Method for preparing amino acid mixture from protein hydrolyzate

FIELD: food industry, medicine.

SUBSTANCE: invention relates to a method for production of highly purified amino acid mixtures. Method involves separation of suspended particles and clearing protein hydrolyzate on clearing sorbent. Ammonia is removed from hydrolyzate by its passing through desalting system at the rate from 2 to 3 volumes per volume of desalting system/h followed by carrying out sorption of amino acids on cation-exchange resin in H+-form. Amino acids are eluted from cation-exchange resin with alkali solution and eluate is subjected for microfiltration and drying. Desalting system represents a system of succession connected ion-exchange column with alternating cation-exchange resin - anion-exchange resin wherein volume of anion-exchange resin provides exceed of its full ion-exchange capacity by 20-25% as compared with that in cation-exchange resin. Method provides enhancing effectiveness of ion-exchange purification of amino acids.

EFFECT: improved preparing method.

6 cl, 2 dwg, 1 tbl, 3 ex

 

This invention relates primarily to the field of food industry and medicine and relates to the production of highly purified amino acid mixtures that are used as food additives and medicines to boost immunity, fight coronary heart disease, diabetes, mental disorders, hepatitis, urogenital infections, etc.

On the market especially appreciated the preparations containing more than 80% of free amino acids with ash content not more than 0.2% and a purity not less than 99%.

High competition of dietary SUPPLEMENTS containing amino acids requires a reduction in the cost and sales price. The necessity of new technological techniques, increasing production efficiency.

The prototype of the invention is the scientific-technical development of the Moscow state University of technologies and management of the Ministry of education and science of the Russian Federation edited by Filatova O.K. (Moscow, 2004)

The disadvantage of the prototype is that, firstly, the use of vacuum evaporation plant to the eluate was evaporated amino acid 1.5 times and to remove ammonia, which is always present in any protein hydrolysate, and to concentrate the solution before drying. Secondly, that the capacity of the cation exchanger is used only by 70-80%. The other exchange places on the resin occupy MIAC and hydrogen. Thirdly, that in the process of elution impossible to separate monoaminoxidase amino acids and diaminocarbenes amino acids, providing a completely different pharmacological effect on the body.

The aim of the present invention is to increase the efficiency of ion-exchange purification of amino acids released from protein hydrolysates, and with it the economic efficiency of the whole production.

To this end, in the method of obtaining amino acid mixture of protein hydrolysate obtained by acid, alkaline or enzymatic hydrolysis of protein materials, separating suspended particles, lighten protein hydrolysate on brightening the sorbent, absorb amino acids on the cation exchanger in the H+form, elute the amino acid with the cation exchange resin with a solution of alkali, microfiltrate and dried product. Before adsorption of amino acids on the cation exchange resin hydrolyzate free from ammonia, passing it through a demineralization system at speeds from 2 to 3 volumes per volume of the demineralizing system/hour, and the desalting system is a system of series-connected ion-exchange columns with alternating cation - anion exchange resin, in which the volume of the anion provides the excess of its total ion exchange capacity compared to the cation exchange resin by 20-25% with speed.

The height of each column or the hazards of ionosorbed demineralizing system is 10% of the total height of the desalting system, but not more than 0.5 m

Preferably, in demineralization system as a cation exchanger is used sulfotyrosine celecrity cation, and anion - srednednevnoy anion exchange resin.

When you do this:

- full exchange capacity of the cation exchanger and the quantity of the alkali solution taken for elution is equivalent to the total number of amino acids contained in the hydrolysate;

the elution of amino acids perform 2-4 h with a solution of alkali, passing it at a speed of not more than 0.5 volume per volume of the cation in 1 hour, the eluate with the concentration of amino acids 25-35% is subjected to filtration and spray drying;

- in the range of pH of the eluate from 3.5 to 8.5 selected fraction moneymoneymoney amino acids, and in the range of pH of the eluate from 8.5 to 10.5 - faction diaminocarbenes amino acids containing arginine, histidine and lysine.

In the process of experimental and theoretical studies conducted by the applicant, it was found that any hydrolysis of the protein together with the cleavage of amino acid chains there is a partial destruction of amino acids with the formation of ammonia and other organic substances, forming a heterocyclic colored compound. During acid hydrolysis of ammonia is in the form of NH4CL and enzymatic hydrolysis in the form of a salt of dicarboxylic amino acids: glutamic and aspartic. P is acesse sorption of ammonia together with amino acids adsorbed on the cation exchanger in the form of ammonium ion, providing competitive effect.

In the process of elution of amino acids with alkali solution with ion exchange columns first go monoaminoxidase amino acids with low selectivity (pH of the eluate 3,5-8,5). Then out ammonia and diaminododecane acid with a pH of 9.5 to 11.5. This process is illustrated in figure 1.

As can be seen from the drawing, the output curve of elution has a smooth character. Therefore, it is impossible to divide diamino - and monoaminoxidase amino acids.

Fraction diaminododecane amino acids containing lysine, arginine and histidine, can be used as a unique tool for the treatment of heart vessels, heart failure and increase potency.

The present invention allows the elution process to select the faction.

To this end, the ammonia is removed from the hydrolysate before sorption of amino acids.

Remove ammonium salt can be made using ion-exchange demineralization. For this purpose a solution of amino acids and ammonia at pH 6-7 is passed through a system of series-connected short columns, in which the cation exchanger is interleaved with the anion exchange resin. Due to the large differences in diffusion coefficients of amino acids and ammonium (2nd order) of the latter is delayed by selectitem sulfotyrosine the cation exchanger, through which amino acids freely pass between the grains of resin.

Released proto is sorbed on a medium or weakly basic ion-exchange resin. Thus, amino acids are transformed into zwitter-ions and are sorbed on the cation exchanger in the H+form according to the principle of chemisorption:

The advantage of this method is that the flow from the column has a neutral reaction and may be discharged into the sewer. In addition, the process of chemisorption is reminiscent of the reaction of neutralization, although thermal effect is only 3.5-4 kcal/mol instead of 13 kcal/mol for the neutralisation of acid and alkali.

Large internal energy process extends the frame of the cation and allows 100% use of its exchange capacity.

The absence of the ion exchanger ammonia, providing a buffer effect on the solution, significantly changes the character of the output curve at alkaline elution of amino acids (see figure 2).

As can be seen from the drawing, the output curve gives the inflection at pH 7-7,5. Thus the graph shows two areas with a sharp boundary at the indicated pH values. In practice, this allows for the elution of amino acids separately to select fractions of mono - and diaminocarbenes acids. The process can be carried out automatically by the command of the pH probe.

Strict equivalence of alkalis taken for elution, the capacity of the resin or, in other words, the number of equivalents of absorbed amino acids, allows to obtain a pure solution of amino acids, the concentration of which PR is portionally concentration of the alkali solution. So, when the alkali concentration of 3 N. the concentration of amino acids in solution is 30-35%. Further increasing the concentration of alkali leads to the fact that monoaminoxidase amino acid crystallizes directly in the output collection.

The essence of the proposed invention is as follows.

Hydrolyzed and filtered neutral protein material to lighten brighten ionosorbed type IA-4, located in the column.

The clarified solution of the hydrolyzate is passed through demineralization system 10 of ion-exchange columns with a cation exchange resin and anion exchange resin at a speed of two or three volumes on the volume of the demineralizing system per hour. The solution was ousted from the column with water. The desalted solution contains almost no inorganic ions and ammonium. Its conductivity does not exceed 0.5 mk8, pH of 9.5 to 10.5. The solution passed through a column of cation exchange resin KU 2×8 H+-the form and then displace in the same direction of the water. The elution of amino acids is carried out, skipping 2-4 N. alkali, the amount of which fully corresponds to the capacity of the cation exchange resin in the column.

The solution was ousted from the column with water. The eluate petitrenaud 36%HCL to a pH of 6.5 to 7.0. Its concentration is 28-35%. Spray drying of the product is carried out at a temperature of entering air 170°and the temperature of the outlet air 70-75°In another embodiment, the elution is collected in two fractions: to pH 7.5 and 7.5 to 10.5. Fractions are dried separately receiving two different drug.

In comparison with the considered the closest analogue of the proposed method is characterized by the following distinctive techniques:

the hydrolysate after purification is subjected to obessolivaniju before the process of adsorption on the cation exchanger;

- amino acids are sorbed to the complete saturation of the cation;

for elution process is used 2-4 N. lye;

- if necessary, meet separately two fractions of amino acids.

The use of these techniques allows to exclude the processes of vacuum residue and the cost of energy to produce new kinds of drugs, amino acids and to make the allocation process more cost effective.

In more detail, the invention is explained with the following examples.

Example 1:

In enameled reactor with a capacity of 630 l load 250 l 18%HCL and 130 kg rogatories flour. The reactor is sealed and the temperature of the contents was raised to 127°using steam in the jacket. This temperature support with stirring for 6 hours. Then the reactor is cooled to 60°and With him pour 290 l of water. Solution (600 l), containing 78 kg amino acid, filtered on a porcelain suction filter through the belting fabric under vacuum. 30 l black oily sediment load in plastic bags, and 570 l of the filtrate, containing 77 kg aminoxy the lot, passed through the ion exchange column bottom, loaded with 700 l of resin EDE-10p HE-form, with a speed of 500 l/h. The hydrolysate was ousted from the column, passing through it 400 l of water. The neutralized fraction of the amino acids are assembled by Refractometer, zero 1,334 and ending with the same value. Collect 600 l of a solution containing 75 kg of amino acids. This solution is lighten on a column loaded with 200 l of resin IA-4 CL--form, flowing with a speed of 150 l/h from the bottom of the column, and then displacing it with water. Get 650 l clarified colorless solution containing amino acids 72 kg solution with a pH of 6.8 is passed from below through a system of series-connected ion-exchange columns with a volume of 20 liters each, alternating as follows: cation exchanger KU 2×20 N+-form anion exchange resin EDE-10p OH--form. The solution is passed at a speed of 400 l/h and then replacing it with water. Control collection of amino acid fractions are also on the Refractometer.

The balance of demineralizing system made in favor of the anion, so the amino acids in the desalted solution are in the form of zwitter-ion, and desalted solution has a pH of 9.8.

800 l obtained desalted solution containing 70 kg amino acids are missing from the bottom of the column, loaded 280 l resin KU 2×8 H+form at the rate of 140 l/h. The solution out to the Onna water. Runoff from the column has a pH of 5.9 and is discharged into the sewer. The column was washed with 300 l of distilled water and then from bottom to let in 180 l 3 n alkali solution of NaOH at the rate of 140 l/h. After the alkali serves distilled water. The collected fractions are Refractometer. Get 226 l of a solution containing 68 kg amino acids (30%solution), which is then passed through a microfiltration installation and dried in the spray dryer, the capacity of 25 l/h of evaporated moisture. Get 65 kg dry white powder, containing 86% of free amino acids, 1% water and 13% of di - and tri-peptides.

Example 2:

8 m lactic acid whey is subjected to nanofiltration for microfiltration installation with membranes ERA-200-1016, with a threshold cut-off of 20 kDa and a surface area of 26 m2. After 5 hours, the whey is concentrated to 300 l of a protein content of 15%. The suspension is loaded into a reactor with a volume of 500 l, contribute 4.5 kg enzyme pretoriana and stirred at a temperature of 50±1°C for 12 hours. The precipitate is removed by microfiltration to install the MPM-2 ceramic filters. While getting 295 l of the filtrate, containing 35 kg of amino acids. The filtrate absoluut on ion-exchange system of serially connected columns with a capacity of 5 l with cationite KU 2×8 H+form and anion exchange resin EDE-10p HE--form. The solution will roboscout bottom at a rate of 100 l/h, he was then ousted from the water column. Get 380 l of a solution with a conductivity of 150 mk8, pH 9.6 and the amino acid content of 34,5 kg

Sorption of amino acids on the cation exchanger KU 2×8 H+-form exercise, flowing the solution from the bottom to the column with 150 l of cation exchanger at a rate of 100 l/h. The solution displace water.

For elution using 100 l 3 n caustic soda solution, which is passed from the bottom of the column at 50 l/h and further displace water. Get 200 l of the eluate with a pH of 8.7, containing 33,4 kg amino acids. The solution petitrenaud to pH 7.0 with 36%HCL and lighten on a column loaded with 50 l of resin IA-4 CL-form.

250 l of a solution is subjected to microfiltration ceramic membranes with a filtration surface 2 m2.

The filtered solution not containing suspended particles, with a concentration of 13% is subjected to spray-drying. Get 30 kg of the drug amino acid with a purity of 98.5% and a content of free amino acids 88,5%.

Example 3:

5 m3suspension of brewer's yeast with a concentration of solids of 10% is loaded into the apparatus with stirrer and jacket with a capacity of 6.3 m3. Then in the device make 10 l of water suspension containing 4 kg of enzyme aminoprotein. The temperature of the mixture was raised to 50°and support her under stirring for 10 hours. At the end of the process of enzymatic hydrolysis of the suspension is subjected to two-stage microfiltration installation with ceramic membranes with a filtration surface 35 m 2.

5.5 m3the suspension containing 130 kg of free amino acids, 50 kg of peptides and 20 kg of ammonia, is passed from the bottom with a speed of 2 m3per hour through a system of series-connected columns containing 100 l of cation exchanger KU 2×20 and of the anion-EDE-10 p. the Solution was ousted from columns 1 m3water at the same speed.

The output of amino acids control the refractive index, the absence of salts by conductivity meter. The maximum allowable rate conductivity is 0.1 mS.

The desalted solution of amino acids and peptides with a pH of 9.7 passed through the ion exchange column loaded with 500 l of cation exchanger KU 2×8 H+form at a speed of 250 l/h. All amino acids are absorbed on the resin. With the column goes neutral runoff containing a small amount of long-chain peptides. After washing the column 1 m3water amino acids elute from the column, passing below 400 l of 2.5 n caustic soda solution at 200 l/h. After the alkali on the column serves deionized water. Collecting fractions of amino acids is controlled by Refractometer and pH meter. Collect 610 liters of 20%solution of amino acids with a pH of 9.5, which neutralize 90 l of 36%HCL (HC) to a pH of 6.0.

700 l of a solution is passed from below through the ion exchange column loaded with 150 l brightening sorbent IA-4 CL-form at 150 l/h. The solution of the expulsion of Aut columns with deionized water. Collecting fractions of amino acids are on the Refractometer. The result is 780 liters of a 15%solution of amino acids. It is passed through the microfiltration installation UMT-6 polymer membrane area of 2 m2to remove microcosmica and microflora.

Sterile solution is subjected to spray drying at a temperature of incoming air 175°and out - 70°C. Powdered particles captured in the baghouse.

Get 138 kg of white powder, odorless, containing:

- moisture - 1%;

- amino acids - 87%;

- 2 - 3-tier peptides - 12%;

- ash - 0,15%.

The product yield from protein contained in yeast, is 69%. The amino acid composition of all the drugs presented in the table.

Table
The composition of highly purified amino acid mixtures obtained by hydrolysis or enzymatic lysis by protein wastes of food industry
Amino acid % of total amino acidsHydrolyzed keratinFermentolizat yeastFermentolizat whey
1245
Aspartic acid8,56,255,1
Glutamic acid14,28,009,8
Series5,26,955,0
Threonine4,36,374,8
Glycine4,82,652,3
Alaninethe 4.78,153,2
Cystine4,01,25-
Valine4,29.007,0
Methionine2,15,202,5
Isoleucine4,58,55a 4.9
Leucine8,213,9211,8
Tyrosinea 4.93,92the 3.8
Phenylalanine6,25,775,8
Histidine4,54,073,2
Lysinethe 5.72,6010,5
Arginine8,83,05the 4.7
Proline5,1to 2.6710,8
Tryptophan---

1. The method of obtaining amino acid mixture of protein hydrolysate obtained by acid, alkaline or enzymatic hydrolysis of protein materials, including the separation of suspended particles, lightening protein hydrolysate on brightening the sorbent, sorption of amino acids on the cation exchanger in the H+form, the elution of amino acids from the cation exchange resin with a solution of alkali, micro filtration and drying of the product, characterized in that before the adsorption of amino acids on the cation exchange resin hydrolyzate free from ammonia, passing it through a demineralization system with a speed of 2 to 3 volumes per volume of the demineralizing system/hour, and the desalting system is a system of series-connected ion-exchange columns with alternating cation - anion exchange resin, in which the volume of the anion provides the excess of its total ion exchange capacity compared to the cation exchange resin by 20-25%.

2. The method according to claim 1, characterized in that the height of each column or layer ionosorbed demineralizing system is 10% of the total height of the desalting system, but not more than 0.5 m

3. The method according to claim 1, characterized in that the desalting system as a cation exchanger is used sulfotyrosine celecrity cation, and anion - srednednevnoy anion exchange resin.

4. The method according to claim 1, characterized in that the total exchange capacity of the cation exchanger and the number is of alkali solution, taken for elution, equivalent to the total number of amino acids contained in the hydrolysate.

5. The method according to claim 1, characterized in that the elution of amino acids perform 2-4 h with a solution of alkali, passing it at a speed of not more than 0.5 volume per volume of cation exchange resin per hour, and the eluate with the concentration of amino acids 25-35% is subjected to filtration and spray drying.

6. The method according to claim 5, characterized in that when the elution in the range of pH of the eluate from 3.5 to 8.5 selected fraction moneymoneymoney amino acids, and in the range of pH of the eluate from 8.5 to 10.5 - faction diaminocarbenes amino acids containing arginine, histidine and lysine.



 

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