JP6617230B2 - Phycoerythrobilin-containing oligopeptide, its production method and its use - Google Patents

Description

  The present invention relates to a phycoerythrobilin-containing oligopeptide (phycoerythrobilin to which an oligopeptide is bound), and more specifically, algae such as red algae and cyanobacteria are hydrolyzed with a protease and then extracted with hot water. The present invention relates to a phycoerythrobilin-containing oligopeptide, a production method thereof, and use thereof as a red dye, a fluorescent dye, a protein saccharification inhibitor, and the like.

  Algae such as red algae and cyanobacteria are known to contain phycoerythrin, which is a kind of red pigment.

For example, B-phycoerythrin of the single-cell red alga Porphyridium Cruentum is a protein in which α and β2 subunits having a molecular weight of about 19 kDa and a protein consisting of a γ subunit having a molecular weight of about 30 kDa and a phycoerythrobilin dye are covalently bound. Specifically, phycoerythrovirin, which is a ring-opened tetrapyrrole, and a cysteine residue of the above protein are covalently bonded.

  Phycoerythrin plays an important role as a photosynthetic pigment (Non-patent Document 1). The maximum absorption wavelength of phycoerythrin is around 540 to 570 nm, and the maximum fluorescence wavelength is around 575 to 578 nm. This phycoerythrin pigment is used in the food industry and the like as a naturally occurring food colorant.

  In addition, phycoerythrin is widely used as a fluorescent dye for labeling various proteins such as antibodies in clinical diagnosis and cytochemical research. Phycoerythrin has a wide range of excitation wavelengths and is characterized by the ability to use various excitation sources.

In addition to its use as a pigment, phycoerythrin contained in the red alga Porphyra yezoensis has antioxidant properties, phycoerythrin and phycoerythrin, which is a pigment component of phycoerythrin. Have been reported to have an inhibitory effect on the inflammatory reaction (Non-patent Document 2).

  By the way, this extraction method of phycoerythrin is extracted into water by crushing algal cells by mechanical means such as salt solution and chemical treatment or ultrasonic treatment, ammonium sulfate salting out, column chromatography, organic solvent A method using the above has been conventionally performed. For example, in Patent Document 1, an algae-derived phycoerythrin pigment solution (A) containing a contaminant pigment and an alkaline earth metal phosphate (B) that aggregates the contaminant pigment are mixed, and the phycoerythris A method for purifying a phycoerythrin dye solution is described in which the contaminating dye in the phosphorus dye solution (A) is aggregated to form an aggregate, and then the aggregate is removed. In addition, it is described that the fiscoerythrin dye solution as a raw material uses a heat-treated one. Patent Document 2 describes a method for producing a natural red pigment, in which a pigment extract obtained from a red pigment-containing algae is heat-treated to separate the red pigment.

  The heat treatment in Patent Documents 1 and 2 is to denature pigments other than red pigments such as phycocyanin and chlorophyll contained in the pigment extract and precipitate as impurities, and the heating temperature is preferably 40 to 40. It is 70 degreeC, Most preferably, it is 50-60 degreeC, and the heating time is 2 to 30 minutes. In general, the higher the heating temperature or the longer the heating time, the higher the purity of the red pigment and the better the color tone, but the problem is that the yield decreases. In addition, when the heating temperature is other than the above temperature range, for example, at 40 ° C. or less, the color tone of the dye solution exhibits a reddish purple color, making it difficult to obtain a desired clear red dye. In addition, it is described in paragraph [0009] of the cited document 2 that the red pigment becomes sedimentation at 70 ° C. or more and the pigment recovery rate decreases.

  By the way, Patent Document 3 describes phycoerythrin derived from seaweeds of the genus Ogonori, including the scorpion gonorrhoeae. The phycoerythrin derived from the seaweed of the genus Ogonori of this invention is a phycoerythrobilin and a protein covalently bound to each other and has the following physical properties.

(1) It has absorption maximum wavelengths at 496 ± 5 nm, 539 ± 5 nm and 566 ± 5 nm.
(2) Fluorescence of 573 ± 5 nm is emitted at excitation wavelengths of 496 ± 5 nm and 539 ± 5 nm.
(3) The molecular weight measured by sodium dodecyl sulfate-polyacrylamide electrophoresis is 95,000 ± 5,000.

  The production method includes a step of crushing seaweeds of the genus Ogonori, mixing with water, stirring the mixture, centrifuging the obtained mixture, and obtaining a supernatant thereof, and In order to purify phycoerythrin, ammonium sulfate was added to the obtained supernatant, the precipitate was recovered, this precipitate was redissolved with a small amount of distilled water, dialyzed with distilled water, ammonium sulfate in the precipitate It is also described that a process other than can be used. Furthermore, this redissolved solution after dialysis contains abundant phycoerythrin and can be used as it is for various applications, but it is further described that it may be purified by known means such as chromatography. Yes.

  On the other hand, as an example of reducing the molecular weight of phycoerythrin, for the purpose of knowing the protein amino acid sequence of the phycobilin binding site, it is from Porphyridium cruentum (hereinafter abbreviated as “P. cruentum”) which is a single-cell red alga chinorimo. There is an example in which phycoerythrobilin tripeptide obtained by purifying B-phycoerythrin by chromatography or the like and digesting it with trypsin was analyzed (Non-patent Document 3). Similarly, P. cruentham's phycoerythrin was purified by chromatography, etc., then trypsinized to purify phycoerythrobilin-binding peptide (6 to 40 amino acids), and the amino acid sequence of the phycobilin binding site was analyzed (Non-Patent Document 4).

As for the formation of a phycoerythrobilin dye having no peptide bond from phycoerythrin, methanolysis in which P. cruentham's phycoerythrin dissociates a covalent bond between phycobilin and a cysteine residue using methanol and HgCl _2. Reported the purification of phycoerythrobilin (Non-patent Document 4). Furthermore, it has been reported that phycoerythrobilin is produced by roasting laver (Non-patent Document 5).

  As described above, a large number of methods for producing phycoerythrin have been reported, but all of them involve complicated production processes and are not suitable for mass production. In addition, as described above, phycoerythrin is a ring-opened tetrapyrrole, phycoerythrovirin, and a subunit protein that are covalently bound to each other, and thus may be affected by a subunit protein-derived portion.

JP-A-2005-75851 JP 07-118555 A JP2011-37714A JP2008-88102

J. Biol. Chem. 259, 5472-5480 (1984) http://bre.soc.i.kyoto-u.ac.jp/~jsfs-kin/History/H19/H19b/1.pdf, "Evaluation of anti-inflammatory properties of seaweed pigment phycoerythrin" 2008 Japan Late Meeting of the Fisheries Society Kinki Branch (Yu Liu, Yuki Nishimura, Tatsuya Sugawara, Takashi Hirata (Kyoto Univ.) J. Am. Chem. SOC. 105, 4072-4076 (1983) J. Biol. Chem. 259, 5472-5480 (1984) http://bre.soc.i.kyoto-u.ac.jp/~jsfs-kin/History/H20/H20b/2.pdf, "Production of phycoerythrobilin by roasting laver", 2008 Japan Late Meeting of the Fisheries Society Kinki Branch (Yu Liu, Yuki Nishimura, Tatsuya Sugawara, Takashi Hirata (Kyoto Univ.) J. Biol. Chem. 267, 14790-14798 (1992)

  The present invention relates to a technology capable of industrially obtaining components that can be used as red pigments and fluorescent pigments from algae containing phycoerythrin, in particular, phycoerythrin that can be advantageously used as a pigment, etc. It is an object of the present invention to provide a technology capable of mass-producing a phycoerythrobilin oligopeptide.

  The inventors of the present invention have been researching technology that can industrially advantageously obtain red pigments and fluorescent pigments (pigment components) from algae containing phycoerythrin. It was found that the pigment component can be easily solubilized by hydrolyzing with a proteolytic enzyme and then extracting the hydrolyzate with hot water.

  The phycoerythrobilin oligopeptide obtained in this step is also novel and has been found to have a protein glycation-inhibiting action that has not been reported with conventional dye components in addition to the action as a red dye and a fluorescent dye.

  The present invention is based on these findings and includes the following contents.

(1) A phycoerythrobilin oligopeptide obtained by hydrolyzing algae containing phycoerythrin with a proteolytic enzyme followed by hot water extraction.
(2) The phycoerythrobilin oligopeptide of (1) having a molecular weight of 700 or more and 10,000 or less.
(3) The phycoerythrovirin oligopeptide of (1) or (2) that is soluble in water.
(4) The following formula (I)
(In the formula, L ₁ represents an oligopeptide residue, L ₂ represents an oligopeptide residue or a hydroxyl group, and the total number of amino acid groups of L ₁ and L ₂ is within 20)
The phycoerythrobilin oligopeptide of any one of (1) to (3) represented by:
(5) L ₁ is Val-Asn-Lys-, and L ₂ is a hydroxyl group (4)
Phycoerythrobilin oligopeptide.
(6) The phycoerythroline oligopeptide according to any one of (1) to (5), wherein the algae containing phycoerythrin is selected from the group consisting of red algae, cyanobacteria, gray algae and crypto algae.
(7) The proteolytic enzyme is selected from the group consisting of pepsin, bromelain, papain, ficin, thermolysin, protease K, filamentous fungus-derived protease, yeast-derived protease, actinomycetes-derived protease, basidiomycete-derived protease, and bacteria-derived protease. The phycoerythrobilin oligopeptide according to any one of (1) to (6).
(8) A method for producing a phycoerythrobilin oligopeptide, comprising hydrolyzing algae containing phycoerythrin with a proteolytic enzyme and then extracting with hot water.
(9) The method for producing a phycoerythrobilin oligopeptide according to (8), wherein the algae containing phycoerythrin is selected from the group consisting of red algae, cyanobacteria, gray algae and crypto algae.
(10) The proteolytic enzyme is pepsin, bromelain, papain, ficin, thermolysin, protease K, filamentous fungus-derived protease, yeast-derived protease,
(8) or (9) a method for producing a phycoerythrobilin oligopeptide selected from the group consisting of actinomycete-derived protease, basidiomycete-derived protease and bacteria-derived protease.
(11) The method for producing a phycoerythrovirin oligopeptide according to any one of (8) to (10), wherein hot water extraction is performed at a temperature of 80 to 135 ° C. and a pressure of 0.1 to 0.22 MPa.
(12) The method for producing a phycoerythrobilin oligopeptide according to (8) to (11), wherein hot water extraction is performed for 1 to 60 minutes.
(13) A protein glycation inhibitor comprising the phycoerythrobilin oligopeptide according to any one of (1) to (7).
(14) A red pigment comprising the phycoerythrobilin oligopeptide according to any one of (1) to (7).
(14) A fluorescent dye comprising the phycoerythrobilin oligopeptide according to any one of (1) to (7).

  According to the present invention, red pigments and fluorescent pigments can be easily mass-produced from algae containing phycoerythrin. A red dye and a fluorescent dye (phycoerythrobilin oligopeptide having a molecular weight of 700 or more and 10,000 or less) obtained by the present invention exhibit a bright red color and emit fluorescence when excited with ultraviolet rays. In addition, phycoerythrobilin oligopeptide has a protein glycation-inhibiting action. Therefore, the dye of the present invention can be used as a red dye or a fluorescent dye in the food field, as a fluorescent labeling reagent for proteins in the medical / research field, or as an object material using fluorescence, etc. It can be used as a health food having a protein saccharification-inhibiting action.

It is a figure which shows solubilization of a red pigment | dye by hydrolyzing and hot-water-extracting Kubireogonori with thermolysin. It is a figure which shows solubilization of a red pigment | dye by hydrolyzing and extracting hot water with protease K. It is a figure which shows solubilization of a red pigment | dye by hydrolyzing and hot-water extracting isonohana with thermolysin. Fraction No. by Sephadex LH-20 column fractionation of Kubireogonori thermolysin hydrolysis / hot water extract FIG. 34 is a diagram showing 34 separated by a C30 reverse phase column. It is the figure which isolate | separated the phycoerythrobilin oligopeptide by loading the peak A to C30 reverse phase column, and the fractionation of the peak A by the C18 column and the Cymmetry prep of Kubireogori thermolysin hydrolysis and hot water extract. It is a figure which shows the isolation | separation by the C18 reverse phase column column of the fraction containing the phycoerythrobilin oligopeptide obtained by processing the thermolysin hydrolysis / hot-water extract of isonohana to the solid phase C18 cartridge.

  Hereinafter, the present invention will be described in detail, but the scope of the present invention is not limited to these descriptions, and other than the following exemplifications, the present invention can be appropriately implemented without departing from the spirit of the present invention.

  The phycoerythrobilin oligopeptide of the present invention can be obtained by hydrolyzing algae containing phycoerythrin with a proteolytic enzyme and then extracting with hot water.

This is, for example, the following general formula (I)
(In the formula, L ₁ represents an oligopeptide residue, L ₂ represents an oligopeptide residue or a hydroxyl group, and the total number of amino acid groups of L ₁ and L ₂ is within 20)
It is represented by

  This has a molecular weight of 700 or more and 10,000 or less, is soluble in water, and functions as a red dye or a fluorescent dye. In addition, being soluble in water means that floating matter and sediment are not visually observed. For example, in Example 5, which will be described later, when the obtained pigment component is an aqueous solution having a concentration of 50 mg / ml, no suspended matter or precipitate is observed, and this is centrifuged at 15,000 rpm for 15 minutes. It can be judged by the fact that no sediment is observed after the treatment.

Algae that are raw materials for producing this phycoerythrobilin oligopeptide are algae containing phycoerythrin. Examples of such algae include red algae and cyanobacteria. More specifically, Gracilaria (Gracilaria tikvahiae), constricted Gracilaria (Gracilaria blodgettii), Isonohana (Halymenia floresia), Chinorimo (Porphyridium cruentum), Porphyra yezoensis (Porphyra yezoensi s), Asakusanori (Porphyra tenera), Gelidium (Gelidiaceae), funori Examples include red algae and cyanobacteria such as ( Gloiopeltis ) and mafnori ( Gloiopeltis tenax (Turner) Decaisne).

  Of the above, Kubireogonori is an algae that is distributed in the Nansei Islands, and is used as a raw material for Moi tofu (agar material), sashimi, and seaweed salad (Okinawa Sukaikai Research Report 96, 50-53). (2008)). Isonohana is an algae found in Ishigaki Island in winter (Okinawa Deep Water Research Bulletin No. 10, pages 19-20). In addition to red algae and cyanobacteria, gray algae and crypto algae may be used as long as they contain phycoerythrin. Furthermore, instead of the algae, purified phycoerythrin or a crude product containing phycoerythrin may be used as a material.

  On the other hand, the proteolytic enzyme used in the present invention is not particularly limited, and various types can be used. Specific examples thereof include pepsin, bromelain, papain, ficin, thermolysin, protease K, other filamentous fungus-derived proteases, yeast-derived proteases, actinomycetes-derived proteases, basidiomycetous-derived proteases, bacterially-derived proteases, and the like.

  In order to obtain the phycoerythrobilin oligopeptide of the present invention, for example, the following may be performed. That is, first, algae containing phycoerythrin (hereinafter sometimes abbreviated as “raw algae”) are suspended in water in a suitable reaction vessel to obtain a suspension.

  The raw algae may be used after being washed with water and then finely chopped, but it is desirable to use after drying with water and pulverizing with a pulverizer. Moreover, containers, such as a flask and a pan, can be utilized as a reaction container.

  Furthermore, as the water used for the suspension, for example, purified water is suitable when the final product is used for food, but this is suitable depending on the type of proteolytic enzyme used in the next step. It may contain a stabilizer such as salts in a buffer solution or purified water.

  The weight (dry weight) of the raw material algae in the suspension is not particularly limited and can be appropriately changed. For example, the concentration is preferably about 2 g per 100 ml of purified water.

  The suspension is preferably homogenized with, for example, polytron, but may be stirred with a magnetic stirrer or the like.

  Next, the above-mentioned proteolytic enzyme is added to the suspension thus prepared to perform hydrolysis.

  The concentration of the proteolytic enzyme used for the hydrolysis is, for example, about 0.1 to 4% by mass (hereinafter referred to as “%”) of the dry weight of the raw algae when using thermolysin (the concentration in the solution is about 0.1). 002% to 0.08%), preferably about 0.5 to 2% (concentration in solution of about 0.01% to 0.04%), more preferably about 0.5% (concentration in solution) It is about 0.01%.

  The temperature for the hydrolysis reaction may be around the optimum temperature for each enzyme. For example, when thermolysin is used, the temperature is in the range of about 30 to 60 ° C. It is short. Furthermore, the time for performing the hydrolysis reaction is not particularly limited. For example, when thermolysin is used, the reaction time is about 1 to 6 hours, and a reaction time of 3 hours is sufficient.

  The hydrolysis reaction product is then subjected to hot water extraction. This hot water extraction can also be performed in conjunction with the previous enzymatic reaction termination step. Specifically, the reaction may be performed by placing a reaction vessel in boiling water and boiling for 1 to 60 minutes, preferably 1 to 20 minutes, more preferably 5 to 15 minutes.

  The hot water extract obtained as described above is then cooled by placing the container in ice water. The obtained hot water extract is recovered as a supernatant by centrifuging after cooling. Moreover, it replaces with this method and may filter with a filter paper etc. and may remove an insoluble matter.

  The dye solution containing the phycoerythrobilin oligopeptide produced by such a process may be used as it is, but may also be used as a powder after being dried by freeze drying, a centrifugal evaporator or the like. Or you may refine | purify by ultrafiltration, chromatography, etc., and may utilize as a high purity sample.

  The phycoerythrobilin oligopeptide of the present invention thus obtained is used in various foods such as soft drinks, lactic acid drinks, seasonings, soups, cheeses, hams, ice creams and confectionery, for example, as red dyes and fluorescent dyes. It can be added to and used. The phycoerythrobilin oligopeptide according to the present invention can be used in the medical and research fields for the purpose of fluorescent labeling of antibodies and various biological proteins.

  In addition, it was found by the present inventor that the phycoerythrobilin oligopeptide and the fraction containing it have a protein glycation-inhibiting action in addition to the action as the dye described above.

  Protein saccharification is a phenomenon in which protein and glucose are combined to produce advanced glycation end products (AGEs) via an Amadori compound, and many substances are known as AGEs. For example, carboxymethyllysine is produced during diabetes, and carboxymethyllysed collagen is said to induce apoptosis of human dermal fibroblasts. Pentosidine is fluorescent with a structure in which a lysine residue and an arginine residue are cross-linked by a pentose sugar, and the blood pentosidine concentration in patients with diabetes or chronic renal failure is said to be high. Moreover, this thing exists also in skin collagen, increases with aging, and is considered as a marker reflecting osteoporosis.

  Many drugs, such as aminoguanidine, are known as drugs that inhibit this protein glycation, but many of them have doubts about their safety when taken orally.

  On the other hand, the phycoerythrobilin oligopeptide of the present invention and the fraction containing the same are derived from food ingredients, and thus are highly safe and added to various foods for the purpose of anti-aging, etc. It can be used as a sex food.

  In addition, as a protein saccharification suppression component derived from seaweed, patent document 4 has a description of a mozuku component (patent document 4). However, since mozuku is a brown algae and does not contain the red pigment phycoerythrovirin, it is judged to be different from the components of the present invention.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Examples 1.
Production of Phycoerythrobilin Oligopeptide from Kubileogonori (1):
( Gracilaria blodgettii ); produced in Okinawa Prefecture, dried and crushed to obtain a Kubireogonori powder, 0.4 g of this powder was placed in a 50 ml centrifuge tube, and 20 ml of distilled water was added and stirred. Furthermore, 2 mg of thermolysin (manufactured by Peptide Institute, Inc.) was added thereto, and the protein was hydrolyzed by incubating at 60 ° C. for 3 hours.

  After the reaction, hot water extraction was performed by holding the centrifuge tube in boiling water for 10 minutes. Immediately after 10 minutes, the mixture was ice-cooled, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. In addition, a comparative supernatant was obtained by performing the same treatment as above except that thermolysin was not added.

  When the supernatant obtained by hot water extraction after treatment with the obtained thermolysin was visually observed, it was solubilized and exhibited a red color, but in the comparative supernatant, almost no red color could be observed. . The absorbance at 540 nm was 0.62 for the supernatant extracted with hot water after thermolysin treatment and 0.036 for the comparative supernatant, confirming that there was a 20-fold difference in absorbance.

  Moreover, it was confirmed that when the supernatant obtained by hot water extraction after hydrolysis with thermolysin was excited with 365 nm ultraviolet light, fluorescence was emitted.

Example 2.
Production of Phycoerythrobilin Oligopeptide from Kubileogonori (2):
In a centrifuge tube having a capacity of 50 ml, 0.5 g of the Kubileogonori powder of Example 1 was added, and 25 ml of distilled water was added and stirred. To this was added 4 mg of protease K (manufactured by Promega Corporation), and the protein was hydrolyzed by incubating at 45 ° C. for 4 hours.

  After the reaction, hot water extraction was performed by holding the centrifuge tube in boiling water for 10 minutes. Immediately after 10 minutes, the mixture was ice-cooled, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected.

  As a result, when the supernatant of the hot water extract after the protease K treatment was visually observed, it was solubilized and had a red color. The absorbance of this supernatant at 540 nm was 0.775. Furthermore, it was confirmed that when the supernatant obtained by the protease K-treated hot water extract was excited with 365 nm ultraviolet light, fluorescence was emitted.

Example 3.
Production of Phycoerythrobilin Oligopeptide from Isonohana (1):
Isonohana ( Halymenia floresia ; produced in Okinawa Prefecture was dried and crushed to obtain isonohana powder. 0.6 g of this powder was placed in a 50 ml centrifuge tube, and 30 ml of distilled water was added and stirred. The protein was hydrolyzed by adding 3 mg and incubating at 60 ° C. for 3 hours.

  After completion of the reaction, hot water extraction was performed by holding the centrifuge tube in boiling water for 10 minutes. After 10 minutes, the mixture was immediately ice-cooled, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. In addition, a comparative supernatant was obtained by performing the same treatment as above except that thermolysin was not added.

  When the obtained supernatant obtained after the thermolysin treatment was subjected to hot water extraction was visually observed, it was solubilized and exhibited a deep red color compared to the comparative supernatant. In contrast, the comparative supernatant is slightly red, but when the temperature is lowered, the remaining polysaccharide mass floats, and this red portion is adsorbed on this polysaccharide and is actually dissolved in water. It was not.

  It was also confirmed that the supernatant obtained by hydrolyzing with thermolysin and then extracted with hot water was excited with 365 nm ultraviolet light to emit fluorescence.

Example 4.
Purification of Phycoerythrobilin Oligopeptide from Kubireogori
Into a 200-ml Erlenmeyer flask, 1 g of the Kubireogonori powder of Example 1 was added, and 50 ml of distilled water was added and stirred. To this, 5 mg of thermolysin was added, and the protein was hydrolyzed by incubating at 60 ° C. for 3.5 hours.

  After the reaction, hot water extraction was performed by holding the conical flask in boiling water for 10 minutes. Immediately after the completion of extraction, the mixture was ice-cooled and centrifuged at 10,000 rpm for 10 minutes to recover approximately 46 ml of the supernatant. Of this supernatant, 40 ml was passed through an ultrafiltration membrane having a fractional molecular weight of 10,000, and this liquid was dried under reduced pressure by a centrifugal evaporator to obtain 344 mg of a solid.

  240 mg of this solid was loaded onto a Sephadex LH-20 column (inner diameter 26 × 900 mm; manufactured by GE Healthcare Japan, Inc.) and eluted with a 30% aqueous methanol solution. Fractionation was carried out at 7 ml / fraction, and the absorbance at 220 nm and 540 nm of each fraction was measured.

  Of the peak with absorption at 540 nm, fraction No. 34 was concentrated by a centrifugal evaporator and then purified by high performance liquid chromatography (HPLC). A C30 reverse phase column (Develosil XG-C30M-3 4.6 × 250 mm, manufactured by Nomura Chemical Co., Ltd.) was loaded, and a linear concentration gradient of an aqueous acetonitrile solution containing 0.1% trifluoroacetic acid (flow rate 1 ml / min, 0 to 0). Elution was performed at a gradient of 52% over 30 minutes), and an absorption peak at 540 nm (arrow in FIG. 4) was collected.

After removing the solvent with a centrifugal evaporator, the peak portion was subjected to Q-TOF LC / MS (Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC / MS system) analysis. Data were obtained for ion, [M + H] ⁺ 1049.5115, MSMS product ion, [M + H] ⁺ 587.2798. Since the monoisotopic mass of phycoerythrovirin is 586.279114, it was estimated that the purified component was a substance in which a peptide having about 4 amino acids was bound to phycoerythrovirin.

In fact, the peak considered to be a peptide, the peak of [M + H] ⁺ 4633.2251 was confirmed. Further, when the amino acid sequence of this substance was analyzed by a protein sequencer (Applied to Nippi Co., Ltd.) of Applied Biosystems, the sequence of Val-Asn-Lys could be confirmed. Since phycoerythrovirin is bound to Cys, when the peptide portion is Val-Asn-Lys-Cys, the molecular weight is 4622.2610, which is consistent with the MSMS product ion of the peptide, [M + H] ⁺ 463.2251. This sequence is present on the 79-82 sequence of R-phycoerythrin, Subunit alpha of Gracilaria tenuistipitata liui, R-phycoerythrin, Subunit alpha. Therefore, phycoerythrovirin and Val-Asn-Lys-Cys were combined in the substance contained in the peak (arrow in FIG. 4) having an absorption at 540 nm on a C30 reverse phase column (Develosil XG-C30M-3 4.6 × 250 mm). It is presumed to be a phycoerythrovirin tetrapeptide having a structure represented by the following formula (II).

  Further, instead of the Sephadex LH-20 column, the phycoerythrobilin oligopeptide can also be purified by a Symmetry Prep C18 column (SymmetryPrep C18 7.8 × 300 mm; manufactured by Waters). That is, 500 mg of a crude pigment obtained by passing through an ultrafiltration membrane with a molecular weight cut-off of 10,000 was dissolved in 1 ml of purified water, 100 μl thereof was loaded onto the column, and contained 0.1% trifluoroacetic acid. Elution was performed with a linear concentration gradient of an aqueous acetonitrile solution (flow rate: 1.5 ml / min, gradient of 0 to 70% was performed for 50 minutes), and peak A in FIG. 5 (A) was collected. This was repeated 8 times.

  For the collected peak A, a C30 reverse phase column (Develosil XG-C30M-3 4.6 × 250 mm) was loaded in six portions, and a linear concentration gradient of an aqueous acetonitrile solution containing 0.1% trifluoroacetic acid (flow rate: 1 ml / And a gradient of 0-52% is performed in 30 minutes), and peaks A1 and A2 in FIG. A2 was identical to the arrow peak in FIG.

On the other hand, when the peak A1 was subjected to Q-TOF LC / MS system analysis after removing the solvent with a centrifugal evaporator, it was further divided into two peaks (amount ratio was about 1: 2). The respective precursor ions were [M + H] ⁺ 1240.5159, [M + H] ⁺ 1311.5525. In addition, the respective MSMS product ions supporting the presence of phycoerythrobilin, [M + H] ⁺ 587.2898, [M + H] ⁺ 587.810 were confirmed. That is, it can be estimated that the peak A1 is composed of a phycoerythrovirin oligopeptide having an estimated molecular weight of 1239 and 1310 having an amount ratio of about 2: 1.

Example 5.
Purification of phycoerythrobilin oligopeptide from isonohana:
3 g of the isonohana powder of Example 3 was placed in a 300 ml Erlenmeyer flask, 150 ml of distilled water was added and stirred, 15 mg of thermolysin was added thereto, and the mixture was kept at 60 ° C. for 3 hours to hydrolyze the protein.

  After the reaction, this centrifugal tube was kept in boiling water for 10 minutes to perform hot water extraction. Immediately after the completion of extraction, the mixture was ice-cooled, centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. 90 ml of this supernatant was passed through an ultrafiltration membrane with a molecular weight cut off of 30,000, and this liquid was dried under reduced pressure with a centrifugal evaporator to obtain 1.35 g of a solid.

  This was dissolved in distilled water at a concentration of 150 mg / ml. Next, fractionation was carried out using a C18 cartridge (ISOLUTE SPE column 500 mg C18 (EC) 3 ml). After washing the cartridge with 1 ml of 0.1% trifluoroacetic acid (TFA), 0.5 ml of the above sample was loaded, and the cartridge was washed with 2 ml of 0.1% TFA. The red dye was eluted by adding 0.5 ml of methanol to the cartridge. This operation was performed a total of 10 times.

  The eluates were combined and methanol was removed under reduced pressure with a centrifugal evaporator to obtain 13 mg of a sample. The dye contained in this sample was purified by HPLC. Loaded onto a C18 reverse phase column (Symmetry C18 3.5 μm 4.6 × 100 mm, manufactured by Waters), a linear concentration gradient of an aqueous acetonitrile solution containing 0.1% trifluoroacetic acid (flow rate 1 ml / min, 0 to 40% gradient 20). Elution). This is shown in FIG.

A plurality of peaks having absorption at 540 nm were collected, and after removing the solvent with a centrifugal evaporator, Q-TOF LC / MS system analysis was performed. As a result, peak 1 in FIG. 6 was [M + H] ⁺ 1063. 5260, MSMS product ion [M + H] ⁺ 587.2816 data.

  Since the monoisotropic mass of phycoerythrovirin is 586.227914, it can be estimated that the purified component is a substance in which a peptide having about 4 amino acids is bound to phycoerythrovirin.

In addition, as for peak 2 in FIG. 6, data of [M + H] ⁺ 850.3775 and MSMS product on [M + H] ⁺ 587.2795 were obtained. Since the isotropic mass of phycoerythrovirin is 586.279114, it can be presumed that the purified component is a substance in which a peptide having several amino acid residues is bound to phycoerythrovirin.

The phycoerythrobilin oligopeptides purified in Examples 4 and 5 are summarized in Table 1 below.

Examples Example 6.
Inhibition of protein glycation of phycoerythrobilin oligopeptide of Kubileogonori
The substance of peak A1 (the phycoerythrovirin oligopeptide having an estimated molecular weight of 1239 and 1310 having a quantitative ratio of about 2: 1) obtained in Example 4 was tested for protein glycation-inhibiting effect.

  In this example, saccharification reaction was performed by incubating glucose and bovine serum albumin (BSA) at 60 ° C. for 3 days, and the protein saccharification inhibitory effect was evaluated by detecting the fluorescence color development.

  Specifically, 0.05 ml of bovine serum albumin 50 mg / ml dissolved in PBS (−) solution, 0.15 ml of 1.7 M glucose dissolved in PBS (−) solution, distilled water or 0.1 ml of the above dye solution diluted. 05 ml was placed in a 1.5 ml tube and allowed to stand at 60 ° C. for 3 days. Next, 0.1 ml of the solution in each tube was transferred to a 96-well microplate, and fluorescence generation (excitation wavelength Ex: 355 nm, fluorescence wavelength Em: 460 nm) was measured with a fluorescence microplate reader. The same test was performed as a positive control when aminoguanidine having a final concentration of 1 mM was added.

  Table 2 shows the results calculated as protein glycation inhibition rate (%) according to the following formula. Each sample addition group was performed in duplicate, and the distilled water addition group was performed in quadruplicate, and the average value of each was recorded as a result.

Protein glycation inhibition rate (%) =
[1- (A ₁ -B _L ) / (A ₀ -B _L )] × 100
A ₁ : Fluorescence measurement value when dye solution is added
A ₀ : Fluorescence measurement value when distilled water is added
B _L : Fluorescence measurement value when distilled water is added and reaction time is zero

  From this result, it was shown that the phycoerythrobilin oligopeptide obtained from Kubireogonori has a protein glycation inhibitory effect.

Example 7.
Protein glycation-inhibiting action of phycoerythrobilin oligopeptide-containing fractions:
By the method of Example 5, the protein saccharification inhibitory effect was tested as an aqueous solution having a concentration of 50 mg / ml with respect to the pigment component fractionated with the C18 cartridge.

  First, 0.1 ml of 50 mg / ml bovine serum albumin dissolved in PBS (−) solution, 0.3 ml of 1.7M glucose dissolved in PBS (−) solution, 0.1 ml of distilled water or the above dye solution diluted to 1 Placed in a 0.5 ml tube and allowed to stand at 60 ° C. for 3 days.

  Next, 0.1 ml of the solution in each tube was transferred to a 96-well microplate, and fluorescence generation (excitation wavelength Ex: 355 nm, fluorescence wavelength Em: 460 nm) was measured with a fluorescence microplate reader. The same test was performed as a positive control when aminoguanidine having a final concentration of 1 mM was added.

  The results were calculated as protein glycation inhibition rate (%) in the same manner as in Example 6, and are shown in Table 3.

As a result, it was revealed that the fraction containing phycoerythrobilin oligopeptide obtained from Isonohana exhibits a protein glycation-inhibiting effect in a concentration-dependent manner.

Claims (10)

A method for producing a protein saccharification inhibitor containing a phycoerythrobilin oligopeptide obtained by hydrolyzing a red algae containing phycoerythrin with thermolysin , followed by hot water extraction. A method for producing a protein saccharification inhibitor, wherein the molecular weight of the phycoerythrobilin oligopeptide is 700 or more and 10,000 or less. The method for producing a protein saccharification inhibitor according to claim 1 or 2, wherein the phycoerythrobilin oligopeptide is soluble in water. The phycoerythrobilin oligopeptide has the following formula (I)
(In the formula, L ₁ represents an oligopeptide residue, L ₂ represents an oligopeptide residue or a hydroxyl group, and the total number of amino acid groups of L ₁ and L ₂ is within 20)
The manufacturing method of the protein saccharification inhibitor of any one of Claims 1 thru | or 3 represented by these. The method for producing a protein saccharification inhibitor according to claim 4 , wherein in formula (1), L ₁ is Val-Asn-Lys- and L ₂ is a hydroxyl group. The method for producing a protein saccharification inhibitor according to any one of claims 1 to 5, wherein the hot water extraction is performed at a temperature of 80 to 135 ° C and a pressure of 0.1 to 0.22 MPa. The method for producing a protein saccharification inhibitor according to any one of claims 1 to 6, wherein the hot water extraction is performed for 1 to 60 minutes. After hydrolysis Rhodophyta containing phycoerythrin by thermolysin, produced how the phycoerythrin lobby phosphorus oligopeptide and extracting with hot water. The hot water extraction, 80 to 135 ° C. temperature, manufacturing how the phycoerythrin lobby phosphorus oligopeptide of claim 8 wherein performing from 0.1 at a pressure of 0.22 MPa. Producing how the phycoerythrin lobby phosphorus oligopeptide of claim 8 or 9, wherein performing the hot water extraction 1-60 minutes.