JPH0515314A - Method for removing bitterness of peptide - Google Patents

Abstract

PURPOSE:To readily remove bitterness of peptides such as proline or aromatic amines by reacting a protein derived from an animal or a plant with a specific protease formulation. CONSTITUTION:A protein derived from an animal or a plant is reacted with a protease formulation containing a prolyl endopeptidase or carboxypeptidase at 20-60 deg.C (preferably 50 deg.C) and pH4-7 (preferably pH5) for 4-5hr to carry out the objective removal of bitterness. Furthermore, the prolyl endopeptidase has the following physico-chemical properties. Relative activity of 0 for prolyl-p- nitroanilide when the carboxyl sides of proline in the peptide and protein are hydrolyzed and the hydrolytic properties for CBZ-Gly-Pro-pNa (CBZ is carbobenzoxy; pNA is p-nitroanilide) is 100, 0.29mM value of the substrate specificity (Km) for the CBZ-GIy-Pro-pNa, optimium pH; about 5, optimum temperature; 37 deg.C, pH stability of >=85% residual activity at pH4-7 when treated at 37 deg.C for 2hr, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for removing the bitterness of a peptide. More specifically, the carboxyl terminal of proline present in the peptide is cleaved by prolyl endopeptidase, and then the carboxyl terminal of the peptide is converted to the carboxyl terminal of the peptide. And a method for reducing the bitterness of peptides by releasing aromatic amino acids and branched chain amino acids.

In the production of peptides, various commercially available proteases are currently used. However, these proteases have a weak ability to cleave before and after proline. Therefore, proline remains in the peptide, but the peptides containing these prolines cause bitterness, which is a problem in peptide production. It has also been reported that aromatic amino acids and branched chain amino acids present at the carboxyl terminus of peptides are also responsible for the bitter taste of peptides.

Regarding the removal of bitterness, in the production of the first peptide, the bitterness is suppressed by changing the combination of proteases used, and the bitterness is generated by allowing various exoproteases to act on the bittered peptide. Is being reduced. However, there is a limit to the reduction of bitterness by these methods. In addition, although attempts have been made to release proline and aromatic amino acids in peptides by using carboxypeptidase, branched chain amino acids, the amount of free amino acids is large in the method using only carboxypeptidase, These free amino acids adversely affect the taste of the peptide.

[0004]

DISCLOSURE OF THE INVENTION The present invention is directed to the reduction of the bitterness of peptides, and in view of the limitations of the conventional methods as described above, the use of a protease preparation containing prolyl endopeptidase and carboxypeptidase It is intended to provide a method for producing a peptide having less bitterness.

[0005]

According to the present invention, there is provided a method for producing a peptide having less bitterness by allowing a protease preparation containing prolyl endopeptidase and carboxypeptidase to act on a protein derived from plants and animals. The prolyl endopeptidase has the following physicochemical properties: (a) Action: Hydrolyzes the carboxyl side of proline present in peptides and proteins. (B) Substrate specificity: (1) CBZ-Gly-Pro-pNA (CBZ: carbobenzoxy, -pN
A: p-nitroanilide) has a hydrolysis activity of 100
, The relative activity to prolyl paranitroanilide is 0. (2) The Km value for CBZ-Gly-Pro-pNA is 0.29 mM. (C) Optimum pH: around 5 (d) Optimum temperature: 37 ° C (e) pH stability: pH at 37 ° C for 2 hours
4-7 shows 85% or more residual activity. (F) Temperature stability: 80% or more of the activity remains after one hour of treatment at 52 ° C at pH5. Have.

The present inventors have investigated methods for reducing the bitterness of various peptides derived from animal and plant proteins. As a result, we obtained information that the main cause of the bitterness of the peptide was due to the bent structure of the peptide derived from proline and the branched chain amino acid and aromatic amino acid existing at the carboxyl terminus of the peptide. The bent side of the peptide is removed by enzymatically cleaving the carboxyl side of proline, and the proline that comes to the carboxyl terminus of the peptide and the branched chain amino acids and aromatic amino acids that originally existed at the carboxyl terminus of the peptide are We have reached the inference that bitterness can be reduced by releasing with peptidases. However, the conventional protease has almost no ability to cleave peptide bonds before and after proline and has no carboxypeptidase activity.Therefore, new prolyl endopeptidase, carboxypeptidase, and protease are simultaneously produced in gabi, The inventors have found a method for producing a peptide having less bitterness by using the enzyme preparation obtained by the above, and completed the present invention.

The enzyme preparation used in the present invention is Aspergillus oryzae FS1.
A protease preparation containing a prolyl endopeptidase and a carboxypeptidase, which is derived from -32.

As the protein derived from animals and plants, soybean protein,
Wheat protein, casein, etc. are used.

The reaction conditions for carrying out the present invention are as follows. Reaction temperature: 20 to 60 ° C. (preferably 50 ° C.) pH: 4 to 7 (preferably 5) Reaction time: It is about 4 to 5 hours, varying depending on the substrate used, reaction temperature, pH, and amount of enzyme. Enzyme concentration: Protease about 650 PU per gram of substrate Carboxypeptidase about 0.01 U Prolyl endopeptidase about 0.03 mU It is possible to shorten the reaction time by increasing the enzyme concentration more than this. In addition, the amount of enzyme added can be reduced instead of lengthening the reaction time, and thus the cost of the enzyme can be reduced.

The enzyme activity used in the method of the present invention is determined by the following method. Prolyl endopeptidase activity It is determined by quantifying the hydrolysis reaction of the carboxyl side of proline by acting on the peptide that is the substrate. The enzyme activity described in this specification was measured by the following method using CBZ-Gly-Pro-pNA as a substrate,
One unit (U) is defined as the enzyme activity that releases 1 μmol of paranitroanilide per minute. CBZ-Gly-Pro-pNA
Degradation activity assay: 2 mM CBZ-G in 40% dioxane solution
0.25 ml of dissolved ly-Pro-pNA was added with 1 ml of 0.1 M citric acid-disodium phosphate buffer (pH 5.0) as a substrate. This was preheated at 37 ° C for 10 minutes, 0.1 ml of the enzyme solution was added, and the mixture was reacted at 37 ° C for 2 hours.
After the reaction, 1M potassium chloride containing 10% Triton-X100-
The reaction is stopped with a hydrochloric acid buffer solution (pH 2) (stop solution), and the absorbance is measured at 410 nm using a solution in which the order of adding the stop solution and the enzyme solution is reversed as a control solution. (CBZ: Carbobenzoxy)

Measurement of carboxypeptidase activity It is determined by quantifying the hydrolysis reaction on the carboxyl side of the peptide which is the substrate. The enzyme activity described in this specification was measured by the following method using CBZ-Glu-Tyr as a substrate, and the enzyme activity releasing 1 μmol of tyrosine per minute was defined as 1 unit (U). There is. CBZ-Glu-Tyr degradation activity assay: 0.5 mM CBZ-Glu-Ty
1 ml of r / 0.05M phosphate buffer (pH 7) is used as a substrate. After preheating this at 37 ° C. for 20 minutes, 50 μl of the enzyme solution is added and reacted at 37 ° C. for 60 minutes. After the reaction, add 500 μl of ninhydrin reagent, heat on a boiling water bath for 15 minutes, and then cool with cold water to obtain 0.1 M disodium phosphate / acetone 5 ml.
Is added and water is added instead of the enzyme solution, and the absorbance at 570 nm is measured using the target solution. This is applied to a standard curve prepared in advance, and the activity of carboxypeptidase is determined from the amount of released tyrosine.

[0012] Protease activity measurement It is determined by quantifying the amount of amino acids released by acting on a protein that is a substrate. The enzyme activity described in this specification was measured by the following method using casein as a substrate, and 1 unit (P) of the enzyme activity releasing 1 μmol of tyrosine equivalent of amino acid per minute was used.
U). Method for measuring casein-degrading activity: 5 ml of 1% casein / 0.05M phosphate buffer (pH 7) is used as a substrate. After preheating this at 37 ° C. for 10 minutes, 1 ml of the enzyme solution was added and reacted at 37 ° C. for 10 minutes. The reaction was stopped by adding 5 ml of 0.44 M trichloroacetic acid solution, allowed to stand at 37 ° C. for 20 minutes, and then filtered with filter paper. To 1 ml of the filtrate, 5 ml of 0.4M sodium carbonate solution and 1 ml of Folin reagent were added, and the mixture was allowed to stand at 37 ° C. for 20 minutes and then the absorbance was measured at 660 nm. This is applied to a standard curve prepared in advance and the casein-degrading activity is determined.

[0013]

EXAMPLE 1 To 300 ml of a solution containing 30 g of soybean protein, 0.166 g of an enzyme preparation containing 4.8 mU / g of prolyl endopeptidase, 2.1 U / g of carboxypeptidase and 124,000 PU / g of protease was added, and 50 A peptide was prepared by reacting at 5 ° C for 5 hours. As a result, it was confirmed that the bitterness was less than that of a peptide prepared by a protease having no prolyl endopeptidase or carboxypeptidase activity as described below. Among 10 panelists Peptides made with normal protease and prolyl endopeptidase, peptides made with protease containing carboxypeptidase 10 people who felt bitterness 3 people 3 people who did not feel bitterness 0 7 people

Example 2 300 ml of a solution containing 30 g of a peptide prepared by reacting a soybean protein with a commercially available protease (Protin AY: manufactured by Daiwa Kasei) was added to Aspergillus oryzae FS1-32 (Microtechnology Research Institute No. 12193). Derived prolyl endopeptidase 4.8 mU / g, carboxypeptidase 2.1 U
0.116g of enzyme preparation containing 100 g / g, 50 ° C
After heating for 5 hours, a sensory test was conducted. As a result, the bitterness was observed to be reduced in those treated with the enzyme as compared with those not treated with this enzyme as follows. 10 panelists Unprocessed peptide Enzyme-treated peptide 10 people who felt bitterness 2 people 2 people who did not feel bitterness 0 8 people

[0015]

EFFECTS OF THE INVENTION By utilizing the present invention, a peptide with less bitterness can be easily prepared. In addition, the bitterness can be reduced by acting an enzyme preparation containing a prolyl endopeptidase or a carboxypeptidase on a peptide having a bitterness. By adding such a peptide having less bitterness to various foods, the protein of the food can be enhanced without impairing the original taste of the food.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masao Hashimoto             396-4 Iwasaku, Matsugasaki, Kashiwa City, Chiba Prefecture             Re-elegance 104 (72) Inventor Tadahisa Shimoda             5-7-1 Kinunodai, Taniwahara Village, Tsukuba-gun, Ibaraki Prefecture

Claims (2)

[Claims] 1. A method for producing a peptide having less bitterness by allowing a protease preparation containing prolyl endopeptidase and carboxypeptidase to act on a protein derived from plants and animals. 2. Prolyl endopeptidase has the following physicochemical properties: (a) Action: Hydrolyzes the carboxyl side of proline present in peptides and proteins. (B) Substrate specificity: (1) CBZ-Gly-Pro-pNA (CBZ: carbobenzoxy, pNA
: P-nitroanilide) has a hydrolysis activity of 100
, The relative activity to prolyl paranitroanilide is 0. (2) The Km value for CBZ-Gly-Pro-pNA is 0.29 mM. (C) Optimum pH: around 5 (d) Optimum temperature: 37 ° C (e) pH stability: pH at 37 ° C for 2 hours
4-7 shows 85% or more residual activity. (F) Temperature stability: 80% or more of the activity remains after one hour of treatment at 52 ° C at pH5. The method of claim 1, comprising: