ES2298071B1 - HYDROLYZED ENZYMATIC COLLAGEN AND PROCEDURE OF OBTAINING. - Google Patents

Abstract

Collagen Enzymatic Hydrolyzate and procurement procedure

The invention relates to a hydrolyzate Collagen enzyme with a molecular weight between 1,000 and 50,000 Daltons, low acidity and conductivity in 1% solution by weight not exceeding 500 microS / cm.

It also refers to a procedure of obtaining a collagen hydrolyzate with low total acidity at from dermal tissue of animal origin, obtained under conditions hygienic, of animal species suitable for human consumption (cattle, pigs, poultry and fish), especially suitable for elaboration of dairy products that contain it. The procedure It includes, among others, the steps of: aqueous extraction of residual byproduct; filtration, demineralization and ultrafiltration of the filtered solution; enzymatic hydrolysis; concentration of the collagen enzyme hydrolyzate solution and drying of the enzymatic collagen hydrolyzate.

The invention also relates to supplements Nutritional and functional foods that comprise the hydrolyzate low acidic collagen enzyme.

Description

Collagen Enzymatic Hydrolyzate and procurement procedure

Technical sector of the invention

The present invention relates to a enzymatic hydrolyzate of collagen with a molecular weight between 1,000 and 50,000 Daltons. For their specials characteristics and conditions of obtaining is especially suitable for use in the production of dairy products.

It also aims at a procedure for obtaining a collagen hydrolyzate from dermal tissue of animal origin, obtained under hygienic conditions, from animal species suitable for human consumption (cattle, pigs, birds and fish), especially suitable for processing of dairy products that contain it. Said tissues can be processed in their native form, or proceed, as by-products, from processes of elaboration of
jelly.

Background of the invention

Collagen is a molecule of nature protein, fibrillar conformation and present in variable quantity in all types of connective tissue, whether skin, muscle, tendons, cartilage, blood vessels, ligaments or others. He collagen is composed of amino acids linked by the bond peptide (HN-C = O) forming sequences of the same that define peptides of different molecular weight and with different structural functions in the organism. In these sequences are all essential amino acids except tryptophan, glycine being especially abundant, the proline and hydroxyproline.

For a long time the usual way of Collagen obtaining has been carried out in two ways. The first pathway consists of the direct extraction of collagen-rich tissues, such as skin, bones and cartilage by direct hydrolysis with proteolytic enzymes, followed by multiple stages of purification, until you reach a final product that undergoes dried Through this first route, color products are obtained clear and with a soft smell and taste. However, the cost of obtaining of the collagen hydrolyzate obtainable by this route is very elevated, which greatly limits its applications.

According to another alternative route, the hydrolyzate of Collagen is obtained from extraction methods in the absence of enzymes by hydrolysis in acidic or basic media starting of skin, bones and / or cartilage that have previously been extracted in aqueous medium the jellies (more valuable products high in the market, employees in food and industry pharmaceutical mainly). Collagen hydrolysates as well obtained have irregular molecular weights, taste and smell unpleasant, dark color and appreciable levels of biogenic amines  like cadaverine or putrescine. The cost of obtaining hydrolyzate of collagen by this second route is lower, but unfortunately, due to the poor organoleptic characteristics of obtained hydrolyzate, the field of application thereof is quite limited.

Another inconvenience of the tracks usual to obtain collagen hydrolyzate is that, in both In cases, the collagen richness of the product obtained is less than 98% on dry product, which makes them generally unprofitable. In addition, taking into account the sources of supply used, preferably bovine and fish skins, it turns out very common that the hydrolyzate is contaminated with impurities and unconventional agents or contaminants.

However, collagen hydrolysates are widely used in many and varied fields, for example in the field of cosmetics, in the paper industry, in the industry food or in the pharmaceutical industry. In the case of pharmaceutical and food industry is required that the Collagen hydrolyzate is as pure as possible, which, as already He has commented before, the process of obtaining remarkably increases. In the particular case of the use of collagen hydrolyzate in the dairy industry, the situation is more unfavorable, due to the sensitivity of milk stability to changes in salinity, pH, acidity and other parameters.

The researchers have developed with previously a procedure according to which, from by-products of processes of obtaining gelatin, you get a enzymatic hydrolyzate of very pure collagen and applicable both in the manufacture of functional foods as in supplements nutritional conditions, allowing an improvement in functional capacity and / or the quality of life of a mammal with functional difficulties derived from alterations of articular cartilage. Without However, when said enzymatic collagen hydrolysates must used in the manufacture of dairy products (milk, yogurts, cheeses, etc.), due to their acidity and / or salinity, problems appear to get products with consistency suitable, for example precipitation problems in acidic medium of milk proteins, or incompatibility problems with other ingredients of the nutritional supplement or food functional.

To date, if you wanted to get a enzymatic hydrolyzed collagen with low acidity, the same they were subject to very high molecular weights, which did not they made them suitable for the preparation of these products Dairy products, mainly due to its high viscosity.

Specifically, the usual collagen hydrolysates, by the process of obtaining them in which the protein molecules are hydrolyzed, releasing the acidic ends of the amino acids that integrate them, have acidity levels so high that they are incompatible with the pasteurization of mixtures of these with milk or milk derivatives, at the concentrations to be used for the manufacture of said mixtures, said concentrations being between 2% and 15% by weight of hydrolyzate with respect to the total weight of the
mixture.

In previous investigations, and in accordance with US Patent 6,211,143 had already been demonstrated with gelatin hydrolysates of lower purity in collagen and with weights molecular molecules (up to 1000000 daltons) than the intake of same for athletes and people with a special effort in their normal activities managed to maintain or improve the integrity of articular cartilage

From patent ES 2087030 it follows that the Gelatin hydrolyzate is also useful as an adjuvant in treatments designed to compensate for calcium deficit.

Another patent application is also deduced ES 2104507, where compositions for the preparation of medicines for the treatment and prevention of dysplasia of hip in dogs

Several recent studies have shown that collagen enzyme hydrolyzate is well absorbed when administered orally and is distributed with great affinity, preferably in the tissues of articular cartilage (Oesser S, et al . "Oral administration of 14 } C Labelled Gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57 / BL) ". J Nutr 1999; 129 : 1891-1895.). This distribution spectrum causes an increase in the volume of the cartilaginous mass, which can develop its action, with the consequent decrease in pain and inflammation and with the improvement of the motility of the said joints. In this regard, the reader refers to the following publications: Adam M. "Therapie der osteoarthrosis. Welche Wirkung haben gelatinepräparate" Therapiewoche 1991, 41 : 2458-2461; Arquer A, et al . "Physical exercise in the elderly people". Selection 1996; 5 (3) : 121-128; Ribas J, et al . "Effects of gelatine hydrolysatesin the prevention of sportsmen and woman injuries". Arch Med Deport 1998; XV No. 66 : 277-282; and Trentham et al . "Effects of oral administration of type II collagen on rheumatoid arthritis". Science 1993; 261 : 1727-1730.

The researchers of the present invention have developed a procedure to obtain a hydrolyzate of collagen with high advantages over procedures current, while it is cheaper, and have discovered a application of the hydrolyzate obtained which is also highly Beneficial for the population.

Increasingly, the population cares about the Dietetics and nutrition, both animal and human. With this they have appeared on the market multiple preparations known as nutritional supplements, which contribute to animals or people  the necessary amount of vitamins, mineral salts or amino acids essential. Also in the field of nutritional supplements has used collagen to obtain preparations that favor cartilage recovery in animals that suffer diseases linked to this type of tissue.

As a nutritional supplement, everything should be understood that additional dietary intake that is consumed orally and that contains a "food ingredient" intended to complement feeding. Some examples of dietary supplements or Nutritional are vitamins, minerals, products vegetables (a single species or a mixture of several), others botanicals, amino acids and food components, Like enzymes Generally, nutritional supplements are distributed in the form of different presentations, such as tablets,  capsules, soft capsules, liquids and powders.

Also in the field of nutrition they are making known lately the so-called functional foods. Functional foods are considered to be those foods that are consumed as part of a normal diet and contain biologically active components, which offer benefits for the health and reduce the risk of illness. Between some examples of functional foods highlight foods that contain certain minerals, vitamins, fatty acids or fiber food, the foods to which substances have been added biologically active, such as phytochemicals or other antioxidants, and probiotics, which have live cultures of microorganisms beneficial. Examples of functional foods are some yogurts with probiotics, some margarines enriched with esters of sterols and eggs rich in fatty acids of the type Omega 3.

Both functional foods and Nutritional supplements are scientifically associated with the improvement of the quality of life of the people or animals that ingest them. They have also been associated with the feeling of general well-being that provoke, taken alone or as a complement to certain therapeutic treatments

Although it is a difficult term to define, quality of life means all that evaluation of the experience that subjects have of their own lives. So, the most authors conceive the quality of life as a construction complex and multifactorial, on which they can develop some forms of objective measurement through a series of indicators, but where it has an important specific weight the experience that the subject may have of himself. Some of the indicators to determine or assess the quality of life of a subject are physical function and role, body pain, health in general, vitality, etc.

Brief description of the drawings

As an example and to better illustrate the object of the present invention, two figures are attached, in the that:

Fig. 1 shows the graph of a chromatogram corresponding to the distribution of the molecular weights of a collagen hydrolyzate according to the state of the art; Y

Fig. 2 represents a chromatogram corresponding to the distribution of the molecular weights of a collagen hydrolyzate according to the invention and obtainable by the described procedure

Explanation of the invention.

The collagen hydrolyzate object of the present invention, with a very low total acidity and with a weight profile molecular molecules, between 1,000 Daltons and 50,000 Daltons, in its essence is characterized because it has an acidity total, expressed in Dornic degrees, less than 25ºD; and one conductivity in 1% solution by weight not exceeding 500 µS / cm.

Dornic grade is understood as sodium hydroxide at a concentration of 0.111 N consumed in a 10 g titration of collagen enzyme hydrolyzate, where the end point mark the turn of phenolphthalein.

According to another feature of the invention, the Total acidity, expressed in Dornic degrees, is less than or equal to 20ºD.

The enzymatic collagen hydrolyzate according to invention is also characterized in that it has a molecular weight between 5,000 and 20,000 Daltons.

According to another feature of the invention, the enzymatic collagen hydrolyzate comprises 99% by weight of hydrolyzed on dry product.

The enzymatic collagen hydrolyzate according to invention is obtained by a combination of reactions of Enzymatic hydrolysis and filtration with membranes that allow achievement of a final product with a very low total acidity without modify the molecular weight profile of the product.

Thus, it is also the object of the present invention, a method of obtaining a low collagen hydrolyzate total acidity according to the invention which, starting from dermal tissue of animal origin, processed in its native form, or previously submitted to aqueous extraction, at different temperatures, one or repeated Sometimes, and after acidic or basic hydrolysis, it is characterized because it includes the stages of:

a) aqueous extraction of dermal tissue from starting at a temperature between 40ºC and 140ºC, during a time between 2 and 8 hours;

b) pH adjustment between 6.0 and 8.0 by solutions of acids or mineral bases;

c) filtration of the solution obtained in the stage b);

d) demineralization of the solution filtered by passing it through ion exchange resins and / or dialysis equipment by membranes;

e) ultrafiltration of the solution demineralized through membranes with a pore diameter of 1,000 to 50,000 Daltons;

f) enzymatic hydrolysis of the solution ultrafiltered, with an amount of enzyme between 1,000 IU and 4,000 IU per kilogram of dry product to hydrolyze and at a pH between the optimal pH of the enzyme ± 0.5 units of pH;

g) filtration of the collagen solution enzymatically hydrolyzed;

h) ultrafiltration through membranes with a pore diameter of 10,000 to 40,000 Daltons;

i) concentration of the hydrolyzate solution Collagen enzyme by heating, at temperatures less than or equal to 70 ° C, at a pressure between 10 and 0.0001 mm Hg, up to a final concentration of hydrolyzate Collagen enzyme 15% to 45% by weight;

j) drying of the enzymatic collagen hydrolyzate until obtaining a dry product with a humidity less than or equal to 8% and a density between 0.3 and 0.8 g / ml.

The process according to the invention is characterized also because in stage f) hydrolysis is performed Enzymatic solution ultrafiltered with an amount of enzyme between 1,500 IU and 2,500 IU per kilogram of product dry to hydrolyze and at a pH between the optimum pH of the enzyme ± 0.5 pH units.

According to another feature of the procedure of Obtaining low acidity collagen enzyme hydrolyzate In total, stage a) of aqueous extraction of the raw material is performed at a temperature between 45 ° C and 100 ° C, specifically between 50 ° C and 80 ° C and preferably for a while between 4 and 6 hours.

The process of obtaining hydrolyzate from low total acidity collagen according to the invention, it is characterized also because after the aqueous extraction the pH of the solution between 7.4 and 7.6.

In the particular case of hydrolyzate of collagen object of the present invention, the step of demineralization through dialysis systems is extended until achieve an absolute conductivity of less than 1000 µS / cm.

The process of obtaining hydrolyzate from collagen is characterized in that the ultrafiltration stage e) of the enzymatically hydrolyzed collagen solution is carried to out through membranes with a pore diameter of 5,000 to 20,000 Daltons.

According to another feature of the procedure of obtaining collagen hydrolyzate according to the invention, the step of concentration i) of the enzyme hydrolyzate solution of Collagen is made at a temperature less than or equal to 60 ° C. Said concentration step i) is carried out, in a preferred manner, at a pressure between 1.0 and 0.01 mm Hg.

Another object of the present invention is a functional food comprising the enzymatic hydrolyzate of collagen described above Said hydrolyzate has a weight molecular range between 1,000 and 50,000 Daltons, preferably between 5,000 and 20,000 Daltons, a total acidity expressed in degrees Dornic, less than 25ºD, a conductivity in solution at 1% in weight not exceeding 500 µS / cm and 99% by weight of hydrolyzate of collagen on dry product.

The functional food according to the invention is characterized in that it comprises raw or treated milk thermally

In addition, the functional food according to the invention It is also characterized by containing a selected compound regardless of: antioxidant compounds, salts minerals, vitamins and / or coenzymes, plant extracts, extracts of animal origin and hydrolyzate adjuvant compounds Collagen enzyme, or a mixture of all of them.

Another object of the present invention is a prepared with milk, or a nutritional supplement, which comprises an enzymatic collagen hydrolyzate, which has a weight molecular range between 1,000 and 50,000 Daltons, preferably between 5,000 and 20,000 Daltons, comprises 99% by weight of Collagen hydrolyzate on dry product, has a total acidity expressed in degrees Dornic, less than 25ºD, and a conductivity in 1% solution not exceeding 500 µS / cm.

The nutritional supplement according to the invention is also characterized because it also comprises a compound independently selected from: antioxidant compounds, mineral salts, vitamins and / or coenzymes, plant extracts, animal extracts and hydrolyzate adjuvant compounds Collagen enzyme, or a mixture of all of them.

The object of the present invention is also the use of an enzymatic collagen hydrolyzate that has a weight molecular range between 1,000 and 50,000 Daltons, preferably between 5,000 and 20,000 Daltons, with 99% by weight hydrolyzate of collagen on dry product, and with a total acidity expressed in Dornic degrees, less than 25ºD, and a conductivity in solution at 1% by weight not exceeding 500 µS / cm, for the preparation of a Dairy product, nutritional supplement or functional food for improve the functional capacity and / or quality of life of a mammal with functional difficulties derived from alterations of articular cartilage

The present invention also aims at the use of collagen enzyme hydrolyzate for preparation of a dairy product, nutritional supplement or functional food to improve physical function.

By dairy product must be understood milk in all its forms (whole, skimmed, semi-skimmed, etc.) and any milk derivative such as cheeses, margarines, creams, etc.

Another object of the present invention is the use of the collagen enzyme hydrolyzate for the preparation of a Dairy product, nutritional supplement or functional food for attenuate the sensation of body ache.

The present invention also aims at the use of collagen enzyme hydrolyzate to prepare a Dairy product, nutritional supplement or functional food for improve overall health

Another object of the present invention is the use of the collagen enzyme hydrolyzate to prepare a product dairy, nutritional supplement or functional food to improve vitality.

The invention also relates to the use of enzymatic collagen hydrolyzate for the preparation of a Dairy product, nutritional supplement or functional food for improve social function, emotional role and / or mental health.

The use according to the invention is further characterized because the daily dose of collagen enzyme hydrolyzate is between 0.1 and 0.25 g / kg body weight.

The use according to the invention is also characterized because it is for a preparation in solution or suspension form oral.

According to another characteristic of the use of enzymatic collagen hydrolyzate according to the invention, it is for a preparation in the form of dispersible tablets or powders in water or milk.

The use of enzymatic collagen hydrolyzate according to the invention, it is characterized in that the mammal with functional difficulties derived from alterations of Articular cartilage is a human.

Detailed description of the invention

The characteristics are detailed below. Essentials of a Low Acid Collagen Enzymatic Hydrolyzate according to the invention, based on a possible method of obtaining it, also object of the invention, highlighting the advantages of both the hydrolyzate and its process of obtaining.

In the aforementioned procedure, tissue is split dermal of animal origin, obtained under hygienic conditions, of animal species suitable for human consumption (cattle, pigs, birds and fish). These tissues can be processed in their form native, or previously subjected to aqueous extraction, at different temperatures, once or repeatedly, and subsequent acid hydrolysis or basic At this point, tissues contain amounts of collagen ranging between 2% and 12%, resulting especially valid those with a residual content of more than 5%.

In the particular case of tissues subjected to previous extraction, it will be by-products of processes of obtaining jellies. The processes of gelatin extraction Classics are broadly based on tissue maceration dermal in conditions of extreme acidity or alkalinity, during a Total time 4 to 24 hours, followed by a water wash demineralized, a decantation of the fat released and a neutralization with inorganic acid or basic solutions. Subsequently, the gelatin is solubilized, by extracting the solid tissue with aqueous solvents, in In our particular case, the solvent is demineralized water. In This point separates the gelatin solution from the solid residue. The aqueous fraction is subjected to selective precipitation of proteins non-collagenic, elimination of these by filtration, demineralization, drying and grinding. The solid fraction, which in normal conditions would represent a major problem due to its high load of organic matter, according to this invention, in the raw material of a process to obtain Collagen hydrolyzate with low total acidity.

In order to obtain a hydrolyzate of collagen with characteristics that make it suitable for production of dairy products, the raw material described previously it is subjected to a plurality of stages until obtaining an enzymatic hydrolyzate of high purity collagen, of weight molecular controlled and with characteristics particular and special physicochemical that make it suitable for use in the production of dairy products, especially those who undergo treatments thermal.

In the first stage, the raw material of heading, both dermal tissue natively, and the by-product of processes of obtaining gelatin previously described, they are subjected to an aqueous extraction at a temperature between 40ºC and 140ºC and for a time between 2 and 8 hours. Preferably, the aqueous extraction is performed between 45 ° C and 100 ° C; and more preferably between 50 ° C and 80 ° C Extraction time may also vary and in a way Preferred usually lasts 2 to 6 hours.

Then, the pH of the extracted solution is adjusts between 6.5 and 8.0, using acid or base solutions minerals For this purpose, for example solutions of hydrochloric acid. In a preferred and more precise way the pH is fits between 7.4 and 7.6.

Once the pH is adjusted, the solution is filtered for example by using porous glass filters, polyvinyl acetate membranes, nylon fibers, carbon activated or rotary diatomaceous earth filters. Evidently, combinations of all these filters can be used until obtaining a transparent product

Said filtered solution is subjected to a subsequent demineralization stage posing as phonic exchange resins or dialysis equipment. To do this, it employ mixed resins, for example, anion exchange followed by cation exchange or vice versa. With this stage you manages to demineralize the filtered solution until obtaining a new solution with an absolute conductivity of less than 1,000 µS / cm and a total content of less than 0.5% mineral salts, or at least a content of mineral salts by weight of less than 1.0%, so that the collagen richness over dry extract is between 99% and 99.5% by weight.

The parameters to control at this stage of demineralization are the amount of solution to demineralize, which must be between 20 and 300 liters per kilogram of resin, with a range of 80 to 120 liters of solution per kilogram of resin. Another important parameter is the speed of passage of the solution to demineralize, which must be between 5 liters / minute / kg of resin and 100 liters / minute / kg of resin. Preferably the speed used It is 20 to 40 liters / minute / kg of resin. In the particular case of use of dialysis equipment, the collagen solution, previously filtered, will be subjected to successive steps through the membranes dialysis, in purge recirculation configuration, up to achieve a steady state in which conductivity absolute of the solution does not exceed 1,000 µS / cm.

After the demineralization stage, the solution is ultrafiltered by passing through membranes with a Pore diameter of 1,000 to 40,000 Daltons. With this stage you aim to eliminate all fractions of low protein residues molecular weight, as well as all those mineral salts and amines biogens that are the cause of bad smells and bad taste, such like putrescine or cadaverine. Preferably, the membranes used for the ultrafiltration stage have a pore size of 20,000 Daltons.

After ultrafiltration, the resulting solution undergoes enzymatic hydrolysis with proteolytic enzymes under adequate temperature and pH conditions for said Enzymes can act.

By way of example, enzymes such as Papain, ficine or bromelain alone or in combination.

The objects of the present invention are also special conditions of enzymatic hydrolysis. For the Obtaining a collagen hydrolyzate of low acidity and weight molecular between 1,000 and 50,000 Daltons, preferably between 5,000 and 20,000 Daltons, controlled doses of enzymes are used proteolytic, at controlled temperature and time and constant pH. Enzyme amounts will be of the order of 1,000 IU to 4,000 IU per kilogram of dry product to hydrolyze. It has been observed that Enzyme concentrations between 1,500 IU and 2,500 IU per kilogram of dry product are preferred for obtaining Collagen hydrolysates with low total acidity.

Working temperatures, as well as Preferred hydrolysis times will depend on the enzyme, or combination of enzymes, used. In turn, the pH will be controlled during the entire hydrolysis to avoid a drift greater than 2 units with respect to the optimum pH of the enzyme, or combination of enzymes, preferably a pH will be maintained, during all hydrolysis, within a range of ± 0.5 units with respect to the optimum pH.

The range of temperatures at which the Enzymatic hydrolysis stage is usually comprised between 15ºC and 80ºC, depending on the optimum working range of the or enzymes Very satisfactory results are obtained in a range of temperatures from 40ºC to 70ºC. Enzymes are left to act over the ultrafiltered solution for 30 to 200 minutes, preferably from 90 to 120 minutes and at a pH of the solution of reaction between 5 and 9, more specifically between 6.8 and 7.8, although This value will depend on the enzymes used.

For a correct evolution of hydrolysis, it performs continuous pH control and natural drift is compensated of this parameter by adding diluted solutions of alkali, for example sodium hydroxide solution.

After the time set for the Enzymatic hydrolysis should be deactivated enzymes, which at being generally thermolabile enzymes is feasible by just increasing the temperature of the product at temperatures  90 ° C for 5 to 30 minutes.

The enzyme hydrolyzate solution of Collagen obtained is subjected to a new stage of filtration with the purpose of ensuring the absence of any type of particles that could destabilize the product, as well as give a total transparency to it. As an example, the filter a use can be frames and plates, deep filtration or porous glass filters.

In any case, this filtration stage of the collagen enzyme hydrolyzate solution is followed by a ultrafiltration stage through membranes with a diameter of pore of 1,000 to 40,000 Daltons. In fact, the objective of this ultrafiltration is to eliminate all those fractions of peptides greater than 20,000 Daltons, as well as fractions of very low molecular weight (<1,000 Daltons). Actually this ultrafiltration stage consists of a filtration or exclusion molecular to select all those fractions of hydrolyzate Collagen enzyme, especially molecular weight between 5,000 Daltons and 20,000 Daltons.

At this point, the solution of enzymatic collagen hydrolyzate is concentrated by heating at temperatures below or equal to 70 ° C with the objective of obtaining a concentrated product that can be dried, for example by atomization.

Preferably, the concentration stage it is carried out at a temperature less than or equal to 60 ° C and by applying vacuum, in order to evaporate the water to temperatures below the boiling temperature of this last. Thus, pressures ranging from 10 to 10 are usually used. 0.0001 mm Hg, preferably 1.0 to 0.01 mm Hg.

This concentration stage is given by finalized when final concentrations of enzymatic collagen hydrolyzate in 15% to 45% solution in weight. Specifically, a range of concentrations of the 30% to 45% by weight of collagen enzyme hydrolyzate in solution.

Finally, the hydrolyzate solution Collagen enzyme with a weight percentage of 30% 45% is dried until a dry product is obtained with a humidity less than or equal to 8% and a density between 0.3 g / ml and 0.8 g / ml, preferably between 0.4 g / ml and 0.6 g / ml.

This final stage of product drying is Performs by lyophilization or by atomization.

The procedure as described allows economically obtain an enzymatic collagen hydrolyzate with constant parameters from batch to batch and highly stable. The stability studies have revealed that the aforementioned Hydrolyzate has a shelf life of about 4 years stored at a controlled temperature of 25 ° C and a relative humidity of environment less than 60%.

The obtainable collagen enzyme hydrolyzate According to the procedure described above, characterized by its high purity and its characteristics Organoleptic suitable for administration in preparations oral Specifically, the enzymatic collagen hydrolyzate It comprises 99% by weight of hydrolyzate on dry product.

Said enzymatic collagen hydrolyzate, also object of the invention, has a molecular weight comprised between 1,000 Daltons and 50,000 Daltons, the rank being preferred molecular weights of 5,000 to 20,000 Daltons.

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The controlled parameters in the final product and its limits are set out below:

Wealth: Must be greater than 99% on product dry.

Humidity: Must be less than 8%.

Bulk density: Must be between 0.3 and 0.8 g / ml, preferably 0.4 and 0.6 g / ml.

pH (20% sol.): Must be between 6.0 and 7.0.

Acidity (Dornic degrees): Less than 25ºD.

Viscosity (20% sol.): Must be included between 15 and 25 mps.

Turbidity (20% sol.): Must be less than 25 NTU

Relative conductivity (1% sol.): Must be less than 500 µS / cm.

Ashes: Must be less than 1%.

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The characteristics of salinity, low acidity and molecular weights of the enzyme hydrolyzate according to the invention make it suitable and optimal for application in products dairy products or containing milk, without causing the degradation of said dairy product or its modifications are modified physical and / or organoleptic characteristics.

They are detailed below, by way of examples non-limiting, variants of the procedure for obtaining collagen hydrolyzate according to the invention. The details are also detailed controlled parameters in the enzymatic hydrolyzate obtainable by The procedure.

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Example one

7,500 kg of dermal tissue of porcine origin, obtained directly, and in hygienic conditions, from room Exploded, and stored at a temperature below 5 ° C, they are subjected to the following stages:

1.- Maceration in hydrochloric acid solution During 4 hours.

2.- Neutralization at pH higher than 4 and lower to 6 with sodium hydroxide.

3.- Extraction at 60ºC for 4 hours.

4.- Adjust pH to 7.5 using hydroxide sodium

5.- Filtration with rotary earth filter diatoms

6.- Demineralization by means of membranes dialysis, in recirculation and purge settings, up to steady state in which the absolute conductivity does not exceed 1,000 µS / cm.

7.- Ultrafiltration through a membrane of 20,000 Daltons.

8.- Enzymatically controlled hydrolysis adding 1,350,000 U.I. of endopeptidase of bacterial origin on the product, at pH 7.2, at 60 ° C for 110 minutes.

9.- Deactivation of enzymes by heat treatment at 90 ° C for 20 minutes.

10.- Filtration through frame filter and 25 plates.

11.- Ultrafiltration through membrane 5,000 Daltons.

12.- Concentration at 45 ° C with a vacuum of 0.15 mm Hg up to a solids concentration of 40 0/0

13.- Spray drying

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The product (collagen enzyme hydrolyzate) obtained with this process (805 Kg) presented the following physicochemical characteristics:

Wealth (on dry product): 99.5%.

Humidity: 4.5%

Bulk density: 0.483 g / ml.

pH (20% sol): 6.08.

Acidity (10 g): 19.8ºD. (According to the scale Dornic)

Viscosity (20% sol.): 20.9 mps.

Turbidity (20% sol.): 24.6 NTU.

Relative conductivity (0.5%): 315 µS / cm.

Ashes: 0.5%.

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Example 2

7,500 kg of dermal tissue of porcine origin, obtained directly, and in hygienic conditions, from room Exploded, and stored at a temperature below 5 ° C, they are subjected to the following stages:

1.- Maceration in hydrochloric acid solution During 4 hours.

2.- Neutralization at pH higher than 4 and lower to 6 with sodium hydroxide.

3.- Extraction at 65ºC for 4 hours.

4.- Adjust pH to 7.5 using hydroxide sodium

5.- Filtration with rotary earth filter diatoms

6.- Demineralization by means of membranes dialysis, in recirculation and purge settings, up to steady state in which the absolute conductivity does not exceed 1,000 µS / cm.

7.- Ultrafiltration through a membrane of 20,000 Daltons.

8.- Enzymatically controlled hydrolysis adding 2,000,000 U.I. of endopeptidase of bacterial origin on the product at pH 7.8 at 60 ° C for 110 minutes.

9.- Deactivation of enzymes by heat treatment at 90 ° C for 20 minutes.

10.- Filtration through frame filter and 10 plates.

11.- Ultrafiltration through membrane 5,000 Daltons.

12.- Concentration at 40ºC with a vacuum of 0.1 mm of Hg up to a solids concentration of 45%.

13.- Spray drying

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The product obtained (enzymatic hydrolyzate of collagen) with this process (950 Kg) presented the following physicochemical characteristics:

Wealth (on dry product): 99.6.

Humidity: 4.9 0/0.

Bulk density: 0.495 g / ml.

pH (20% sol): 6.12.

Acidity (10 g): 16,9ºD. (According to the scale Dornic)

Viscosity (20% sol.): 21.1 mps.

Turbidity (20% sol.): 18.5 NTU.

Relative conductivity (0.5%): 210 µS / cm.

Ashes: 0.4 0/0.

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Obviously, the enzymatic collagen hydrolyzate according to the invention, with a total acidity, expressed in Dornic degrees, less than 25 ° D; and a conductivity in 1% solution not exceeding 500 µS / cm, can be obtained by other procedures, such as biotechnological procedures, in which the immobilization of enzymes can be considered in removable and easily recoverable supports, or by the contribution of the enzymes of interest working " in situ " within cell units for example bacteria.

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Example of the evidence obtained through Application of the method according to the disclosed invention previously:

Under the conditions detailed above, obtains a low acidity collagen hydrolyzate, thus being compatible with the manufacture of nutritional supplements in which is understood as the main ingredient milk, or derivatives dairy products, and in which collagen hydrolyzate is present in concentrations between 2% and 15% by weight, without overcome a total acidity of 50º on the Dornic scale. In these conditions, mixtures of collagen and milk hydrolyzate, or Dairy derivatives, in the doses detailed above, are stable to the pasteurization and sterilization processes, and to the subsequent fermentation, in the production of supplements Food based on milk, or milk derivatives, enriched with enzymatic collagen hydrolyzate.

As an example, they are presented in Figs. 1 and 2 two chromatograms corresponding to the weight distribution Molecules of collagen hydrolysates, the first (Fig. 1) corresponds to a collagen hydrolyzate prepared according to the process classic, the second (Fig. 2) corresponds to the preparation according to process conditions detailed above. They are also cited the corresponding acid values, expressed in degrees of Dornic scale It can be seen that acidity has been achieved compatible with the use of collagen hydrolyzate in the formulation of pasteurized and fermented dairy products, at once the original molecular weight profile is preserved. The acidity reduction is 38%, while observing that the Molecular weight profile is equivalent.

Thus, in Fig. 1 the chromatogram of a collagen enzyme hydrolyzate, prepared by the procedure according to the state of the art, with acidity total of 36.37ºDornic.

Alternatively, in Fig. 2, which corresponds to a collagen hydrolyzate according to the invention and obtained by the process according to the invention, whose total acidity is of 22.56ºDornic.

Examples of functional foods and supplements Nutritional:

According to another of the objects of the present invention, examples of functional foods and nutritional supplements that comprise the low acidity collagen enzyme hydrolyzate according to the invention, obtainable according to the procedure above described

These functional foods or supplements Nutritional can also comprise a selected compound regardless of: antioxidant compounds, salts minerals, vitamins and / or coenzymes, plant extracts, extracts of animal origin and hydrolyzate adjuvant compounds Collagen enzyme. You can also use a mixture of all they.

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Example one

Functional food: yogurt

A yogurt has been prepared with good results with the following composition:

100

The taste of yogurt is pleasant, as well as its appearance and texture.

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Example 2

Functional food: Chocolate dessert

A chocolate dessert has been prepared with the following composition:

101

Both extensibility and viscosity, the texture and taste are not altered, while improving nutritional capacity of the food.

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Example 3

Functional food: drinkable yogurt

A drinkable yogurt has been prepared with the following composition:

102

This preparation admits different flavorings to get a range of drinkable yogurts with properties functional.

Example 4

Functional food: Milkshake and chocolate

A chocolate milkshake has been prepared without the addition of collagen enzyme hydrolyzate modifying the organoleptic characteristics of it.

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103

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Example 5

Functional food: Molten cheese

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104

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The addition of collagen hydrolyzate does not appreciably modifies the taste, extensibility or Creaminess of the product.

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Example 6

Nutritional supplement: Energy bars

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105

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Modifying some of the components is feasible to prepare bars to help control weight.

In all these examples it is observed that for the development of nutritional supplements or functional foods in Those that include milk, or a dairy derivative, total acidity of the product, expressed as the amount of sodium hydroxide consumed for the neutralization of 10 grams of hydrolyzate of collagen, according to the Dornic unit scale, must be less than 50. Preferably less than or equal to 40.

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Clinical trial to determine the improvement in quality of life and functional capabilities in humans

According to another of the objects of the In the present invention, the inventors have determined a new use of enzymatic collagen hydrolyzate obtainable by the process previously described. Specifically, the enzymatic hydrolyzate of Collagen is used for the preparation of a nutritional supplement or functional food to improve functional capacity and / or quality of life of a mammal, specifically a human, with functional difficulties derived from alterations of articular cartilage

So, in order to study the possible application of the new high collagen enzyme hydrolyzate purity and better bioavailability (lower molecular weight, between 5,000 and 20,000 Daltons) as functional food or as a component of functional foods or nutritional supplements, it was decided conduct a study on 100 volunteers, to whom 50 of they would administer this collagen enzyme hydrolyzate, in a dose of 10 g daily, equivalent to a dose between 0.1 and 0.25 g per kg of body weight of enzymatic collagen hydrolyzate and at the other 50 a daily dose of 1.5 g of sulfate glucosamine, equivalent to a dose between 0.015 and 0.0375 g per kg body weight Glucosamine Sulfate is widely used, both in Europe and in the United States, at these doses for the treatment of joint disorders in general and several studies validate that the administration of glucosamine is a means effective to improve pain and joint mobility with consecutive dysfunctions to alterations of the cartilage articular

The purpose of this trial was to assess the effect of the new enzymatic collagen hydrolyzate in those conditions where the known non-steroidal anti-inflammatory drugs (NSAIDs) are usually used, specifically in those disorders of functional capacity or quality of life derived from alterations of the cartilage.
cular

On the other hand, glucosamine sulfate is also widely used in Europe, and especially in states United, as a functional food or as a food member functional and nutritional supplements (the monograph of USP 24 it is included in nutritional supplements) at these same doses (1.5 g daily) and ensures that functions improve overall bodily, mainly mechanical (mobility, agility, ....) and that improve the general state and quality of life.

The clinical trial consisted of a study multicentre, double blind, parallel and comparative between the new Enzymatic hydrolyzate of collagen and glucosamine sulfate. The purpose was to compare the improvement in the quality of life (according to the generic SF-36 questionnaire (The MOS 36-item short-form health survey (SF-36). I. Conceptual framework and item selection. Med care 1992 Jun; 30 (6): 473-483) as well such as the effectiveness, safety and tolerability of both treatments administered orally for 90 days consecutive.

The study was conducted in 100 volunteers (of which 93 were evaluable) affected by alterations joints that induced pain, decreased mobility and notable alteration and loss of quality of life.

The subjects were distributed in two groups of 50 individuals, of which 47 were evaluable (hydrolyzed Collagen enzyme) and 46 (glucosamine sulfate) which, after washing week, received either 10 g of enzymatic hydrolyzate of collagen or 1.5 g of glucosamine sulfate daily during 90 consecutive days, therefore the duration of the study 3 months for each subject.

The quality of life was measured at the beginning and at finish both treatments, specifically, by generic SF-36 questionnaire, validated in a multitude of bibliographic studies and reviews and having been used in more than 4000 scientific articles over the last 20 years, originally from the Health Institute, New England Medical Center, Boston (Massachusetts, USA). In Spain, said questionnaire It has been validated by Alonso et al. (11) at the IMIM (Institute Municipal Medical Research) in Barcelona.

The SF-36 represents for everyone the authors who have carried out various validations of the questionnaire, an adequate, truthful and effective instrument very useful in Clinical research and practice.

With the term "quality of life", with the SF-36, was intended to assess aspects of the joint dysfunctions following alterations of articular cartilages that are not strictly clinical, but also aspects related to the daily life of the patient. It was also intended to assess how life was affected of the patient due to the presence of these joint dysfunctions, always from the point of view of the patient himself. This valuation was multidimensional and it was generally accepted to include, at least eight sections or parameters that act as indicators of functional capacity and / or quality of life.

The parameters evaluated correspond to the facets of daily life most commonly affected by the functional difficulties derived from alterations of the articular cartilage. The division of these parameters allows us to clearly differentiate activities and moments in the lives of people in which the pain derived from a joint alteration affects, or can affect, the correct development of activities and tasks. It also distinguishes between the affectation of the individual quality of life, the relationship with other people, or the performance of a
profession.

The parameters evaluated are, and are defined, as follow.

1. Physical function: Physical function is understood as the ability to perform basic activities of daily life such as personal care, basic mobility and the ability to make small or moderate efforts. This parameter allows us to assess the degree to which the alteration of articular cartilage limits us individually.
dual.

2. Physical role: For its part, the physical role is defines as the parameter that evaluates the ability to perform the activities of our job. It allows us to identify if our lack of health affects our work performance, or limits or prevents us from performing normally the tasks associated with our work. This parameter allows us assess how lack of health affects physical activities related to our environment and work performance.

3. Body pain: This parameter gives us the measure of perceived pain and its effect, both at work and in the relationship with our environment, as in individual activities Basics of everyday life.

4. General health: Parameter that values, by part of the evaluated, the perception of the state of health that you have of oneself As well as future perspectives and perception individual resistance to sickness.

5. Vitality: It is defined, for interpretation of this study, like the feeling of energy and vitality that It has of oneself. It also includes the individual perception of the resistance to tiredness and discouragement.

6. Social function: Parameter thanks to which we can assess how the alteration of the cartilage of the joints, and the problems derived from such alterations, They affect the development of a satisfactory social life. This parameter encompasses both physical and emotional problems. Us lets assess how these problems interfere with social life habitual.

7. Emotional role: It is understood by emotional role, and thus the degree to which the problems are defined for this study emotional derivatives of pain and capacity impairment physics affect work performance as well as completion satisfactory of other daily activities. The role assessment emotional gives us very valuable information about such factors important as performance or dedication at work.

8. Mental health: Parameter that allows us assess general mental health, considering depression, anxiety, self-control and general well-being.

Then, as a summary, it describe the eight sections or parameters that act as indicators of functional capacity and / or quality of life. (Table 1)

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TABLE 1 Parameters and meaning studied to determine the quality of life of a human with functional difficulties derived from alterations of the cartilage articular

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one

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The results obtained can be seen in the Table 2.

TABLE 2 Results of the generic questionnaire SF-36 applied to 100 volunteers affected by joint alterations that induced significant loss of quality of lifetime

2

From Table 2 it can be extracted that both the enzymatic hydrolyzate of collagen such as glucosamine sulfate, administered for 90 consecutive days at doses of 10 g and 1.5 g / day respectively, significantly improve the quality of life in Its different aspects.

But you can check that, unexpectedly, the values of the absolute differences between the initial visit and final are always more extensive with the enzymatic hydrolyzate of collagen than with glucosamine sulfate.

In aspects as important as health general, vitality, social function, physical function, physical work, emotional function and mental health we can check that the collagen enzyme hydrolyzate is always more effective in inducing the improvement of the different parameters investigated that glucosamine sulfate.

From the analysis of the global results obtained in this clinical study, the Advantages of using the object collagen enzyme hydrolyzate of the invention for the preparation of a functional food or nutritional supplement, to improve functional capacity and / or quality of life of people with functional difficulties derived from alterations of articular cartilage.

With the collagen enzymatic hydrolyzate of high purity object of the invention within the doses per Kg of body weight used in the clinical study (0.1 - 0.25 g / kg weight body / day) can therefore be prepared, due to its good taste and good organoleptic characteristics, different types of nutritional supplements or supplements, as solutions or drinkable suspensions, mixtures of water dispersible powders, tablets, capsules In the same way food can be prepared functional, such as juices, yogurts, liquid yogurts, custard, energy bars, slimming bars, etc., at the same time as the same collagen enzyme hydrolyzate, because of its good taste, can be itself the functional food itself.

In conclusion, it is firmly demonstrated that, in addition, the process of obtaining a hydrolyzate of collagen object of the invention, which valorizes the by-products Residual pollutants from obtaining gelatins, Not only does it allow profitable costs derived from treatment of these environmentally harmful by-products, it also allows to obtain a new enzymatic hydrolyzate of high purity and low molecular weight collagen which, due to its good taste and organoleptic characteristics, can be used both in Functional foods, such as yogurts, juices, desserts, bars energy, etc., as in nutritional supplements of the type multivitamin complexes, in the form of tablets, solutions, suspensions, etc.

In addition, the inventors have discovered a new use for this collagen enzyme hydrolyzate, which allows the quality of life of people or animals affected by dysfunctions derived from alterations in the cartilage of the joints, can be significantly improved in a way equivalent and more pleasant to the administration of anti-inflammatory, eliminating unwanted side effects of the latter.

Claims (29)

1. Collagen enzyme hydrolyzate, with a molecular weight between 1,000 and 50,000 Daltons, characterized in that it has a total acidity, expressed in Dornic degrees, less than 25ºD; and a conductivity in solution at 1% by weight not exceeding 500 µS / cm. 2. Enzymatic collagen hydrolyzate according to claim 1, characterized in that the total acidity, expressed in Dornic degrees, is less than 25 ° D, preferably less than or equal to 20 ° D. 3. Collagen enzyme hydrolyzate according to any one of the preceding claims, characterized in that it has a molecular weight between 5,000 and 20,000 Daltons. 4. Enzymatic collagen hydrolyzate according to any one of the preceding claims, characterized in that it comprises 99% by weight of hydrolyzate on dry product. 5. Procedure for obtaining a collagen hydrolyzate of low total acidity from dermal tissue of animal origin, in its native form, or from by-products of processes of obtaining gelatin, in which after extraction in aqueous medium of The gelatins, the described tissues, rich in collagen, are subjected to successive stages of aqueous extraction and subsequent acidic, basic or enzymatic hydrolysis, finally obtaining a residual by-product, characterized in that it comprises the steps of: a) aqueous extraction of the residual by-product a a temperature between 40 ° C and 140 ° C for a while between 2 and 8 hours; b) pH adjustment between 6.5 and 8.0 by solutions of acids or mineral bases; c) filtration of the solution obtained in the stage b); d) demineralization of the solution filtered by passing it through ion exchange resins and / or dialysis equipment by membranes up to a steady state in that the absolute conductivity of the solution does not exceed 1,000 µS / cm; e) ultrafiltration of the solution demineralized through membranes with a pore diameter of 1,000 to 40,000 Daltons; f) enzymatic hydrolysis of the solution ultrafiltered with an amount of enzyme between 1,000 IU and 4,000 IU per kilogram of dry product to hydrolyze and at a pH between the optimal pH of the enzyme ± 0.5 units of pH; g) filtration of the collagen solution enzymatically hydrolyzed; h) ultrafiltration through membranes with a pore diameter of 10,000 to 40,000 Daltons; i) concentration of the hydrolyzate solution Collagen enzyme by heating at temperatures below or equal to 70 ° C at a pressure between 10 and 0.0001 mm Hg to a final concentration of hydrolyzate Collagen enzyme 15% to 45% by weight; j) drying of the enzymatic collagen hydrolyzate until obtaining a dry product with a humidity less than or equal to 8% and a density between 0.3 and 0.8 g / ml. 6. Method according to claim 5, characterized in that in step f) the enzymatic hydrolysis of the ultrafiltered solution is carried out with an amount of enzyme between 1,500 IU and 2,500 IU per kilogram of dry product to be hydrolyzed and at a pH between pH Optimum enzyme ± 0.5 pH units. 7. Method for obtaining collagen hydrolyzate according to any one of claims 5 or 6 above, characterized in that step a) of aqueous extraction of the residual by-product is carried out at a temperature between 45 ° C and 100 ° C, specifically between 50 ° C and 80 ° C for a period of time between 4 and 6
hours. 8. Method for obtaining collagen hydrolyzate according to any one of claims 5 to 7 above, characterized in that after the aqueous extraction the pH of the solution is adjusted between 7.4 and 7.6. 9. Method for obtaining collagen hydrolyzate according to any one of claims 5 to 8 above, characterized in that the ultrafiltration step of the enzymatically hydrolyzed collagen solution is carried out through membranes with a pore diameter of 5,000 to 20,000 Daltons.

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10. Method for obtaining collagen hydrolyzate according to any one of claims 5 to 9 above, characterized in that the step of concentration i) of the collagen enzyme hydrolyzate solution is carried out at a temperature less than or equal to 60 ° C. 11. Method for obtaining collagen hydrolyzate any one of claims 5 to 10 above, characterized in that the concentration step i) of the collagen enzyme hydrolyzate solution is carried out at a pressure between 1.0 and 0.01 mm of Hg. 12. Functional food comprising the enzymatic collagen hydrolyzate according to any one of the claims 1 to 4 above. 13. Functional food according to claim 12, characterized in that it comprises raw or heat treated milk. 14. Functional food according to claims 12 or 13 further comprising a compound independently selected from: antioxidant compounds, mineral salts, vitamin and / or coenzymes, plant extracts, animal extracts and adjuvant compounds of enzymatic collagen hydrolyzate, or a mixture of all they. 15. Milk-based nutritional supplement that comprises the enzymatic collagen hydrolyzate according to a any one of claims 1 to 4 above. 16. Nutritional supplement according to claim 15 further comprising a selected compound regardless of: antioxidant compounds, salts minerals, vitamin and / or coenzymes, plant extracts, extracts of animal origin and hydrolyzate adjuvant compounds Collagen enzyme, or a mixture of all of them. 17. Use of enzymatic collagen hydrolyzate according to any one of claims 1 to 4 for the preparation of a dairy product, nutritional supplement or functional food to improve functional capacity and / or quality of life of a mammal with functional difficulties derived from alterations of articular cartilage. 18. Use according to claim 17 for the preparation of a dairy product, nutritional supplement or Functional food to improve physical function. 19. Use according to claim 17 for the preparation of a dairy product, nutritional supplement or Functional food for the improvement of the physical role. 20. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve the sensation of body pain. 21. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve overall health. 22. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve vitality. 23. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve social function. 24. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve emotional role. 25. Use according to claim 17 for the preparation of a dairy product, a nutritional supplement or Functional food to improve mental health. 26. Use according to any one of the claims 17 to 25 above, wherein the daily dose of enzymatic collagen hydrolyzate is between 0.1 and 0.25 g / kg body weight. 27. Use according to any one of the preceding claims 17 to 25, characterized in that it is for a preparation in the form of oral solutions or suspensions. 28. Use according to any one of the preceding claims 17 to 25, characterized in that it is for a preparation in the form of tablets, capsules or water dispersible powders. 29. Use according to any one of the claims 17 to 28 above, wherein the mammal with functional difficulties derived from alterations of Articular cartilage is a human.