DE3741469C2 - Process for the fractionation of protein hydrolyzates - Google Patents

Description

The invention relates to a method for fractionation ion neutral protein hydrolyzates exclusion chromatography.

In practice, protein hydrolyzates are used in industry ellal scale by hydrolysis of vegetable or animal proteins. This hydrolysis takes place either by means of acids or enzymes or in the special case of yeast by autolysis. In the acid hydrolysis, the proteins become almost complete cleaved into the free amino acids, while at enzymatic hydrolysis still shares of peptides are present in the hydrolyzate.

Preferred raw materials for the manufacture of industri ellen protein hydrolyzates are herbal products like soybean meal, peanut meal or the various Grain glue. So the well-known soy sauce goes through Fermentation of cooked and crushed soy beans and wheat made. Here, as a micro organisms which provide proteases or amylases,  Mushrooms, yeasts and bacteria are used. To avoid Foreign infections become larger amounts of table salt added. The protein hydrolyzates have as a food medium or food ingredients a large economical meaning. In addition to use as Liquid condiments are an important part of protein hydrolyzates Part of soups, bouillon cubes and sauces.

In the manufacture of protein hydrolyzates for the Use as a seasoning in the kitchen or in the Food industry will be the vegetable raw substances hydrolyzed with hydrochloric acid. For a quanti tative cleavage of the proteins into the individual amino a considerable excess of acid is required.

Depending on the hydrolysis time and temperature, per mol Nitrogen 1.5 to 3 mol hydrochloric acid used. To Hydrolysis is ended with hydrochloric acid with sodium carbonate or sodium hydroxide neutralized, and after Separation of the humine formed during the hydrolysis substances you get liquid wort with about 50% sodium chloride based on dry matter.

As flavoring ingredients are in these Protein hydrolyzates in addition to the amino acids still cook salt and some decomposition or reaction products present, which in the hydrolysis from carbohydrates or amino acids arise.

The taste of a protein hydrolyzate becomes like the current state of the art by selecting the Raw materials, hydrolysis conditions and by night treatment determined.  

This is the free choice of raw materials for manufacturers position of protein hydrolyzates limited, and quali fluctuations in the raw materials lead to un wished taste changes in the fer products.

The protein hydrolysates obtained by hydrolysis contain a large proportion of table salt. This high salinity limits the use of the Hydro lysates in the preparation of food. This applies particularly to the affluent countries in which because of the common disease of hypertension a strict reduction in table salt or sodium content in the food as a precautionary or healing measure acceptance is required.

It has already been described (US Pat. No. 3,045,026) Ion exclusion chromatography in the sodium cycle cook to win low-salt protein hydrolyzates. In the pre proposed chromatographic separation of the Na hydrochloric acid or sodium carbonate neutralized hydro However, lysates remain the taste-active mono sodium salts of glutamic acid and aspartic acid in the saline fraction, and the basic amino acids Lysine, histidine, arginine are in the ion exchanger bound. This separation gives a solution the neutral amino acids with low taste value, which is hardly to be used for seasoning purposes.

According to another proposal (US Pat. No. 2,937,199) the chromatographic separation in the isoelectric Point of glutamic acid, d. H. at a pH of 3.2.

At this pH the glutamic acid is in their slightly dissociated hermaphrodite form, but  the neutral amino acids are already 40 to 50% converted into their cation form. With this chroma The glutamic acid can be separated graphically Saline are separated, but then remains large part of the neutral amino acid in the salt fracture tion. In addition, the glutamic acid in their iso electrical point a solubility minimum of 0.8%, and the chromatographic separation so diluted Solutions are uneconomical.

In the known principle of ion exclusion rens becomes a cation exchanger with the ions of saturated solution to be filtered. In this system added strong electrolytes pass unchanged the exchanger while weak electrolytes or Non-electrolytes from the matrix of the exchanger ad be sorbed. By subsequent elution with Water becomes the adsorbed substances in the series follow their polarity again from the matrix of the out exchangers solved. Alternating between loading with substance and elution with water can thus separate from Electrolytes and non-electrolytes can be achieved.

When using the known ion exclusion ver driving in the sodium cycle for the fractionation of Protein hydrolyzates become the ingredients of the Hydro lysates according to their degree of ionization in the following Groups separated:

• 1. The strongly ionized compounds sodium chloride, Sodium glutamate and sodium aspartate are from Ion exchanger not adsorbed and in the first Fraction enriched. Belong to this group also the sodium salts of some nitrogen-free ones organic acids, which are by-products of  the hydrolysis have arisen. In this fraction are also some ionized dyes ent hold.
• 2. In a transition fraction, in addition to shares of acidic amino acids with neutral amino acids polar side groups such as serine and threonine act.
• 3. In the following fraction are the neutral ones Amino acids with non-polar or weakly polar sides groups strongly enriched. These include glycine, Alanine, proline, valine, leucine, isoleucine, methionine as well as the aromatic amino acids phenylalanine and Tyrosine.

In this known method, the former is mentioned Fraction the seasoning fraction, which, however, very much cook contains salt from the spice substances in the extraction this fraction is not separable.

The invention is based, low salt or sodium-free protein hydrolyzates with certain Flavor properties for the preparation of Nah to produce foodstuffs and sodium-free amino acid Remixes for dietetic or pharmaceutical purposes to win and furthermore the pure amino acid from protein isolate inhydrolysates.

This characterizes the solution to the above task initially mentioned method in that the protein hydrolyzate neutralized with magnesium or calcium salts and subsequently via a cation exchanger in Mag Chromatographically separated nesium or calcium form becomes.  

The invention is based on the knowledge that one can Neutralization of an acidic protein hydrolyzate with Calcium or magnesium salts and subsequent chro matographic separation via cation exchanger in Calcium or magnesium form the taste-active gluta minic acid and aspartic acid together with the neutral ones Can separate amino acids in a salt-free fraction. This effect that the Monokalzium- or Monomag nesium salts of glutamic acid and aspartic acid chromatographic separation like neutral amino acids behavior was unpredictable and forms the basis for a number of new technical applications.

The acidic hydrolysates can be used with inexpensive cal zium carbonates, magnesium carbonates or the hydroxides these alkaline earth metals are neutralized. After this prior art had sodium carbonate for this or caustic soda can be used as calcium chloride or magnesium chloride in the protein hydrolyzates tasty is not acceptable.

By using the cation exchanger in the cal cium or magnesium form is the ion exchange prevents basic amino acids.

The glutamic acid and is in the salt-free fraction Aspartic acid in the form of its mono-calcium or mono magnesium salts, and these can be obtained by simple Reaction with potassium carbonate or sodium carbonate in the corresponding alkali salts are transferred. Thereby there is the possibility of any mixing ratios of calcium, magnesium, potassium and sodium glutamate and To produce aspartate. This is particularly the case with the Potassium and magnesium glutamate and aspartate a we considerable advantage, because these connections due to their easy solubility are very difficult to produce.  

By dividing the salt-free amino acid fraction can be any mixture of glutamate, aspartate and neutral amino acids with different Ge taste.

When concentrating the salt-free amino acid fracti ons crystallize the sparingly soluble amino acids, such as cystine, tyrosine, methionine, leucine, isoleucine, Phenylalanine and valine. Have these amino acids a bitter taste, while the more easily dissolves neutral amino acids, such as glycine, alanine, Serine, threonine and proline, a pleasant sweet have sour taste. By separating the sparingly soluble amino acids result in further Va Ration possibilities in taste value.

From the separated sparingly soluble amino acids can pure L-cystine, L-tyrosine, L-leucine, L-isoleucine, L-phenylalanine and L-valine be won.

From the solution of the easily soluble amino acids can Glutamic acid and aspartic acid in their isoelek trical points. By further Fractionate using ion exclusion or ion exclusion exchange chromatography in connection with known Precipitation or extraction processes can be isolate individual amino acids and in pure form win. Because these solutions are of very low order contain some foreign ions, mainly stand for ion exchange chromatography pure amino acid solutions without disturbing mineral components Available, and the separation operations can be very effi be carried out efficiently.  

Through these numerous variation options in Regarding the composition of the amino acid fraction and the flavor compositions with which he process according to the invention differ a large number whose raw materials are used. Preferably be however, vegetable raw materials, such as protein-rich glues of wheat, corn and rice or high-protein oil press residues of soy, peanut, sunflowers, etc. ver turns. In contrast to the many described Amino acid separations using ion exchange chromatography With this new process, the graphics become the amino do not acid into the active groups of ion exchange Schers bound, but they pass through diffusion sion in the pores of the matrix of the exchanger and are eluted by simply washing with water. In contrast to ion exchange chromatography no regenerant needed, and this process is therefore very environmentally friendly.

Further details of the invention are in the sub claims 2 to 8 specified.

The present invention is described in the following examples play described:

Embodiment 1 hydrolysis

1000 g of corn glue are mixed with 2000 ml of 6NHCI for 5 hours hydrolyzed at 120 ° C in an agitator autoclave. After hydrolysis has ended, the acidic hydrolyzate in portions with solid calcium hydroxide up to one pH from 5.0 to 6.0 neutralized and the water insoluble residue from humic substances and insoluble Calcium salts filtered off. This filtered protein hydrolyzate is the feed solution for the following chromatographic separation.  

Chromatography

A double-walled column column made of glass with one pass knife of 25 mm is up to a height of 1000 mm filled with a cation exchanger. Is used a polystyrene resin with 6% DVB crosslinking and one average grain size of 0.40 mm. The double coat the separation column is via a circulation thermostat heated to 85 ° C, and the cation exchanger is then with 1000 ml of a 10% calcium chloride solution regenerated, washed with 5000 ml of water and backwashed. On the separation column prepared in this way with a dosing pump and a speed of 4 ml / minute the following solutions:

Cycle 1

100 ml protein hydrolyzate
450 ml of water

Subsequent cycles

• 1. 40 ml return 1 (RF 1)
• 2. 80 ml protein hydrolyzate
• 3. 150 ml return 2 (RF 2)
• 4. 180 ml of water.

In the outlet of the separation column, a density meter the dry substance (TS) and with a registering conductivity meter Conductivity (LF) measured. In individual fractions the amino acid content is determined by means of an automatic Amino acid analyzer determined, and depending on these The following fractions are measured values in the outlet of the column deducted:

• 1. 120 ml salt fraction (SF)
• 2. 40 ml return 1 (RF 1)
• 3. 70 ml product fraction 1 (PF 1)
• 4. 70 ml product fraction 2 (PF 2)
• 5. 150 ml return 2 (RF 2)

Processing the product fractions

In product fraction 1 (PF 1) 0.35% Ca and found in the product fraction 2 (PF 2) 0.05% Ca. From 1000 ml product fraction 1 (PF 1) with the stoichiometric amount of 12 g K₂CO₃ at 80 ° C Calcium carbonate precipitated and filtered off. The fil is then in a vacuum rotary evaporator to 100 ml evaporated, and after cooling they are off filtered crystallized sparingly soluble amino acids and dried.

1000 ml of product fraction 2 (PF 2) become the same if concentrated to 100 ml, the sparingly soluble Amino acids separated and dried. The severed sparingly soluble amino acids of the product fraction 1 and 2 have different compositions with different tastes and can for the fro dietary foods.

The easily soluble amino acids from the PF 2 with a very high proportion of proline are made with carbon dioxide treated, the precipitated calcium carbonate is removed separates and the remaining solution for insulation pure amino acids used.

Embodiment 2

The hydrolysis and chromatographic separation are carried out as in Example 1, and the entire product fraction is separated into 4 parts and treated as follows:
500 ml product fraction are heated to 70 to 80 ° C and it is precipitated with a 10% sodium carbonate solution calcium as calcium carbonate and filtered off. The stoichiometric amount of sodium carbonate required for the precipitation is calculated from the previously determined calcium content of the solution.

500 ml of product solution are removed via a cation Exchanger in magnesium form and calcium ions exchanged for magnesium ions.

500 ml of product solution at 70 to 80 ° C with a 10% potassium carbonate solution and the calcium precipitated as calcium carbonate and filtered off. The for this precipitation required stoichiometric potassium kar The quantity of bonate is made from the calcium determined beforehand content of the solution calculated.

500 ml of product solution remain in the calcium form.

These differently treated parts of the product fraction are mixed, concentrated in half and cooled down. A 1st fraction of sparingly soluble amino acids, which essentially Lichen consists of cystine and tyrosine. This amino Acids are filtered off, and the remaining solution will return to a quarter of their original volume concentrated and cooled. One crystallizes 2. Fraction of the sparingly soluble amino acids from which essentially from leucine, isoleucine, phenylalanine, Valine and methionine exist. This amino acid mixture is filtered off and for the production of pure amino acids used. The remaining solution can be as liquid seasoning or as a litter after drying wort are used and contains the same amount divide the monosodium, potassium, magnesium and Calcium salts of glutamic acid and aspartic acid.

Embodiment 3 hydrolysis

1000 g of peanut extract meal are mixed with 2000 ml of 6NHCI hydrolyzed in a stirrer autoclave at 120 ° C for 5 hours.  After hydrolysis has ended, the acidic hydrolyzate in portions with magnesium carbonate up to a pH Value from 5.0 to 6.0 neutralized and the insoluble Huminstoff separated. The filtered neutral hydro lysate is the starting solution for the following chro matographic separation.

Chromatography

It will have the same column and the same arrangement as used in embodiment 1. The cations exchanger is, however, with 1000 ml of a 10% Magnesium chloride solution regenerated. The task on the separation column and the deduction of the individual fractions from the outlet take place analogously to the embodiment 1.

Processing the product fractions

The product fractions 1 and 2 (PF 1 and PF 2) are evaporated together to 1/10 of their original volume, and the crystallized sparingly soluble amino acids are separated. From the severed severely Soluble amino acids are made by a known method L-tyrosine, L-leucine, L-isoleucine and L-phenylalanine won. The concentrated mother liquor is dried and is a sodium-free seasoning powder with a high Magnesium glutamate and aspartate content for the manufacturer suitable dietary seasonings.

Claims (8)

1. A process for the fractionation of neutral protein hydrolyzates by ion exclusion chromatography, characterized in that the protein hydrolyzate is neutralized with magnesium or calcium salts and subsequently separated chromatographically via a cation exchanger in magnesium or calcium form. 2. The method according to claim 1, characterized ge indicates that on the separation column neutral protein hydrolyzate, water and recycle abandoned and a salt at the bottom of the separation column fraction, low ash amino acid fractions and re guides are deducted. 3. The method according to claim 1 and 2, characterized characterized in that the chromato graphic separation at temperatures from 70 to 90 ° C and the fractionation depending density and / or conductivity the expiring solution is controlled. 4. The method according to claim 1 to 3, characterized characterized that by addition of alkali carbonates, alkali hydroxides and carbons dioxide alkaline earth carbonates or hydroxides from the Product fraction to be precipitated.   5. The method according to any one of claims 1 to 4, there characterized by that the amino acid fractions after ion exclusion via an ion exchanger to exchange the cal cium ions against other cations. 6. The method according to any one of claims 1 to 5, there characterized by that the amino acid fractions concentrated and the heavy soluble amino acids are gradually separated. 7. The method according to claims 4 and 5, characterized characterized in that after the Claims 4 and 5 pretreated amino acid oil solutions for adjusting the magnesium content and / or calcium and / or potassium and / or sodium be mixed. 8. The method according to any one of claims 1 to 7, there characterized in that the separated sparingly soluble amino acids and the Solution of the easily soluble amino acids for the Production of pure amino acids can be used.