JPS6350998B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing an oligopeptide mixture with excellent nutritional value. More specifically, the present invention provides an oligopeptide mixture that has excellent nutritional improvement effects not only for malnourished people but also for normal people. (Prior Art) The nutritional value of proteins is limited by the protein's limiting amino acids. Therefore, conventional efforts have been made to improve the nutritional value of proteins by adding limiting amino acids or other proteins containing the limiting amino acids. However, some limited amino acids have a bad flavor, cause coloration, and are unstable in themselves, so mere addition is often inconvenient. Furthermore, when other proteins containing limiting amino acids are added, the amino acid composition other than the limiting amino acids changes. Therefore, the present inventor solved this inconvenience by first treating the protein with an enzyme under special conditions and then utilizing the reverse reaction that occurs at that time to prepare an enzyme-modified protein in which a limiting amino acid is covalently introduced. We succeeded in improving the nutritional value. these are,
J.Agric.Food Chem. by the present inventors, 27 ,
52−56 (1979), Agric.Biol.Chem., 43 , 1065−
1068 (1979), the same volume, pages 1069-1074, discloses the means. Later, the invention of the same means was disclosed in Japanese Patent Application Laid-Open No. 57-50900. Furthermore, although methods for hydrolyzing proteins to obtain oligopeptides by changing or combining enzymes have been known, the yield is poor or the amino acid composition is different from that of the raw protein. However, there were problems such as containing a large amount of free amino acids and polypeptides, and having inconsistent molecular weights. On the other hand, as an invention that is different from the oligopeptide of the present invention and obtains a di- or tripeptide in high yield, for example, in Japanese Patent Publication No. 57-45560, a protein raw material (5 to 20 W/V) is Low molecular weight peptides with an average molecular weight of 700 or less (free amino acids and molecular weight
A method for obtaining peptides of 700 or more (20% or less) with a yield of 80% or more is disclosed. Further, to the same purpose, there is an invention in JP-A-58-152498 in which the raw protein is limited in the same way as that of JP-A-57-50900. As disclosed in the aforementioned Japanese Patent Publication No. 57-45560, even if proteins are hydrolyzed using trypsin or alkaline protease, molecular weights of 820 or 950
oligopeptides can only be obtained with yields of 16.5% or 20.0%. There is no known method for obtaining oligopeptide mixtures with uniform molecular weights and high levels of limiting amino acids in high yield as in the present invention. At the same time, it is not known that such oligopeptide mixtures have excellent nutritional improvement effects. (Purpose) The present inventor has developed oligopeptides that are distinguished from amino acids, di- or tripeptides, which are somewhat unnatural for use in diets that require many enzymes in the digestive system and macromolecular proteins that require few digestive enzymes. researched on the nutritional value of At the same time, we also conducted research on the effects of limiting amino acids. As a result, as shown in Table 1 below, oligopeptide mixtures with a molecular weight of 500 to 1,500 daltons with covalent attachment of limiting amino acids have a higher We have obtained findings that have a particularly excellent nutritional improvement effect not only on malnourished people but also on normal people. Therefore, as a result of conducting research with the aim of obtaining such oligopeptide mixtures in high yield, the first step was to aminolyze proteins and amino acid esters in an aqueous system, resulting in covalent addition of amino acids with a molecular weight of 1000 to 9000. Knowledge that it is possible to obtain a target oligopeptide mixture in a previously unknown high yield by preparing an enzyme-modified protein centered on Dalton and, in the second step, specifically hydrolyzing this enzyme-modified protein. With this, we have completed the present invention.

[Table] (Structure) The present invention (1) produces an oligopeptide mixture with a molecular weight of 500 to 1,500 daltons, (a) aminolyzes proteins and amino acid esters in an aqueous system at pH 7 to 11 using thiol protease. This is a method for producing an oligopeptide mixture, comprising the steps of: obtaining a modified protein; and (b) specifically hydrolyzing the obtained enzyme-modified protein with a proline-specific peptidase. The proteins used in the present invention include egg white, lactalbumin, fish or livestock meat, animal proteins such as casein, vegetable proteins such as soybean protein, wheat protein, oilseed protein, and leaf protein, and microorganisms such as yeast and algae. Proteins etc. can be used.
Generally, the higher the limiting amino acid level of a protein, the better, but the protein used in the present invention does not matter whether the limiting amino acid level is high or low. The enzyme used in step (a) of the present invention can be an enzyme that causes aminolysis in the presence of amino acids in an aqueous system, such as thiol proteases such as papain, bromelain, and fuicin. Can be used. It is important that the operating conditions of the enzyme be such that aminolysis occurs. For this purpose, the substrate concentration can be relatively high compared to normal hydrolysis conditions, typically 5 to 40% w/w. Aminolysis is difficult to occur when the substrate concentration is less than 5%,
Mere hydrolysis occurs, which is undesirable. If the concentration exceeds 40%, it becomes difficult to uniformly dissolve the protein substrate in the aqueous system. The optimum pH for hydrolysis of the enzyme used in the present invention is mostly 5, but for aminolysis in the present invention it is appropriate to set the pH to 7 to 11, preferably 8 to 10. If the pH is less than 7, aminolysis is difficult to occur and hydrolysis occurs, which is not preferable.
Moreover, if the pH exceeds 11, aminolysis is difficult to occur, which is not preferable. Temperature, enzyme/substrate ratio, etc. can be the same as those for normal hydrolysis. Since the thiol protease used in the present invention is a reducing enzyme, it is preferable to use a reducing agent (for example, L-cysteine) in combination. In step (a) of the present invention, it is appropriate to use the required amount or more of the limiting amino acid in the form of an ester depending on the level of the limiting amino acid in the raw protein. That is, it can be used in the form of esters such as methyl, ethyl, propyl, butyl, pentyl, and hexyl. Furthermore, in the present invention, L-type amino acids and L
The amino acid ester is characterized in that it can be used not only as a racemic form of these amino acid esters, but also as a racemic form thereof. This will be explained in detail. Generally, only L-type amino acids exist in nature. Non-naturally occurring D-form amino acids are harmful and undesirable. Therefore, the amino acid covalently added by aminolysis must be in the L-form. However, commercially available amino acids are D-type and L-type.
It is a type of racemate. From now on, dividing only the L-type will not only be industrially difficult but also expensive. Furthermore, L-type amino acids obtained by fermentation methods and the like are expensive. In this regard, in the present invention, economically inexpensive racemic commercially available amino acids are used, and known methods (e.g.
Method of Boissonas et al.: Helv.Chem.Acta. 39, 1421
(1956)), only the L-type can be preferentially and selectively covalently added to proteins using these D- and L-type amino acid esters. That is, as a result of deep research into aminolysis, the present inventors found that the aminolysis reaction used in the present invention is accompanied by a D-, L-racemic resolution reaction on the other hand. Therefore, D-, L-
Among amino acid esters, only L-type amino acid esters are preferentially covalently added to proteins as amino acids, while D-type amino acid esters remain (divide) in the system. This situation is schematically shown in FIG. Therefore, it is not necessary to use only L-type amino acids or L-type amino acid esters. In step (a) of the present invention, unreacted amino acid esters and amino acid esters in the enzyme-modified protein (approximately 15% present) are saponified and decomposed by alkaline decomposition. The saponified product can be removed together with other low-molecular substances using separation means such as membrane separation. Particularly when racemic amino acid esters are used, residual D-
type amino acids need to be removed. In the case of membrane separation, the molecular size of the membrane is preferably about 1000. In addition, known separation means using acids, organic solvents, etc. can be used. In this way, the enzyme-modified protein can be obtained in high yield (95% or more) in step (a). The enzyme-modified protein usually has a molecular weight of 1,000 to 9,000 daltons, and can be an enzyme-modified protein that has only the limiting amino acid content increased compared to the raw protein, with the other amino acid composition remaining almost unchanged. It can also be diluted, concentrated or dried. Unlike general hydrolysis, there is almost no generation of free amino acids. In step (b) of the present invention, the previously obtained enzyme-modified protein can be specifically hydrolyzed using a proline-specific peptidase. As is well known, the proline-specific peptidase is an enzyme that specifically hydrolyzes the N-terminal side of proline, and those derived from bacteria such as Flavobacterium meningosepticum or those derived from animals such as calf kidney can be used. Flavobacterium
Proline-specific peptidase originating from Menigosepticum Yoshimoto T. and Tsuru D., Agr. Biol.
Chem. 42 ． 2417 (1978) and Yoshimoto T and
Walter R and Tsuru D., J.Biol.Chem , 255 ,
4786 (1980) etc. The conditions for specific hydrolysis are the same as for normal hydrolysis: enzyme/substrate ratio of 0.01 to 5, protein concentration of 1 to 10%, temperature of 10 to 60°C, and time depending on the enzyme titer. Another feature of the present invention is that the time can be shortened, and can be set to 1 to 300 minutes, preferably 3 to 120 minutes. In the present invention, the desired oligopeptide can be obtained in high yield only by combining step (a) and step (b). The former special public service 1977-
45560, hydrolysis with trypsin alone produces an oligopeptide with an average molecular weight of 820.
According to the method of the present invention, an oligopeptide mixture with a molecular weight of 500 to 1500 (average molecular weight 900) can be obtained with a yield of 55% or more, and if desired, the following decomposition-fractionation operation can be performed. By repeating this method, it is possible to obtain an oligopeptide mixture with a high yield of over 80%. In step (a) of the present invention, after specific hydrolysis, if desired, enzyme deactivation (for example, at 90 to 100°C)
The mixture can be separated into an oligopeptide mixture with a molecular weight of 500 to 1500 and a non-oligopeptide mixture. As a method for fractionation, known methods such as membrane separation and precipitation methods can be used, but from an industrial standpoint, fractionation using an organic solvent is preferable. In this case, after adding an organic solvent (for example, a polar organic solvent such as ethanol) so that the concentration of the entire system is 50 to 80 W/W%, the precipitate and supernatant are separated using separation means such as centrifugation. The supernatant can be subjected to solvent removal (for example, evaporation), and optionally concentrated or dried to obtain an oligopeptide mixture. Furthermore, if desired, by repeating the specific hydrolysis of the precipitate in the same manner as in the case of the enzyme-modified protein, the amount of the precipitate can be reduced and the yield of the supernatant, ie, the oligopeptide mixture, can be increased. That is, in step (b), the obtained enzyme-modified protein is specifically hydrolyzed with a proline-specific peptidase, separated into an oligopeptide mixture and a non-oligopeptide mixture, and the non-oligopeptide mixture is again hydrolyzed with a proline-specific peptidase. By repeating the specific hydrolysis and fractionation operation once or twice, we can create an oligopeptide mixture with uniform molecular weight that produces little free amino acids (5% or less) and does not contain polypeptides. It can be obtained in high yield. The oligopeptide mixture thus obtained can contain a desired level of limiting amino acids that is higher than the source protein. This oligopeptide mixture is more effective in malnourished animals than raw protein or amino acid mixtures with the same amino acid composition as the oligopeptide mixture, in which limiting amino acids are simply added so that the limiting amino acids are at the same level as the oligopeptide mixture. Not only that, but it also has a significant nutritional improvement effect on normal animals. (Example) Embodiments of the present invention will be described below with reference to Examples. Example 1 Using Fujipro-R manufactured by Fuji Oil Co., Ltd. as isolated soybean protein (abbreviated as SPI), an oligopeptide mixture was obtained in a good yield (87%/SPI) through the manufacturing process shown below. In addition, in this example, papain was (1.16×10BAPA
unit), Spectra-por tube (Spectrum Medical
Industry, Inc.) had a molecular size of 1000, and commercially available D- and L-racemic methionine were used. Below, step (a) of covalently adding methionine, which is the first limiting amino acid of soybean protein, and then
This will be explained separately in the step (b) of specific hydrolysis, and finally the nutritional improvement effect of the obtained oligopeptide mixture will be explained. In addition, D-, L-
Methionine ester was prepared by boiling D-,L-methionine in hydrochloric acid and ethanol according to the method of Boissonas et al. Hydrochloric acid was easily scattered by drying and did not remain in the D-, L-methionine ester.

[Table] ↓

[Table] ↓
freeze drying
↓
Papain modified protein (9.5g)

[Table] ↓ ↓
Freeze-dried precipitate supernatant
↓
↓
Oligopeptide mixture deethanol

Claims (1)

[Claims] 1. When producing an oligopeptide mixture with a molecular weight of 500 to 1500 Daltons, (a) a step of aminolyzing proteins and amino acid esters in an aqueous system using thiol protease at pH 7 to 11 to obtain enzyme-modified proteins; and (b) a method for producing an oligopeptide mixture, comprising the steps of specifically hydrolyzing the obtained enzyme-modified protein with a proly-specific peptidase. 2. The production method according to claim 1, wherein in step (a), unreacted amino acid esters and some amino acid esters in the enzyme-modified protein are saponified and decomposed to remove amino acids and low-molecular substances. 3 In the step (b), the enzyme-modified protein obtained in the step (a) is specifically hydrolyzed with a proline-specific peptidase, and if necessary, separated into an oligopeptide mixture and a non-oligopeptide mixture. 2. The production method according to claim 1, wherein the specific hydrolysis and fractionation operation in which the mixture is specifically hydrolyzed again with a proline-specific peptidase is repeated once or twice or more.