Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
2001-03-14
2002-09-24
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C426S052000, C426S056000, C426S063000, C426S046000, C435S068100
Reexamination Certificate
active
06455273
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing a protein hydrolysate with low bitterness. More specifically, the present invention relates to a method for producing a protein hydrolysate with low bitterness, by a simple procedure of enzymatic hydrolysis using a protease having a low specificity to cleave a site of a hydrophobic amino acid residue in the protein, with no further need of any procedure to reduce bitterness.
2. Discussion of the Background
Various proteases are used to modify the properties of food proteins. The resulting protein hydrolysates have various functions and properties and are used in diversified manners, depending on the purpose. For example, because the enzymatic protein hydrolysates contain less free amino acids, as compared with HVP (acid-hydrolyzed vegetable proteins) and HAP (acid-hydrolyzed animal proteins), they are used as seasoning materials, due to their mild taste and thickness. As nutrient materials, peptides are rapidly absorbed, as compared with free amino acids. In a peptide form, branched chain amino acids are also absorbed well. Furthermore, corn proteins, which are difficult to digest, can be digested and absorbed as a nutrient when hydrolyzed to peptides. As a food material, a peptide form is superior to a protein form, because modification of proteins into peptides involves the improvement of solubility at a wide range of pH, the decrease of viscosity, the improvement of hygroscopicity and moisture, and the modification of emulsifiability, foaming potency, and gelatinizability. Furthermore, it is reported that a soybean protein hydrolysate has physiological activities, such as a hypocholesterolemic activity, inhibition of cholesterol absorption, a bile acid-binding activity, suppression of platelet aggregation, and an anti-oxidative activity. Additionally, the allergenecity of a protein is reduced or eliminated when the protein is hydrolyzed into a low molecular weight peptide composition.
As has been described above, enzymatic protein hydrolysates have such excellent functions and properties. However, they have strong bitterness. Currently, therefore, the protein hydrolysates have only limited applicability as foodstuffs. Thus, as an approach to reduce the bitterness, a method for eliminating the bitterness by using an aminopeptidase specifically cleaving the N-terminal hydrophobic amino acid residues (Japanese Patent Application, Laid-Open No. 173168/1996) has been described. However, this method disadvantageously involves an increase in free amino acids. Other methods, restricting substrate proteins, have also been reported (Japanese Patent Application, Laid-open Nos. 344847/1993 and 23863/1998), but the applicable range thereof is limited. Furthermore, the use of agents which mask bitterness (Japanese Patent Application. Laid-open Nos. 162/1997 and 100297/1997), a method by fractionating and removing peptides with bitterness (Japanese Patent Application, Laid-open No. 244978/1993), polymerization with a plastein reaction (J. Agric. Biol. Chem. 34, 1484 (1970)), and the utilization of enveloping compounds (Japanese Patent Application, Laid-open No. 283246/1990) have also been 1 g reported. However, all these methods have various problems, including a loss of essential functions of peptides, a low recovery yield, a need for specific equipment, and high cost.
Accordingly, there remains a need for a protein hydrosylate which overcomes the disadvantages discussed above.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for producing a protein hydrolysate with low bitterness, without the problems associated with the materials described above.
The protein hydrolysate prepared in accordance with the present invention tastes less bitter than hydrolysates prepared by enzymes used conventionally in the food industry. Accordingly, the protein hydrolysates with less free amino acid content are readily ingestible. Additionally, the essential nutrient value of the protein can sufficiently be utilized. Hence, the protein hydrolysates prepared in accordance with the invention may be used in numerous applications, such as foodstuffs, infant formulas, medicinal diets, seasonings, flavor-modifying materials, food property-modifying materials, food additives, and feeds.
Thus, the present invention relates to a method for producing a protein hydrolysate with low bitterness, comprising contacting a protein with a protease having a low specificity to cleave a site of a hydrophobic amino acid residue in the protein.
In another embodiment, the present method relates to a method for producing a protein hydrolysate with low bitterness where the protease has a specificity to cleave C-terminals of hydrophilic amino acid residues adjacent to C-terminals of hydrophobic amino acid residues in the protein.
In another embodiment, the present method relates to a method for producing a protein hydrolysate with low bitterness wherein the protease is a thiol protease derived from germinating soybean cotyledons.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have focused their attention on the mechanism by which peptides exert a bitter taste. More specifically, proteases conventionally used in the industry have a substrate specificity to selectively cleave a site of hydrophobic amino acids. Thus, hydrophobic amino acids are very likely to be present at the N-terminals and/or C-terminals of the peptides formed by the hydrolysis. Additionally, it is known that once the protein hydrolysates with such strong bitterness are treated with a peptidase specific to hydrophobic amino acid residues, the bitterness can be eliminated. Thus, the bitterness of the protein hydrolysates is considered to be due to the presence of hydrophobic amino acids at the C-terminals and/or N-terminals of the peptides contained therein.
The present inventors have discovered that a protein hydrolysate with low bitterness can be obtained with no further need of any treatment to reduce bitterness, by contacting a protein with a protease having a low specificity to cleave a site of a hydrophobic amino acid residue in the protein.
The protease having low specificity to cleave a site of a hydrophobic amino acid residue in the protein used in the present invention is one having a cleavage tendency to reduce the probability of the presence of hydrophobic amino acid residues at the C-terminals and/or N-terminals of the peptides formed by the hydrolysis. By hydrolyzing a protein using a protease having such a substrate specificity, a protein hydrolysate with less free amino acids and with weak bitterness can be obtained.
In accordance with the present invention, at least one type of protease having a low specificity to cleave a site of a hydrophobic amino acid residue in the protein is used. Furthermore, at least one type of protease having a low specificity to cleave a site of a hydrophobic amino acid residue in the protein may be used in combination with at least another type of protease having a substrate specificity to cleave a site of a hydrophobic amino acid residue in the protein, as long as the combination can comply with the purpose to provide a protein hydrolysate with low bitterness with no need of any additional process for reducing the bitterness. The proteases may satisfactorily be in the form of a crude enzyme separated from a natural origin in a purified form thereof or in an expression product of genetically recombinant organisms.
The activity of the proteases to cleave a site of a hydrophobic amino acid residue in the protein can be assayed, for example, by the following method. However, the method for estimating the activity is not limited to the method described below.
1. The enzyme is added to a peptide hormone (50 mmol) as a substrate to 0.1% by weight and is then mixed wit
Asano Minao
Kodera Tomohiro
Miwa Tetsuay
Nio Noriki
Ajinomoto Co. Inc.
Weber Jon P.
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