JP3010795B2 - Peptide bitterness removal method - Google Patents

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 bitter taste of a peptide, and more particularly to a method for cleaving the carboxyl terminus of proline present in a peptide with prolyl endopeptidase and then bringing the proline to the carboxyl terminus of the peptide with carboxyl peptidase. And a method for reducing the bitterness of a peptide by releasing aromatic amino acids and branched-chain amino acids.

[0002] In the production of peptides, various commercially available proteases are currently used. However, these proteases have a weak ability to cut before and after proline. For this reason, proline remains in the peptide, but the peptide containing the proline causes bitterness, which is a problem in the production of the peptide. It has also been reported that aromatic amino acids and branched chain amino acids present at the carboxyl terminus of a peptide are also responsible for the bitter taste of the peptide.

[0003] With respect to the removal of bitterness, in the production of peptides for the first time, the generation of bitterness is suppressed by changing the combination of proteases to be used, or by the action of various exoproteases on the peptide that has generated bitterness. Has been reduced. However, there is a limit to the reduction of bitterness by these methods. Attempts have also been made to release proline, aromatic amino acids, and branched-chain amino acids in peptides by using carboxypeptidase, but the method using only carboxypeptidase results in a large amount of released amino acids, These released amino acids adversely affect the taste of the peptide.

[0004]

SUMMARY OF THE INVENTION In the present invention, in reducing the bitter taste of a peptide, in view of the limitations of the conventional method as described above, the use of a protease preparation containing prolyl endopeptidase and carboxypeptidase is considered. An object of the present invention is 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 animals and plants. Here, 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: Hydrolysis activity against p-nitroanilide) is 100
And 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 when treated at 37 ° C for 2 hours
4 to 7 show a residual activity of 85% or more. (F) Temperature stability: 80% or more of the activity remains at pH 5 at 52 ° C. for 1 hour. Having.

The present inventors have studied 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 bitter taste of the peptide was due to the bent structure of the peptide derived from proline and the branched and aromatic amino acids present at the carboxyl terminus of the peptide. The proline at the carboxyl terminus of the peptide is removed by enzymatic cleavage of the proline at the carboxyl terminus, thereby removing the proline at the carboxyl terminus of the peptide and the branched and aromatic amino acids originally present at the carboxyl terminus of the peptide. It was concluded that bitterness could be reduced by peptidase release. However, conventional proteases have little ability to cleave peptide bonds before and after proline, and do not have carboxypeptidase activity, so that new prolyl endopeptidase, carboxypeptidase, and protease are produced simultaneously by gabi. The present inventors have found a method for producing a peptide having less bitterness by using the enzyme preparation obtained by the above method, and have completed the present invention.

[0007] The enzyme preparation used in the present invention is Aspergillus oryzae FS1.
This is a protease preparation containing prolyl endopeptidase and carboxypeptidase derived from -32 (Microtechnical Laboratory No. 12193).

[0008] Proteins derived from animals and plants include soy protein,
Wheat protein, casein and the like 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, depending on the substrate, reaction temperature, pH and amount of enzyme used. Enzyme concentration: About 650 PU of protease per 1 g of substrate About 0.01 U of carboxypeptidase About 0.03 mU of prolyl endopeptidase It is possible to shorten the reaction time by increasing the enzyme concentration further. In addition, instead of lengthening the reaction time, the amount of the enzyme to be added can be reduced, thereby reducing the cost for the enzyme.

The enzyme activity used in the method of the present invention is determined by the following method. It is determined by quantifying the hydrolysis reaction on the carboxyl side of proline by acting on the peptide which is the active substrate of prolyl endopeptidase . The enzyme activity described in this specification was measured by the following method using CBZ-Gly-Pro-pNA as a substrate,
The enzyme activity that releases 1 μmol of paranitroanilide per minute is defined as 1 unit (U). CBZ-Gly-Pro-pNA
Degradation activity measurement method: 2 mM CBZ-G in 40% dioxane solution
A substrate is prepared by adding 1 ml of 0.1 M citric acid-disodium phosphate buffer (pH 5.0) to 0.25 ml of ly-Pro-pNA dissolved therein. After preheating at 37 ° C. for 10 minutes, 0.1 ml of the enzyme solution is added, and the mixture is reacted at 37 ° C. for 2 hours.
After the reaction, 1 M potassium chloride containing 10% Triton-X100-
The reaction is stopped with a hydrochloric acid buffer (pH2) (stop solution), and the absorbance is measured at 410 nm using the control solution as the stop solution and the enzyme solution in the reverse order. (CBZ: Carbobenzoxy)

[0011] The activity of carboxypeptidase is determined by quantifying the hydrolysis reaction on the carboxyl side of the peptide, which is a substrate for measuring the activity of carboxypeptidase . The enzyme activity described in this specification was measured by the following method using CBZ-Glu-Tyr as a substrate. I have. 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 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, 500 μl of a ninhydrin reagent was added, and the mixture was heated on a boiling water bath for 15 minutes and then cooled with cold water.
Is added, and the absorbance at 570 nm is measured using a solution obtained by adding water instead of the enzyme solution as a target solution. This is fitted to a previously prepared standard curve, and the activity of carboxypeptidase is determined from the amount of released tyrosine.

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

[0013]

EXAMPLE 1 0.166 g of an enzyme preparation containing 4.8 mU / g of prolyl endopeptidase, 2.1 U / g of carboxypeptidase and 124000 PU / g of protease was added to 300 ml of a solution containing 30 g of soybean protein. The peptide was prepared by reacting at 5 ° C. for 5 hours. As a result, it was confirmed that the bitter taste was less than that of a peptide prepared by a protease having no prolyl endopeptidase or carboxypeptidase activity as described below. Out of 10 panelists Peptides prepared with prolyl endopeptidase in addition to ordinary proteases and peptides prepared with proteases containing carboxypeptidase Number of people who felt bitter 10 3 Number of people who did not feel bitter 0 7

Example 2 To 300 ml of a solution containing 30 g of a peptide prepared by allowing a commercially available protease (protin AY: manufactured by Daiwa Kasei) to act on soybean protein, use Aspergillus oryzae FS1-32 (Microtechnical Laboratories No. 12193). Derived prolyl endopeptidase 4.8 mU / g, carboxypeptidase 2.1 U
/ G containing 0.116 g of the enzyme preparation at 50 ° C.
After heating for 5 hours, a sensory test was performed. As a result, a reduction in bitterness was observed in the case where the enzyme treatment was performed as compared with the case where the treatment with the present enzyme was not performed. Untreated peptide Enzyme-treated peptide Out of 10 panelists Number of people who felt bitterness 10 2 Number of people who did not feel bitterness 0 8

[0015]

According to the present invention, a peptide having a low bitterness can be easily prepared. In addition, bitterness can be reduced by causing an enzyme preparation containing prolyl endopeptidase and carboxypeptidase to act on a peptide having bitterness. By adding such a less bitter peptide to various foods, the protein of the food can be enhanced without impairing the original taste of the food.

Continuation of the front page (72) Inventor Yukio Hashimoto 396-4 Wakure Elegance 104, Matsugasaki, Kashiwa-shi, Chiba 104 (72) Inventor Tadahisa Shimoda 5-7-1, Kinudai, Yawahara-mura, Tsukuba-gun, Ibaraki Prefecture JP-A-60-192599 (JP, A) JP-A-60-98993 (JP, A) JP-A-2-39896 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A23J 3/34 C12P 21/06 A23J 3/16 C12N 9/62 BIOSIS (DIALOG)

Claims (1)

(57) [Claims] To 1. A protein derived from plants and animals, due to the action of prolyl endopeptidase and carboxypeptidase protease preparation containing peptidase, a method of manufacturing a small peptide bitter, the Puroriruendo
Peptidase has the following physicochemical properties: (a) Action: proline present in peptides and proteins
Hydrolyze the carboxyl side of (B) Substrate specificity: (1) CBZ-Gly-Pro-pNA (CBZ: carbobenzoxy, pNA
: P-nitroanilide) with a hydrolysis activity of 100
Phase for prolyl paranitroanilide
The activity is 0. (2) The Km value for CBZ-Gly-Pro-pNA is 0.29 mM.
You. (C) Optimum pH: around 5 (d) Optimum temperature: 37 ° C (e) pH stability: pH when treated at 37 ° C for 2 hours
4 to 7 show a residual activity of 85% or more. (F) Temperature stability: treatment at 52 ° C. for 1 hour at pH5
At 80% or more. A method comprising: