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The present invention relates to hydrolysed collagen products or collagen hydrolysates, a process for their preparation and their uses as anti-inflammatory and/or analgesic active ingredients for topical applications such as for example as a cream, and for oral applications, such as for example as food or food supplement.

A collagen hydrolysate, belonging to the class of hydrolysed animal protein compounds, is a collagen that has been processed or subjected to a chemical and/or physical process to obtain hydrolysed collagen products characterized, for example, by the presence of small-to-medium peptides or peptide chains of small-to-medium length, and, for example, by a lower molecular weight than normal or native collagen.

These characteristics of collagen hydrolysates may facilitate the use of collagen, for example in cosmetic formulations and oral sports nutrition, and may also enhance its properties.

A hydrolysed collagen is one of the most common forms of collagen used nowadays, for example, in cosmetic formulations and is a completely harmless compound from a health point of view and with no known harmful side effects. Its use has long been known, particularly in the form of a dietary supplement for oral administration.

Patent application WO 2017/050775 discloses a method for the preparation of hydrolysed collagen from chicken foot powder. The hydrolysates obtained (< 3 KDa) show ACE inhibition activity.

Patent application WO 2020/128070 (WC'070) describes a collagen hydrolysate for use as an active substance in the treatment of inflammatory skin disorders and intestinal disorders and/or skin disorders and intestinal disorders accompanied by inflammation. According to WC'070, the hydrolysed collagen derives from a collagen-containing starting material that could be, for example, skin of pigs, and is obtained through a process that involves enzymatic hydrolysis of a collagen-containing starting material. The collagen hydrolysate according to WO'070 typically has a mean molecular weight of from 500 to 15,000 Da, preferably from 1,000 to 8,000 Da, more preferably from 1,500 to 5,000 Da, most preferably from 1,800 to 2,200 Da.

Patent application WO 2019/122376 (WO'376) describes a collagen hydrolysate and a process for making such collagen hydrolysate, said collagen hydrolysate being characterized by the following technical parameters: a. A moisture content comprised from 4 to 12 percent, by weight. b. A protein content of 85 percent or more, by weight. c. A hydration time of 7 seconds, or less. d. A dissolution time of 250 seconds, or less.

Said composition comprising protein according to WO'376 may be an aqueous composition wherein protein is freely available, dissolved and/or not dissolved, such as for example an aqueous composition comprising gelatin and/or collagen.

Bello A et al. in "Collagen hydrolysate for the treatment of osteoarthritis and other joint disorders: A review of the literature”, Current Medical Research and Opinion, Hants, GB, vol 22, no 11, 1 Nov 2006, generally discloses the uses of collagen hydrolysates in the treatment of osteoarthritis and symptoms thereof. Collagen hydrolysates on the market today still present several limitations in their use due to the processes used for their preparation and, in addition, they also present major drawbacks due to the lack of standardisation and repeatability.

Therefore, to date, there remains a high interest among industry players for new processes to prepare collagen hydrolysates that are easy to implement and repeatable to produce hydrolysates with improved chemical and physical properties, and high efficacy and absorption capacity, as well as stable and repeatable.

The Applicant, after intensive and prolonged research and development, has developed an improved preparation process for collagen hydrolysates that can produce standardised, repeatable, effective, and low-cost hydrolysates.

Therefore, the present invention relates to hydrolysed collagen products and a process for obtaining them, said process being characterized by the following steps: a) submitting a starting raw material comprising collagen or Type A pig gelatin to a solubilisation step in warm water under stirring, thus obtaining a dissolved solution, b) preferably, submitting said dissolved solution from step a) to a pre-treatment step, where the dissolved solution is heated up, preferably under stirring, obtaining a dissolved and pre-treated solution, c) preferably, cooling down said dissolved and pre-treated solution, obtaining a dissolved, pre-treated and cooled solution, d) submitting said dissolved solution from step a), or said solution from steps b) or c), to a hydrolysis step treating the dissolved solution from step a), or said solution from steps b) or c) with at least one enzyme, thus obtaining a hydrolysed collagen and enzyme mixture, e) heating the hydrolysed collagen and enzyme mixture from step d) for inactivating the enzyme, obtaining a mixture of hydrolysed collagen and inactivated enzyme, f) preferably, cooling down said mixture of hydrolysed collagen and inactivated enzyme from step e), thus obtaining a cooled hydrolysed collagen and inactivated enzyme mixture, g) submitting said mixture of hydrolysed collagen and inactivated enzyme from step e), or said cooled hydrolysed collagen and inactivated enzyme mixture from step f) to at least one ultrafiltration step, obtaining a hydrolysed collagen, h) submitting the hydrolysed collagen from step g) to a drying step, thus obtaining dried hydrolysed collagen products in powder form.

Preferably, before carrying out the drying step h) a reduction of the microbial content which is present in the hydrolysed collagen obtained by step g) can be foreseen, as an additional optional step g’).

According to the present invention, said starting raw material comprises or, alternatively, consists of collagen or gelatin, preferably a Type A pig gelatin.

In the context of the present description, the terms gelatin and gelatine may have the same meaning and both of them may be used. Preferably, in the context of the present invention the starting raw material may be chosen from a native gelatin or collagen, for example from pig. This is because generally a gelatin's composition is usually a mixture of collagen fibres (or chains having different lengths and average molecular weights). The longer collagen chain is, the higher viscosity of the gelatin is achieved. During the hydrolyzation step process, these collagen fibres or chains are reduced in length to smaller peptides, and the gelifying properties of the native collagen disappear.

Preferably, according to the present invention, in said solubilisation step a) said starting raw material is slowly added (i.e., in a period of time from about 1 minute to about 30 minutes, preferably from about 5 minutes to about 15 minutes) under stirring to warm water (from about 30°C to about 60°C, preferably from about 45°C to about 50°C) and kept stirred for a period of time comprised from about 10 minutes to about 60 minutes, preferably about 30 minutes.

Preferably, according to the present invention, said pre-treatment step b) is carried out at a temperature comprised from about 35°C to about 95°C, more preferably from about 60°C to about 80°C, for a period of time preferably comprised from about 30 minutes to about 4 hours, more preferably from 1 hour to 3 hours, for example 2 hours.

Preferably, according to the present invention, said step c) is carried out as fast as possible until a temperature comprised from about 30°C to about 80°C, more preferably from about 50°C to about 60°C. It is important to assure that the dissolved solution from step a), or said and pre-treated solution from step b) has reached a homogeneous temperature of about 50°C ± 5°C, before being further processed.

Preferably, according to the present invention, said hydrolysis step d) is carried out at a temperature comprised from about 30°C to about 70°C, more preferably from about 40°C to about 60°C, for example about 50°C ± 5°C, for a period of time preferably comprised from about 4 hours to about 36 hours, more preferably from about 12 hours to 32 hours, for example 16 hours or 24 hours, and said enzyme is preferably a serine endoprotease selected as Alcalase ® 2,4 L, also named subtilisin Carlsberg, which is a serine protease of Bacillus licheniformis (E.C. 3.4.21.62).

Preferably, according to the present invention, it might be possible that said enzyme is preferably selected as Protamex® corresponding to proteolytic enzymes of Bacillus licheniformis and Bacillus amyloliquefaciens (E.C. 3.4.21.62 and 3.4.24.28). Preferably, according to the present invention, said enzyme is selected as Flavourzyme® which corresponds to an aminopeptidase of Aspergillus oryzae (E.C. 3.4.11.1); Neutrase® 0,8L which is a zinc dependent metal loprotease of Bacillus amyloliquefaciens (E.C. 3.4.24).

All said enzymes are currently purchased from Novozyme, Nordisk.

Preferably, according to the present invention, said step e) is carried out by heating said hydrolysed collagen and enzyme mixture from step d) at a temperature comprised from about 75°C to about 98°C, more preferably from about 80°C to about 90°C, for a period of time preferably comprised from about 5 minutes to about 60 minutes, more preferably from about 10 minutes to about 30 minutes, for example about 20 minutes, for inactivating the enzyme. This latter mixture is then preferably cooled down, preferably according to said step f) at a temperature comprised from about 30°C to about 70°C, more preferably from about 40°C to about 60°C, for example about 50°C ± 5°C, for a period of time comprised from about 10 minutes to 60 minutes, more preferably from about 20 minutes to about 50 minutes, for example about 35 minutes. It is important to assure that the cooled hydrolysed collagen and inactivated enzyme mixture has reached a homogeneous temperature comprised from about 35°C to about 70°C, more preferably from about 45°C to about 60°C, for example about 50°C ± 5°C, before being further processed.

Preferably, according to the present invention, said step g) is preferably carried out at a temperature comprised from about 30°C to about 70°C, more preferably from about 40°C to about 60°C, for example about 50°C, utilizing a membrane having a molecular weight cut-off comprised from about 5,000 Da to about 20,000 Da, more preferably from about 8,000 Da to about 15,000 Da, for example about 10,000 Da, obtaining a hydrolysed collagen fraction characterized by a molecular weight profile equal to or below of 10,000 Da, preferably equal to or below of 8,000 Da or 5,000 Da.

Preferably, said drying step h), according to the present invention, is preferably carried out, for example, through spray-drying technique.

Preferably, an additional optional step g') can be carried out before said drying step h). This additional step g') is set out to reduce the microbial content of the hydrolysed collagen fraction, for example characterized by a molecular weight profile preferably equal to or below of 10,000 Da, or preferably equal to or below of 8,000 Da or 5,000 Da, obtained according to step g) as above indicated. Step g') can be, for example, carried out by a UHT (Ultra High Temperature) step, a pasteurization step or a step involving a gamma ray treatment of the hydrolysed collagen fraction as obtained according to step g). In any case, optional step g') is carried out to reduce or eliminate the microbial content of the product, if needed.

Preferably, according to the present invention, an additional optional step I) can be carried out after said drying step h), for reducing or eliminating the microbial content of the dried hydrolysed collagen product obtained according to step h) of the above process. Step I) could be, for example, an irradiation step carried out directly on the already dried hydrolysed collagen product obtained according to step h).

Preferably, according to the present invention, step g') and step I) can be both present in the whole process or only step g') or only step I) can be carried out. In any case, both steps g') and I) are to be considered as optional. Preferably, said pre-treatment step b), as indicated above, allows to obtain a partial denaturalization of the starting raw material, for example collagen or gelatin, preferably a Type A pig gelatin, so that proteases action during hydrolysis step (step d)) results more efficient and quicker. Before using proteases during the hydrolysis step d), the dissolved solution from step a), or dissolved and pre-treated solution from step b) has to be, therefore, cooled down, preferably according to said step c) until a temperature comprised from about 30°C to about 80°C, more preferably from about 50°C to about 60°C, for example about 50°C ± 5°C. After this step c), the dissolved, pre-treated and cooled solution can be added with proteases, preferably according to step d), thus avoiding the risk of denaturizing them because of too high temperatures. Another important aspect is related to the enzyme inactivation and subsequent cooling down of the obtained hydrolysed collagen and inactivated enzyme mixture. In this case, according to step e) of the above-indicated process, the enzyme inactivation is important because if the enzyme is not inactivated, hydrolysis can proceed further, and bioactivity of the resulting products can significantly change in an uncontrolled way.

The subsequent cool down, according to said step f), allows to avoid problems with the subsequent ultrafiltration step indicated as step g). For example, if the temperature of the cooled hydrolysed collagen and inactivated enzyme mixture would be cooler than about 40°C, the density of the mixture could increase, and the ultrafiltration step would not be enough efficient in obtaining the desired products. Therefore, to maintain the temperature at a value of from about 40°C to about 70°C, preferably from about 50°C to about 60°C, during the ultrafiltration process, it might be advisable to add for example some extra water to the mixture in order to maintain a low viscosity value to allow the ultra-filtration process to take place in an efficient way.

According to another embodiment of the present invention, the hydrolysed collagen fractions characterized by a molecular weight from about bigger than zero to about lower than 10,000 Da (Dalton), preferably from about bigger than zero to about lower than 3,000 Da (Dalton) for example from about 2,000 Da to about 3,000 Da or from about 500 Da to about 3,000 Da,, and obtained preferably:

I) according to a process that comprises or, alternatively, consists of steps from a) to h) [a)+b)-0)-Ki)-^)+f)-^)+h)]; or

II) according to a process that comprises or, alternatively, consists of steps [a)+b)+c)+d)-^)+f)-^)+g')+h)]; or ill) according to a process that comprises or, alternatively, consists of steps [a)+b)-Kj)+d)+e)+f)+g)+h)+i)]; or iv) according to a process that comprises or, alternatively, consists of steps [a)+b)-Kj)+d)+e)+f)+g)+g')+h+i))]; have been isolated and identified, and it has been found that they have an anti-inflammatory and analgesic activity, preferably after topical applications.

It is known that inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and initiate tissue repair.

However, during this process the body generates different molecules that affects to blood flow, vascular permeability, accumulation of leukocytes and inflammatory mediator production, such as interleukin-1 (IL-1), tumour necrosis factor-o (TNF-o), IL-6, IL-1, IL-8, y interferon (IFNy) and other chemokines that could produce some sensation of pain. Some of these cytokines can activate nuclear factor KB (NFKB) expression, a transcription factor that can mediate the inflammatory response by regulating the transcription of many acute phase proteins and a large variety of stress response genes, such as inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2). In fact, cyclooxygenases (COXs) are a family of oxidoreductase enzymes that catalyse eicosanoids biosynthesis through arachidonic acid (AA) oxidation and there are three isoforms described to date, COX-1, COX-2 and COX-3. Focusing on COX-2, the inducible isoform, is only expressed in some stages of cell differentiation or during replication. COX-2 plays an important role during inflammatory response because it is the major contributor enzyme to prostanoid synthesis in inflammatory processes and its expression is upregulated by inflammatory mediators, and it has been observed in pathological processes such as inflammation and angiogenesis, among others.

For this reason, it is very important to have specific and selected hydrolysates available, free from side effects, but at the same time effective and easy to prepare. In particular, it is necessary to have available specific and selected hydrolysates having anti-inflammatory and/or analgesic activity for oral use, to prepare a food or food supplements, and for external use to prepare finished products or compositions for topical or cosmetic applications.

According to an embodiment of the present invention, one hydrolysed collagen product obtained according to the process steps from a) to h) above was named G71 hydrolysate (also indicated alternatively as G71 PLUS or G71+). G71 PLUS or G71+ is the G1 fraction containing peptides with a molecular weight from about bigger than zero to about lower than 10 KDa.

In the context of the present description, the skilled person in the art is well aware that values of molecular weights may be considered to have a certain tolerance for instance up to around ±10%, e.g., a value of 10 KDa may be considered as 10 KDa ± 0.1 KDa or 10 KDa ± 0.2 KDa or 10 KDa ± 0.5 KDa or 10 KDa ± 1 KDa, depending on the method and instruments used.

Another embodiment of the present invention relates to a composition comprising a mixture that consists or, alternatively, comprises of at least one hydrolysed collagen product which is selected from G71 plus and, optionally, excipients and carrier pharma or food grade.

Another embodiment of the present invention relates to a composition comprising a mixture that consists or, alternatively, comprises of at least one hydrolysed collagen product which is selected from G71 plus and, optionally, excipients and carrier pharma or food grade, wherein said composition being for use as antiinflammatory and analgesic active ingredients for topical applications, such as for example a cream, and for oral applications, such as for example as a food or food supplements.

Another embodiment of the present invention relates to a composition comprising a mixture that consists or, alternatively, comprises of at least one hydrolysed collagen product which is selected from G71 plus and, optionally, excipients and carrier pharma or food grade, wherein said composition being for use in a method for the treatment of neuroinflammation and neurodegeneration, in particular for use in a method for treating pain, muscular aches, joint inflammation, and muscular pain.

The following examples are intended as exemplifying but not limiting the scope of the present invention.

EXAMPLE 1 : Process for obtaining hydrolysed collagen products starting from collagen

First, collagen solubilization was performed. To do this, 0.2-0.5 g/mL of collagen was dissolved in water and kept at 40-45°C for 30 min at 250 rpm in a water bath. Once the collagen was solubilized, the solution was heated to 60°C or 80°C for 2 h at 100 rpm. Subsequently, the solution was allowed to cool to 50°C (for treatment at 80°C) and the enzyme preparation was added at an enzyme/protein concentration of 0.1, 0.2 and 0.4 AU or 10, 20 and 40 LAPU as indicated in Table I. Hydrolysis was performed with 4 types of enzymes: Alcalase® 2.4 L, Neutrase® 0.8 L, Protamex® and Flavourzyme® 1,000 L (Novozyme) separately or by combining some of them. These enzymes show different activities (2.4, 0.8, 1.5 Anson Units (AU)/g and 1,000 Leucine aminopeptidase Units (LAPU)/g, respectively) therefore the added enzyme volume varied depending on the enzyme and enzyme/protein concentration tested. For this reason, the final volume was adjusted with distilled water so that all reached the same dilution. The final hydrolysis volume was 34.85 mL. The enzyme Protamex®, sold in solid state, was reconstituted in distilled water to the desired concentration prior to its addition. Hydrolysis was performed at 50°C with constant stirring at 200 rpm in orbital, for 4 and 24 h at the solution's pH. Once the hydrolysis time was completed, the reaction was completed by heat treatment at 90°C for 20 min. Finally, all samples were centrifuged at 4,500 x g, 10 min, 40°C to remove non-soluble compounds. The supernatants obtained are what we will call hydrolysates, which were frozen at -20°C until their subsequent analysis. Table I lists the hydrolysis conditions of the eleven hydrolysates obtained.

Table I. Hydrolysis treatments performed on collagen. AU: Anson Units; LAPU: Leucine aminopeptidase units EXAMPLE 2: Selection of hydrolysed collagen products obtained starting from collagen

Once the hydrolysates were prepared, their COX-2 inhibitory activity was determined, following the procedure described in example 3. The COX-2 inhibitory activity of the different hydrolysates varied widely, presenting inhibition percentages between 33 and 60% when tested at their 1/10 dilution. Hydrolysates G33, G50, G51 and G71 showed the greatest activity, showing inhibition values greater than 50% (Table II). These 4 hydrolysates were selected to evaluate their anti-inflammatory activity in macrophages.

Table II. Inhibitory activity of the enzyme Ciclooxigenase-2 (COX-2). Dilution used: 1/10.

Data are expressed as mean (n=3). EXAMPLE 3: Cyclooxygenase-2 enzyme inhibitory activity measure

In vitro anti-inflammatory activity was determined by the ability of samples to inhibit the pro-inflammatory enzyme Cyclooxygenase-2 (COX-2). The Cayman Chemical (Michigan, USA) commercial kit was used, and the manufacturer's instructions were followed. The primary rationale for the kit is to measure the ability of COX-2 to transform arachidonic acid into prostaglandins, using N,N,N'N'-tetramethyl-p-phenylenediamine (TMPD) as a co- substrate, which oxidizes to form a measurable compound at 590 nm wavelength. Briefly, the assay was performed on a 96-well plate and consisted of mixing 10 pL of the sample with 10 pL of enzyme solution, 150 pL of assay buffer and 10 pL of hemin (cofactor) and the resulting solution was incubated for 5 minutes at room temperature. Subsequently, 20 pL TMPD and 20 pL Arachidonic acid (final concentration in 100 pM well) are added and absorbance was measured at 590 nm 2 min after starting the reaction. The negative (white) control was performed following the same procedure described above by substituting the sample and enzyme volume for the same volume of assay buffer and the positive control (100% activity) by substituting the sample volume for the same volume of assay buffer.

The inhibition percent (Inhibition %) was calculated by applying the following formula:

(Sample Abs) - (White)

Inhibition % = 1- x lffl

(Abs Positive Control) - (White)

EXAMPLE 4: Determination of hydrolysed collagen products anti-inflammatory activity on macrophages

The anti-inflammatory effect of the four selected hydrolysates were carried out in RAW 264.7 macrophage cell line. The RAW 264.7 cells (Sigma Aldrich Chemical, Madrid, Spain) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-Glutamine (2 mM), 1% Penicillin/Streptomycin (100U/mL) and 2.5% (v/v) HEPES (25 mM) in an incubator at 37 °C in a 5% CO2- humidified atmosphere. Then, they were cultured in 24-well plates at a density of 50,000 cells/well. The macrophages were stimulated with supplemented red phenol-free DMEM containing lipopolysaccharide (LPS; 0.25 pg/mL) 24 h after platting. The LPS-stimulated cells were treated during 24 h with the positive hydrolysates concerning the in vitro COX-2 inhibitory assay (250 pg/mL). Hydrolysates were previously lyophilized and then, diluted with phosphate-buffered saline (PBS) and filtrated with 0.22 pm filters. After the treatment incubation, the supernatants were removed and kept at -20°C.

The anti-inflammatory effect of the hydrolysates was evaluated by the nitric oxide production. It was determined by measuring the nitrites levels in the culture supernatants. Nitrite levels were quantified using the Griess reagent by monitoring the appearance of azo dye at 540 nm. Sodium nitrite was used as a standard curve from 0 to 200 pM. In 96-well plates, 50 pL of the removed supernatants and the standard dilutions were mixed with 100 pL of 1% sulphanilamide in 0.5 M HCI. Plates were incubated in the dark 10 min at 4°C and 50 pL of N-1- naftiletilendiamine (NED) were added in each well. Plates were incubated again in the dark during 30 min at 4°C and 5 min at room temperature. The absorbance was read at 540 nm. Results are shown as mean ± SD. Differences between treatments were analyzed by one way ANOVA (post-hoc test Bonferroni).

Figure 1 shows the content of nitrites levels found in the supernatant of the cells treated with the four selected hydrolysates at a concentration of 250 pg/mL. Cells treated G33, G50 and G51 showed levels of nitrites like the ones produced by LPS treatment. However, the treatment with G71 produced a significant reduction of the production of nitric oxide in the macrophages in comparison with the LPS-treatment. This result indicated that G71 contain peptides that produce a reduction of the inflammation induced by LPS administration to the macrophages. Considering the evidence, G71 was selected to evaluate its potential anti-inflammatory effect in vivo. (EXAMPLE 5)

EXAMPLE 5: Determination of anti-inflammatory activity of hydrolysed collagen products characterized by a Molecular Weight profile under 10,000 Da after topical administration on rats

Regarding the Example 2, 3 and 4 it is described that G71 contain peptides that produce a reduction of the inflammation, selecting this G71 to evaluate its potential anti-inflammatory effect in vivo. Peptides of small sizes (3-12 amino acids residues) are usually the peptides showing the highest effects inhibiting enzymes (Bravo et al 2019. Molecular Nutrition and Food Research, 63, 3: 1801176). Thus, G71 peptides were filtered through a hydrophilic membrane with cut-off values of 10 kDa, getting a G71 fraction enriched in small peptides (from about bigger than zero to about lower thanlO KDa of molecular weight). This hydrolysed collagen product, containing peptides with a molecular weight from about bigger than zero to about lower than 10 KDa, is named G71 plus or G71+.

The measure of the anti-inflammatory effect of the treatments was carried out following the method described by Fehrenbacher et al. 2012 (Current Protocols in Pharmacology, 2012, 56:5.4.1-5.4.7.). The method consisted of producing local oedema in the soles of the animal's hind paws and quantifying possible changes in paw volume caused by the treatments to be tested. 8-14-week-old Wistar strain male rats (Rattus norvegicus) from Envigo (Spain) were used for the study. Rats remained at a stable ambient temperature of 23°C, and with 12-hour lightdark cycles, ingesting freely on-demand food and water.

The procedure was as follows: after cleaning the animals' legs with a cloth soaked with 70% ethanol, 300 mg of cream containing the different treatments was applied, rubbing gently using a thumb, on the plantar area about 50 times and repeatedly, to promote its absorption. 30 min after the application of the treatments, the oedema in the animals' paws was induced by a single injection of carrageenan, 0.1 mL of 1% w/v carrageenan (Sigma-Aldrich) diluted in saline. To do this, the needle (27G gauge) was inserted with the bevel facing up, in the metatarsal region near the distal calluses. Base cream was used as negative control, base cream incorporating hydrolysate G71 (Ex. 1) (1-10%) and base cream incorporating the lesser fraction of 10,000 Da of hydrolysate G71 plus or G71+ (1-10%) (Ex. 5).

The anti-inflammatory activity of the treatments (n=10 per treatment group) was performed by determining the volume of oedema in the animals' hind paws with a digital plethysmometer (Panlab) and with the animals anaesthetized by inhaled anaesthesia (2% isoflurane; RWD Life Science). Leg volume measurement was performed before and after 1, 2 and 3 h of carrageenan injection. These actions were done in duplicate. Result analyses were performed using an initial analysis to rule out discrepant points within groups. To this end, the Grubbs statistical test was used, using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, USA). All data are expressed as means ± standard error of the mean (SEM) and are represented as the difference in leg volume at various times from that observed at the initial time (0 h). To analyse differences between the different treatments, a non-parametric test was performed using the Kruskal-Wallis test. Additionally, the area under the curve obtained by representing the paw volumes of the animals subjected to the different treatments was calculated. The evaluation of the differences between treatments was performed using a 1-way Anova with the Bonferroni post-hoc test.

The experimental procedure used was approved by the Eurecat Animal Experimentation Ethics Committee and the Catalonian Regional Government's Animal Experimentation Commission. The study was conducted following the Guidance for the Care and Use of Laboratory Animals and in accordance with the European provisions of Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes.

Figure 2A shows the effect of the different treatments applied topically to the animals' paws. An increase in paw volume can be observed in all treatments at 1 h due to the administration of carrageenan, which causes oedema (inflammation). Administration of G71 hydrolysate resulted in a slight decrease in inflammation at 1 and 2 h after inducing oedema (11% and 23% reduction from the effect observed with the base cream). Regarding the fraction from about bigger than zero to about lower than 10,000 Da obtained from the G71 hydrolysate (i.e., G71 plus or G71+), a significant reduction in leg volume was observed at 1 and 2 h (48% and 49% reduction compared to the effect observed with the base cream), showing its great anti-inflammatory potential.

Figure 2B shows the area under the curve calculated from the curve plotted with the volumes of the animals' paws, in which oedema had been induced with carrageenan, after the application of base cream (control), base cream with 5% of the hydrolysate G71 and base cream with 5% of the fraction from about bigger than zero to about lower than 10,000 Da of the hydrolysate G71 (i.e., G71 plus or G71+). The observed area under the curve was significantly lower in the animals that had the base cream applied containing the fraction from about bigger than zero to about lower than 10,000 Da of the hydrolysate G71 (i.e., G71 plus or G71+) than that observed in the animals treated only with the base cream. No significant differences were observed between the effect of the application of the cream containing the hydrolysate G71 on oedema with respect to the application of the base cream.

EXAMPLE 6: Determination of analgesic effect of hydrolysed G71 and of the fraction characterized by MW lower than 10,000 Da (G71 plus or G71+)

The analgesic effect of the G71 hydrolysate and the from about bigger than zero to about lower than 10,000 Da fraction of the same hydrolysate (i.e., G71 plus or G71+) was evaluated in prolonged and acute pain produced in the tail of the animals.

8-week-old male Wistar rats were used to conduct the analgesia assay study. After one week of acclimatization of the animals, they became accustomed to the procedure, immersing the most distal part of the animal's tail (3 cm) in warm water (25°C; for 2 minutes) daily for one week.

Briefly, the trial consisted of cleaning the animals' legs with a cloth soaked with 70% ethanol, after this, 300 mg of base cream, base cream containing the hydrolysate G71 (5%) or base cream containing the fraction from about bigger than zero to about lower than 10,000 Da of the hydrolysate G71 (i.e., G71 plus or G71+) was applied to the animals' distal part, gently and repeatedly rubbing to promote the absorption thereof. 30 minutes after the creams were applied, the animals' tails were immersed in water at 48°C to generate prolonged pain or at 55°C to generate acute pain. The evaluation of analgesic effect of treatments was performed by timing the time it took for the animal to show pain by moving its tail. The process was performed in triplicate for each animal, immersing the animal's tail at 48°C or 55°C until it showed pain. Between the three measurements the animal's tail was left in water at 25°C for two minutes. Results are shown as mean ± SEM. For each parameter, an initial analysis was performed to rule out discrepant points within groups. To this end, the Grubbs statistical test was used, using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, USA). For comparisons between two groups, Student's t-test was used using GraphPad Prism 9 software.

The experimental procedure used has been approved by the Eurecat Animal Experimentation Ethics Committee and the Catalonia Regional Government's Animal Experimentation Commission. The study was conducted following the Guidance for the Care and Use of Laboratory Animals and in accordance with the European provisions of Directive 86/609/EEC on the Protection of Animals Used for Experimental and Other Scientific Purposes.

Figures 3A and 3B show the time it takes for animals to move their tails in response to prolonged and acute pain, respectively, to which base cream (control), base cream containing hydrolysate G71 and base cream containing the fraction from about bigger than zero to about lower than 10,000 Da of the same hydrolysate (i.e., G71 plus or G71+) had been previously applied. Regarding the results in prolonged pain, neither of the two treatments produced analgesic effects with respect to the effect observed by the application of the base cream (Figure 3A). However, regarding acute pain, it was observed that the animals to which the cream containing the from about bigger than zero to about lower than 10,000 Da fraction of the G71 hydrolysate (i.e., G71 plus or G71+) was applied took longer to withdraw the tail from the water than the control animals. This fact indicates that the animals treated with this peptide fraction, had more resistance to pain and therefore is indicative of an analgesic effect.

EXAMPLE 7: Isolation, identification, and synthesis of peptides having anti-inflammatory activity

Finally, the aim of the example 7 is identify the most important peptides inside the active subtraction < 10,000Da (G71 PLUS or G71 +).

Analytical (Nano-HPLC Easy-LC from ThermoFisher Scientific) and semi-preparative (Agilent Technologies 1260 series) high-performance liquid chromatography (HPLC) equipment as well as mass spectrometry equipment (NanoLC-Orbitap-MS/MS from Thermo Scientific) that allowed peptide sequencing were used to perform this work. The following steps were performed.

7. A. obtainment of hydrolysed product and of the proteic fraction characterized by MW lower than 3000 Daltons.

Following the procedure described in Example 1, the fraction from about bigger than zero to about lower than 10,000 Da of the hydrolysate selected in pilot scale powder format was obtained (Example 5). The product was dissolved 10-30% in milli-Q water and centrifuged at 1,000 to 4,000 x g, from 60 to 180 min, from 2°C to 10°C °C in filter devices (Centripep, Amicon Inc) with 2,000-3,000 Daltons (Da) pore size hydrophilic membrane, preferably 3,000 Daltons (Da). The permeate obtained (fraction from about bigger than zero to about lower than 3,000 Da) was collected, lyophilized, and stored -10°C to -20°C until its subsequent fractionation.

7.B HPLC fractions on reverse phase on semi-preparative scale

Fractionation of the fraction from about bigger than zero to about lower than 3,000 Da obtained from hydrolysate G71+ was performed by semi-preparative HPLC. The equipment used was an Agilent 1260 (Agilent Technologies) with a quaternary pump, gradient controller, injector, DAD detector, fraction collector, and data acquisition and processing software (Agilent OpenLab CDS ChemStation Edition for LC). The sample was injected at a concentration of 100-300 mg/mL dissolved in water and the peptides were separated by reversed phase chromatography using a Europa peptide column (120A. 25x1mm. 5pm. Teknokroma). The solvents used were water: trifluoroacetic acid (1000:1) solvent A and acetonitrile: trifluoroacetic acid (1000:0.8) solvent B. Elution was performed at a flow of 4 mL/min using the following gradient: 0 to 23.5% B in 59.2 min. 23.5-90% B in 5.8 min and 90-0% B in 2 min. Subsequently, the column was allowed to reach an equilibrium with 100% phase A for 13 min. The injected volume of the sample was 400 pL and the absorbency was 214 nm.

Twenty-two different fractions (Figure 4) named F1-F22 were collected and lyophilized and reconstituted in Milli Q water. The protein content of these reconstituted fractions was determined by the bicinchonicic acid method (BCA ^TM Protein Assay Kit. Thermo Scientific) following the manufacturer's instructions. Subsequently, these fractions were adequately diluted in water so that they all had the same protein content (1 mg/mL) and were determined for COX-2 inhibitory activity, according to the protocol described in Example 3 (final concentration in well 45.5 pg/mL).

The fraction that showed the greatest activity was F3, which at a concentration of 45.5 pg/mL produced 93% inhibition of the enzyme, followed by F20 with 48% inhibition (Table III). F3 was obtained between minute 8 and 10 of the chromatogram when a solvent B gradient in A from 0% to 23.5% was used, corresponding to a percentage of B between 1.4 and 4% and F21 was obtained between min 44-46, corresponding to a percentage of B between 17.5 and 18.3 (Figure 4).

Subsequently, the fractions were analysed by mass spectrometry as explained in Example 6C, in order to identify the peptides contained therein and that could be responsible for the COX-2 inhibitory and anti-inflammatory activity of the fraction from about bigger than zero to about lower than 10,000 Da of the G71 hydrolysate.

Table III. COX-2 inhibitory activity of the RP-HPLC fractions obtained from the selected hydrolysate.

Fraction COX-2 inhibitory activity (%)

F.1 26.54 ± 4.84

F.2 5.79 ± 0.9 F.3 92.58 ± 0.32

F.4 12.24 ± 6.59

F.5 23.42 ± 1.88

F.6 8.29 ± 4.46

F.7 33.49 ± 2.30

F.8 27.46 ± 5.37

F.9 3.36 ± 2.37

F.10 0.90 ± 1.49

F.11 1.90 ± 0.30

F.12 15.11 ± 7.84

F.13 6.90 ± 3.20

F.14 4.50 ± 0.40

F.15 4.30 ± 0.90

F.16 3.80 ± 1.70

F.17 31 .56 ± 7.50

F.18 20.19 ± 8.19

F.19 38.97 ± 2.83

F.20 47.54 ± 2.03

F.21 22.35 ± 4.77

F.22 1.74 ± 2.46

7.C. Identification of hydrolysed peptides obtained from collagen through MS spectrometry (MS/MS)

Fractions F3 and F20 were analysed by UHPLC-Orbitrap MS/MS (LTQ-Orbitrap Velos Pro mass spectrometer (ThermoFisher Scientific, CA, USA)). The sample was separated onto a Peptide BEH C18 Reverse Phase column (Waters, Massachusetts, USA) coupled to an UHPLC (Rapid Resolution Liguid Chromatograph, Agilent Technologies, California, USA) using a 70 min acetonitrile gradient (A = water, 0.1% formic acid; B = acetonitrile). The flow rate during elution gradient was 0.400 mL/min.

For real time ionization and peptide fragmentation, an enhanced FT-resolution spectrum (resolution = 30,000 FHMW) followed by a data dependent FT-MS/MS scan from most intense ten parent ions with a charge state of one or more, using a HCD fragmentation with a normalized collision energy of 40% and dynamic exclusion of 0.5 min.

Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer version 1.4.0.288 (ThermoFisher Scientific, CA, USA). All MS and MS/MS samples were analysed using Mascot (Version 2.5) search engine using one node with the proteome of Sus Scrofa (26103 entries) from UniProt. This search assumed no enzyme digestion and an error of 40 mmu for fragment ion mass and 20 ppm for precursor ions. Oxidation of methionine and acetylation of N-termini were specified as variable modifications. For the peptides identified visual verification of fragmentation spectra was done and only these peptides found in both replicates were considered.

These 9 peptides (namely from P1 to P9) were identified in the fractions. P1-P3 were identified in F3 and peptides P4-P9 in the F20. Table IV shows information about the amino acid sequence, charge state, mass and m/z of the identified peptides.

Table IV. Peptides identified in the two selected UHPLC-fractions.

Peptide Amino acid Charge MH+ m/z sequence (Da)

P1 AGERGEO 2 746.33 373.67

P2 GSKGRPG 2 658.36 329.68

P3 GPAGSVG 2 544.27 272.30

P4 AGPAGKP 2 597.33 299.17

P5 AGPPGAK 2 597.33 299.17

P6 GPAGPAGPRG 2 836.43 418.72

P7 GPAGPAG 1 526.26 526.26

P8 PGPAGPV 1 594.32 594.32

P9 TGPIGPPGPA 1 863.47 863.47 7.D Peptides synthesis and determination of inhibitory COX-2 activity of those identified peptides fractions.

Once the peptides in the fractions, obtained by liquid chromatography from the bioactive hydrolysate were identified, the COX-2 inhibitory activity of the peptides was determined in order to identify which of the peptides could be responsible for the bioactivity of the hydrolysate. To do this, the peptides were sent to be chemically synthesized to Casio ApS (Lyngby, Denmark) using the phase Fmoc method with a synthesizer (Model 431 A; Applied Biosystems Inc., Uberlingen, Germany).

It has also been tested by the Applicant the purity of the synthesized peptides, which has been determined by Casio ApS by reversed phase HPLC at analytical scale and mass spectrometry using Esquire-LC equipment (Bruker Daltonik GmbH. Bremen, Germany). All peptides tested had a purity greater than 90-98%.

Table V. Purity and COX-2 inhibitory activity of identified peptides in fractions obtained from anti-inflammatory hydrolysate. These synthetic peptides were diluted in Mill! Q water and determined for COX-2 inhibitory activity following the method described in Example 3. The final concentration tested in all peptides was 9.09 pg/mL. Table V shows the results obtained in the determination of the aforementioned activity, represented as a percentage of inhibition. All peptides showed a certain COX-2 inhibitory activity, ranged between 14.5 and 41.3% when they were tested at the concentration of 9.09 pg/mL. The peptides with the best activity were peptides P1, P2 and P3 from F3 fraction and P8 from F21 fraction, which inhibited the COX-2 between 37 and 41%.

Table V. Purity grade and COX-2 inhibitory activity of the peptides identified in the hydrolysate.

Peptides Purity (%) Inhibitory

COX-2 activity (%)*

P1 96.69 37.6 ± 0.3

P2 96.93 37.5 ± 3.8

P3 97.45 40.1 ± 6.5

P4 97.73 32.2 ± 7.9

P5 97.23 27.5 ± 4.4

P6 97.01 30.3 ± 3.2

P7 98.98 29.1 ± 4.2

P8 96.13 41.3 ± 5.0

P9 98.92 14.5 ± 6.2

* Not determined

*COX-2 inhibitory activity of peptides was tested at 9.09 pig/mL

Additional tests have been carried out in order to evaluate hydration and dissolution time of hydrolysed collagen G71 PLUS and products according to the following technical indication.

The hydration time (H) represents the time needed for the protein hydrolysate to fully sink to the bottom of a recipient filled with water, it is measured as follows.

The dissolution time (D) means the time needed for substantially all the particles of the protein hydrolysate to be dissolved in water. It is measured according to method described further below.

Hydration and dissolution time are measured according to following methods:

Method A/B

Sample: weigh a sample of 2.5g protein hydrolysate.

A 400ml beaker is filled with 300ml tap water having a temperature of from 15°C to 20°C.

The protein hydrolysate sample is added all at once in one go to the water without stirring. Start chronometer and observe behaviour of the protein hydrolysate.

After 1 minute, stir 3x with a tablespoon. Evaluation:

Method A - Hydration time (H) or wettability: the time needed for the whole protein hydrolysate sample to sink to the bottom of the beaker filled with water such that no more lumps are floating on the surface of the water, corresponds to the hydration time of that sample.

If the sample sinks to the bottom immediately after addition to the water, H= 0 seconds

If the sample does not immediately sink to the bottom, note the number of seconds until the sample sinks to the bottom of the beaker until no more lumps are floating on the surface of the water, and H corresponds to said number of seconds.

Method B — Dissolution time (D) or dissolution rate: the time needed for the whole 20 protein hydrolysate sample to be dissolved in the water, meaning no visible particles remain in the solution:

If the sample is dissolved without the need of stirring, D = 0 seconds

If stirring after 1 minute is needed for the sample to dissolve, D corresponds to the number of seconds needed for the sample to dissolve and is computed from the start of the chronometer. From the start of the chronometer, the sample is stirred 3x with a table spoon every minute until dissolved.

DISSOLUTION TIME (D):

The powder remains floating and starts moisturizing and falling to the bottom of the beaker as starts to dissolve. After the first minute, the solution is shaken with a table spoon 3 times. Afterwards, every minute this step is repeated.

Results: 10 min and 20 sec.