JP2958801B2 - Method for producing debittered functional peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Use Classification) The present invention relates to a peptide having good taste and new or improved functional properties.

(Problems of the Prior Art) Limited hydrolysis (fragmentation) using a protease or an acid is performed as one of the means for newly expressing and improving the functional properties of food proteins. The peptide thus obtained is added to foods such as beverages, seasonings and emulsions from the viewpoint of enhancing nutritional properties and improving physical properties such as emulsifying properties. However, it is pointed out that a bitter peptide having bitterness and harshness is produced by the limited hydrolysis (fragmentation) operation, which results in a material having poor taste and difficulty in palatability,
This limits their use.

Thus, generation of unpleasant tastes such as bitterness and astringency is the greatest difficulty in using proteases and acids in food processing, and development of a method that does not generate bitterness is expected.

Well, Fujimaki, Arai, Ishii et al. Studied the amino acid sequence and bitterness in the pepsin degradation product of soy protein, and found that the bitterness of the peptide was attributed to hydrophobic amino acids such as Leu, Val, Ile, and Phe in the peptide chain. Further, it is found that the closer they are to the C-terminus of the peptide chain, the stronger the bitterness, and using Streptomyces or Asporgillus-derived exopeptidase to release hydrophobic amino acids from the N-terminus or C-terminus. (J. Food Sci., 35, 215-218, (197
0), Food Journal, 23,524-530, (1976), Food Journal, 24,
171-178, (1976), Food Journal, 26,168-174 (1976),
Proceedings of the 17th Annual Conference of the Japan Food Industry Association 26-35 (1970
Year)). Further, as another method of removing bitterness, a method of extracting and removing a bitter peptide in a protein hydrolyzate with an organic solvent (J. Agric. Biol. Chem., 26, 742-749, (197
8)), a method of removing peptides exhibiting bitterness, harshness, etc. by hydrophobic chromatography using a resin (J. Fo
od Sci., 43, 1491-1493, (1978)) and the like. However, a peptide from which bitterness and astringency are completely removed has not been obtained by any of the methods.

(Problems to be Solved by the Present Invention) Accordingly, an object of the present invention is to provide a peptide from which bitterness and astringency have been completely removed by using a novel method which has not been used before.

(Means for Solving the Problems) The present inventors have conducted intensive studies in order to achieve the above object, and as a result, transglutaminase acts on glutamine residues and lysine residue side chains in the peptide chain to form a chemical compound on both side chains. Utilizing the modification of the structure, it was found that the bitter peptide produced by limited hydrolysis was debittered with transglutaminase, and the present invention was completed.
Furthermore, it has been found that the bitter peptide can be debittered by an acid treatment with dilute hydrochloric acid or dilute sulfuric acid.

That is, the present invention converts food proteins into proteases and / or
Alternatively, it is a functional peptide obtained by subjecting a hydrolyzed peptide obtained by limited hydrolysis with an acid to a treatment with transglutaminase or dilute acid.

The type of food protein used in the present invention is not particularly limited, for example, soybeans, wheat, plant proteins such as corn protein, gelatin, animal meat,
Animal proteins such as fish meat and casein can be exemplified. One type of protein to be subjected to the limited hydrolysis described below may be used, or two or more types of proteins may be used in combination.

When a protein is subjected to limited hydrolysis with an acid and / or a protease, conditions for the limited hydrolysis are not particularly limited. When an acid is used, it is usually carried out using an inorganic acid such as hydrochloric acid or sulfuric acid. Although the concentration of the acid is not particularly limited, it is generally used at a concentration of 0.001 to 12N, preferably 0.1 to 7N.
That is, the protein is dissolved in the acid in the above concentration range and hydrolyzed. The hydrolysis conditions are not particularly limited, but are usually 4 to 200 ° C., preferably 60 to 120 ° C. for about 1 to 3000 minutes, preferably 1 to 150 minutes, and the pressure during the reaction is not particularly limited. Usually, it may be carried out under normal pressure to 2 atm.

The protease used in the limited hydrolysis by the protease is not particularly limited, but usually, it is desirable to use a protease such as papain, promelain, trypsin, pepsin, actinase or the like in accordance with the functionality. For example, by utilizing protease specificity, fragmentation such that an aromatic amino acid such as phenylalanine comes to the C-terminal portion of the peptide chain with pepsin, and then cleaving the C-terminal from the C-terminal with actinase to obtain a peptide A peptide having no phenylalanine in the chain can be obtained.

The concentration of the protease is usually 0.001 to 50,000 per 1 part by weight of the food protein to be treated.
Units, preferably in a concentration range from 0.01 to 6000 units. The conditions for the limited hydrolysis using a protease are not particularly limited, but are usually 0 to 90 ° C, preferably 4 to 60 ° C.
C. for about 0.01 to 3000 minutes, preferably 1 to 1500 minutes. The concentration of the starting protein when performing the limited hydrolysis is not particularly limited, but is usually 0.01 to 90% by weight, preferably 1 to 50% by weight.

When performing limited hydrolysis, it may be carried out using only one kind of protease, or two or more kinds of proteases may be combined. Further, the inorganic acid treatment and the protease treatment may be performed in combination.

In addition, a solvent for dissolving the protein, that is, limited hydrolysis in this solution, but as this solution,
Various buffers such as water, ethanol, and phosphate buffers may be used. That is, any solvent may be used as long as the protein is dissolved and the protease activity is not inactivated.

Before proceeding to the next debittering operation (transglutaminase treatment / dilute acid treatment), if necessary, the hydrolyzed peptide subjected to limited hydrolysis treatment may be subjected to gel filtration chromatography or the like, which may cause unnecessary or adverse effects. Amino acids, salts, and the like may be removed. However, this operation is indispensable when the hydrolyzed peptide is used for patients whose intake of specific amino acids is restricted, such as phenylketonuria patients and hepatic encephalopathy patients.

Next, the hydrolyzed peptide subjected to such limited hydrolysis treatment is treated with a dilute acid or transglutaminase to obtain a desired functional peptide having no bitterness.

Although the kind of the dilute acid used in the present invention is not particularly limited, an inorganic acid such as hydrochloric acid and sulfuric acid is usually used. The origin of the transglutaminase used in the present invention is not particularly limited. That is, transglutaminase derived from guinea pig liver (see JP-A-58-149645), transglutaminase derived from microorganisms (see JP-A-1-27471), and transglutaminase derived from cod (Japanese Society of Fisheries Science, 55, 18).
67, (1989)) and those produced by genetic engineering (see JP-A-1-300889), so long as they have transglutaminase activity.

The details of the enzymological properties of transglutaminase are specified in the above-mentioned publication, but are briefly described as follows.

Transglutaminase is an enzyme that catalyzes an acyl transfer reaction of a γ-carboxamide group of a glutamine (Gln) residue in a peptide chain. When the ε-amino group of a lysine (Lys) residue in a protein acts as an acyl acceptor, ε- (γ-Gl
u) Lys crosslinks are formed. Further, when water functions as an acyl acceptor, it is an enzyme that promotes a reaction in which a glutamine residue is deamidated into a glutamic acid residue.

Due to such properties of transglutaminase, protein-containing solutions or slurries can be gelled using transglutaminase. Transglutaminase is known to be derived from animals such as those derived from guinea pig liver, but those derived from animals are Ca-dependent. On the other hand, microbial transglutaminase exhibits enzyme activity in the absence of Ca or in the presence of Ca.
In the present invention, transglutaminase derived from microorganisms is abbreviated as BTG, and transglutaminase derived from guinea pig liver is abbreviated as MTG.

Now, under the conditions of transglutaminase treatment,
Usually, 0.001 to 1000 units, preferably 1 to 100 units of transglutaminase may be added to 1 part by weight of the hydrolyzed peptide. The reaction conditions are not particularly limited, but are usually 0 to 90 ° C., preferably 4 to 60 ° C., and 0.
The reaction may be performed for 01 to 6000 minutes, preferably for 1 to 1500 minutes. The pH of the reaction solution is usually pH 2 to 10, preferably pH 4
It may be adjusted to ~ 8.

When transglutaminase treatment is performed, the γ-carboxamide group of the Gln residue in the bitter peptide and the ε-amino group of the Lys residue located in the vicinity thereof cause an intramolecular or intermolecular cross-linking reaction and become a polymer. It is thought that the molecular structure of the bitter peptide changes due to this, and the side chain of the Gln residue in the bitter peptide is deamidated to eliminate bitterness.

Alternatively, the hydrolyzed peptide obtained by the limited hydrolysis treatment may be subjected to a dilute acid treatment to eliminate bitterness and the like. The type of the dilute acid used is not particularly limited, but inorganic acids such as dilute hydrochloric acid and dilute sulfuric acid may be used.

The reaction conditions are not particularly limited, but usually, the hydrolyzed peptide obtained by the limited decomposition is dissolved in 0.01 to 80% by weight of an inorganic acid, and is dissolved at 0 to 200 ° C, preferably 40 to 150 ° C for 1 to 20%.
The heat treatment may be performed for 0 minute, preferably for 10 to 120 minutes. The pressure during the reaction is not particularly limited, but usually,
What is necessary is just to carry out under normal pressure and 2 atmospheres.

As described above, by treating the hydrolyzed peptide obtained by limited hydrolysis under mild conditions, the peptide bond of the peptide chain is not cleaved, but the
Since deamidation of the side chain acid amide of Gln and / or Asp occurs, a functional peptide having no bitterness, astringency or the like can be obtained.

As described above, the functional peptide having no bitterness or astringency obtained by the present invention is a useful food material that can be widely used in various functional foods such as beverages.

 Hereinafter, the present invention will be described based on examples.

<Example 1> Zein of a maize protein having a high content of branched-chain amino acids and a relatively low content of aromatic amino acids was prepared by adding 10% EtOH to 0.1% zein.
It was dissolved in 02M NaOH to 1%. Alkalophilic proteinase (22.3U) produced by actinomycetes is an enzyme that hydrolyzes zein and positions aromatic amino acids near the ends.
/ mg, Toyobo) with a substrate / enzyme ratio (weight ratio) of 500/1 (substrate 1
Enzyme amount is 44.6U when converted to g).
Stirred at 11, 37 degrees for 8 hours.

Next, at pH 6.5, aromatic amino acids near the terminals were released by exo-type hydrolysis using actinase E (1000 U / mg, Kaken Pharmaceutical).

The reaction was carried out at a substrate / enzyme ratio (weight ratio) of 50/1 (20,000 U of enzyme per 1 g of substrate) at 37 ° C. for 4 hours. The resulting peptide from which the aromatic amino acids had been removed had a bitter taste.

Then, the substrate / enzyme ratio was 100/1 with BTGasc (1 U / mg).
(The BTG amount per 1 g of the substrate was 10 U) The treatment was carried out at pH 8, 37 ° C. for 24 hours, and the taste of the bitter peptide was greatly improved.

According to the above method, it became possible to prepare a peptide meal material for hepatic encephalopathy patients with good taste.

<Example 2> As in Example 1, zein having a high content of branched-chain amino acids and a relatively low content of aromatic amino acids was replaced with 10% EtOH and 0.02MN.
Dissolved in aOH to 1%. Alkalophilic proteinase (22.3U / mg, Toyobo) produced by actinomycetes is used as an enzyme that hydrolyzes zein to position aromatic amino acids near the ends, so that the substrate / enzyme ratio becomes 500/1, and pH1
The mixture was stirred at 1, 37 degrees for 8 hours.

Next, at pH 6.5, aromatic amino acids near the terminals were released by exo-type hydrolysis using actinase. The reaction was carried out at a substrate / enzyme ratio of 50/1 at 37 ° C. for 4 hours.
The resulting peptide from which the aromatic amino acids had been removed had a bitter taste.

Subsequently, the taste of the bitter peptide was improved by heating and refluxing with 1N (3.65% by weight) hydrochloric acid for 2 hours.

<Example 3> Papain (100,000 U / g, Nagase Seikagaku Corporation), trypsin (3000 U / mg, Novo), and protease P (10000 U / g, Amano Pharmaceutical) were used to hydrolyze skim milk, respectively. Weight ratio) is 100/2 (in terms of the amount of enzyme per 1g of substrate, papain 2000U, trypsin 6000U, protease
p200U) and stirred at room temperature for 24 hours.

Next, with the BTGase used in Example 1, the substrate / enzyme ratio was
By treating under conditions of 100/1, room temperature, 24 hours,
Bitterness was reduced.

It is known that the skim milk hydrolyzed to peptides reduces the argenicity. However, conventionally, it was in a state of being commercialized with bitterness, but it is possible to prepare good-tasting, low-argen (for milk-allergic patients) milk with reduced bitterness by treating with the above method. It became.

Example 4 Gluten was dissolved in water to 2%. For the hydrolysis of gluten, the papain used in Example 3 (100,000 U / m
g, Nagase Seikagaku), trypsin (3000U / mg, Novo), protease P (10000U / g, Amano Pharmaceutical)
The mixture was used at a substrate / enzyme ratio of 100/2 and stirred at room temperature for 24 hours.

Next, with the BTGase used in Example 1, the substrate / enzyme ratio was
By treating under conditions of 100/1, room temperature, 24 hours,
Bitterness was reduced.

The solubility of gluten limitedly hydrolyzed to peptides was improved. However, although bitterness has been maintained until now, the treatment described above has made it possible to prepare highly soluble and highly soluble gluten with reduced bitterness.

(Effect of the present invention) Conventionally, a new functional property has been obtained by performing a hydrolysis treatment in order to expand the use of food proteins and to improve the use of the same. ing. The debittered peptide food material according to the present invention is obtained by modifying the chemical structure of a bitter peptide to a form that does not produce bitterness by acid treatment or transglutaminase treatment after limited hydrolysis. It gives a great way to the place where it was suppressed.

──────────────────────────────────────────────────続 き Continuation of front page Preliminary examination (58) Investigated field (Int. Cl. ⁶ , DB name) A23J 3/32-3/34 A23L 1/305 C12P 21/06 WPI (DIALOG) CA (STN) JICST file (JOIS) JAFIC file (JOIS)

Claims (1)

(57) [Claims] 1. A method for producing a debittered functional peptide obtained by subjecting a food protein to limited degradation with a protease and then subjecting the obtained hydrolyzed peptide to a treatment with transglutaminase or dilute acid.