ES2412961B1 - Procedure for the production of bleached peptides from proteins of animal origin - Google Patents

Abstract

Procedure for the production of decolorized peptides from proteins of animal origin in a reactor with stirring, temperature and pressure control, using high temperatures (180ºC) and moderate pressures (40 atmospheres) under an atmosphere controlled by injection well of oxygen or nitrogen. This process provides low molecular weight peptides without the addition of enzymes or chemicals. In addition, a discoloration is achieved simultaneously with respect to the original product, which facilitates the use of the peptides obtained as a food ingredient. # Application in obtaining peptides and amino acids from proteins of animal origin, mainly in the sectors of agriculture, chemistry and pharmacy, environmental or food, and in particular in the agri-food or meat industries that generate an excess of residues rich in proteins and that require an efficient method for the post-processing of by-products.

Description

  PROCEDURE FOR THE PRODUCTION OF COLORED PEPTIDES FROM PROTEINS OF ANIMAL ORIGIN

S 10

The invention relates to a process for the hydrolysis of proteins of animal origin using high temperatures (180 OC) and moderate pressures (40 atmospheres) under a controlled atmosphere, by the injection of either oxygen or nitrogen. This process is intended to provide a method of protein hydrolysis to obtain peptides of low molecular weight and that does not involve the addition of enzymes or chemicals. In addition, a decolorization is achieved simultaneously with respect to the original product, which facilitates the use as a food ingredient of the peptides thus obtained.

fifteen

The invention is applicable in obtaining peptides and amino acids from proteins of animal origin, mainly in the sectors of agriculture, chemical and fanatic, environmental ° food, and in particular in those agri-food or meat industries that generate an excess of residues rich in proteins and that require an efficient method for post-processing of by-products.

STATE OF THE TECHNIQUE

twenty

At present, proteins of animal origin can be hydrolysed following enzymatic processes, chemical hydrolysis (acidic or basic) or with very high hydrostatic pressures together with very high temperatures (HHP).

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In the first case, peptides are obtained in a predictable manner in terms of size and sequence, without losses by degradation of amino acids; however, the concentration of protein that can be treated is usually in the range of 5-50 gIL (Yike Y., Jianen H., Xuefeng B., Yuguang D. & Bingcheng L., "Preparation and function of oligopeptide- enriched hydrolysate from globin by pepsin ", Process Biochem., 2006, 41, 1589-1593; Tauzin, J., Mielo, L., Roth, S., Mollé, D. & Gaillard, J.-L.," Tryptic hydrolysis of bovine cr.S2-casein: identification and release kinetics ofpeptides ", [nI. Dairy J., 2003,13,15-27; Su, R.-X., Qi, W. & He, Z.-M ., "Time-dependent nature peptic hydrolysis of native bovine hemoglobin. Eur. Food Research Tech.", 2007, 225, 637-647).

very strict and continuous of the enzyme / substrate ratio and the pH of the medium, which varies constantly as hydrolysis progresses. Temperature is another essential parameter, since only within a narrow range of temperature values are good hydrolysis yields obtained. Once the reaction is over, the enzyme used must be neutralized at a later step in the process and further removed from the medium.

In the case of using chemical agents to hydrolyze proteins, degradation of certain amino acids occurs. In the case of using acids, asparagine and glutamine are transfonnate in aspartic and glutamic acid, while tryptophan and cysteine are completely destroyed (Fountoulakis M., Hans-Wemer L., "Hydrolysis and amino acid composition analysis of proteins ", Journal of Chromatography A, 1998, 826, 109-134). If alkalis are used, the degradation loss of serine, threonine, arginine and cysteine is caused; and asparagine and glutamine are converted equally to aspartate and glutamate (Ravindran, G. & Bryden, W.L., "Tryptophan arresting in proteins and feedstuffs by ion exchange chromatography", Food Chemistry, 2004, 89, 309-314). This loss of amino acids is a serious inconvenience, since some of them are essential and their lack decreases the nutritional value of the hydrolyzate obtained. These processes are capable of managing high substrate concentrations, but are usually only used to produce free amino acids, without the possibility of obtaining peptides (US 2,657,232 A; US

4,181,651 A; US 4,874,893 A). Once the process has been finalized, the hydrolyzing agent must be neutralized, with the concomitant production of salts that have to be eliminated in later steps of the process.

When high pressures and high temperatures are used (between 15 and 27 MPa and between 250 and 300 OC), the final product is mainly composed of amino acids and degradation products, such as organic acids and ammonia; and therefore it is not possible to obtain peptides (Rogalinski, T., Herrmann, S., Brunner, G., "Production of amino acids from bovine serurn albumin by continuous sub-critical water hydrolysis", Journal of Supercritical Fluids, 2005 , 36: 49-58). In addition, the rate of protein transfer in amino acids stands at 65%, with the remaining 35% being residues. (Esteban, M. B .; García, A, J .; Ramos, P .; Márquez, M. C.,

S

"Subcritical water hydrolysis of heg hair for amino acid productioo", Bioresour. Technol 2010, 101, 2472-2476; Rogalinski, T .; Herrrnann, S .; Brunner, G., "Production of aminoacids from bovine serum albwnin by continuous sub-critical water hydrolysis", J. Supereri !. Fluids 2005,36,49-58; Xiao, l .; Chao, l .; Liaog, l .; . Cheng, H., "AmiDa acids production from fi sh proteins hydrolysis in subcritical water.", Chino J. Chem. Eng. 2008, 16,456-460).

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In specific cases, the discoloration of the hydrolyzate is desired so that it does not change the color of the product to which it is to be added. This fact is especially relevant in the use of hemoglobin hydrolysates, whose use is restricted to strongly colored products. Until now, the bleaching of hemoglobin hydrolysates was produced using hydrogen peroxide or strong oxidants and joint treatments of enzymes and HHP (Toldrá, M .; Paré s, D .; Saguer, E .; Carretero, e., "Hemoglobin hydrolysates from porcine blood obtained through enzyyrnatic hydrolysis assisted by high hydrostatic pressure processing ", lnnovative Food Science Emerging Technologies 20 11, 12, 435-442; Oord van den, AHA; Wesdorp,! .J. (1979)" Decolouration of slaugbterbouse blood by trealmen ! with hydrogen peroxide ", Proceedings of the 25th European Meeting of Meat Research Workers, Budapest, Hungary. 827-828).

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With none of the aforementioned methods, good performance, controlled production of low molecular weight peptides, processing of large amounts of substrate and decolorization of the final product are achieved simultaneously. With the present invention all these objectives can be achieved in a single step.

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DESCRIPTION OF THE INVENTION

The present invention relates to a process for the hydrolysis of proteins of animal origin through the use of high temperatures and moderate pressures, under an atmosphere controlled by the injection of a detained gas: oxygen or nitrogen.

30

The process for hydrolysis of proteins of animal origin object of the invention comprises the following steps:

to.

The substrate protein is introduced into a reactor with stirring, with internal pressure and temperature control, and water is added to facilitate the hydrolysis process. The reactor with mechanical agitation usable in the method also provides means for the control of temperature and pressure, means for the injection of a gas for the control of the atmosphere, such as valves, pressure control valves, ports of Entry and exit or fittings. For the purposes of this invention and its description, the total volume of protein plus water is considered the reaction volume.

b.

The reactor is heated maintaining a constant stirring until reaching the temperature of 180 oC and simultaneously a gas that can be oxygen or nitrogen is injected, preheated to the same temperature as the reactor, until reaching a reaction pressure of 40 atmospheres. To maintain the internal pressure of the reactor, any pressure regulating means can be used, such as pressure control valves that open when the internal pressure reaches or exceeds a certain limit, evacuating excess gas. Preferably, the injected gas is previously saturated with moisture and preheated, using for example a thermostated humidifier, to avoid temperature variations and evaporations within the reactor.

C.

The constant temperature is maintained once 180 oc is reached and the gas injection is maintained at the same flow rate as stage b) between 180 minutes up to 360 minutes. Once the desired temperature and pressure is reached, the reaction is maintained under these conditions for the time necessary to obtain the production of low molecular weight peptides (typically this time depends on the temperature used), During this stage of the process gas continues to be injected in the reactor for the decolorization process to take place, and the gas stream that would generate an overpressure is expelled by means of a valve that allows this excess gas to be released, thus keeping the internal pressure of the reactor constant,

d.

At the end of stage c) the hydrolysis has already tended and the product obtained is extracted through a heat exchanger to reduce its temperature, thus the hydrolysis process is stopped immediately.

s

This excess heat can be used for preheating the injected gas or reactor jacket. The product obtained is a solution rich in soluble peptides with the presence of free amino acids. and. The extracted product is filtered or centrifuged to separate possible solid residues. F. Excess water is removed by drying.

In a previously degreased embodiment.

preferred the protein substratum is from Animal origin,

10

The substrate protein used can be purified hemoglobin, blood cell fraction, blood plasma, liquid or coagulated whole blood, or any mixture of these fractions from any animal source, as well as whole sliced plots or a mixture of blood derivatives and feathers.

In another preferred embodiment, the amount of water added in step a) is that necessary to have a protein concentration between 50 and ISO gIL.

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In another preferred embodiment, the reaction temperature of step b) is 180 ° C and is reached in the first 60 minutes of the process.

In another preferred embodiment, the reaction pressure of step b) is 40 atmospheres and is reached in the first 30 minutes of the process.

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In another preferred embodiment, the gas introduced in step b) is nitrogen, with a flow rate equivalent to once the volume of the original protein solution per minute. In a more preferred embodiment, the injection time of the nitrogen gas during step e) of the process is 360 minutes.

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In another preferred embodiment, the gas introduced in step b) is oxygen, using a flow equivalent to once the volume of the original protein solution per minute. In a more preferred embodiment, the injection time of the oxygen gas during step e) of the process is 180 minutes.

In a specific embodiment, the excess gas is evacuated from the reactor with stirring by a pressure control valve and is recirculated during stages b) and e) of the process, in a proportion equivalent to 70% of the total gas flow

injected. This excess gas can be recirculated into the reactor, the input current being composed of a part of recirculated gas and another of new gas.

In another specific embodiment, step e) of the filtration process is carried out with a filter whose pore size is 20 microns.

In another specific embodiment, step e) of the centrifugation process is carried out for 10 minutes with a force of 10,000 g.

A general objective of the method of the present invention is to obtain low molecular weight peptides from proteins of animal origin, avoiding the addition of chemicals or enzymes, and in turn improving the yield obtained to date with the use of methods based on HHP, which only manage to transfer the substrate protein into free amino acids.

The hydrolyzate thus obtained is composed of JX> r peptides of low molecular weight (between 1 and 3 kDa of average weight according to the conditions used), and a yield of 83% is obtained using high concentrations of protein as substrate, which can reach up to 400 gil. In addition, in the case of hemoglobin, a color reduction ranging from 80010 is achieved when nitrogen is injected and 95% when the gas injected is oxygen.

With the present invention it is possible to avoid the use of enzymes or other chemical products, necessary until now, to produce low molecular weight peptides. Thus, the advantages that the present invention provides are the following:

Production of low molecular weight peptides without the use of enzymes or chemical compounds. Application of pressure and temperature to obtain peptides and not only amino acids with a substantial improvement in the yield obtained so far. It goes from obtaining a 60% conversion into amino acids «Rogalinski, T., Hemnann. S., Brunner, G., "Production of amino acids trom bovine serum albumin by continuous sub-critical water hydrolysis", Joumal of Supercritical Fluids, 2005, 36: 49-58) to obtain 83% peptides. The production of peptides is desired since they have functional properties that amino acids lack: emulsifiers, foaming agents, antioxidants or gelling agents. The average molecular weight of the peptides obtained is a function of the temperature and the injected gas, being smaller at a higher temperature and in the presence of oxygen. Discoloration of the hydrolyzate obtained simultaneously with the hydrolysis of the substrate protein. Very low degradation of the amino acids present in the original protein, which improves the nutritional quality with respect to the use of acids or alkalis as hydrolyzing agents. Ability to process higher protein concentrations than enzymatic processes. The intensity of the discoloration, as well as the final size of the

Peptides are dependent on the type of gas injected, being smaller and more discolored when oxygen is used instead of nitrogen.

The invention is applicable in sectors that require a method of hydrolysis of proteins to obtain peptides of low molecular weight, in which subsequent processes of purification, separation, inactivation or discoloration are not desired, as for example in the agriculture sectors. Chemical and pharmacy, environmental or food.

EXPLANATION OF A PREFERRED EMBODIMENT

For a better understanding of the present invention, two preferred embodiments, described in detail, are described below, which should be understood without limitation of the scope of the invention.

Example 1: Hydrolysis of purified hemoglobin with oxygen or nitrogen injection. A small volume was used to perform low scale hydrolysis. The substrate used was a 50 gil purified hemoglobin solution.

In a volume of 400 rnL, 20 grams of hemoglobin of 95% purity were dissolved and subsequently the solution was introduced into a sealed hermetic steel reactor. The reactor was placed inside a jacket capable of raising the temperature inside the reactor to the desired temperature. In addition, the reactor had a temperature controller, an internal pressure controller, mechanical agitation, an air inlet port injected and an outlet port of excess gas controlled by a valve dependent on internal pressure. Finally, it had an outlet port through which to extract the sample through a heat exchanger to cool the sample.

The solution was kept under constant stirring at a speed of 500 rpm and was heated to 180 ° C, a process that lasted approximately 60 minutes. During this period, nitrogen or oxygen was injected through the inlet port at a rate of 1 liter per minute until a pressure was reached within the reactor of 40 atmospheres. This value was reached during the first 30 minutes of reaction and at that time the valve that automatically controls the pressure went into operation and a continuous injected gas was established that kept the pressure constant and did not stop the gas injection. The gas, before being introduced into the reactor, was preheated to 150 oC in a humidifier to saturate with water vapor

These conditions were maintained for 6 hours (for the case in which nitrogen was injected) or 4 hours (for the case in which oxygen was injected). After this time, the heat supply and gas injection were disconnected, but stirring was continued.

Then, through the outlet port, the product was slowly extracted by passing it through a heat exchanger with liquid cooling to leave the sample at room temperature.

Approximately 400 mL of the original sample was obtained. The analysis of the peptides obtained was performed by gel filtration chromatography. The results obtained were the following:

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In the case of using nitrogen, after 6 hours of reaction, between 16 and] 7 grams of soluble peptides of an average molecular weight of 3.2 kDa and with a color reduction measured at 407 nm of 80% were obtained, accompanied by a production of 1.5 grams of free amino acids.

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In the event that the gas injected was oxygen, after 4 hours of reaction, the same peptide production was obtained but with an average molecular weight of between 1 and 2 kDa, with a discoloration of 95% and 0.2 grams of free amino acids.

It was found that the peptides thus obtained improve the solubility of purified hemoglobin. Emulsifying properties and antioxidant capacity.

Example 2: Hydrolysis of bird feathers along with blood clots of the same species with oxygen injection.

A small-scale test was performed with fresh samples of chicken blood clots and whole feathers. This mixture of raw materials is carried out because the plwnas, formed by keratin, do not have certain essential amino acids within their composition, which are contributed by the added clots. In this way it was possible to have a final product without nutritional deficiencies.

200 grams of chicken blood clots (100 grams of dry weight) were weighed, 40 grams of dried crushed bird feathers were added and water was added to complete a volume of 400 roL, which in the final solution involved 140 grams of dry matter per liter. Said solution was placed in the previously described reactor and the hydrolysis conditions were fixed under constant stirring, 180 oC, pressure of 40 atmospheres and an oxygen flow of 1400 mUmin. The reaction time in this case was set at 270 minutes. After this period the product obtained was extracted, whose final volume was 600 mL. The feathers and clots had been completely solubilized, resulting in a solution rich in soluble peptides and residues of suspended matter. To remove these possible aggregates and suspended matter, centrifugation of the product was carried out for 10 minutes with a force of 10,000 g. The supernatant obtained consisted of a light amber solution with a concentration of 120 giL of peptides in solution, which resulted in a yield of 85%. The remaining 15% was composed of suspended solids removed during centrifugation.

The peptides were analyzed by size exclusion chromatography to determine their concentration and molecular weight distribution. Under the conditions described, peptides of an average molecular weight were obtained

2-3 kDa, although their range ranged from 0.5 to 0.5 kDa. The

Discoloration that was obtained was 85%.

Claims (12)

1. Method for the hydrolysis of proteins of animal origin comprising the following stages:

to.

introduce the substrate protein into a reactor with stirring. with internal pressure and temperature control and add water to facilitate the hydrolysis process;

b.

heat the reactor while maintaining constant stirring until it reaches a temperature of 180 oC and simultaneously inject a gas that can be oxygen or nitrogen. preheated to the same temperature as the reactor, until reaching a reaction pressure of 40 atmospheres;

C.

keep the temperature constant once 180 OC has been reached and keep the gas injection at the same flow rate as stage b) between 180 minutes up to 360 minutes;

d.

extraction of the product obtained through a heat exchanger to reduce its temperature;

and.

filter or centrifuge the extracted product to separate possible solid residues;

F.

removal of excess water by drying.

2.

Method according to claim 1, characterized in that the substrate protein is of animal origin, previously defatted

3.

Method according to claim 1, characterized in that the amount of water added in step a) is that necessary to have a protein concentration of between 50 and 150 gIL.

Four.

Method according to claim 1, characterized in that the reaction temperature of step b) is 180 OC and is reached in the first 60 minutes of the process.

5.

Method according to claim 1, characterized in that the reaction pressure of step b) is 40 atmospheres and is reached in the first 30 minutes of the process.

6.

Method according to claim 1, characterized in that the gas introduced in step b) is nitrogen, with a flow rate equivalent to once the volume of the original protein solution per minute.

7. Method according to claim 6, characterized in that the time of S injection of the nitrogen gas during step e) of the process is 360 minutes. Method according to claim 1, characterized in that the gas introduced in step b) is oxygen, using a flow equivalent to once the volume of the original protein solution per minute. Method according to claim 8, characterized in that the injection time of the oxygen gas during step e) of the process is 180 minutes. Method according to claim 1, characterized in that the excess gas is evacuated from the reactor with stirring by a pressure control valve and is recirculated during steps b) and c) of the process, in a proportion that 15 equals 70% of the total flow of the injected gas. 11. Method according to claim 1, characterized in that in step e) of the process the filtration is carried out with a filter whose pore size is 20 microns. 12. Method according to claim 1, characterized in that in step e) 20 of the process the centrifugation is carried out for 10 minutes with a force of 10,000 g