JP2014180216A - Food product functional material derived from lk fibroin and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of efficiently producing a food functional material comprising peptide with relatively low molecular weight and suitable amount of amino acid, having no bitterness characteristic of peptide and having functionality such as ACE inhibitory activity or the like derived from peptide from silk fibroin.SOLUTION: In the case silk fibroin is subjected to enzymolysis with proteolysis enzyme to produce a food functional material, neutral protease derived from Aspergillus is used as the proteolysis enzyme.

Description

  The present invention is obtained by using silk fibroin as a raw material and enzymatically degrading it with a proteolytic enzyme, and has a functionality such as angiotensin converting enzyme (ACE) inhibitory activity showing a blood pressure lowering action, and is used as a food material. The present invention relates to a food functional material derived from fibroin and a method for producing the food functional material for producing the food functional material from silk fibroin.

  Silk is formed from two types of proteins, fibroin and sericin, and these silk proteins are composed of various useful amino acids. Accordingly, various attempts have been made to effectively use silk protein as a food material and the like, and some have been put into practical use. Since this silk protein is unlikely to be decomposed by digestive enzymes such as pepsin and trypsin unlike general proteins, various methods for hydrolyzing silk protein with strong acids, strong alkalis, and proteolytic enzymes have been proposed. For example, fibroin, which is the main ingredient of silk, is hydrolyzed with strong alkali such as sodium hydroxide or strong acid such as phosphoric acid, or silk fibroin is enzymatically degraded with proteases such as papain, samoyase, elastase, and buncreatine. A method for obtaining food containing a silk protein hydrolyzate having a molecular weight in the range of 300 to 20,000 has been proposed and put into practical use (for example, see Patent Document 1). In addition, Patent Document 1 discloses a neutral region from the viewpoint of eliminating the complexity of performing a desalting step after enzymatic degradation of silk fibroin and suppressing a decrease in the yield of hydrolyzate by performing the desalting step. It is shown that it is preferable to use a proteolytic enzyme having activity in

Japanese Patent No. 2737790 (pages 3-6)

  As shown in Patent Document 1, in the method of hydrolyzing silk fibroin using strong acid or strong alkali, it is necessary to neutralize the reaction solution with alkali or acid after hydrolysis. Is produced. For this reason, if it is going to utilize the product by hydrolysis as a foodstuff material, the desalting process for removing the salt produced | generated by neutralization will be needed, and a manufacturing process will become complicated. Moreover, since a part of the product by hydrolysis is lost together with the salt in the desalting step, the yield is lowered. This problem also applies to the case where silk fibroin is enzymatically degraded using a proteolytic enzyme having activity in an acidic region or an alkaline region.

  In addition, a proteolytic enzyme that has been studied for use so far does not have a very high reaction rate of enzymatic degradation using it regardless of whether it is active in an acidic, alkaline or neutral region. For this reason, for example, in a reaction time of about 24 hours, the main component of the product obtained by enzymatic degradation is a peptide, and the finally obtained food material has a bitterness peculiar to peptides. This problem can be solved to some extent by prolonging the time for enzymatic degradation or increasing the amount of proteolytic enzyme to partially degrade the peptide to amino acids. However, this deteriorates production efficiency and cost effectiveness and cannot be employed industrially.

  The present invention has been made in view of the above circumstances, and is obtained without performing a desalting step after enzymatic degradation, and is produced by a rapid enzymatic degradation reaction and is suitable together with a relatively low molecular weight peptide. Providing a silk fibroin-derived food functional material comprising an amount of amino acids and thus having no peptide-specific bitterness, while having ACE inhibitory activity derived from peptides and functionality derived from specific amino acids, and so on An object of the present invention is to provide a method for producing a food functional material capable of efficiently producing a simple food functional material from silk fibroin.

  In order to achieve the above-mentioned problems, a neutral protease was used as a proteolytic enzyme for hydrolyzing silk fibroin. Furthermore, among various neutral proteases, when silk fibroin is enzymatically degraded using neutral protease derived from Aspergillus oryzae, the reaction rate increases, for example, suitable for a relatively low molecular weight peptide with a reaction time of about 24 hours. It has been found that a functional material containing an amount of amino acids can be obtained efficiently, and thus the invention has been completed.

  That is, the invention according to claim 1 is obtained by using a neutral protease derived from Aspergillus as a proteolytic enzyme in a food functional material derived from silk fibroin obtained by enzymatic degradation of silk fibroin with a proteolytic enzyme, It is characterized by containing a peptide mainly having a molecular weight of 1,000 to 2,200 and free amino acids mainly containing glycine, alanine, serine and tyrosine.

  The invention according to claim 2 is characterized in that in the method for producing a silk fibroin-derived food functional material by enzymatic degradation of silk fibroin with a proteolytic enzyme, a neutral protease derived from Aspergillus is used as the proteolytic enzyme. To do.

  The invention according to claim 3 is the method for producing a functional food material according to claim 2, wherein the refined silk thread is poured into an aqueous calcium chloride solution and boiled to dissolve the silk thread, and the obtained fibroin aqueous solution is desalted. It is characterized by preparing silk fibroin by treatment and using it as a raw material for silk fibroin.

  The invention according to claim 4 is the method for producing a food functional material according to claim 3, wherein the silk fibroin raw material is dissolved in distilled water, and a buffer solution and an enzyme of neutral protease derived from Aspergillus are added to the obtained fibroin aqueous solution. Silk fibroin is enzymatically decomposed by adding a solution and reacting.

  The invention according to claim 5 is characterized in that, in the method for producing a food functional material according to claim 4, the reaction liquid after enzymatic decomposition of silk fibroin is dried to form a powder.

  Since the silk fibroin-derived food functional material of the invention according to claim 1 is obtained by enzymatic degradation of silk fibroin using a neutral protease, it can be produced without performing a desalting step after enzymatic degradation. it can. In addition, this food functional material is produced by a rapid enzymatic degradation reaction using a neutral protease derived from Aspergillus oryzae and contains an appropriate amount of amino acid together with the peptide. It has functionalities derived from amino acids such as ACE inhibitory activity and promoting action of alcohol metabolism by alanine.

  According to the method for producing a food functional material of the invention according to claim 2, the food functional material can be produced without performing a desalting step after enzymatic decomposition of silk fibroin. Therefore, the manufacturing process is simplified, and a decrease in yield due to the desalting process can be suppressed. Moreover, since silk fibroin can be rapidly enzymatically degraded, a food functional material containing an appropriate amount of amino acid together with a relatively low molecular weight peptide can be efficiently produced. Therefore, there is obtained a food functional material that does not have the bitterness peculiar to peptides, and has functions derived from amino acids such as ACE inhibitory activity derived from peptides and the promotion of alcohol metabolism by alanine.

  In the method for producing a functional food material of the invention according to claim 3, the raw material is a silk thread that has been refined to remove sericin from the surface, and is obtained by dissolving the silk thread and desalting the food. Functional materials are manufactured.

  In the method for producing a food functional material of the invention according to claim 4, the enzymatic degradation of silk fibroin by a proteolytic enzyme is favorably performed.

  In the method for producing a food functional material of the invention according to claim 5, powdery food functional materials that can be easily used as various food materials can be obtained.

It is a figure which shows the gel filtration chromatogram of the reaction liquid after carrying out the enzymatic decomposition of silk fibroin using the neutral protease derived from Aspergillus.

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.
The functional food material according to the present invention is obtained by enzymatic degradation of silk fibroin with a neutral protease derived from Aspergillus oryzae and mainly comprises peptides having a molecular weight of 1,000 to 2,200 and glycine, alanine, serine and tyrosine. Containing free amino acids.

  Silk fibroin used as a raw material is refined by a predetermined method such as silk thread, jade ball, and scrap rice cake, that is, refined by immersing silk thread etc. in boiling sodium carbonate aqueous solution, and removed sericin from the surface of silk thread etc. Prepared from. This refined silk thread is poured into, for example, an aqueous calcium chloride solution and boiled to dissolve the silk thread. And the silk fibroin is prepared by carrying out the desalination process of the obtained fibroin aqueous solution, and let this be a silk fibroin raw material. This silk fibroin is easily soluble in water. As the desalting method, a reverse osmosis method, an electrodialysis method, an ion exchange method, or the like is used. In addition, you may make it filter the fibroin aqueous solution after a desalination process with a membrane filter. Further, after the desalting treatment, the fibroin aqueous solution may be dried by a freeze-drying method or the like to prepare a cotton-like or powdery silk fibroin, which may be used as a silk fibroin raw material. As described above, when the drying treatment is performed after the desalting treatment, the concentration of the aqueous solution can be determined based on the dry weight of the raw material when the fibroin raw material is prepared by dissolving the silk fibroin raw material in distilled water.

  In order to enzymatically decompose silk fibroin, a silk fibroin raw material is dissolved in distilled water, a buffer solution and an enzyme solution are added to the fibroin aqueous solution, and the reaction is performed under predetermined conditions, for example, for about 24 hours. As the buffer solution, for example, a phosphate buffer solution having a pH of 6 to 7 is used. And a neutral protease derived from Aspergillus is used as a proteolytic enzyme. As the neutral protease derived from Aspergillus oryzae, a commercially available product sold by enzyme manufacturers may be used. The substrate concentration of silk fibroin, the amount of enzyme, and the reaction conditions such as enzyme decomposition temperature, time, and pH are appropriately determined according to the type of enzyme. The reaction solution after enzymatic degradation of silk fibroin is dried by a method such as freeze drying or spray drying. Thereby, the powdery food functional material which can be used as various food materials can be obtained. In addition, after filtering the reaction liquid after enzymatic decomposition with a membrane filter, the filtrate may be dried.

[Preparation of silk fibroin raw material]
A refined silk thread is put into a 40 w / v% calcium chloride aqueous solution, and this aqueous solution is boiled to dissolve the silk thread. The obtained fibroin aqueous solution was passed through a dialysis cellulose tube (manufactured by Sanko Junyaku Co., Ltd., dialysis membrane UC18-32-100), and desalted. Next, the fibroin aqueous solution after dialysis was freeze-dried. Thereby, soft cotton-like silk fibroin was obtained.

[Enzymatic degradation of silk fibroin]
The silk fibroin obtained above was used as a raw material, and the silk fibroin was dissolved in distilled water to prepare a fibroin aqueous solution having a concentration of 3 mg / mL. In addition, as the enzyme, neutral proteases derived from four types of Aspergillus oryzae, namely peptidase R, proteax, protease M [Amano] SD and protease P [Amano] 3SD (all of which are products of Amano Enzyme Co., Ltd.) Each enzyme was dissolved in distilled water to prepare enzyme solutions having a concentration of 10 mg / mL. Then, 700 μL of a buffer solution and 100 μL of an enzyme solution were added to 400 μL of the fibroin aqueous solution, and an enzyme decomposition reaction was performed at a predetermined temperature for 24 hours. As the buffer, 50 mM phosphate buffer (pH 7.0) or 50 mM phosphate buffer (pH 6.0) was used. The reaction conditions at this time are shown in Table 1. For peptidase R, the optimum temperature for enzyme activity was 37 ° C. However, since the spoilage occurred at the reaction temperature of 37 ° C, the reaction temperature was set to 50 ° C, thereby preventing spoilage.

  As comparative examples, bromelain F, which is an alkaline protease, Neulase F3G, which is an acidic protease, Samoaase PC10F, which is a thermostable protease derived from high-temperature bacteria, protease N Amano G and protin NY100, which are neutral proteases derived from bacteria, and papaya In the same manner as above, 700 μL of a buffer solution and 100 μL of an enzyme solution were added to 400 μL of an aqueous fibroin solution for 24 hours, using papain W-40 (a trade name of Amano Enzyme), which is a neutral protease derived from The enzymatic degradation reaction was performed at temperature. As the buffer, 50 mM Tris-HCl buffer (pH 8.5), 50 mM acetate buffer (pH 3.0), or 50 mM phosphate buffer (pH 7.0) was used. The reaction conditions at this time are also shown in Table 1 above.

[Amino acid analysis of enzyme digestion solution]
For each reaction solution after enzymatic decomposition, a strongly acidic ion exchange resin column Shim-pack Amino-Na was used in a liquid chromatograph Prominence manufactured by Shimadzu Corporation, and a trisodium citrate solution was used as the mobile phase, o-phthalaldehyde Was used as a reaction reagent, and amino acid analysis was performed at a fluorescence wavelength [Ex = 348 nm, Em = 450 nm]. The analysis results are shown in Table 2. The numerical values in the table indicate the weight ratio of each amino acid to the amount of fibroin before the reaction. In the table, “Gly” represents glycine, “Ala” represents alanine, “Ser” represents serine, and “Tyr” represents tyrosine amino acid. These four amino acids occupy 90% or more of the total amino acid. In addition, “−” in the analysis result when using Samoaase PC10F indicates that it was below the detection limit value.

  As can be seen from the analysis results shown in Table 2, the degradation rate for enzymatic degradation of silk fibroin to amino acids is determined by neutral proteases (peptidase R, proteax, protease M [Amano] SD and protease P [Amano] derived from 4 types of koji molds. ] Both increase when 3SD) is used. In particular, when silk fibroin was enzymatically degraded using proteax, the degradation rate up to amino acids was the highest. In contrast, alkaline protease (bromelain F), acidic protease (neurase F3G), heat-resistant protease derived from high-temperature bacteria (Samoase PC10F), bacterial-derived neutral proteases (protease N Amano G and protin NY100), and papaya When enzymatic degradation was performed using a neutral protease derived from the disease (papain W-40), the degradation rate to amino acids was low. From this result, it is possible to obtain an enzymatic degradation product containing a relatively low molecular weight peptide and an appropriate amount of amino acid by enzymatic degradation of silk fibroin using a neutral protease derived from Aspergillus oryzae. I understand.

[Measurement of molecular weight by gel filtration of enzymatic degradation solution]
For the reaction solution after enzymatic degradation of silk fibroin using Proteax, silica gel filtration column Shim-pack Diol-150 was used in a liquid chromatograph LC-10 manufactured by Shimadzu Corporation, and 10 mM phosphate buffer. Using the solution (pH 7.0, containing 0.1 M NaCl) as a mobile phase, the molecular weight was measured by gel filtration at an absorption wavelength of 280 nm. A gel filtration chromatogram of this enzyme decomposition solution is shown in FIG. Calibration curve prepared using Bio-Rad standard for gel filtration (Thyroglobulin (670kDa), γ-globulin (158kDa), ovalbumin (44.0kDa), Myoglobin (17.0kDa), Vitamin B12 (1.35kDa)) Thus, the molecular weight of the enzyme degradation product component was determined.

  As shown in FIG. 1, the enzyme degradation product contained a peptide (molecular weight 1,000-2,200) and free amino acids mainly composed of glycine, alanine, serine and tyrosine. Moreover, the peptides in the enzymatic degradation product were relatively low molecular weight peptides, mainly about 1,000. The enzyme degradation product did not contain residual fibroin.

[Measurement of ACE inhibitory activity of enzyme digestion solution]
The ACE inhibitory activity of the silk fibroin enzymatic degradation solution was measured using a measurement kit (manufactured by Dojindo Laboratories, Inc., ACE Kit-WST). The measurement was performed on two enzymatic degradation solutions obtained by reacting silk fibroin for 13 hours and 24 hours, respectively, using proteax having the highest degradation rate to amino acids. As a result of the measurement, in a reaction solution with a reaction time of 13 hours, the value of IC50 (concentration required to inhibit ACE activity by 50%) is 0.05 mg / mL, and the reaction time is 24 hours. It was 0.064 mg / mL.

  As shown by the above results, ACE inhibitory activity was confirmed regardless of whether the enzymatic degradation reaction time was 13 hours or 24 hours, but higher inhibitory activity was obtained when the reaction time was 13 hours. . This ACE inhibitory activity is considered to be caused by silk peptide produced by enzymatic degradation of silk fibroin. The reaction time of 13 hours showed higher inhibitory activity than that of 24 hours. When the reaction time was increased, the peptide having ACE inhibitory activity was further decomposed into amino acids, and the inhibitory activity was increased. This is thought to be due to the decline.

  The reaction solution obtained after the enzymatic decomposition obtained above was freeze-dried to obtain a powdery material, which was easily dissolved in water. Moreover, since a part of the peptide was decomposed into amino acids, the obtained material did not feel any bitterness peculiar to the peptide.

[Enzymatic degradation of silk fibroin]
Silk fibroin prepared in the same manner as in Example 1 was used as a raw material, and the silk fibroin was dissolved in distilled water to prepare a 3 mg / mL aqueous fibroin solution. Moreover, commercially available proteax (trade name of Amano Enzyme Co., Ltd.) was used as the enzyme, and the enzyme was dissolved in distilled water to obtain 5.0 mg / mL, 1.0 mg / mL, and 0.4 mg / mL 3 Different concentrations of enzyme solutions were prepared. Further, a 20 mM phosphate buffer solution (pH 7.0) was prepared as a buffer solution. Then, 700 μL of a buffer solution and 100 μL of an enzyme solution were added to 400 μL of the fibroin aqueous solution, and an enzyme decomposition reaction was performed at a temperature of 50 ° C. for 5 hours.

[Amino acid analysis of enzyme digestion solution]
For each reaction solution after enzymatic decomposition, a strongly acidic ion exchange resin column Shim-pack Amino-Na was used in a liquid chromatograph Prominence manufactured by Shimadzu Corporation, and a trisodium citrate solution was used as the mobile phase, o-phthalaldehyde Was used as a reaction reagent, and amino acid analysis was performed at a fluorescence wavelength [Ex = 348 nm, Em = 450 nm]. The analysis results are shown in Table 3. The numerical values in the table indicate the weight ratio of each amino acid to the amount of fibroin before the reaction.

  As can be seen from the analysis results shown in Table 3, when the proteax was used in Example 1 described above (enzyme concentration of the enzyme solution: 10 mg / mL), the amino acid concentration was reduced by the decrease in the enzyme concentration. Decomposition rate became low. However, in Example 1, the enzymatic degradation reaction time was 24 hours, but in this example, the reaction time was 5 hours, and by increasing the reaction time, the degradation rate to amino acids was the value shown in Table 3. It is considered to be larger. In addition, coloring was observed in the enzyme degradation product at an enzyme concentration of 5.0 mg / mL, but coloring was hardly observed at an enzyme concentration of 1.0 mg / mL or less. Here, when the amount of the enzyme used increases, the production cost of the enzyme degradation product increases. Therefore, the enzyme concentration in the industrial production of food functional materials is determined by the suppression of the production cost and the properties and quality of the product. What is necessary is just to set suitably by balance. Moreover, even if the concentration of the phosphate buffer used is lowered from 50 mM (Example 1) to 20 mM, the enzymatic degradation reaction proceeds. Therefore, the enzyme degradation can be achieved by appropriately setting the concentration of the phosphate buffer. The flavor of things can be improved.

[Measurement of molecular weight by gel filtration of enzymatic degradation solution]
About the reaction liquid after enzymatic decomposition, silica-based gel filtration column Shim-pack Diol-150 is used for liquid chromatograph LC-10 manufactured by Shimadzu Corporation, and 10 mM phosphate buffer (pH 7.0, containing 0.0. 1M NaCl) was used as the mobile phase, and the molecular weight was measured by gel filtration at an absorption wavelength of 280 nm. As a result, the molecular weight of the peptide in the enzyme degradation product when using an enzyme solution having an enzyme concentration of 5.0 mg / mL is 1,260, and when the enzyme concentration is 1.0 mg / mL, 1,270, and the enzyme concentration is 0.4 mg. In each case, the peptide was a relatively low molecular weight peptide.

[Measurement of ACE inhibitory activity of enzyme digestion solution]
The ACE inhibitory activity of the silk fibroin enzymatic degradation solution was measured using a measurement kit (manufactured by Dojindo Laboratories, Inc., ACE Kit-WST). As a result of the measurement, in the reaction solution using the enzyme solution having an enzyme concentration of 5.0 mg / mL, the IC50 value is 0.15 mg / mL, and at the enzyme concentration of 1.0 mg / mL, 0.23 mg / mL, and the enzyme concentration is 0. It was 0.28 mg / mL at 4 mg / mL. Although the ACE inhibitory activity was reduced as compared with the case where the enzyme concentration of the enzyme solution was 10 mg / mL (Example 1), even when the enzyme solution having an enzyme concentration of 0.4 mg / mL was used, ACE inhibition of the enzyme degradation solution was performed. Activity was confirmed.

  According to the present invention, a novel food functional material having functions such as an ACE inhibitory activity and an alcohol metabolism promoting action without a bitter peculiar to peptides is provided, and this food functional material is used as a new food material in the food related industry There is expected. In addition, the present invention may also be used in silk related industries.

Claims (5)

In the functional food material derived from silk fibroin obtained by enzymatic degradation of silk fibroin with proteolytic enzyme,
A peptide having a molecular weight of 1,000-2,200 and a free amino acid mainly comprising glycine, alanine, serine and tyrosine, obtained by using a neutral protease derived from Aspergillus as the proteolytic enzyme A functional food material derived from silk fibroin. In a method of producing a silk fibroin-derived food functional material by enzymatic degradation of silk fibroin with a proteolytic enzyme,
A method for producing a functional food material derived from silk fibroin, wherein a neutral protease derived from Aspergillus is used as the proteolytic enzyme. 3. The silk fibroin is prepared by putting a refined silk thread into a calcium chloride aqueous solution, boiling it to dissolve the silk thread, and desalting the resulting fibroin aqueous solution, which is used as a silk fibroin raw material. For producing functional food material derived from silk fibroin. Enzymatic degradation of silk fibroin by proteolytic enzyme is performed by dissolving the silk fibroin raw material with distilled water, adding a buffer solution and an enzyme solution of neutral protease derived from Aspergillus to the resulting aqueous fibroin solution, and reacting them. The manufacturing method of the food functional material derived from the silk fibroin of Claim 3. The method for producing a silk fibroin-derived food functional material according to claim 4, wherein the reaction liquid after enzymatic decomposition of silk fibroin is dried to form powder.

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