JP2010183918A - Production method of tripeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing tripeptide Val-Pro-Pro and/or Ile-Pro-Pro useful as a hypotensive agent, an anti-stress agent and the like, wherein the production method is improved in yield, and composed of an easy and stabilized production step. <P>SOLUTION: There is provided a production method of tripeptide, providing tripeptide Val-Pro-Pro and/or Ile-Pro-Pro by acting proteinase and peptidase on a material containing milk casein, wherein the proteinase contains one or more proteinase selected from papain and proteinase derived from Aspergillus, producing an intermediate peptide containing a specific peptide listed in the sequence table when acting the proteinase to the milk casein, and the peptidase contains a peptidase selected from the group consisting of aminopeptidase, carboxypeptidase and a combination thereof. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a tripeptide capable of efficiently producing the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro.

Conventionally, it is known that various peptides having a specific sequence have utility such as having various physiological activities. Examples of such peptides include the tripeptides Val-Pro-Pro and Ile-Pro-Pro. These tripeptides can be found in lactic acid bacteria fermented milk, have strong ACE inhibitory activity, have strong antihypertensive action in spontaneously hypertensive rats (SHR), and have antihypertensive effects on hypertensive patients It is shown. ( Non-Patent Document 1 ). Furthermore, it has been reported that the tripeptides Val-Pro-Pro and Ile-Pro-Pro have an anti-stress effect ( Patent Document 1 ).

Regarding the production method of the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro, a high-efficiency production example by lactic acid bacteria fermentation method has been reported ( Patent Document 2 ). However, when lactic acid bacteria fermentation is performed, the fermentation is stopped midway due to a decrease in pH accompanying lactic acid fermentation, and a lot of undegraded casein remains. Moreover, since many lactic acid produces | generates in the culture solution obtained by lactic-acid-bacteria fermentation, when producing into various product forms, trouble arises. For example, when it is processed into a dry powder, pulverization is difficult due to the coexisting lactic acid, and deoxidation treatment is essential.

As another method for producing the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro, a method using an enzyme can be considered. For example, as suggested in Patent Document 1 , it is conceivable to obtain tripeptide Val-Pro-Pro and / or Ile-Pro-Pro by treating milk casein with proteinase and further with carboxypeptidase.

  In the case of production by the enzymatic method, advantages such as improvement of peptide yield, stabilization of the production process, reduction of production man-hours and manpower, and the absence of lactic acid production can be obtained compared with the lactic acid fermentation method. It is expected. However, when cleaving proteins by enzymatic methods, the degradation reactivity by peptidases is extremely low at amino acid sequence sites including Pro, such as Xaa-Pro or Pro-Xaa (Xaa represents any amino acid). End up. Therefore, in the production of the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro by the enzymatic method, there is a problem that it is difficult to cleave the sequence Pro-Xaa. Therefore, the production method by the enzymatic method of the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro, in which the yield is improved over the lactic acid fermentation method and the advantages such as stabilization of the production process are obtained, Is not found.

In addition, as a method for producing useful peptides other than tripeptide Val-Pro-Pro and / or Ile-Pro-Pro by degrading protein in combination with proteinase and peptidase, tripeptide Leu-Pro having ACE inhibitory activity from γ-zein A method for obtaining P-Pro ( Patent Document 3 ), a method for obtaining peptide Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Xaa-Asn and the like from β-casein ( Patent Document 4 ) and the like have been proposed. However, the yield and stability that can be made industrially useful production methods are not obtained, and they are not put into practical use.

Japanese Patent Laid-Open No. 11-100328 Japanese Patent Laid-Open No. 11-98978 Japanese Patent No. 2873327 JP-A-6-128287

J. Dairy Sci. 1995, 78: 777-783; J. Dairy Sci. 1995, 78: 1253-1257; Am. J. Clin. Nutr. 1996, 64: 767-771

  The object of the present invention is to improve the yield and facilitate the production process in a method for producing tripeptides Val-Pro-Pro and Ile-Pro-Pro useful as antihypertensive agents, antistress agents and the like by an enzymatic method. Another object of the present invention is to provide a production method that is stabilized.

  According to the present invention, there is provided a method for producing a tripeptide, wherein a proteinase and a peptidase are allowed to act on a material containing milk casein to obtain a tripeptide Val-Pro-Pro and / or Ile-Pro-Pro by an enzyme treatment method, The proteinase is selected from a proteinase derived from papain, Aspergillus, which produces an intermediate peptide comprising at least one peptide of SEQ ID NOs: 1-27 described in the sequence listing when acting on milk casein There is provided a method for producing a tripeptide, comprising one or more proteinases, wherein the peptidase comprises a peptidase selected from the group consisting of aminopeptidases, carboxypeptidases, and combinations thereof.

  The method for producing a tripeptide of the present invention easily produces the tripeptides Val-Pro-Pro and Ile-Pro-Pro, which are useful as antihypertensive agents, antistress agents, etc., at high yields and at low cost by enzyme treatment. And is extremely valuable industrially.

In the method for producing a tripeptide of the present invention, a triprotein Val-Pro-Pro and / or Ile-Pro-Pro is obtained by allowing a specific proteinase and peptidase to act on a material containing milk casein.
In consideration of the tripeptide content, the material price, the ease of industrialization, etc., the milk casein-containing material is milk such as animal milk, skim milk, skim milk powder, milk casein and processed products thereof, such as β casein and κ casein. A material containing a large amount of casein can be appropriately selected and used.
Among milk caseins, those containing the amino acid sequences Val-Pro-Pro and Ile-Pro-Pro are particularly β-casein and κ-casein. Therefore, the material containing these milk caseins preferably contains these.

  In general milk casein, β-casein is contained in an amount of 25 to 30% by weight and κ-casein is contained in an amount of 10 to 15% by weight, and β-casein is more contained. Therefore, in the production method of the present invention, β-casein can be used as the main substrate source.

  The specific proteinase is a proteinase that produces a specific intermediate peptide when allowed to act on milk casein.

  The specific intermediate peptide includes at least one peptide of SEQ ID NOs: 1 to 27 described in the sequence listing, and includes a peptide Gln-Asn-Ile-Pro having a sequence included in the β casein sequence. -Pro-Leu-Thr-Gln-Thr or any of the peptides from this peptide to Ile-Pro-Pro obtained by removing one amino-terminal or carboxy-terminal amino acid one by one (SEQ ID NOs: 1 to 15 below) ), Or the peptide Val-Val-Val-Pro-Phe-Leu-Gln having a sequence included in the β casein sequence or Val-Pro-, which is obtained by removing one amino-terminal or carboxy-terminal amino acid from this peptide. It includes peptides up to Pro (SEQ ID NOs: 16 to 27 below).

Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr (SEQ ID NO: 1)
Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr (SEQ ID NO: 2)
Ile-Pro-Pro-Leu-Thr-Gln-Thr (SEQ ID NO: 3)
Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln (SEQ ID NO: 4)
Asn-Ile-Pro-Pro-Leu-Thr-Gln (SEQ ID NO: 5)
Ile-Pro-Pro-Leu-Thr-Gln (SEQ ID NO: 6)
Gln-Asn-Ile-Pro-Pro-Leu-Thr (SEQ ID NO: 7)
Asn-Ile-Pro-Pro-Leu-Thr (SEQ ID NO: 8)
Ile-Pro-Pro-Leu-Thr (SEQ ID NO: 9)
Gln-Asn-Ile-Pro-Pro-Leu (SEQ ID NO: 10)
Asn-Ile-Pro-Pro-Leu (SEQ ID NO: 11)
Ile-Pro-Pro-Leu (SEQ ID NO: 12)
Gln-Asn-Ile-Pro-Pro (SEQ ID NO: 13)
Asn-Ile-Pro-Pro (SEQ ID NO: 14)
Ile-Pro-Pro (SEQ ID NO: 15)
Val-Val-Val-Pro-Pro-Phe-Leu-Gln (SEQ ID NO: 16)
Val-Val-Pro-Pro-Phe-Leu-Gln (SEQ ID NO: 17)
Val-Pro-Pro-Phe-Leu-Gln (SEQ ID NO: 18)
Val-Val-Val-Pro-Pro-Phe-Leu (SEQ ID NO: 19)
Val-Val-Pro-Pro-Phe-Leu (SEQ ID NO: 20)
Val-Pro-Pro-Phe-Leu (SEQ ID NO: 21)
Val-Val-Val-Pro-Pro-Phe (SEQ ID NO: 22)
Val-Val-Pro-Pro-Phe (SEQ ID NO: 23)
Val-Pro-Pro-Phe (SEQ ID NO: 24)
Val-Val-Val-Pro-Pro (SEQ ID NO: 25)
Val-Val-Pro-Pro (SEQ ID NO: 26)
Val-Pro-Pro (SEQ ID NO: 27)

  Specific examples of the proteinase include papain, protease A (manufactured by Amano Pharmaceutical Co., Ltd.), protease M (manufactured by Amano Pharmaceutical Co., Ltd.), protease P (manufactured by Amano Pharmaceutical Co., Ltd.), or a combination thereof. Etc.

  As the peptidase, casein cannot be directly decomposed, but the intermediate peptide produced by the proteinase can be decomposed to produce the tripeptide Val-Pro-Pro and / or Ile-Pro-Pro. Of peptidases can be used. Specifically, for example, exo-type peptidases such as aminopeptidase and carboxypeptidase, endo-type peptidases such as oligopeptidases, and combinations thereof can be used.

  Examples of the peptidase include carboxypeptidase and / or endo-type peptidase that cleave the bond between Pro and Xaa in the sequence Val-Pro-Pro-Xaa and / or Ile-Pro-Pro-Xaa. It is particularly preferable to use one. More specifically, for example, one capable of cleaving the bond of Pro-Leu and / or Pro-Phe of the sequence Ile-Pro-Pro-Leu and / or Val-Pro-Pro-Phe contained in milk casein. Preferably, a peptidase having specificity for this binding is more preferably used.

  The peptidase can be appropriately selected according to the substrate specificity of the proteinase used together. For example, depending on the substrate specificity of the proteinase, either aminopeptidase or carboxypeptidase may be used, but in general, peptidases that cleave peptides such as aminopeptidases from the N-terminus and peptides such as carboxypeptidases are C It is preferable to use in combination with a peptidase and / or an oligopeptidase that cleave from the end. Specifically, for example, papain, protease A (manufactured by Amano Pharmaceutical Co., Ltd.), protease M (manufactured by Amano Pharmaceutical Co., Ltd.), protease P (manufactured by Amano Pharmaceutical Co., Ltd.), or a mixture thereof is used as the proteinase. In this case, it is preferable to use aminopeptidase together with carboxypeptidase and / or oligopeptidase as the peptidase.

  Examples of the aminopeptidase include aminopeptidase I derived from Streptomyces griseus I (manufactured by Sigma), aminopeptidase derived from Aerononas proteolytica (manufactured by Sigma), leucine amino acid derived from porcine kidney cytoplasm. Examples include peptidase and porcine kidney endoplasmic reticulum leucine aminopeptidase.

  Examples of the carboxypeptidase include carboxypeptidase Y (manufactured by Sigma), carboxypeptidase A (manufactured by Sigma), carboxypeptidase B (manufactured by Sigma), and cathepsin G (manufactured by Sigma).

  As the peptidase, in addition to those exemplified above, microorganisms such as lactic acid bacteria, Escherichia coli or Bacillus subtilis, or enzymes derived from animal tissues or plants can be used.

  For example, a peptidase derived from the lactic acid bacterium Lactobacillus helveticus can be used. The peptidase derived from Lactobacillus helveticus is, for example, collected from the culture solution of the lactic acid bacterium Lactobacillus helveticus by centrifugation, etc., then disrupted by centrifugation of cells such as ultrasonic treatment, and centrifuged. Separation is performed, the precipitate is removed, the supernatant is recovered as a crude enzyme extract, and the crude extract is fractionated using an adsorption separation column such as DEAE-sepharose (Pharmacia).

  Peptidase derived from lactic acid bacteria Lactobacillus helveticus acts on the intermediate peptide without combining with other peptidases to obtain the tripeptide Ile-Pro-Pro and / or Val-Pro-Pro in high yield However, other peptidases such as aminopeptidase and / or carboxypeptidase can be used in combination.

  The step of allowing the proteinase and the peptidase to act on the material containing the milk casein is a two-stage degradation in which the proteinase is allowed to act on the material containing the milk casein, or the proteinase and the peptidase are simultaneously reacted. It can be carried out by a one-stage decomposition.

  In the case of performing the two-stage decomposition, the first-stage decomposition in which the proteinase is allowed to act on the milk casein-containing material has an addition ratio of the proteinase to the milk casein of 1/100 to 1/10000, pH and temperature. The reaction is preferably carried out for 3 to 24 hours at 5 to 9 and 20 to 40 ° C., respectively, preferably at the optimum pH and temperature of the enzyme.

  In the case of performing the two-stage degradation, after the first-stage degradation, before the second-stage degradation in which the peptidase is allowed to act on the obtained intermediate peptide, proteinase depletion is performed as necessary. Various operations such as activation, removal of undegraded protein, concentration of intermediate peptide, removal of solvent and the like can be performed.

  The inactivation of the proteinase can be usually performed by a heat treatment at 60 ° C to 100 ° C. By carrying out such inactivation, the subsequent reaction with peptidase can be carried out efficiently.

  The undegraded protein can be removed, for example, by removing the precipitate by centrifuging for 3 to 10 minutes at a rotational speed of 5,000 to 20,000 rpm.

Concentration of the intermediate peptide can be performed using a hydrophobic resin or the like. For example, as a hydrophobic resin, specifically, a silica-based resin bonded with a group containing a cyano group such as a propionitrile group, a phenyl group, or an alkyl group having 1 to 18 carbon atoms, specifically a trade name “Amberlite XAD-7”, “Amberlite XAD-2” (all manufactured by Organo Corporation), Sep-Pak cartridge (manufactured by Waters) and the like can be used. These hydrophobic resins are prepared by adsorbing the intermediate peptide by the column method or batch method and then eluting with a solvent such as a polar solvent such as methanol, ethanol, 1-propanol, 2-propanol and acetonitrile. Then, the intermediate peptide can be concentrated and the next enzyme reaction can be carried out more efficiently.
The removal of the solvent can be performed by a vacuum concentration treatment or the like.

  After performing the first step of degradation and further performing various operations such as inactivation of proteinase, removal of undegraded protein, concentration of intermediate peptide, removal of solvent, etc., if necessary, the peptidase is allowed to act. Stage decomposition can be performed. The second stage decomposition can be preferably carried out by adjusting the pH to 4.0 to 7.0, more preferably 4.5 to 6.5, and the temperature to 25 to 50 ° C, more preferably 30 to 45 ° C. Moreover, when using a some enzyme as a peptidase, you may divide the decomposition | disassembly of a 2nd step into several for every enzyme to be used, and may perform reaction on the optimal conditions of each enzyme.

  When the step of allowing the proteinase and the peptidase to act on the material containing the milk casein is performed by a one-step decomposition in which the proteinase and the peptidase are allowed to act simultaneously, the reaction conditions are pH 4.5 to 7.0, temperature 25 to 50 ° C. Can be done.

  The reaction mixture after the proteinase and peptidase are allowed to act is usually a mixture containing other peptide components in addition to the tripeptides Val-Pro-Pro and Ile-Pro-Pro. This reaction mixture can be made into a product as it is or by concentrating and purifying the tripeptides Val-Pro-Pro and Ile-Pro-Pro. Tripeptides Val-Pro-Pro and Ile-Pro-Pro can also be made into products as industrially acceptable salts such as hydrochloride, succinate, citrate, and tartrate. .

  The product containing the tripeptide obtained by the production method of the present invention can be used as it is or mixed with other food or pharmaceutical materials to form a liquid, powder, granule, tablet, etc. It can be made into dairy products such as yogurt and dairy drinks, general foods and drinks, foods for specified health use, health foods, pharmaceuticals, etc. having an action and an antistress action.

  Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples, but the present invention is not limited thereto.

Experimental Examples 1-1 to 1-21
(Selection of peptidase preparations that produce tripeptides Ile-Pro-Pro and Val-Pro-Pro from intermediate peptides and examination of optimal reaction conditions)
Synthetic peptides based on the amino acid sequence of β-casein, which contained Ile-Pro-Pro and Val-Pro-Pro in the amino acid sequence and had no Pro at other sites, were chemically synthesized (Table 1). These synthetic peptides were all synthesized by an automatic peptide synthesizer PPSM-8 (Shimadzu Corporation).

The enzyme reactivity of various commercially available peptidase preparations against these synthetic peptides was examined (Table 1). Synthetic peptides were adjusted to 100 mM phosphate buffer pH 5.3 for Experimental Examples 1-1 to 1-4, 1-10 to 1-12, 1-16 and 1-21, and 50 mM Tris HCl pH 8 for other experimental examples. 0.0 was dissolved to 10 μg / ml. Various peptidase preparations were added thereto at 0.01 μg / ml and reacted at 37 ° C. for 3 hours. After enzymatic reaction, analyzed by reversed-phase high performance liquid chromatography (HPLC), the peak derived from the tripeptide Val-Pro-Pro detected at 11.0 minutes elution time and the tripeptide Ile-Pro- detected at 13.7 minutes Peaks derived from Pro were analyzed. The detected peak is fractionated and subjected to amino acid sequence analysis with an automatic peptide analyzer model PPSQ-10 (Shimadzu Corporation). The amino acid sequence of the peptide in the fractionated peak is Ile-Pro-Pro or Val-Pro- Confirmed to be Pro. The HPLC analysis conditions were as follows.
Pump: L6200 intelligent pump (Hitachi) , L6000 pump (Hitachi)
Detector: L4000UV detector (manufactured by Hitachi, Ltd.)
Column: Microbonder sphere, 5μC18 (Φ3.9x150mm) (manufactured by Waters)
Eluent: Liquid A; 0.1 wt% trifluoroacetic acid (TFA) aqueous solution
Solution B: acetonitrile containing 0.1 wt% TFA Elution condition: Solution B 0% (solution A 100%) to solution B 40% (solution A 60%) linear concentration gradient elution (40 minutes)
Flow rate: 1ml / min

In addition, the symbol of the enzyme in Table 1 shows the following enzymes, respectively.
A: Aminopeptidase (Sigma)
LC: Leucine aminopeptidase, cytosol (Aminopeptidase derived from porcine kidney cytoplasm, manufactured by Sigma)
LM: leucine aminopeptidase, microsomal (aminopeptidase derived from porcine kidney endoplasmic reticulum, manufactured by Sigma)
I: aminopeptidase I (manufactured by Sigma)
Y: Carboxypeptidase Y (manufactured by Sigma)
B: Carboxypeptidase B (manufactured by Sigma)
A: Carboxypeptidase A (Sigma)
G: Cathepsin G (manufactured by Sigma)
As shown in Table 1, it was found that any aminopeptidase can cleave and remove the amino terminal amino acid down to the sequence just before the first amino acid residue of the Pro residue. In addition, when carboxypeptidase Y was allowed to act at pH 5.3 as a carboxypeptidase, it degraded the Pro-Leu bond of -Pro-Pro-Leu and the Pro-Phe bond of -Pro-Pro-Phe particularly well. It was recognized that

  Enzymes with aminopeptidase and carboxypeptidase Y using the longer peptides Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr and Val-Val-Val-Pro-Pro-Phe-Leu-Gln In the case of treatment (Experimental Examples 1-11 and 1-21), the generation of tripeptides Ile-Pro-Pro and Val-Pro-Pro was confirmed. That is, a peptide having a casein sequence containing the sequence Ile-Pro-Pro- or Val-Pro-Pro in the internal sequence and no other Pro is used in combination with aminopeptidase and carboxypeptidase to cause It has been shown that the peptides Ile-Pro-Pro and / or Val-Pro-Pro are produced.

Experimental example 2
(Production of intermediate peptide by proteinase)
A synthetic peptide consisting of 28 amino acids including the Ile-Pro-Pro and Val-Pro-Pro sequences in the casein sequence was chemically synthesized by an automatic peptide synthesizer model PPSQ-10. The sequence of the synthetic peptide was as follows.
Leu Pro Gln Asn Ile Pro Pro Leu Thr Gln Thr Pro Val Val Val Pro Pro PheLeu Gln Pro Glu Val Met Gly Val Ser Lys (SEQ ID NO: 28)
Proteinases that produce various short-chain peptides that contain the sequence Ile-Pro-Pro or Val-Pro-Pro in the amino acid sequence of the peptide and do not contain Pro residues at other sites by allowing various synthetic proteinase preparations to act on this synthetic peptide Explored.

  For the reaction, various enzymes were added to the above synthetic peptide 10 μg / ml (100 mM phosphate buffer, pH 6.1) so as to be 0.1 μg / ml, and reacted at 37 ° C. for 5 hours to obtain a reaction solution. The reaction solution was analyzed by reversed-phase high performance liquid chromatography (HPLC), the detected peptide peak was collected, and the amino acid sequence was analyzed by an automatic peptide analyzer model PPSQ-10.

  As a result, when papain (Sigma) was used, Val-Pro-Pro-Phe-Leu as a peptide containing the sequence Val-Pro-Pro and Asn-Ile- as a peptide containing the sequence Ile-Pro-Pro It was confirmed that Pro-Pro-Leu-Thr and Ile-Pro-Pro-Leu-Thr were produced in the reaction solution.

Experimental Example 3
(Processing of peptide intermediates with peptidases)
In Experimental Example 2, the proteinase was inactivated by heat-treating the reaction solution obtained using papain as the proteinase at 100 ° C. for 5 minutes. Thereafter, aminopeptidase A (manufactured by Sigma) was added to a concentration of 0.1 μg / ml and reacted at 37 ° C. for 3 hours. After completion of the reaction, 1N hydrochloric acid was added to adjust to pH 5.3, carboxypeptidase Y was added so as to be 0.1 μg / m1, and reacted at 37 ° C. for 10 hours. The obtained reaction solution was analyzed by reversed-phase high performance liquid chromatography (HPLC) under the same conditions as in Experimental Example 1. As a result, peptide peaks thought to be tripeptide Val-Pro-Pro and Ile-Pro-Pro were detected. As a result, the detection peak was collected, and the amino acid sequence was determined with an automatic peptide analyzer PPSQ-10, and confirmed to be tripeptides Ile-Pro-Pro and Val-Pro-Pro.

Example 4
(Screening of lactic acid bacteria peptidases that produce tripeptides Ile-Pro-Pro and Val-Pro-Pro from intermediate peptides)
Among various peptidases contained in lactic acid bacteria Lactobacillus helveticus, those that can be used in the method of the present invention were screened by the method described below.
(Extraction and fractionation of Lactobacillus helveticus enzyme) Inoculate a milk medium containing 9% by weight of skim milk powder with Lactobacillus helveticus CM4 strain ( patent microorganism deposition center deposit number: FERM BP-6060) And cultured at 37 ° C. for 24 hours. The culture was inoculated into 500 ml of fresh 9% by weight milk medium and 5% by weight, and pH stat culture was performed at 37 ° C. while maintaining the pH at 6.5. Five hours after the start of stat culture, sodium citrate was added to a final concentration of 2% by weight, and the mixture was stirred at room temperature for 30 minutes. After confirming that the culture solution became clear, the cells were collected by centrifugation at 5,000 g for 10 minutes. After washing twice with 50 mM phosphate buffer, 150 mM NaCl, pH 6.8, suspended in 20 ml of 50 mM Tris-HCl, pH 8.0, and the cells were disrupted with an ultrasonic crusher (manufactured by Otsuchi Seisakusho, model 5203). . Centrifugation was performed at 15,000 g for 10 minutes to remove the precipitate, and the supernatant was recovered as a crude extract. 5 ml of the crude extract was passed through 1 ml of DEAE-sepharose (Pharmacia) previously equilibrated with the above Tris buffer, and after washing the column with 5 ml of Tris buffer, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM and Washing and fractionation with 3 ml of Tris buffer containing 500 mM NaCl gave six fractions (fractions 1 to 6) shown in Table 2.
(Intermediate peptide cleavage)
The intermediate peptide Val-Pro-Pro-Phe-Leu was chemically synthesized. This synthetic peptide was dissolved in 100 mM phosphate buffer (pH 6.1) to a concentration of 10 μg / ml. This solution and any of the above fractions 1 to 6 were mixed at a volume ratio of 9: 1 and reacted at 37 ° C. for 30 minutes. After completion of the reaction, the reaction solution was analyzed by reverse phase high performance liquid chromatography (HPLC) to confirm the production of the tripeptide Val-Pro-Pro. The tripeptide Val-Pro-Pro production amount is calculated by multiplying the peptide peak height (mm) derived from the tripeptide Val-Pro-Pro detected at an HPLC elution time of 11.0 minutes and the eluate volume (ml). Calculated as a unit. The results are shown in Table 2.

  As a result, in analysis of the liquid reacted with fractions 2 to 5, a peptide peak having peptidase activity to produce the tripeptide Val-Pro-Pro was observed. These fractions contain endo-type peptidases involved in the reaction to produce the tripeptide Val-Pro-Pro from the peptide Val-Pro-Pro-Phe-Leu. In particular, fraction 4 contains about 0.01 μg endo-peptidase was included.

  Next, fraction 4 with the strongest peptidase activity was reacted in the same manner as above using various chemically synthesized synthetic intermediate peptides as substrates, and the reaction solution was analyzed by HPLC, and the tripeptide Ile-Pro- Production of Pro or Val-Pro-Pro was confirmed. As a result, it was confirmed that the tripeptide Val-Pro-Pro was generated from the peptides Val-Pro-Pro-Phe-Leu and Val-Pro-Pro-Phe-Leu-Gln. In addition, generation of the tripeptide Ile-Pro-Pro was confirmed from the peptides Ile-Pro-Pro-Leu-Thr-Gln-Thr and Ile-Pro-Pro-Leu-Thr (Table 3).

Experimental Example 5
(Production of tripeptides Ile-Pro-Pro and Val-Pro-Pro by combining proteinase and peptidase enzyme derived from Helveticas)
To the synthetic peptide consisting of 28 amino acids used in Experimental Example 2 10 μg / ml (100 mM phosphate buffer, pH 6.1), the commercially available proteinase enzymes for food processing shown in Table 4 were each 0.1 μg / ml. The mixture was added and reacted at 37 ° C. for 5 hours. After completion of the reaction, the reaction solution was heated at 100 ° C. for 5 minutes to inactivate the proteinase, then reacted with the fraction 4 obtained in Experimental Example 4, and the tripeptide Ile-Pro-Pro or HPLC was analyzed by HPLC analysis. The production of Val-Pro-Pro was analyzed. Table 4 shows the presence or absence of tripeptide production in each reaction solution.

  As a result, in addition to papain, as a proteinase, protease A (Aspergillus oryzae origin, Amano Pharmaceutical Co., Ltd.), protease M (Aspergillus oryzae origin, Amano Pharmaceutical Co., Ltd.), protease P (Aspergillus melleus origin, Amano Pharmaceutical) In the case of the production of Tripeptide Ile-Pro-Pro and Val-Pro-Pro, the two-step reaction with fraction 4 obtained in Experimental Example 4 was confirmed. When other proteinases were used, the production of tripeptides Ile-Pro-Pro and Val-Pro-Pro was not observed.

Example 1
(Generation of tripeptides Val-Pro-Pro and Ile-Pro-Pro from casein-1)
Casein (Sanlacto S, Taiyo Kagaku Co., Ltd.) 50 mg was dissolved in 10 ml of 50 mM phosphate buffer (pH 6.5), 0.2 mg of papain (Sigma) was added, and reacted at 37 ° C. for 12 hours. The reaction-terminated liquid was heat-treated at 100 ° C. for 3 minutes and then rapidly cooled to deactivate the enzyme. After passing 10 ml of the reaction completion solution through a Sep-pak C18 cartridge (Waters), the adsorbed peptide was eluted with 5 ml of 30% by volume acetonitrile. Acetonitrile was distilled off from the eluted peptide fraction, dried, dissolved in 1 ml of 50 mM Tris-HCl (pH 8.0), and aminopeptidase I (Amano Pharmaceutical Co., Ltd.) and carboxypeptidase Y (Amano Pharmaceutical Co., Ltd.) were respectively added. It was added at a concentration of 0.1 μg / ml and reacted at 37 ° C. for 12 hours. In order to quantify the tripeptides Val-Pro-Pro and Ile-Pro-Pro contained in the reaction-finished solution, they were analyzed by the following HPLC method.
(Quantitative determination of Val-Pro-Pro and Ile-Pro-Pro by HPLC)
Peaks corresponding to tripeptides Ile-Pro-Pro and Val-Pro-Pro were prepared by sequentially diluting the sample with HPLC eluent (0.3M NaCl, 0.05 wt% TFA aqueous solution) and using synthetic peptides as standards. Was quantitatively analyzed.

The HPLC analysis conditions for quantitative analysis were as follows.
Model used: Hitachi L4000 UV detector (215nm) , L6200 intelligent pump.
L5030 Column oven (35 ℃)
Separation conditions: flow rate; 0.5 ml / min
Eluent: 0.3M NaCl, 0.05wt% TFA aqueous solution Column: Asahipak GS320 (Φ3.9x600mm) (manufactured by Showa Denko KK)
As a result, it was found that 50 μg of tripeptide Val-Pro-Pro and 50 μg of tripeptide Ile-Pro-Pro per 50 mg of casein were contained in the reaction completion solution. In casein, the sequences of Val-Pro-Pro and Ile-Pro-Pro exist mainly one by one in β-casein, and the tripeptides Ile-Pro-Pro and Val-Pro-Pro are 100% efficient from these sequences. The theoretical value is about 4 mg per gram of casein, the recovery rate of this experiment for this theoretical value is recovery rate = (actual value / theoretical value) x100 (%)
Val-Pro-Pro = 0.05mg / (4mgx50mg / 1000mg) x100 (%) = 25%
Ile-Pro-Pro = 0.05 mg / (4 mg × 50 mg / 1000 mg) × 100 (%) = 25%.

Example 2
(Generation of tripeptides Val-Pro-Pro and Ile-Pro-Pro from casein-2)
50 mg of casein (Sanlacto S, Taiyo Kagaku Co., Ltd.) was dissolved in 10 ml of 50 mM phosphate buffer (pH 6.5), 0.2 mg of papain (Sigma) was added, and reacted at 37 ° C. for 12 hours. The reaction-terminated liquid was heat-treated at 100 ° C. for 3 minutes and then rapidly cooled to deactivate the enzyme. After passing 10 ml of the reaction completion solution through a Sep-pak C18 cartridge (Waters), the adsorbed peptide was eluted with 5 ml of 30% by volume acetonitrile. After evaporating acetonitrile from the eluted peptide fraction and drying, 1 ml of the Lactobacillus helveticus extracted and purified enzyme fraction (Fraction 4) obtained in Experimental Example 4 was added and reacted at 37 ° C. for 12 hours. The reaction completed solution contained 50 μg of tripeptide Val-Pro-Pro (recovery rate 25%) and 195 μg of tripeptide Ile-Pro-Pro (recovery rate 97.5%) per 50 mg of casein.

Example 3
(Generation of tripeptides Val-Pro-Pro and Ile-Pro-Pro from casein-3)
50 mg of casein (Sanlacto S, Taiyo Kagaku Co., Ltd.) was dissolved in 10 ml of 50 mM phosphate buffer (pH 6.5), 0.2 mg of papain (Sigma) was added and reacted at 37 ° C. for 12 hours. The reaction-terminated liquid was heat-treated at 100 ° C. for 3 minutes and then rapidly cooled to deactivate the enzyme. 1 ml of the Lactobacillus helveticus extracted and purified enzyme fraction (Fraction 4) obtained in Experimental Example 4 was added and reacted at 37 ° C. for 12 hours. The reaction completed solution contained 50 μg of tripeptide Val-Pro-Pro (recovery rate 25%) and 50 μg of tripeptide Ile-Pro-Pro (recovery rate 25%) per 50 mg of casein.

Examples 4-6
(Generation of tripeptides Val-Pro-Pro and Ile-Pro-Pro from casein-4)
150 mg of casein (Sanlacto S, manufactured by Taiyo Kagaku Co., Ltd.) was dissolved in 30 ml of 50 mM phosphate buffer (pH 6.5) and then divided into 10 ml portions. To these, 0.2 mg of either protease A (Example 4), protease M (Example 5) or protease P (Example 6) (both trade names, manufactured by Amano Pharmaceutical Co., Ltd.) were added, The reaction was carried out at ° C for 12 hours. The reaction-terminated liquid was heat-treated at 100 ° C. for 3 minutes and then rapidly cooled to deactivate the enzyme. 1 ml of the Lactobacillus helveticus extracted and purified enzyme fraction (Fraction 4) obtained in Experimental Example 4 was added and reacted at 37 ° C. for 12 hours. The content ratios of tripeptide Val-Pro-Pro and Ile-Pro-Pro per 50 mg of casein in the reaction-finished solution were 40 μg (recovery rate 20%) and 45 μg (recovery rate 22.5%) in Example 4, respectively. In Example 5, they were 37 μg (recovery rate 18.5%) and 50 μg (recovery rate 25%), and in Example 6 they were 42 μg (recovery rate 21%) and 45 μg (recovery rate 22.5%).

Claims (4)

A method for producing a tripeptide in which a proteinase and a peptidase are allowed to act on a material containing milk casein to obtain a tripeptide Val-Pro-Pro and / or Ile-Pro-Pro by an enzyme treatment method,
The proteinase is selected from proteinases derived from papain and Aspergillus which, when allowed to act on milk casein, produce an intermediate peptide containing at least one peptide of SEQ ID NOs: 1 to 27 described in the sequence listing. A method for producing a tripeptide, comprising one or more proteinases, wherein the peptidase comprises a peptidase selected from the group consisting of aminopeptidases, carboxypeptidases, and combinations thereof.   The peptidase is a carboxypeptidase, which cleaves the bond between Pro and Xaa in the sequence Val-Pro-Pro-Xaa and / or Ile-Pro-Pro-Xaa (Xaa represents any amino acid) The method for producing a tripeptide according to claim 1, comprising: The method for producing a tripeptide according to claim 1 or 2 , wherein the peptidase contains a peptidase derived from a living lactic acid bacterium Lactobacillus helveticus. The bird according to any one of claims 1 to 3, wherein the proteinase is allowed to act on the material containing milk casein, the obtained intermediate peptide is concentrated with a hydrophobic resin, and then the peptidase is allowed to act. A method for producing a peptide.