WO2007090504A1

WO2007090504A1 - Hydrolysed collagen and uses therof

Info

Publication number: WO2007090504A1
Application number: PCT/EP2007/000330
Authority: WO
Inventors: Julian Agut Sanchez; Carlos Fernandez Navarro; Josep Junca Busquets; Miguel Junca Riuro; Ferran Junca Riuro; Gabriel Espelleta Gil; Jaime MELENDO BAÑOS
Original assignee: Masterfam, S.L.; Quimera Ingenieria Biomedica, S.L.
Priority date: 2006-02-10
Filing date: 2007-01-16
Publication date: 2007-08-16
Also published as: ES2288791A1; ES2288791B1

Abstract

Production procedure for hydrolysed collagen according to the invention using by-products from gelatin-producing processes, wherein after aqueous extraction of gelatins, tissues rich in collagen are subjected to successive steps of extraction in aqueous solution and subsequently to acid, base or enzymatic hydrolysis, finally producing a residual by-product. The procedure comprises, among others, the following steps: aqueous extraction of the residual by-product; filtration, demineralisation and ultrafiltration of the filtered solution; enzymatic hydrolysis; concentration of the enzymatically-hydrolysed collagen solution and drying of the enzymatically-hydrolysed collagen. The invention also relates to enzymatically-hydrolysed collagen produced by the procedure and to uses thereof.

Description

D E S C R I P T I O N

HYDROLYSED COLLAGEN AND USES THEROF

Technical field of the invention

The present invention relates to a procedure for producing a hydrolysed collagen from by-products of gelatin-producing processes, wherein after extracting gelatins in aqueous solution, tissues rich in collagen are subjected to successive steps of extraction in aqueous solution and subsequently to acid or base hydrolysis, finally producing a residual by-product. Another object of the invention is an enzymatically-hydrolysed collagen and uses thereof.

Background of the invention

Collagen is a fibrous protein molecule that is present in varying quantities in all types of connective tissue, be this skin, muscle, tendons, cartilage, blood vessels, ligaments or other tissues. Collagen is made up of amino acids linked by the peptide bond (HN-C=O) to form sequences thereof that define peptides of different molecular weights with different structural functions in the organism. All the essential amino acids are found in these sequences, except tryptophan, of which glycine, proline and hydroxyproline are particularly abundant.

For a long time the usual production process of collagen has been carried out using two methods. The first consists of directly extracting tissues rich in collagen, such as skin, bones and cartilage, by direct hydrolysis using proteolytic enzymes, followed by several purification steps until an end product is achieved, which is then dried. Using this first method, it is possible to produce light-coloured products with a mild smell and flavour. However, it is very expensive to produce hydrolysed collagen by this method, which greatly limits its applications. According to an alternative method, hydrolysed collagen is produced using extraction methods free from enzymes by hydrolysis in acid or base solutions using skin, bones and/or cartilage from which the gelatins have previously been extracted in aqueous solution (products with a higher market value, used chiefly in food and in the pharmaceutical industry). Hydrolysed collagen produced in this manner presents irregular molecular weights, an unpleasant flavour and smell, a dark colour and considerable levels of biogenic amines such as cadaverine and putrescine. The cost of producing hydrolysed collagen using this second method is lower, but unfortunately, due to the poor organoleptic characteristics of the hydrolysate produced, its field of application is quite limited.

Another of the drawbacks of the usual methods of obtaining hydrolysed collagen is that, in both cases, the collagen content of the end product is lower than 98% of the dried product, which generally means that they are not very profitable. Moreover, bearing in mind the supply sources used, preferably bovid and fish skins, it is very common for the hydrolysate to be contaminated with unconventional or contaminant impurities and agents. However, hydrolysed collagen is widely used in a large variety of fields, e.g. in the field of cosmetics, in the paper industry, in the food industry or in the pharmaceutical industry. In the case of the pharmaceutical and the food industry, the hydrolysed collagen is required to be as pure as possible, which, as has been mentioned above, makes the production process considerably more expensive. In earlier research, and according to patent US 6211143, it has already been shown that when hydrolysed gelatin with a lower collagen purity and a higher molecular weight (up to 10,000 Da) was taken by athletes and people whose normal activities involved particular efforts, they managed to maintain or improve the integrity of their joint cartilage. It can be deduced from patent ES 2087030 that hydrolysed gelatin is also useful as an adjuvant in treatments designed to compensate for a calcium deficiency.

Another application can also be deduced from patent ES 2104507, which discloses compositions for the preparation of medicinal products for the treatment and prevention of hip dysplasia in dogs.

Several recent studies have shown that enzymatically-hydrolysed collagen is well absorbed when administered orally and is distributed with great affinity preferably in the tissues of the joint cartilage (Oesser S, et al. "Oral administration of ¹⁴C Labelled Gelatin hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/BL)". J Nutr 1999; 129: 1891-1895.). This distribution spectrum results in an increase in the volume of cartilaginous mass, the action of which can reduce pain and inflammation and improve the motility of said joints. See the following publications that refer to this aspect: Adam M. Therapie der osteoarthrosis. Welche Wirkung haben gelatinepraparate" Therapiewoche 1991 , 41: 2458-2461 ; Arquer A, et al. "Physical exercise in the elderly people". Selecciόn 1996; 5 (3): 121 -128; Ribas J₁ et al. "Effects of gelatine hydrolysates in 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 Il collagen on rheumatoid arthritis." Science 1993; 261: 1727-1730.

The researchers of the present invention have developed a procedure for producing a hydrolysed collagen with significant advantages over the current procedures, whilst also being more economical, and they have discovered an application for the resulting hydrolysate that is also highly beneficial for the population.

People are becoming increasingly concerned with animal and human dietetics and nutrition. As a result, many preparations have come onto the market known as nutritional supplements, which provide animals or people with the necessary amount of vitamins, mineral salts or essential amino acids. Collagen has also been used in the field of nutritional supplements to produce preparations that favour the recovery of cartilage in animals suffering from diseases related to this type of tissue.

The term "nutritional supplement" refers to any additional dietary contribution that is taken orally and which contains a nutritious ingredient designed to complement the normal diet. Some examples of dietary or nutritional supplements are vitamins, minerals, herbs (a single herb or a mixture of several herbs), other botanical products, amino acids and components of foodstuffs such as enzymes. Nutritional supplements tend to be distributed in different forms, such as pills. capsules, soft gelatin capsules, gelatin capsules, liquids and powders.

Also in the field of nutrition, so-called functional foods have become increasingly well known recently. Functional foods are considered to be foods that are consumed as part of a normal diet and which contain biologically active components that are beneficial to one's health and reduce the risk of suffering diseases. Examples of functional foods are those containing certain minerals, vitamins, fatty acids or carbohydrates, foods to which biologically active substances, such as phytochemical or other antioxidants, have been added and probiotics, which contain live cultures of beneficial microorganisms. Some yoghurts with probiotics are examples of functional foods, as are some margarines enriched with sterol esters, and eggs rich in omega-3 type fatty acids.

Both functional foods and nutritional supplements have been scientifically proven to improve the quality of life of the people or animals that consume them. They have also been linked to the feeling of overall well-being that they bring about, whether taken alone or as a complement to certain therapeutic treatments.

Although it is a difficult term to define, "quality of life" means any assessment of the subject's experience of his or her own life. Therefore, most authors see quality of life as a complex and multi-factor construction for which some forms of objective measurement can be developed by means of a number of indicators, but where a great deal of emphasis is placed specifically on the subject's own experience. Some of the indicators used in determining or assessing a subject's quality of life are physical functioning and role, body pain, general state of health, vitality, etc.

Explanation of the invention

The procedure for producing a hydrolysed collagen according to the invention, using by-products of gelatin-producing processes, wherein after aqueous extraction of gelatins, tissues rich in collagen are subjected to successive steps of extraction in aqueous solution and subsequently to acid or base hydrolysis, finally producing a residual by-product, is characterised in that it comprises the following steps: a) aqueous extraction of the residual by-product at a temperature between 65⁰C and 14O⁰C for between 2 and 8 hours; b) adjustment of the pH to between 6.5 and 8.0 using mineral acid or base solutions; c) filtration of the solution produced from step b); d) demineralisation of the filtered solution by passing it through ion exchange resins; e) ultrafiltration of the demineralised solution through membranes with a pore diameter of 1 ,000 to 40,000 Da; f) enzymatic hydrolysis of the ultrafiltered solution; g) filtration of the enzymatically-hydrolysed collagen solution; h) ultrafiltration through membranes with a pore diameter of 1 ,000 to 40,000 Da; i) concentration of the enzymatically-hydrolysed collagen solution by heating it at temperatures of 7O⁰C or lower at a pressure of between 10 and 0.0001 mm Hg to a final concentration of enzymatically-hydrolysed collagen of 15% to 35% by weight; j) drying of the enzymatically-hydrolysed collagen to produce a dry product with a humidity of 8% or less and a density of between 0.3 and 0.8 g/ml.

According to another characteristic of the production procedure for enzymatically-hydrolysed collagen, step a) aqueous extraction of the residual byproduct is carried out at a temperature of between 65⁰C and 100⁰C, specifically between 85⁰C and 95⁰C and preferably for between 4 and 6 hours.

The production procedure for hydrolysed collagen according to the invention is also characterised in that, after aqueous extraction, the phi of the solution is adjusted to between 7.4 and 7.6.

According to another aspect of the production procedure for hydrolysed collagen according to the invention, step e) ultrafiltration of the demineralised solution is carried out using membranes with a pore diameter of 20,000 Da. The production procedure for hydrolysed collagen is characterised in that the ultrafiltration step of the enzymatically-hydrolysed collagen solution is carried out using membranes with a pore diameter of 5,000 to 20,000 Da.

According to another characteristic of the production procedure for hydrolysed collagen according to the invention, the concentration step i) of the enzymatically-hydrolysed collagen solution is carried out at a temperature of 6O⁰C or lower. Said concentration step i) is preferably carried out at a pressure of between

1.0 and 0.01 mm Hg.

Another object of the present invention is an enzymatically-hydrolysed collagen that can be produced by the above-described procedure. The enzymatically-hydrolysed collagen according to the invention is characterised in that it has a molecular weight of between 1 ,000 and 50,000 Da.

According to another characteristic of the enzymatically-hydrolysed collagen according to the invention, the molecular weight is between 5,000 and 20,000 Da.

The enzymatically-hydrolysed collagen according to the invention is also characterised in that the hydrolysate comprises 99% of the weight of the dry product.

Another object of the present invention is a functional food that comprises the enzymatically-hydrolysed collagen produced by the above-described procedure, the hydrolysate having a molecular weight of between 1 ,000 and 50,000 Da, preferably between 5,000 and 20,000 Da, and comprising 99% of the weight of the dry product.

The functional food according to the invention is characterised in that it also comprises a compound chosen independently from: antioxidant compounds, mineral salts, vitamins and/or coenzymes, plant extracts, extracts of animal origin and adjuvant compounds of the enzymatically-hydrolysed collagen, or a mixture of all of these. Another object of the present invention is a nutritional supplement that comprises the enzymatically-hydrolysed collagen produced by the above-described procedure, the hydrolysate having a molecular weight of between 1 ,000 and 50,000

Da, preferably between 5,000 and 20,000 Da, and comprising 99% of the weight of the dry product.

The nutritional supplement according to the invention is characterised in that it also comprises a compound chosen independently from: antioxidant compounds, mineral salts, vitamins and/or coenzymes, plant extracts, extracts of animal origin and adjuvant compounds of the enzymatically-hydrolysed collagen, or a mixture of all of these.

Another object of the present invention is the use of an enzymatically- hydrolysed collagen produced by the above-described procedure, the hydrolysate having a molecular weight of between 1 ,000 and 50,000 Da, preferably between 5,000 and 20,000 Da, and comprising 99% of the weight of the dry product, for the preparation of a nutritional supplement or functional food to improve the functional capacity and/or quality of life of a mammal with functional difficulties deriving from articular cartilage disorders.

Another object of the present invention is the use of the enzymatically- hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve physical functioning.

The invention also relates to the use of the enzymatically-hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve the physical role.

Another object of the present invention is the use of the enzymatically- hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve the sensation of body pain.

Another object of the present invention is the use of the enzymatically- hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve general health. Another object of the present invention is the use of the enzymatically- hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve vitality.

The invention also relates to the use of the enzymatically-hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve social functioning.

Another object of the present invention is the use of the enzymatically- hydrolysed collagen for the preparation of a nutritional supplement or functional food to improve the emotional role.

The use according to the invention is also characterised in that the daily dose of enzymatically-hydrolysed collagen is between 0.1 and 0.25 g/kg of body weight. The use according to the invention is also characterised in that it is for a preparation in the form of an oral solution or suspension.

According to another characteristic of the use of the enzymatically- hydrolysed collagen according to the invention, it is for a preparation in the form of tablets or water-soluble powders. The use of the enzymatically-hydrolysed collagen according to the invention is characterised in that the mammal with functional difficulties deriving from articular cartilage disorders is a human.

Detailed description of the invention The production procedure for hydrolysed collagen according to the invention is given below, highlighting the advantages thereof and specifying its essential characteristics.

Said procedure begins with a residual by-product produced from repeated aqueous extractions of tissues rich in collagen, from which gelatins are first extracted and then subjected to subsequent aqueous extraction steps followed by acid or base hydrolysis until said residual by-product is produced. Until now, this residual by-product was disposed of because gelatin could no longer be produced therefrom, and furthermore, due to its high organic content it was a contaminant.

In order to reuse the residual by-product of the gelatin production processes whilst also recycling it to avoid harming the environment, it is subjected to a number of steps until a high-purity enzymatically-hydrolysed collagen with a controlled molecular weight is produced.

In the first step, the residual by-product is subjected to an aqueous extraction at a temperature of between 65⁰C and 14O⁰C for between 2 and 8 hours. Preferably, the aqueous extraction is carried out between 75⁰C and 100⁰C; and more preferably between 85⁰C and 95⁰C. The extraction time can also vary and preferably lasts for 4 to 6 hours.

The pH of the extracted solution is then adjusted to between 6.5 and 8.0, using mineral acid or base solutions. Hydrochloric acid solutions, for example, can be used to do this. Preferably, and more exactly, the pH is adjusted to between 7.4 and 7.6. Once the phi has been adjusted, the solution is filtered, e.g. using porous glass filters, polyvinyl acetate membranes, nylon fibres, activated carbon or rotating diatomaceous earth filters. Obviously, combinations of all these filters can be used to achieve a transparent product. Said filtered solution is subsequently subjected to a demineralisation step, passing it through ion exchange resins. Mixed resins are used for this, e.g. anionic exchange followed by cationic exchange or vice versa. This step makes it possible to demineralise the filtered solution in order to achieve a new solution with 0.5% mineral salts, or at least a weight content of mineral salts of less than 1.0%, so that the collagen content is between 99% and 99.5% of the weight of the dry product.

The parameters that must be controlled in this demineralisation step are the quantity of solution to be demineralised, which must be between 20 and 300 litres per kilogram of resin, preferably within the range of 80 to 120 litre of solution per kilogram of resin. Another important parameter is the flow rate of the solution to be demineralised, which must be between 5 litres/minute/kg of resin and 100 litres/minute/kg of resin. The rate used is preferably from 20 to 40 litres/minute/kg of resin.

This demineralisation step can be repeated according to the mineral content or charge that the starting raw material, i.e. the starting residual by-product, is supposed to have.

After the demineralisation step, the solution is ultrafiltered by passing it through membranes with a pore diameter of 1,000 to 40,000 Da. The purpose of this step is to remove all fractions of protein moieties with a low molecular weight, and all the mineral salts and biogenic amines that cause a bad smell and flavour, such as putrescine or cadaverine. Preferably, the membranes used for the ultrafiltration step have a pore size of 20,000 Da.

After ultrafiltration, the resulting solution is subjected to enzymatic hydrolysis with proteolytic enzymes at a suitable temperature and pH to enable said enzymes to act. For instance, enzymes such as papain, ficin or bromelain could be used alone or in combination.

Quantities of 1 ,000 I. U. to 6,000 I. U. of enzyme per kilogram of the dry product to be hydrolysed are used to carry out the enzymatic hydrolysis. It has been observed that an enzyme concentration range of 1 ,500 I. U. to 3,500 I. U. per kilogram of dry product is preferable and optimum. The range of temperatures at which the enzymatic hydrolysis step is carried out is usually between 15⁰C and 8O⁰C, depending on the optimum working range of the enzyme(s). Very satisfactory results are achieved within a temperature range of 4O⁰C to 70⁰C. The enzymes are allowed to act on the ultrafiltered solution for 30 to 200 minutes, preferably for 90 to 120 minutes, the pH of the reaction solution being between 5 and 9, and more specifically between 6.8 and 7.8.

At the end of the time allowed for enzymatic hydrolysis, the enzymes must be deactivated, which is possible in the case of generally thermolabile enzymes simply by increasing the temperature of the product to 90° C for between 5 and 30 minutes. The resulting enzymatically-hydrolysed collagen solution is subjected another filtration step in order to ensure the absence of any kind of particles that could destabilise the product, and to ensure that it is completely transparent. For example, the filter that is used could be with frames and plates, deep filtration or porous glass filters. In any case, this filtration step of the enzymatically-hydrolysed collagen solution is followed by an ultrafiltration step through membranes with a pore diameter of 10,000 to 40,000 Da. In fact, the purpose of this ultrafiltration is to remove all peptide fractions of more than 20,000 Da and fractions with a very low molecular weight (< 5,000 Da). This ultrafiltration step actually consists of molecular filtration or exclusion to select all the fractions of enzymatically-hydrolysed collagen that have a molecular weight of between 5,000 Da and 20,000 Da.

Having reached this point, the enzymatically-hydrolysed collagen solution is concentrated by heating it to temperatures of 7O⁰C or less in order to achieve a concentrated product that can be dried, e.g. by spray drying. The concentration step is preferably carried out at a temperature of 6O⁰C or less, by applying a vacuum to evaporate the water at temperatures lower than the boiling temperature of the water. Pressures of between 10 and 0.0001 mm Hg, preferably 1.0 to 0.01 mm Hg, can therefore be used.

Said concentration step is completed when end concentrations with a 15% to 35% weight of enzymatically-hydrolysed collagen in solution are achieved. Specifically, a preferred range of concentrations is a 20% to 25% weight of enzymatically-hydrolysed collagen in solution.

Finally, the enzymatically-hydrolysed collagen solution, whose weight percentage thereof is 15% to 35%, is dried until a dry substance is achieved with a humidity of 8% or less and a density of between 0.3 g/ml and 0.8 g/ml, preferably between 0.4 g/ml and 0.6 g/ml. This final drying step is carried out by freeze-drying or spray drying.

The procedure described above enables the economical production of an enzymatically-hydrolysed collagen that has constant parameters from one batch to another and is highly stable. Stability studies that have been carried out have shown that said hydrolysate has a shelf-life of about 4 years when stored at a controlled temperature of 25°C and at a relative atmospheric humidity below 60%.

The enzymatically-hydrolysed collagen produced according to the above- described procedure is characterised in that it has high purity and its organoleptic characteristics are suitable for administration in oral preparations. Specifically, the enzymatically-hydrolysed collagen comprises 99% hydrolysate in the weight of the dry product.

Said enzymatically-hydrolysed collagen, which is also the object of the invention, has a molecular weight of between 1 ,000 Da and 50,000 Da, the preferred range being molecular weights of 5,000 to 20,000 Da. The parameters controlled in the end product and the limits thereof are described below:

Assay: Must be above 99% of the dry product.

Humidity: Must be less than 6%.

Apparent density: Must be between 0.3 and 0.8 g/ml, preferably between 0.4 and 0.6 g/ml. pH 20% sol.): Must be between 6.0 and 7.0.

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

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

Relative conductivity (0.5% sol): Must be lower than 500 μS/cm. Ashes: Must be less than 1%.

Some non-limiting examples of variants of the production procedure for hydrolysed collagen according to the invention are given below. The parameters controlled in the enzymatic hydrolysate produced by the procedure are also described.

EXAMPLE 1

7,500 kg of residual by-products from the production of gelatins are subjected to the following steps:

1.- Extraction at 85° C for 4 hours. 2.- Adjustment of the pH to 7.5 using hydrochloric acid.

3.- Filtration with rotating diatomaceous earth filter. 4.- Demineralisation using first an anionic resin and then a cationic resin at a rate of 40 litres/minute and passing 90 litres per kg of resin. 5.- Ultrafiltration through a 20,000 Da membrane.

6.- Controlled enzymatic hydrolysis by adding 1 ,350,000 I. U. of Bromelain to the product at pH 7.2 and at 60° C for 110 minutes.

7.- Deactivation of the enzymes through heat treatment at 90° C for 20 minutes.

8.- Filtration through a 25 μ frames and plates filter. 9.- Ultrafiltration through a 5,000 Da membrane. 10.- Concentration at 45° C with a 0.15 mm Hg vacuum to a 22 % concentration of solids. 11.- Spray drying.

The product (enzymatically-hydrolysed collagen) produced by this process (98.9 kg) presented the following physicochemical characteristics:

Assay (of the dry product): 99.5 %

Humidity: 4.5 %

Apparent density: 0.483 g/ml pH (sol. at 20%): 6.08

Viscosity (sol. at 20%): 20.9 mps

Turbidity (sol. at 20%): 24.6 NTU

Relative conductivity (0.5%): 315 μS/cm

Ashes: 0.5 %

EXAMPLE 2

1.- Extraction at 90° C for 4 hours. 2.- Adjustment of the pH to 7.5 using hydrochloric acid.

3.- Filtration using a nylon fibre cartridge filter followed by a rotating diatomaceous earth filter.

4.- Demineralisation using an ion exchange mixed resin at a rate of 30 litres/minute and passing 120 litres per kg of resin. 5.- Ultrafiltration through a 20,000 Da membrane.

6.- Controlled enzymatic hydrolysis by adding 1 ,700,000 I. U. of Papain to the product at pH 7.6 and at 50° C for 90 minutes.

8.- Filtration through a 10 μ frames and plates filter. 9.- Ultrafiltration through a 5,000 Da membrane.

10.- Concentration at 40° C with a 0.1 mm Hg vacuum to a 25 % concentration of solids. 11.- Spray drying.

The product (enzymatically-hydrolysed collagen) produced by this process

(101.8 kg) presented the following physicochemical characteristics:

Assay (of the dry product): 99.6 %

Humidity: 4.9 % Apparent density: 0.495 g/ml pH (sol. at 20%): 6.12

Viscosity (sol. at 20%): 21.1 mps

Turbidity (sol. at 20%): 18.5 NTU

Relative conductivity (0.5%): 210 μS/cm Ashes: 0.4 %

According to another object of the present invention, examples of functional foods and nutritional supplements that comprise enzymatically-hydrolysed collagen, produced according to the above-described procedure, are given below.

Examples of functional foods and nutritional supplements:

These functional foods or nutritional supplements can also comprise a compound independently selected from: antioxidant compounds, mineral salts, vitamins and/or coenzymes, plant extracts, extracts of animal origin and adjuvant compounds of the enzymatically-hydrolysed collagen. A mixture of all of them can also be used.

EXAMPLE 1. Functional food: Yoghurt A yoghurt has been prepared with good results with the following composition: MiIk 73.9 g

Enzymatically-hydrolysed collagen 10.0 g

Skimmed milk powder 12.0 g

Cream 4.0 g Lactic ferments 0.1 g

The flavour of the yoghurt is pleasant, as are its appearance and texture.

EXAMPLE 2: Functional food: Chocolate dessert

A chocolate dessert has been prepared with the following composition: Skimmed milk 34.8 g

Enzymatically-hydrolysed collagen 10.0 g

Sugar 15.0 g

Lactose 10.0 g

Cream 5.0 g Pregelatinised corn starch 12.0 g

Defatted cocoa 10.0 g

Carrageenan 2.0 g

Gelatin 1.0 g

Emulsifier 0.2 g We manage to improve the nutritional function of the food without affecting the spreadability, viscosity, texture and flavour.

EXAMPLE 3: Nutritional supplement in liquid form

Enzymatically-hydrolysed collagen 24.00 g

Fructose 30.00 g

Anhydrous Citric Acid 4.00 g

Magnesium gluconate 2.00 g

Tomato extract 2% 0.25 g Potassium sorbate 0.20 g

Pineapple flavour 0.14 g

Acesulfame 0.01 g

Vitamin B1 (Pyridoxine H Cl) 0.01 g

Vitamin B2 (Riboflavin) 0.01 g Vitamin B6 (Pyridoxine H Cl) 0.01 g

Vitamin B5 (Panthenol) 0.05 g Vitamin E 50% water-soluble 0.07 g

Purified water 39.26 g

This liquid presentation is designed to be taken directly in a dose of 30 g or diluted in water. It provides magnesium, antioxidants and vitamins and it is characterised by its pleasant flavour.

EXAMPLE 4: Nutritional supplement in the form of a water-soluble powder.

Enzymatically-hydrolysed collagen 85.4O g

Magnesium carbonate 6.00 g

Vitamin B1 (Thiamine H Cl) 0.02 g Vitamin B2 (Riboflavin sodium phosphate) 0.02 g

Vitamin B6 (Pyridoxine H Cl) 0.02 g

Vitamin B5 (Calcium Pantothenate) 0.06 g

Strawberry flavour 1.12 g

Aspartame 0.14 g Fructose 5.39 g

Anhydrous Citric Acid 1.83 g

This powdered mixture can be packaged in containers with a scoop to measure out 12 g doses and the product is consumed after dissolving it in half a glass of water. It can also be packaged in single-dose paper-aluminium-polythene sachets that help provide the correct daily dose to be taken.

Clinical trial to determine improvements to quality of life and functional capacities in humans

According to another object of the present invention, the inventors have determined a new use for enzymatically-hydrolysed collagen produced by the above-described procedure. Specifically, enzymatically-hydrolysed collagen is used to prepare a nutritional supplement or functional food to improve the functional capacity and/or quality of life of a mammal, specifically a human, with functional difficulties deriving from articular cartilage disorders.

Therefore, in order to study the possible application of the new high purity enzymatically-hydrolysed collagen with better bioavailability (lower molecular weight, between 5,000 and 20,000 Da) as a functional food or as a component of functional foods or nutritional supplements, it was decided that a study should be carried out on 100 volunteers, 50 of whom would be administered this enzymatically-hydrolysed collagen in a daily dose of 10 g, equivalent to a dose of between 0.1 and 0.25 g of enzymatically-hydrolysed collagen per kg of body weight and the other 50 would be administered a daily dose of 1.5 g of glucosamine sulphate, equivalent to a dose of between 0.015 and 0.0375 g per kg of body weight. Glucosamine sulphate 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 the fact that the administration of glucosamine is an effective means of improving pain and mobility in joints with dysfunctions caused by cartilage disorders.

The purpose of this trial was to evaluate the effect of the new enzymatically- hydrolysed collagen on complaints where known non-steroidal anti-inflammatory drugs (NSAIDs) are usually used, specifically in disorders affecting functional capacity or quality of life deriving from articular cartilage disorders.

Glucosamine sulphate is also widely used in Europe and particularly in the

United States as a functional food or as part of functional foods and nutritional supplements (USP monograph 24 includes it in nutritional supplements) at these same doses (1.5 g per day) and it is said that they improve body functions in general, mainly mechanical functions (mobility, agility, etc.), and that they improve the patient's overall state of health and quality of life.

The clinical trial consisted of a multi-centre, double blind, parallel study comparing the new enzymatically-hydrolysed collagen with glucosamine sulphate. The purpose was to compare improvements in the quality of life (based on the SF-

36 generic survey (The MOS 36-item short-form health survey (SF-36). I.

Conceptual framework and item selection. Med care 1992 Jun; 30 (6): 473-483), and the efficacy, safety and tolerability of the two treatments administered orally for

90 days consecutively. The study was carried out on 100 volunteers (of whom it was possible to evaluate 93) affected by joint disorders that cause pain, a reduction in mobility and considerable impairment to and loss of quality of life.

The subjects were distributed into two groups of 50 individuals, of whom it was possible to evaluate 47 (enzymatically-hydrolysed collagen) and 46 (glucosamine sulphate). After a washout week, they received either 10g of enzymatically-hydrolysed collagen or 1.5g of glucosamine sulphate per day for 90 days consecutively, the study therefore lasting for 3 months for each subject.

Quality of life was measured at the beginning and end of both treatments, specifically by using the SF-36 generic survey, which has been validated by many studies and bibliographical reviews and has been used in over 4000 scientific articles over the last 20 years, although it was originally designed by the Health Institute, New England Medical Center, Boston (Massachusetts, USA). In Spain, said survey has been validated by Alonso et al. (11 ) at the IMIM (Municipal Institute of Medical Research) in Barcelona.

For all the authors who have validated the survey, the SF-36 is considered a suitable, truthful and effective instrument that is very useful in research and clinical practice.

In terms of "quality of life", the purpose of the SF-36 was to evaluate aspects of joint dysfunctions caused by articular cartilage disorders that are not strictly clinical, but also aspects related to the patient's daily life. Another of the aims was to evaluate how the patient's life was affected by the presence of these joint dysfunctions, always from the patient's point of view. This was a multidimensional evaluation and it was generally accepted as including at least eight sections or parameters that serve as indicators of the functional capacity and/or quality of life, as described in Table 1 below.

Table 1. Parameters and meaning studied to determine the quality of life of a human with functional difficulties deriving from articular cartilage disorders.

The results of this survey can be seen in Table 2. TaWe 2: Results of the SF-36 generic survey applied to 100 volunteers affected by joint disorders that caused considerable loss of quality of life.

Enzymatically-hydrolysed collagen Glucosamine sulphate m SD me Range n m SD me Range n

Initial visit (day -7)

Physical functioning 56.0 20.6 55.0 20.0-100.0 47 58.2 21.5 52.5 20.0-100.0 44

Physical role 36.4 34.8 25.0 0.0-100.0 46 34.8 37.1 25.0 0.0-100.0 46

Body pain 52.5 14.8 52.0 20.0-100.0 46 53.4 14.5 52.0 22.0-84.0 46

General Health 53.1 16.1 52.0 20.0-97.0 47 54.7 17.1 52.0 25.0-97.0 45

Vitality 52.9 15.5 52.5 25.0-100.0 46 53.7 14.2 52.5 30.0-100.0 46

Social functioning 72.3 18.0 75.0 25.0-100.0 46 71.5 20.1 75.0 25.0-100.0 43

Emotional role 82.6 34.2 100.0 0.0-100.0 46 90.6 22.9 100.0 0.0-100.0 46

Mental health 66.8 15.5 64.0 28.0-100.0 45 68.9 12.5 68.0 44.0-96.0 44

Rate of physical health 36.0 7.7 34.2 23.8-52.8 45 35.8 9.8 34.5 20.4-56.8 39

Rate of emotional health 50.1 9.1 52.1 25.6-66.9 45 51.8 6.8 52.7 29.4-64.9 39

Final visit (day 90)

Physical functioning 68.2² 21.4 \ 70.0 20.0-100.0 47 65.2² 23.6 75.0 15.0-100.0 45

Physical role 61.2² 38.2 75.0 0.0-100.0 47 48.4² 42.0 50.0 0.0-100.0 46

Body pain 67.1¹'² 17.0 74.C 22.0-100.0 47 62.7¹'² 16 6 64. 0 22.0-84.0 46

General Health 56.8² 13.7 57.0 30.0-97.0 47 57.6² 16.0 52.0 25.0-97.0 45

Vitality 57.0² 16.7 55.0 25.0-100.0 47 57.1² 15.7 55.0 30.0-100.0 46

Social functioning 81.6² 15.2 87.5 50.0-100.0 47 76.1² 19.8 75.0 25.0-100.0 44

Emotional role 89.4² 28.7 100.0 0.0-100.0 47 92.8² 25.3 100.0 0.0-100.0 46

Mental health 70.3² 13.4 68.0 36.0-100.0 47 70.6² 13.3 68.0 44.0-96.0 45

Rate of physical health 42.0² 8.3 44.9 24.8-53.1 47 40.0² 9.9 41.0 21.8-56.8 41

Rate of emotional health 50.7 7.7 52.1 25.6-66.5 47 51.1 7.1 52.9 29.6-61.6 41

1.- Statistically significant differences between colatech and glucosamine (GLM, repeated values; p<0.05).

2.- Statistically significant differences between the initial and final visits of the same treatment (GLM, repeated values; p<0.05).

No statistically significant differences were found between the values of the two groups for each visit (GLM, repeated values; p<0.05). Table 2 shows that both enzymatically-hydrolysed collagen and glucosamine sulphate, administered for 90 days consecutively at doses of 10g and 1.5g /day, respectively, considerably improve the different aspects of the patient's quality of life. But it can be seen that, unexpectedly, the values for the absolute differences between the initial and final visit are always broader with enzymatically-hydrolysed collagen than with glucosamine sulphate.

In such important aspects such as general health, vitality, social functioning, physical functioning, physical work, emotional functioning and mental health, we can see that enzymatically-hydrolysed collagen is always more effective than glucosamine sulphate in bringing about improvements in the different parameters applied.

From analysing the overall results achieved in this clinical trial, it can be seen that there are clear advantages of using the enzymatically-hydrolysed collagen that is the object of the invention for the preparation of a functional food or nutritional supplement to improve the functional capacity and/or quality of life of people with functional difficulties deriving from articular cartilage disorders.

With the high purity enzymatically-hydrolysed collagen that is the object of the invention at the doses per kg of body weight used in the clinical trial (0.1 - 0.25 g/kg body weight/day), due to its pleasant flavour and good organoleptic characteristics, it is possible to prepare different types of nutritional complements or supplements such as drinkable solutions or suspensions, water-soluble powdered mixtures, tablets and capsules. Functional foods such as juices, yoghurts, yoghurt drinks, desserts, energy bars, slimming bars, etc. can also be prepared, whilst the enzymatically-hydrolysed collagen can be a functional food in itself thanks to its pleasant flavour.

It can therefore be concluded that it has been firmly shown that the production procedure for hydrolysed collagen that is the object of the invention, which reuses the contaminant residual by-products from the production of gelatins, not only makes it possible to achieve a return on the costs deriving from the treatment of these environmentally harmful by-products, but also makes it possible to achieve a new high purity enzymatically-hydrolysed collagen with a low molecular weight that, due to its pleasant flavour and good organoleptic characteristics, can be used in both functional foods, such as yoghurts, juices, desserts, energy bars, etc. and nutritional supplements of the multivitamin complex type in tablet form, solutions, suspensions, etc. Furthermore, the inventors have discovered a new use for this enzymatically- hydrolysed collagen, which makes it possible to considerably increase the quality of life of people or animals affected by dysfunctions deriving from cartilage disorders of the joints, in an equivalent and more pleasant manner than the administration of anti-inflammatory drugs, as it avoids their undesired side effects.

Claims

C L A I M S

1.- Production procedure for hydrolysed collagen according to the invention using by-products of gelatin-producing processes, wherein after aqueous extraction of gelatins, tissues rich in collagen are subjected to successive steps of extraction in aqueous solution and subsequently to acid, base or enzymatic hydrolysis, finally producing a residual by-product, characterised in that it comprises the following steps: a) aqueous extraction of the residual by-product at a temperature between 65⁰C and 14O⁰C for between 2 and 8 hours; b) adjustment of the pH to between 6.5 and 8.0 using mineral acid or base solutions; c) filtration of the solution produced from step b); d) demineralisation of the filtered solution by passing it through ion exchange resins; e) ultrafiltration of the solution demineralisation through membranes with a pore diameter of 1 ,000 to 40,000 Da; f) enzymatic hydrolysis of the ultrafiltered solution; g) filtration of the enzymatically-hydrolysed collagen solution; h) ultrafiltration through membranes with a pore diameter of 10,000 to 40,000

Da; i) concentration of the enzymatically-hydrolysed collagen solution by heating it at temperatures of 7O⁰C or lower at a pressure of between 10 and 0.0001 mm Hg to a final concentration of enzymatically-hydrolysed collagen of 15% to 35% by weight; j) drying of the enzymatically-hydrolysed collagen to obtain a dry product with a humidity of 8% or less and a density of between 0.3 and 0.8 g/ml.

2.- Production procedure for enzymatically-hydrolysed collagen according to claim 1 , characterised in that step a) aqueous extraction of the residual by-product is carried out at a temperature of between 65⁰C and 100⁰C, specifically between 85⁰C and 95⁰C for between 4 and 6 hours.

3.- Production procedure for hydrolysed collagen according to claim 1, characterised in that, after aqueous extraction, the pH of the solution is adjusted to between 7.4 and 7.6.

4.- Production procedure for hydrolysed collagen according to claim 1 , characterised in that step e) ultrafiltration of the demineralised solution is carried out using membranes with a pore diameter of 20,000 Da.

5.- Production procedure for hydrolysed collagen according to claim 1 , characterised in that the ultrafiltration step of the collagen enzymatically-hydrolysed solution is carried out using membranes with a pore diameter of 5,000 to 20,000 Da.

6.- Production procedure for hydrolysed collagen according to claim 1 , characterised in that the concentration step i) of the enzymatically-hydrolysed collagen solution is carried out at a temperature of 6O⁰C or lower.

7.- Production procedure for hydrolysed collagen according to claim 1 , characterised in that the concentration step i) of the enzymatically-hydrolysed collagen solution is carried out at a pressure of between 1.0 and 0.01 mm Hg.

8.- Enzymatically-hydrolysed collagen produced by the procedure according to any of claims 1 to 7.

9.- Enzymatically-hydrolysed collagen according to claim 8, characterised in that it has a molecular weight of between 1 ,000 and 50,000 Da.

10.- Enzymatically-hydrolysed collagen according to claim 9, characterised in that it has a molecular weight of between 5,000 and 20,000 Da.

11.- Enzymatically-hydrolysed collagen according to any of claims 8 to 10 above, characterised in that it comprises 99% hydrolysate in the weight of the dry product.

12.- Functional food that comprises the enzymatically-hydrolysed collagen according to any of claims 8 to 11 above.

13.- Functional food according to claim 12 that also comprises a compound independently selected from: antioxidant compounds, mineral salts, vitamin and/or coenzymes, plant extracts, extracts of animal origin and adjuvant compounds of the enzymatically-hydrolysed collagen, or a mixture of all of these.

14.- Nutritional supplement that comprises the enzymatically-hydrolysed collagen according to any of claims 8 to 11 above.

15.- Nutritional supplement according to claim 14 that also comprises a compound independently selected from: antioxidant compounds, mineral salts, vitamin and/or coenzymes, plant extracts, extracts of animal origin and adjuvant compounds of the enzymatically-hydrolysed collagen, or a mixture of all of these.

16.- Use of the enzymatically-hydrolysed collagen according to any of claims 8 to 11 for the preparation of a nutritional supplement or functional food to improve the functional capacity and/or quality of life of a mammal with functional difficulties deriving from articular cartilage disorders.

17.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve physical functioning.

18.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve physical role.

19.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve the feeling of body pain.

20.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve general health.

21.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve vitality.

22.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve social functioning.

23.- Use according to claim 16 for the preparation of a nutritional supplement or functional food to improve emotional role.

24.- Use according to any of claims 16 to 23 above, wherein the daily dose of enzymatically-hydrolysed collagen is between 0.1 and 0.25 g per kg of body weight.

25.- Use according to any of claims 16 to 24 above, characterised in that it is for a preparation in the form of oral solutions or suspensions.

26.- Use according to any of claims 16 to 24 above, characterised in that it is for a preparation in the form of water-soluble tablets or powders.

27.- Use according to any of claims 16 to 26 above, wherein the mammal with functional difficulties deriving from articular cartilage disorders is a human.