dilution ratio
W: Amount of test sample collected (mg)

Operating conditions:
Detector: Differential refractometer (RID-10A, Shimadzu Corporation)
Column: gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation)
Column tube: Stainless steel tube with inner diameter of about 7.8 mm and length of 30 cm Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5mL/min Injection volume: 50μL

The results of this measurement are shown in Figure 5. The arrow in Figure 5(A) indicates the elution position (13 min to 15 min) of proteoglycan in the sample solution (proteoglycan-containing solution of Sample 3-1). Although not shown, in HPLC analysis using the standard solution, the elution position of proteoglycan was around 13 min to 15 min, which was almost the same as the position indicated by the arrow in Figure 5(A).
On the other hand, in FIG. 5(B), in the sample solution (solution of sample 3-2 not containing proteoglycan), the elution position of the proteoglycan could not be clearly confirmed due to the presence of other components such as pectin, and the amount of the proteoglycan in the sample solution (3-2) could not be quantified.

(Example 4) Quantitative analysis of proteoglycan in the composition 1. Preparation of soft capsules (samples 4-1 and 4-2)
Two types of soft capsules (sample 4-1 and sample 4-2, gelatin-containing soft capsules) were prepared to prepare samples used in the measurements shown in FIG.

Sample 4-1: 470.00 mg of a soft capsule containing approximately 190 mg of vegetable oil and 5.5 mg of proteoglycan was prepared.
Sample 4-2: Approximately 190 mg of vegetable oil and 470.00 mg of a soft capsule not containing proteoglycan were prepared.

2. Preparation of standard solution A standard solution was prepared in parallel with 3. below. Approximately 2 mg of a proteoglycan standard sample (Fujifilm Wako Pure Chemical Industries) was precisely weighed, and approximately 2 mg of powder was prepared. Phosphate buffer (pH 6.8) was added to dissolve the powder, and phosphate buffer (pH 6.8) was added again to prepare a standard solution.

The phosphate buffer solution (pH 6.8) used in this Example 4 is in the "Food Additives Official Standards, General Testing Methods, Reagents and Test Solutions."

3. Preparation of sample solutions The following sample solutions (Solution 4-1-1, Solution 4-1-2, Solution 4-2-1, Solution 4-2-2) were prepared from the soft capsules.
Solution 4-1-1: A solution obtained by adding a chloroform solution (Kishida Chemical, 282-16013) and dissolving sample 4-1. Solution 4-1-2: A solution obtained by adding a chloroform solution (Kishida Chemical, 282-16013) and dissolving sample 4-2. Solution 4-2-1: A solution obtained by adding an acetone solution (Kishida Chemical, 170-00305) and dissolving sample 4-1. Solution 4-2-2: A solution obtained by adding an acetone solution (Kishida Chemical, 170-00305) and dissolving sample 4-2.

In preparing the sample solution, the soft capsule was dissolved in a solvent as follows. The soft capsule was divided into two pieces using scissors or the like. The two pieces were placed in a centrifuge tube (50 mL capacity) and 10 mL of each solvent (chloroform solution, acetone solution, etc.) was added. Next, the solution was treated in an ultrasonic cleaner for 15 minutes.
When a hexane solution (Kishida Chemical, 140-36763) was used as the solvent, the above capsules (samples 4-1 and 4-2) did not dissolve.

The supernatant (the solvent) after the centrifugation was removed by centrifugation (3000 rpm, 5 minutes). 20 mL of 100% ethanol solution was added to the pellet after the centrifugation to prepare a new solution for each sample. The solution was then subjected to the same treatment again in the centrifuge. This process was repeated five times, and the solvent used was replaced with ethanol.

The precipitate obtained was dissolved by adding 15 mL of 4 M guanidine hydrochloride solution, and the proteoglycan fraction was concentrated and purified using an Amicon Ultra centrifugal filter unit (molecular weight cutoff 100K, Merck Ltd.). After washing and permeating the stock solution, the centrifuge tube was rinsed with 15 mL of purified water, and the fractionation membrane was washed once using this rinse water. Phosphate buffer (pH 6.8) was added to the purified proteoglycan concentrate to make exactly 50 mL, and the filtrate filtered through a 0.45 μm membrane filter was used as the sample solution, and the proteoglycan in the measurement sample was quantitatively analyzed.

3. Measurement by liquid chromatography The standard solution and the sample solutions were tested by liquid chromatography under the following operating conditions, and the proteoglycan concentration in the test solution was calculated from the calibration curve. The proteoglycan content in the standard solution and the sample solutions (sample solution 4-1-1, sample solution 4-1-2, sample solution 4-2-1, and sample solution 4-2-2) was calculated by the following formula.

Calculation method:
Proteoglycan content (%) = (A x V x N x 1/W) x 100
A: Proteoglycan concentration in the test solution (mg/mL)
V: constant volume (mL)
N: Dilution ratio
W: Amount of test sample collected (mg)

Operating conditions:
Detector: Differential refractometer (RID-10A, Shimadzu Corporation)
Column: gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation)
Column tube: Stainless steel tube with inner diameter of about 7.8 mm and length of 30 cm Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5mL/min Injection volume: 50μL

The results of this measurement are shown in FIG.
The arrow in Fig. 6(A) indicates the elution position (13 min to 15 min) of the proteoglycan from the sample solution 4-1-1 (containing proteoglycan, soft capsule dissolved in chloroform solution).Compared with the measurement results shown in Fig. 6(C), the detection of impurities was confirmed near the elution position (16 min) indicated by the dotted arrow in Fig. 6(A).
FIG. 6(B) shows the measurement results for the sample solution 4-1-2 (not containing proteoglycan, soft capsule dissolved in chloroform solution), but no proteoglycan peak was confirmed at the position indicated by the arrow (13 min to 15 min).
The arrow in Fig. 6(C) indicates the elution position (13 min to 15 min) of the proteoglycan in the sample solution 4-2-1 (containing proteoglycan, soft capsule dissolved in acetone solution).Compared with the measurement results shown in Fig. 6(A), no impurities were detected near the elution position (16 min).
FIG. 6D shows the measurement results for sample solution 4-2-2 (not containing proteoglycan, soft capsule dissolved in acetone solution), but no proteoglycan peak was confirmed at the position indicated by the arrow (13 min to 15 min).

A composition containing proteoglycan and a protein (such as milk protein) is expected to be a functional food that, for example, fixes calcium in bones and has the effect of promoting cartilage differentiation and improving knee joints. According to the method of the present invention, the content of proteoglycan in a composition containing proteoglycan and a protein can be easily and precisely quantified, and therefore, the method can be used for quality control of such functional foods.

Claims (4)

extracting the composition containing proteoglycans and proteins with a sodium PIPES buffer and/or a potassium PIPES buffer ;
recovering the water-soluble fraction after the extraction;
measuring the proteoglycan concentration of the water-soluble fraction using a color reagent containing methylene blue or a derivative thereof;
calculating the proteoglycan content in the composition from the proteoglycan concentration in the water-soluble fraction;
A method for quantifying proteoglycan in a composition, comprising: extracting the composition containing proteoglycans and proteins with a sodium PIPES buffer and/or a potassium PIPES buffer ;
recovering the water-soluble fraction after the extraction;
A step of adsorbing the recovered water-soluble fraction onto an ion exchange resin;
Eluting the adsorbed proteoglycan from the ion exchange resin with an elution solution containing a PIPES buffer;
measuring the molecular weight of the eluted proteoglycan by HPLC;
A method for analyzing proteoglycan in a composition, comprising: The method according to claim 1 or 2 , wherein the concentration of sodium ions and/or potassium ions in the PIPES sodium buffer and/or the PIPES potassium buffer is 5 mM or more . The method according to any one of claims 1 to 3 , wherein the protein is a milk protein, a plant-derived protein and/or a seafood-derived protein .

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