GB2249101A

GB2249101A - Process for preparing peptide and/or protein products enriched with covalently bound amino acids

Info

Publication number: GB2249101A
Application number: GB9122663A
Authority: GB
Inventors: Gyorgy Hajos; Anna Halasz; Laszlo Pallos; Frigyes Goergenyi; Tibor Szarvas; Eva Gelencser
Original assignee: Egyt Gyogyszervegyeszeti Gyar
Current assignee: Egyt Gyogyszervegyeszeti Gyar
Priority date: 1990-10-26
Filing date: 1991-10-25
Publication date: 1992-04-29
Also published as: DK177991D0; HUT69773A; DK177991A; JPH0595A; ITMI912837A0; DE4135535A1; IT1251427B; GB9122663D0; ITMI912837A1; HU906902D0

Abstract

A process for preparing peptide and/or protein products enriched with covalently bound amino acids comprises the addition of the amino acid (which may be in an aligopeptide) to be incorporated in the form of an active derivative to the reaction mixture containing the protein(s) or peptide(s) to be enriched as substrate(s), where enzymatic peptide modification is accomplished through catalysis by means of protease enzyme, and the product(s) obtained and the excess of the amino acid derivative are removed from the reaction mixture. The process renders possible the improvement of the food-value of proteins of some foods and dietetic products.

Description

2 2 4 91 -') 1 1.

PROCESS FOR PREPARING PEPTIDE AND/OR PROTEIN PRODUCTS ENRICHED WITH COVALKWMY BOUND AMINO ACIDS The invention relates to a process for preparing peptide and/or protein products with covalently bound amino acids.

Enzyme-catalysed modification of proteins, apart from their enzymatic hydrolysis, has for long been called and is still being called "plastein" reaction. This reaction can be developed for the following purposes, according to literature data:

- to enhance the solubility of proteins, modifying their physical properties, eliminating bitter peptides, to build in limiting essential amino acids, for removal of unwanted colour, smell and taste components, to eliminate undesired amino acids, and to prepare surface-active agents (J.R. Whitaker and A. J. Puigserver: Advances in Chemistry Series, 198, pp. 57-87 (1982); M. Fujimaki, S. Arai and N. Yamashita: Advances in Chemistry Series, 160, pp. 156-184 (1977)).

The reaction is usually carried out at 37 OC and lasts 24 hours. The concentration of proteins or peptides is generally in the range of 25-50% by weight, as applied in the reaction mixture.

Enzymatic modification of proteins with the aim of increasing their foodvalue has been described in the literature. For instance, the nethionine content of soybean proteins has thus been increased by 7 %, L-lysine has been incorporated into gluten, and into proteins of photosynthetic origin L-lysine, L-methionine and L- tryptophane have simultaneously been incorporated through covalent bonds [R.D. Phillips and L.R. Beuchat:

ACS Symposium Series, No. 147, pp. 275-298, (1981); M. Fujimaki et al. in "Food Proteins: Improvement Through Chemical and Enzymatic Modification", p. 156, (1977)].

In case of soybean protein a one-step method for incorporating methionine has been described by S. Arai et al. in Agric. Biol. Chem., A3 (5), pp. 1069-1074 (1979).

The U.S. patent No. 4,016,147 discloses an enzymatic process by which proteins and peptides of low phenylalanine content can be prepared. The same kind of phenylalanine-impoverished proteinaceous dietetic product is prepared for patients suffering from phenylketonuria according to the Hungarian patent specification No. 194,487.

L-methionine plays an especially important role in living organisms, partly as an - in food proteins frequently limiting - essential amino acid, partly as a sole biological methylating agent. Due to this fact, special care must be brought upon making up of the L-methionine deficiency, according to the age and health state requirements and in situations accompanied by stress, and that dietetic supplement should be given in time, in appropriate quantity and manner. This apparent need led us to investigate the possibility of developing a protein-based dietetic product f amily containing easily digestible proteins ofmodified amino acid compo- sition, consciously designed following the above viewpoints.

Physiological experiments demonstrate that when essential amino acids added with the purpose of enrichment (in order to increase the food value) are only in loose association with the basic substrate proteins, their resorption and bioavailability are unsatisfactory. on the contrary, if these amino acids are even covalently bound to and built in the said proteins, their bioavailability - optionally their utilization as food-stuffs - reaches maximum level.

The aim of the present invention was therefore to work out a method, whose application renders possible to ameliorate the nutritive value of food protein and die- tetic products, to modify their amino-acid composition and structure according to a predetermined plan, and to produce target proteins that may have a significant role in conservation of health and in safe alimentation.

The invention is based on the recognition that the above aim can fully be realized and a covalent aminoacid incorporation can be achieved when the amino acid(s) to be built in is (are) added in the form of an activated derivative to the reaction mixture containing the proteins and/or peptides to be enriched, where enzymatic peptide modification is carried out through directed protease catalysis by using protease enzymes.

Accordingly the invention relates to a process for preparing peptide and/or protein products enriched with covalently bound amino acids. The process according to the invention is accomplished by the addition of the amino acid to be incorporated in the form of its active derivative to the reaction mixture containing the protein(s) or peptide(s) to be enriched as substrate(s), where enzymatic peptide modification is carried out through controlled protease catalysis by means of pro tease enzyme, and the product(s) obtained and the excess of the amino-acid derivative are removed from the reac tion mixture.

Using the process according to the invention the amino acids or the peptides comprising these amino acids, applied for completion or enrichment, are in serted into the basic protein chain of the substrate.

According to prior art methods, the enrichment in methionine content has always been made exclusively with the aim of increasing the nutritive value of the proteins concerned. on the other hand, we are suggesting 5 these methionine-enriched proteins also as exclusive methylating agent sources for the body, that can be administered perorally, through alimentation. This is of special importance with certain ages and illnesses, when the human organism suffers from lack of methylat'ing agents (e.g. in situations accompanied by stress, in elderly age, in weakness conditions, etc.).

The process according to the invention is, contrary to the plastein reaction as described in the literature, called enzymatic peptide modification (EPM).

In the "plastein products" belonging to the prior art the cohesive molecular forces acting among the primary and complementary amino acids and among the peptide chains are, according to pertinent literature data, of the nature of hydrophobic, van der Waals and ionic (salt bond) forces, while in the products prepared according to the present invention the building-in of amino acids by covalent bonds can be verified by several methods, including analytical tests. In contrast to the frequently observable, at the "plastein products" char- acteristically expected increase in the average molecular mass (as compared to the average molecular mass of the starting hydrolysate), no significant increase has been found in the average molecular mass jof the products prepared under the reaction conditions according to the invention.

In the process according to the invention, as sub strates in the EPM-reaction, favourably proteins of plant origin, single cell proteins, milk proteins (or llpre-digestedll milk proteins obtained by partial hydrolysis), protein by-products from industrial produc tion and unconventional proteins can be used. From the point of view of implementing the process according to the invention it is advantageous if partially hydro lyzed, llpre-digestedll milk proteins or native milk pro teins (casein, whey proteins) are used as substrates.

To carry out the enzymatic peptide modification (EPM) reaction one may use different endo- and/or exo peptidases andlor transpeptidases, preferably proteases (proteolytic enzymes) approved in the food industry or for manufacturing dietetic products. Such preferable en zymes include a-chymotrypsin, papain, trypsin, pepsin, pronase, alcalase, carboxypeptidase. These enzymes may be applied separately or in deliberate combinations with each other as to their mass ratio and sequence of use.

Any amino acid can be used but essential amino acids are of advantage, favourably in the form of an oligopeptide containing the particular amino acid.

The amino acids to be incorporated are to be applied in the form of an active derivative. These derivatives include the esters, thioesters, anhydrides and other ac tive acyl derivatives of the corresponding amino acids.

According to a preferred embodiment of the process of the invention Lmethionine ethyl ester is used for the amino-acid incorporation. The reaction is preferably carried out at a temperature of 370 15 OC, at a pH value from 4.0 to 8.5 for 2 to 16 hours, whereas the ratio of the substrate to the reaction mixture amounts to 10 to 25 % by mass, and the mass ratio of the amino acid(s) to be incorporated and the substrate, calculated on the mass of the amino acid, is between h and 1/40.

Removal of the products of the process according to the invention and of the excess amino acid derivatives used from the reaction mixture is accomplished by known separation techniques such as dialysis, lyophilization, ultrafiltration, membrane separation or by the combination of these methods.

By using the EPM-method according to the invention a number of different products can be prepared, the main fields of application whereof can be summarized as follows:

- preparation of proteins with a harmonic amino acid composition to selected purposes (providing so- -called "target proteins").. primarily for dietetic products (infant's and baby food formulas); - preparation of methionine-enriched protein prod- ucts a) for persons affected by stress; b) as natural radical traps, detoxicants for prevention and cure when harms of carcinogenic c) character are considered; for treatment of liver patients and alcoholics; d) for providing the necessary methylating agent for the human body; preparation of dietetic products for special purposes and of medicinal foods a) as easily digestible proteins of suitable amino-acid composition for post-operat:ive patients; b) as a protein ingredient causing retardation of drug absorption; C) as dietetic product for the elderly; d) for sportsmen as a general roborant, improving the physical fitness; - preparation of food additives.

In addition to all these, some products made by the EPM-method are useful in animal breeding,, for instance as food-stuffs for pheasants and calves, or in the breeding of fur-bearing animals.

The main advantages of the process according to the invention and of the products prepared by that process are listed hereinbelow:

a) It affords a covalent incorporation of the added amino acids whereby the bioavailability of the product, i.e. its utilization as a food-stuff, is remarkably improved.

b) It enables the improvement of the food-value of -g- c) e) f) proteins of some foods and dietetic products. It provides a planned, purpose-oriented modification of the amino-acid composition and structure of food and dietetic-product proteins, that is the preparation of socalled "target" proteins is afforded, primarily for use in infants' and baby-food formulas.

d) The products may play a significant role in the conservation of health and in safe alimentation. It affords the manufacturing of protein products enriched with methionine, which are favourably applicable for treating patients with liver diseases and for the treatment of alcoholics and patients suffering from some forms of cancer. It enables the application of food-stuffs and feed additives which are favourably used in animal breeding.

g) The process can be implemented also on an industrial scale.

The process according to the invention as well as the products obtained by this process are presented hereinbelow in further details through the examples, without limiting the scope of protection to what is contained in these 25 examples.

ExamDle 1 8 g of casein (product of Hammersten) were dissolved in distilled water, the pH of the solution was adjusted to 7.5 by adding about 75 ml of 0,1 M NaOH; the end volume was 800 ml. 80 mg/of a-chymotrypsine (Sigma, St. Louis, USA) were then added, and the suspension was stirred for 1 hour at 37 OC with a magnetic stirrer, followed by freezing of the liquid and lyophilization.

g of hydrolyzate prepared according to the above method were suspended in distilled water, then 21.1 g of methionine methylester hydrochloride were added, the resulting mixture was thoroughly admixed, a few ml-;.s of distilled water were added and the pH was adjusted to 6.0 by adding about 20 ml I M NaOH, followed by filling up to 100 ml end volume. 250 mg of achymotrypsin (Serva, Heidelberg, Germany) were added and the suspens ion was mixed up and then incubated at 37 OC f or 16 hours without stirring. The resulting gelatinized product was dialyzed for 48 hours, followed by freezing and lyophilizing in order to remove excess of the methionine derivative.

Example 2 g of casein (Reanal, Hungary) were first sus pended in 1 m3 of water. then the pH was set to 1.6 by diluted hydrochloric acid. It was followed by hydrolysis with 100 g of pepsin at 37 OC for 2 hours. There was a continuous stirring provided for the whole reaction period. Then the pH of the suspension was adjusted to 6.0 by using sodium hydroxide, and the next hydrolysis was made by papain under conditions as described herein- above.

The concentrationof the substrate, prepared according to the method referred to hereinabove, was 300 kgm-3, the enzyme/substrate mass ratio was 1:100, while the incubation temperature was 37 OC, the pH 6.0 and the time of incubation - without agitating and stirring - 16 hours. The mass ratio of the methionine methyl ester/substrate was 5:100 for the buildingin of the amino acid. The incorporation of methionihe was accomplished by using several kinds of proteolytic enzymes, namely by "Pronase" (made by Calbiochem, Palo Alto, USA and by Serva, respectively), by a-chymotrypsin (Sigma) and by papain. In the last case L-cysteine was used as papain activator.

Example 3 16 g of casein (Hammersten) were dissolved in distilled water.. the pH of the solution was adjusted to 7. 5 and its end volume to 800 ml, then it was hydrolysed by adding 160 mg of a-chymotrypsin (Serva). The reaction tine was 1 hour. the temperature was 37 OC.

g of hydrolyzate prepared as above were suspended in distilled water, then 21.1 g of methionine methylester hydrochloride were added, the pH was adjusted to 6.0 and the end volume of the mixture was completed to 100 ml, then the suspension was incubated with 250 mg of achymotrypsin at 37 "C for 16 hours.

The resulting gelatinized product was dialyzed and afterwards lyophilized.

Examnle 4 Casein was hydrolyzed in a 1 per cent solution by alcalase (Novo, Nordisk, Denmark) at 55 OC for 2 hours at a pH value of 8.5. After 2 hours of stirring the pH of the mixture was adjusted with hydrochloric acid to 6.0. After a heat treatment of 5 minutes at 85 OC the mixture was lyophilized.

While carrying out the EPM-reaction, the pH was ad justed to 6.9 and the hydrolyzate prepared as described above was applied in a concentration of 12.7% by mass.

Calculated on the 330.2 g dry weight of the hydrolyzate, 63.9 g of L-methionine ethyl ester and 8 ml of alcalase (AlcalaseR, Novo) were added to the hydrolyzate. Both the enzyme and the ester were added in the form of an aqueous solution.

ExamDle 5 The papainic hydrolyzate of egg yolk was used as substrate. The egg yolk used was in some cases fresh, in other experiments it was lyophilized, dried, deep frozen or irradiated.

The EPM-reaction was carried out in such a way that 15 g of egg yolk hydrolyzate were incubated for 6 hours at 37 OC in 100 ml of a buffer solution of pH 6.0, in the presence of papain and 9,75 g of L-methionine ethyl ester.

Example 6

16 g of Prominei-E 500 (Purina Proteins B.V., Puurs, Belgium) were suspended in 800 ml of distilled water, and the digestion was carried out at a pH value of 7.5 with 80 mg of a-chymotrypsin for 2 hours. The obtained product was lyophilized.

EPM-reaction: 2.0 g of the above product, 1.8 g of methionine ethyl ester and 20 mg of a-chymotrypsin were incubated at pH 6.0 for 10 hours at 37 OC without 10 stirring.

Example 7

In order to prepare a dietetic food of harmonic essential amino-acid composition, based on milk protein, we used as substrate of the EPMreaction the enzymatically pre-digested milk protein preparate called SportrobiR (Egis Lacta, K8rmend, Hungary). The purpose of the methionineenrichment of the enzymatically pre-digested milk protein preparate was to further enhance the dietary advantage of an enzymatically pre-digested protein by approaching the harmonic essential amino-acid composition thereof.

Experiments were carried out both with a) previously not hydrolyzed milk protein preparate and b) milk 25 protein preparate subjected to another hydrolysis of 2 hours by papain. The amino-acid derivative to be incorporated was in both cases L-methionine ethyl ester which was added to the reaction mixture. Papain or a- -chymotrypsin was used as enzyme catalyst. The degree of the achieved enrichment,,in the methionine content is summarized in the following Table 1.

Table 1

Methionine content of Droducts DreDared bv EPM-method Initial applied in the EPM-reaction Met-content of substrate the product amino acid given in per enzyme derivative cent of total amino-acid con tent Pre-di- -gested 2.2 milk pro tein Pre-di- a-chimotrypsin 2.1 -gested milk pro tein Pre-dia-chimotrypsin Met ethyl 4.6 -gested ester milk pro tein Pre-di -gested milk pro- tein after papain 2.2 hydrolysis of 2 further hours by papain Table 1 (continued) Initial 5 substrate applied in the EPM-reaction enzyme amino acid derivative Met-content of the product given in per cent of total amino-acid content Pre-di-gested milk pro- tein after hydrolysis of 2 further hours by papain papain Met ethyl ester 5.9 As seen from the Table, although the greatest degree of methionine- enrichment was achieved with the preparate subjected to 2 hours additional hydrolysis and later used as substrate of the EPM-reaction, the optimum Met-content - as given in the joint FAO-WHO recommendation for food-stuffs - has already been surpassed by the 4.6% Met-ratio obtained when pre-digested milk protein was applied as substrate.

Example 8

L-DOPA (3,4-dihydroxy-L-phenylalanine) was built as L-DOPA ethyl ester into casein hydrolyzates. The reaction mixture contained 1/5 mass parts of L-DOPA ethyl ester, related to the substrate.

The experiments were carried out by using two kinds of enzymes, wherein a) into a 4-hour a-chymotrypsin-hy- drolyzate of casein by a-chymotrypsin and by alcalase and b) into an 1- hour and a 2-hour alcalase-hydrolyzate of casein L-DOPA was built in.

ExamDle 9 Partially hydrolyzed brewer's yeast protein obtained through directed autolysis, or a water-soluble protein fraction obtained therefrom by ultrasound cell dis integration, was applied in the experiments. Concentra- tion of the solutions made from the lyophilized substrate was 200 g/dm3 and 400 g/dm3, respectively. The methionine, was applied in the form of its ethyl ester; a-chymotrypsin, carboxypeptidase and the own carboxypeptidase enzyme of yeast served as catalysts. The not incorporated portion of methionine was removed from the product by dialyzis.

Claims

1. A process for preparing peptide and/or protein products enriched with covalently bound amino acids, c o m p r i s i n g the addition of the amino acid to be incorporated in the form of its active derivative to the reaction mixture containing the protein(s) or peptide(s) to be enriched as substrate(s), where enzymatic peptide modification is accomplished through controlled protease catalysis by means of protease enzyme, and the product(s) obtained and the excess of the amino acid derivative are removed from the reaction mixture.

2. The process as claimed in claim 1, c o m - p r i s i n g the use of essential amino acid as the amino acid to be incorporated.

3. The process as claimed in claim 1 or 2, i n w h i c h the amino acid to be incorporated is brought into the reaction mixture in the form of an oligopeptide.

4. The process as claimed in claim 1 or 2, i n w h i c h the amino acid to be incorporated is ap- plied in the form of its ester, thioester, anhydride or some other active acyl derivative.

5. The process as claimed in any of claims 1, 2 and 4, c o m p r i s i n g the use of L-methionine ethyl ester as the active derivative for the amino-acid in corporation.

6. The process as claimed in any of claims 1 to 5, i n w h i c h the substrates to be used in the amino acid incorporation process are proteins of plant origin, single cell proteins, milk proteins, protein by-products deriving from industrial manufacturing processes, or non-conventional proteins.

7. The process as claimed in any of claims I to 6, i n w h i c h endoand/or exopeptidases and/or transpeptidases are applied as protease enzymes.

8. The process as claimed in any of claims 1 to 7, c o m p r i s i n g the use of a protease enzyme ap proved in the food industry or for manufacturing die tetic products, including a-chymotrypsin, papain, trypsin, pepsin, pronase, alcalase and/or carboxy- peptidase.

9. A process of preparing peptide and/or protein products enriched with amino acids substantially as hereinbefore described in any one of Examples 1 to 9.

10. Amino acid enriched products obtained by the process claimed in any one of claims 1 to 9.

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