ES2412961A1 - Procedure for the production of discolored peptides from proteins of animal origin (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Procedure for the production of bleached peptides from proteins of animal origin in a stirred reactor, temperature and pressure control, using high temperatures (180ºC) and moderate pressures (40 atmospheres) under a controlled atmosphere 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 with respect to the original product is simultaneously achieved, which facilitates the use of the peptides obtained as a food ingredient. Applicable in obtaining peptides and amino acids from proteins of animal origin, mainly in the agriculture, chemical and pharmaceutical, environmental or food sectors, and particularly 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

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 discoloration is achieved simultaneously with respect to the original product, which facilitates the use as a food ingredient of the peptides thus obtained.

The invention is applicable 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 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

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).

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 aS2-casein: identification and release kinetics of peptides ", Int. 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). In addition, a control is necessary

very strict and continuous of the enzyme / substrate ratio and the pH of the medium, which varies constantly as the hydrolysis progresses. Temperature is another essential parameter, since only within a narrow range of temperature values are good hydrolysis yields obtained. Once the

5 reaction, 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 a degradation of certain amino acids occurs. In the case of using acids, asparagine and glutamine are transformed into aspartic and glutamic acid, while the

Tryptophan and cysteine are completely destroyed (Fountoulakis M., Hans-Werner L., "Hydrolysis and amino acid composition analysis of proteins", Journal of Chromatography A, 1998, 826, 109-134). In case of alkalis, the loss due to degradation of serine, threonine, arginine and cysteine is caused; and asparagine and glutamine are converted equally to aspartate and glutamate (Ravindran, G. &

15 Bryden, W.L., "Tryptophan determination 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 alias substrate concentrations, but are usually only used for

20 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 is finished, 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 ° C), 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 serum albumin by continuous sub-critical water hydrolysis", Journal of Supercritical Fluids, 2005 , 36: 49-58). In addition, the rate

The transformation of protein into amino acids is around 65%, with the remaining 35% being residues. (Esteban, M. B .; Garcia, A. J .; Ramos, P .; Marquez, M. C.,

"Subcritical water hydrolysis of hog hair for amino acid production", Bioresour. Technol 2010, 101, 2472-2476; Rogalinski, T .; Herrmann, S .; Brunner, G., "Production of aminoacids from bovine serum albumin by continuous sub-critical water hydrolysis", J. Supercrit. Fluids 2005, 36, 49-58; Xian, Z .; Chao, Z .; Liang, Z .; 5

 Cheng, H., "Amino acids production from fish proteins hydrolysis in subcritical water.", Chin. J. Chem. Eng. 2008, 16, 456-460).

In specific cases, the discolouration 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 10 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 (Toldra, M .; Pare s, D .; Saguer, E .; Carretero, C., "Hemoglobin hydrolysates from porcine blood obtained through enzymatic hydrolysis assisted by high hydrostatic pressure processing ", Innovative

15 Food Science Emerging Technologies 2011, 12, 435-442; Oord van den, A.H.A .; Wesdorp, J.J. (1979) "Decolouration of slaughterhouse blood by treatment with hydrogen peroxide", Proceedings of the 25th European Meeting of Meat Research Workers, Budapest, Hungary. 827-828).

With none of the methods mentioned above is achieved in a way

20 simultaneous good performance, the controlled production of low molecular weight peptides, the processing of large amounts of substrate and a discoloration of the final product. With the present invention all these objectives can be achieved in a single step.

25 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 particular gas: oxygen or nitrogen.

The process for the 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 temperature and temperature control.

 pressure, means for injecting a gas for the control of the atmosphere, such as valves, pressure control valves, inlet and outlet ports or fittings. For the purposes of this invention and its description, the total volume of protein plus water is considered the reaction volume.

b. Heat the reactor while maintaining constant agitation until

10 to reach a temperature of 180 ° C and simultaneously a gas that can be oxygen or nitrogen is injected, preheated to the same temperature as the reactor, until a reaction pressure of 40 atmospheres is reached. To maintain the internal pressure of the reactor, any pressure regulating means can be used, such as pressure control valves that are

15 when the internal pressure reaches or exceeds a certain limit, evacuating the 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.

20 c. The constant temperature is maintained once the 180 ° C is reached and the gas injection is maintained with the same flow rate of 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.

25 (typically this time depends on the temperature used). During this stage of the process, gas continues to be injected into the reactor so that the decolorization process takes 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 maintaining the internal pressure of the reactor.

30 d. At the end of stage c) the hydrolysis is over and the product obtained is extracted through a heat exchanger to reduce its temperature. This stops the hydrolysis process immediately.

This excess heat can be used for preheating the injected gas or the jacket of the reactor. 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 remains.

solid.

F. Seel excess water by drying.

In a preferred embodiment, the substrate protein is of animal origin, previously defatted.

The substrate protein used can be purified hemoglobin, fraction

10 blood cell, blood plasma, liquid or coagulated whole blood, or any mixture of these fractions from any animal source, as well as sliced whole feathers or a mixture of blood derivatives and feathers.

In another preferred embodiment, the amount of water that is allied in step a) is that necessary to have a protein concentration of between 50 and 150 g / L.

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.

In another preferred embodiment, the gas introduced in step b) is nitrogen °, with

20 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 c) of the process is 360 minutes.

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 25 per minute. In a more preferred embodiment, the time of

Injection of oxygen gas during stage c) of the process is 180 minutes.

In a specific embodiment, the excess gas is evacuated from the reactor with agitation by a pressure control valve and is recirculated during stages b) and c) of the process, in a proportion that is equivalent to 70% of the total flow of the injected gas. This excess gas can be recirculated inside 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 process is filtered with a filter whose pore size () is 20 microns.

5 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 until

10 date with the use of HHP-based methods, which only transform the substrate protein into free amino acids.

The hydrolyzate thus obtained is composed of low molecular weight peptides (between 1 and 3 kDa of average weight according to the conditions used), and a yield of 83% is obtained using high protein concentrations.

15 as a substrate, which can reach 400 g / l. In addition, in the case of hemoglobin, a color reduction ranging from 80% is achieved when nitrogen is injected and 95% when the injected gas 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 tidos without the use of enzymes or

chemical compounds.

Pressure and temperature application to obtain peptides and not only

25 amino acids with a substantial improvement in the yield obtained so far. It goes from obtaining a 60% conversion in amino acids ((Rogalinski, T., Herrmann, S., Brunner, G., "Production of amino acids from bovine serum albumin by continuous sub-critical water hydrolysis", Journal of Supercritical Fluids , 2005, 36: 49-58) to obtain

 83% peptides. Peptide production 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 injected gas, being smaller to higher

 temperature and in the presence of oxygen. Discoloration of the hydrolyzate obtained simultaneously with the hydrolysis of the protelna substrate. Very low degradation of the amino acids present in the original protein, which improves nutritional quality with respect to use

10 deacides or aticalis 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 more

15 smaller and more discolored when oxygen is used instead of nitrogen.

The invention results from application in sectors that require a protein hydrolysis method to obtain low molecular weight peptides, in which subsequent purification, separation, inactivation or discoloration processes are not desired,

20 as for example in the agriculture, chemical and pharmaceutical, environmental or food sectors.

EXPLANATION OF A PREFERRED EMBODIMENT

For a better understanding of the present invention, two examples of preferred embodiment are described below, described in detail, which should be understood without limiting catheter of the scope of the invention.

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

In a volume of 400 mL, 20 grams of hemoglobin of 95% purity were dissolved and subsequently the solution was introduced into a tightly closed steel reactor. The reactor was placed inside a jacket capable of

raise the temperature inside the reactor to the desired temperature. In addition, the

The reactor had a temperature controller, an internal pressure controller, mechanical agitation, an injected air inlet port and an excess gas outlet port controlled by an internal pressure dependent valve.

Finally, it had an outlet port through which to extract the sample through a heat exchanger to cool the sample.

10 Ladisolucion the kept under constant agitation at a speed of 500 rpm and was heated to a temperature of 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 40 atmosphere reactor. This value is

15 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 stream of injected gas was established that allowed to keep the pressure constant and not stop the gas injection. The gas, before being introduced into the reactor, is preheated to 150 ° C in a humidifier to saturate with water vapor.

20 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 the stirring was continued. Then, through the outlet port, the product 25 was slowly extracted by passing it through a heat exchanger with liquid refiteration to leave the sample at room temperature.

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

30 -In the case of using nitrogen, after 6 hours of reaction between 16 and 17 grams of soluble peptides of an average molecular weight of

3.2 lcDa and with a color reduction measured at 407 nm of 80%, accompanied by a production of 1.5 grams of free amino acids.

-

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 between 1 and 2 lcDa, 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.

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

A small-scale test was performed with fresh samples of polio blood clots and whole feathers. This mixture of raw materials is carried out because the feathers, formed by keratin, do not have in their composition certain

15 essential amino acids, which are contributed by the added clots. In this way it was possible to have a final product without nutritional deficiencies.

200 grams of polio blood clots (100 grams dry weight) were weighed, 40 grams of dried crushed bird feathers were added and water was added until a volume of 400 mL was completed, which in the final solution involved 140

20 grams of dry matter per liter. Said solution was placed in the reactor previously described and the conditions of the hydrolysis were fixed at constant agitation, 180 ° C, pressure of 40 atmospheres and an oxygen flow of 1400 mL / min. 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. Feathers and clots

25 had been completely solubilized, resulting in a solution rich in soluble peptides and suspended matter residues. To eliminate these possible aggregates and suspended matter, the product was centrifuged for 10 minutes with a force of 10,000 g. The supernatant obtained consisted of a clear amber solution with a concentration of 120 g / L of peptides in

30 solution, which meant a yield of 85%. The remaining 15% was composed of suspended solids removed during centrifugation.

The peptides were analyzed by tatngio exclusion chromatography to determine their concentration and molecular weight distribution. Under the conditions described, peptides of an average molecular weight of 2-3 l (Da, were obtained, although the range thereof ranged from 10 to 0.5 IcIDa.

5 discoloration that was obtained was 85%.

Claims (10)

1. Procedure for the hydrolysis of proteins of animal origin comprising the following stages:  a. introduce the substrate protein into a reactor with agitation, with internal pressure and temperature control and add water to facilitate the hydrolysis process; b. heat the reactor maintaining a constant stirring until reaching the temperature of 180 ° C and simultaneously inject a gas that 10 can be oxygen or nitrogen, preheated to the same temperature as the reactor, until a reaction pressure of 40 atmospheres is reached; C. keep the temperature constant once it reaches 180 ° C and keep the gas injection at the same flow rate as stage b) between 180 minutes up to 360 minutes; 15 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. Procedure, 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 between 50 and 150 g / L.

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

5. Procedure according to claim 1, characterized in that the pressure of the reaction of stage b) is 40 atmospheres and is reached in the first 30 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. Procedure, according to claim 6, characterized in that the time of 5  Injection of nitrogen gas during stage c) of the process is 360 minutes. 8. 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. 9. Procedure according to claim 8, characterized in that the injection time of the oxygen gas during step c) of the process is 180 minutes. 10. 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 stages b) and c) of the process, in a proportion that 15 equals 70% of the total flow of the injected gas. 11. Procedure, 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