ES2931084B2 - Integrated process and equipment for obtaining type II or native collagen from cartilage of animal origin by contact with CO2 - Google Patents

Description

DESCRIPTION

Integrated process and equipment for obtaining type II or native collagen from cartilage of animal origin by contact with CO2

TECHNICAL SECTOR

The present invention describes an integrated process for obtaining native collagen from cartilage using only a supercritical fluid, particularly supercritical CO2. The collagen obtained is suitable for medical and nutraceutical use.

BACKGROUND OF THE INVENTION

Collagen is the most abundant protein structure in the extracellular matrix of connective tissue and basement membranes. Collagen fibers are made up of fine filamentous subunits called fibrils. The fibrils, in turn, are made up of collagen molecules that are grouped one after the other in such a way that two zones are formed, an overlap zone and a gap zone, which gives rise to the characteristic striation or banding of collagen. Covalent bonds are established between the collagen molecules that give strength to these fibrils and therefore to the collagen.

To date, 28 different types of collagen have been characterized, which form fibrils, microfibrils and connecting structures. Collagen types are classified according to the homologous structure of their domains and are assigned Roman numerals from I to XXVIII based on their chronological order of discovery. For example, the structure of type II collagen is made up of three a1 chains (a1 (II)3) and it was the second to be discovered [1,2].

In addition to the molecular composition, Table 1 shows some structural characteristics and the location of the three most important types of collagen. Collagen type II molecules consist of a central COL domain and two domains at the ends consisting of non-collagenous sequences called telopeptides; at the C-terminal end they have a telopeptide and a partially processed N-terminal end [3].

Table 1. Summary of the main characteristics of collagen types I, II and III. [1,2]

Type II or native collagen is the main structural component of cartilage. Together with other tissue-specific collagen types, it is randomly oriented in a matrix of viscous proteoglycans. The rigidity of the fibrils of this type of collagen imparts strength and compressibility. This property allows joints to absorb shock. Therefore, it is used as a dietary supplement to promote joint health. In addition, it has been proven to suppress rheumatoid arthritis. Other applications of type II collagen are in the food industry (as a clarifying agent, emulsifier or whipping agent), in pharmaceutical applications (as a tissue engineering material, for microencapsulation and as a coating) and in the cosmetics industry, for the manufacture of shampoos and lipsticks. However, today, the greatest demand is for its use in the formulation of products related to joint health.

Traditional collagen production procedures include several stages: first, the preparation of the raw material (which usually includes mechanical cleaning, washing or even cooking to disinfect), followed by a grinding stage and an acid or alkaline digestion (to eliminate the non-collagenous fraction and increase the extraction yield); second, the extraction of the collagen, which can be carried out by enzymatic and/or chemical hydrolysis, mainly with strong acids, where a diluted collagen suspension is obtained; and, finally, the concentration and purification of the diluted suspension (Cao, H. and Xu, S. Y. Purification and characterization of type II collagen from chick sternal cartilage, Food Chem. 108 (2008) 439-445; Deyl, Z et al., Preparative procedures and purity assessment of collagen proteins, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. (2003), 245-275).

These methods, in addition to being very time-consuming, requiring several days, include numerous steps, some of which (such as centrifugation or chromatography) are very expensive, as well as treatment with various solvents and aggressive agents such as acids or salts that can remain in the collagen as residues. Acid hydrolysis could also cause damage to the sensitive three-dimensional structure of native collagen.

However, there is currently a need to make chemical processes more sustainable and simple. In this sense, supercritical CO2 extraction is proposed as a promising technology for collagen processing (Harris, M. et al. Developments for Collagen Hydrolysate in Biological, Biochemical, and Biomedical Domains: A Comprehensive Review. Materials, 14(2021), 2806).

There are patented processes that include a stage with supercritical CO2 in the production of collagen, but only for the purpose of extracting impurities. Patent ES2238926 proposes degreasing pig skin using compressed carbon dioxide to subsequently obtain collagen paste for food use. The method includes successive compressions and decompressions of the extractor to cause the rupture of the adipocyte membranes, thus facilitating fat-solvent contact. However, this method only degreases the skin from which type I collagen could be obtained, not type II or native collagen. It would require a subsequent extraction, concentration and purification procedure.

On the other hand, patent WO2018030748 relates a method to extract collagen from liposuction effluent, using supercritical CO2; and patent CN102633877A describes a complex process to obtain collagen from fish skin and scales in which supercritical CO2 is only used in one stage to extract oil as a secondary product. Document ES2105323 includes a procedure for tanning, dyeing and waterproofing natural skins containing collagen in which supercritical CO2 is used as the fluid in all these stages instead of water to minimize the environmental impact and the consumption of raw materials.

In addition, there are publications in which sub or supercritical CO2 is used as a sterilization agent for collagen-based materials (Herdegen, V. et al. Sterilization of medical products from collagen by means of supercritical CO2, Chem. Eng. Technol. 37 (2014) 1891-1895; Bernhardt, A. et al. Improved sterilization of sensitive biomaterials with supercritical carbon dioxide at low temperature, PLoS One.

10 (2015) 1-19; Santos-Rosales, V. et al. Sterile and Dual-Porous Aerogels Scaffolds Obtained through a Multistep Supercritical CO2-Based Approach, Molecules 24 (2019) 1-17). In these descriptions, collagen has already been processed and obtained by conventional methods; it is a mineralized and shaped collagen to generate scaffolds for implants, obtained from the dermis of the pig and from the submucosa or intestine of the pig. Therefore, CO2 is not used in its production nor is native collagen obtained.

For all the above reasons, there is still a need for new, sustainable and simple alternative processes for the production of type 2 or native collagen, which use smaller amounts and types of solvents, milder temperature conditions to reduce energy requirements and with the recirculation of unprocessed raw materials and solvents used. Additionally, the current market for drugs and functional foods increasingly demands minimally processed products with the least amount of solvent or chemical agent residues possible.

The present invention proposes a clean and simple, integrated process to obtain a native collagen type II since, from the grinding of the natural raw material, only a treatment with supercritical CO2 is needed to obtain a native collagen that maintains its three-dimensional structure intact and is free of solvent, which would allow its use in medical and nutraceutical applications. After the procedure, only grinding and sieving would be required.

EXPLANATION OF THE INVENTION

This invention describes a method for obtaining native collagen from collagen of animal origin by using supercritical or subcritical CO2. The process is carried out under moderate pressure and temperature conditions, for a time sufficient for effective contact between the collagen and the CO2. A degreased and sterile native collagen is obtained that preserves its three-dimensional structure and its quality properties and with an appropriate humidity for its storage.

Collagen can be used as raw material with its original water content or drained, partially dried at moderate temperature (e.g. in a vacuum dryer or with intermittent drying) or partially dehydrated by a pretreatment with a solvent such as acetone or ethanol. Although the water content of the raw material to be treated with CO2 can range from the original to the degree of moisture in equilibrium with the environment, preferably the water content is 20 - 30% or higher.

The raw material is put in contact with subcritical or supercritical CO2 under pressure conditions of 50 to 500 bar and a temperature of 20 to 100°C. The CO2 flow rate is between 5 and 120 KgCO2/h/kg of raw material, for a time between 1 and 24 hours.

To remove CO2 and recover the treated collagen, depressurization is carried out at a speed of less than 20 bar/min. The removed CO2 is recirculated to the process.

Optionally, to reduce the operating pressure, increase the process efficiency, decrease CO2 consumption or contact time, a modifier such as ethanol, ethyl acetate, ethyl lactate, alcohols, acetone or compounds that are commonly known as co-solvents in the state of the art can be added to the CO2.

In a preferred embodiment of the invention, the raw material is placed in contact with CO2 at temperatures between 38 and 43 °C and depressurization rates of < 10 bar/min to preserve the quality of the collagen obtained, as well as using pressures between 150 and 350 bar to maintain high yields without increasing energy costs or equipment costs.

The equipment where the process is carried out comprises a raw material input system, a container (R1) to contain the raw material to be treated, a CO2 supply line, a CO2 recovery line, devices for controlling and reading the pressure and temperature, safety elements, a depressurization system and a discharge system for the collagen obtained.

The CO2 supply line uses a refrigerant (E1) to sub-cool it before compressing it in a pump (P1) and adjusting its temperature in a heat exchanger (E2).

After contact with the collagen paste, the CO2 is recirculated, previously reducing its pressure, cleaning it of impurity residues and reconditioning it to operating conditions.

Optionally, the equipment includes a co-solvent supply line. Also to improve the effective contact between the raw material and the CO2, distributors can be introduced if the raw material is arranged in a fixed bed or a rotating drum that allows the movement of the raw material.

The native collagen obtained maintains its three-dimensional structure intact and is free of solvent. It can reach a humidity of less than 10%. All of this would allow its use in medical and nutraceutical applications. After the procedure, it would require at most grinding and sieving.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the impact of CO2 consumption on the collagen process performance measured as mass loss and on the moisture content of the collagen obtained at 350 bar and 40 °C.

Figure 2 shows TEM images of samples obtained at 350 bar. A) 40 °C, 14 h of treatment; B) 40 °C, 65 h, 9 compression/decompression cycles; C) 17 min at 45 °C out of 8 h of treatment. The scale bar is 200 nm.

Figure 3 shows a longitudinal and cross section of the collagen obtained from chicken sternum cartilage pre-dried and treated with supercritical CO2 at 350 bar and 40 °C.

Figure 4 shows the equipment for obtaining collagen from cartilage using supercritical CO2.

Figure 5 shows the impact of operation time and flow rate on the collagen production process performance measured as mass loss of chicken sternum cartilage paste. Supercritical CO2 was supplied at 350 bar and 40 °C. The error bar shows the average standard deviation. The solid lines show the polynomial fit of the experimental data.

Figure 6 depicts the impact of temperature on processing rate as the CO2/pulp ratio was increased. The pressure was 350 bar and the flow rate was 60 kg CO2/h/kg pulp. The error bar shows the average standard deviation. The solid lines show the polynomial fit to the experimental data.

Figure 7 shows TEM images of the collagen structure obtained with supercritical CO2 at 350 bar and 40 °C (A) or 43 °C (B).

PREFERRED EMBODIMENT OF THE INVENTION

The present invention is illustrated by the following examples, which are not intended, in any case, to be limiting of its scope.

Example 1

This example concerns the production of native collagen from chicken sternal cartilage using only supercritical CO2.

Chicken sternal cartilage is ground to produce a paste. An approximate quantity of 20 g of paste is introduced into the 100 mL high-pressure stainless steel vessel, forming a fixed bed with a length to diameter ratio of approx. 6:1. A metal mesh at the bottom and head of the bed prevents the collagen from being carried away during operation. As it has a tendency to compact, favouring the formation of preferential channels, redistributors are used. The vessel is then closed and preheated to operating temperature. CO2 pumping is then started and, when the pressure of interest is reached, the back pressure valve (BPR) is opened, providing a continuous flow of CO2 through the bed at an average of 4 g/min giving a mass flow rate of 16 g CO2/h/g paste. After the valve, the CO2 is depressurized and therefore becomes gaseous, which causes what had been extracted or carried to precipitate into the collector. Once the operating time has elapsed, the equipment is depressurized. The total flow of CO2 passed is read on the flow meter. The container is separated from the equipment and the product is discharged for subsequent analysis.

Example 2

This example describes the analysis of the obtained collagen.

The effectiveness of the treatment described in example 1 is determined from the data on moisture content and microbial load of the product obtained, as well as from transmission electron microscope (TEM) images of the structure of the final product and its proportion of native collagen.

Microbiological analysis is carried out on both the treated samples and the starting paste to establish the degree of inactivation during treatment. Five grams of sample are mixed with 45 ml of peptone water under sterile conditions. Serial dilutions are then prepared and 1 ml of each dilution is cultured in Petri dishes containing 20 ml of the appropriate specific medium. Aerobic mesophilic microorganisms are counted after cultivation at 30 °C for 24 h in PCA (plate count agar) medium. Anaerobic microorganisms are counted in SPS (Polymyxin Sulfite Sulfadiazine) medium after cultivation in an anaerobic jar at 37 °C for 48 h. Results are expressed in colony forming units per gram (cfu/g) and the degree of inactivation is defined as the logarithm of the count after treatment (N) with respect to the initial count (No). The maximum standard deviation in these measurements is ± 0.3 logarithmic cycles.

First, ground cartilage is analyzed to establish the baseline for the collagen production method. The anaerobic microorganism count in this product is in the range of 104 cfu/g. Typically, chicken has anaerobic contamination with Clostridium species, although it may also have Staphilococcus aureus or other obligate or facultative anaerobes. The total aerobic count is in the range of 105-106 cfu/g depending on the batch (average 7.4x105 cfu/g).

The moisture content of the raw material and the product is determined by weight loss in an oven at 105°C until a constant reading is obtained. The standard deviation of these measurements is ± 3%.

The product collected after the treatment of the container is ground and sieved on a 180-micron sieve to be structurally characterized by transmission electron microscopy (TEM). Observation of the samples by TEM allows the degree of heterogeneity of the samples to be observed, that is, whether there is the presence of another type of tissue, the state of the collagen fibrils (whether they are defined or not, their length) and the presence or absence of the characteristic banding in them.

In order to determine the native collagen content in the product obtained after CO2 treatment, ELISA (Enzyme-linked immunosorbent assay) analyses are performed based on specific enzymatic immunosorption tests. In a first stage, the sample is digested with pepsin, which separates the collagen molecules whose triple helix structure is maintained from those collagen molecules that are degraded. The sample is then degraded with proteinase K for subsequent analysis with ELISA. The kit used determines the degradation products of the C-terminal telopeptides of type II collagen.

Example 3.

This example describes the use of untreated ground chicken sternal cartilage.

The cartilage used is ground, with an average water content of 68% and a fat content of 2-3%. This primary material, which is highly microbiologically contaminated, very moist and contains some fat, is tested at two operating pressures: 100 bar and 350 bar. The treatment temperature is kept constant at 40°C. These tests are also carried out at increasing operating times, in order to increase the CO2 ratios per unit of pulp mass. A summary of the results is shown in Table 2 and Figure 1.

Table 2. Conditions and results of microbial inactivation measured as total aerobic count of supercritical CO<2> treatment of chicken sternum cartilage paste.

(*) In the data reported with a logarithmic reduction of -5.9 in the aerobic count and -4.4 in the anaerobic count, there was no growth in any dilution; therefore, they represent a degree of sterility.

At low pressure, little precipitate is collected in the CO2 outlet manifold, while in the tests carried out at 350 bar, a yellowish, gummy, moist precipitate is collected. On the other hand, the product collected from the high-pressure vessel after treatment is a white porous solid. After grinding, its native collagen content, which is typically 60%, is analysed and its structure is observed. Using TEM images, it can be observed that during the procedure the typical three-dimensional helical structure of native type II collagen is preserved in excellent conditions. As an example, Figure 2A shows images taken from Sample 4 of Table 2 in which a very high degree of drying (<5%) is achieved and sterility is achieved. No degraded fibres are observed, the striation is marked and the fibrils remain elongated with a similar size, the characteristic banding of collagen is well defined.

In contrast, Figure 2B shows the TEM image of Sample 5 obtained under similar conditions, but which was subjected to a very long treatment time (65 hours) and nine compression/decompression cycles. It clearly shows signs of degradation with more broken fibres and less defined banding. Figure 2C also includes observations of a sample in which the temperature was raised to 45 °C for a period (17 min) of the total treatment (8 h). The condition of the fibres is very deteriorated due to the high thermo-sensitivity of collagen.

In the tests carried out at higher pressure, the product was also sterile, with zero colonies counted in all dilutions of both aerobic and anaerobic species. This is because high-pressure CO2 has antimicrobial activity [13,14]. In a previous work carried out on a natural product, cocoa, it was shown that, with this reduction in the total microbial load, biocontamination such as enterobacteria and staphylococcus aureus is also eliminated [15]. In addition, other authors have shown under similar operating conditions that viruses causing human pathologies are also inactivated, a fact that is very relevant in the case of chicken or bird derivatives in general, due to the health problem of avian flu. For example, in experiments performed in a pilot plant for the preparation of bone allografts, it was shown that four types of viruses (HIV-1, Sindbis, Polio Sabin type I and Pseudorabies) were inactivated at values below the detection limit by applying CO2 at 250 bar, 50°C and 10 min per g of bone [16]. Also, disinfection of several viruses (Coronavirus “TEGV”, Herpes ivurs “PRV”, Flaviviridae “JEV”, Arterivirus ‘PRRSV’) was achieved in a representative temperature-sensitive biomaterial (different cell lines), with CO2 at 40-50°C, 160 bar and 5 min without causing significant damage to the bioactivity of the biomaterial [17].

Example 4

This example describes the use of ground cartilage pre-dried under vacuum to an initial water content of 34%. Microbial contamination is 480 cfu/g of total aerobic count. In this case, the tests are carried out at 350 bar and 40°C and for a time of 14 hours. A collagen with a final water percentage of 5% is obtained, which is also sterile, with a good collagen structure, with long, structured, parallel fibres and well-defined banding, as shown in Figure 3.

Example 5

The production of native collagen from chicken sternum cartilage pretreated with acetone or ethanol before processing with supercritical CO2 is shown.

The paste is first treated with acetone or ethanol to dehydrate, remove fats and thus reduce the time and pressure of subsequent treatment with supercritical CO2. The acetone residue in the collagen obtained is measured. In the samples treated with ethanol, the residual content was not measured because this solvent is recognized as safe, GRAS (Generally Recognized As Safe).

The work is carried out on similar raw material to example 1, although because they are from different batches, the initial water content is 77% for the tests with acetone (samples 6 to 9) and 81% for the tests with ethanol (samples 10 and 11). Acetone and ethanol are supplied by Scharlau Chemie, S.A. with a purity of >99.8% and >99.9%, respectively. The CO2 is the same as in the previous example.

Pre-treatment with acetone and ethanol. The mixture is treated with a ratio of 270 ml of acetone or ethanol per 100 g of pulp. The mixture is vacuum filtered using a Büchner funnel. The process is repeated at a rate of 5 ml per gram of pulp obtained from the filtrate, followed by a new vacuum filtration and then immediately introduced into the high-pressure vessel to be treated with CO2.

Treatment with CO2. The tests are carried out operating with CO2 at 150 bar and 38-40°C in the same way as in example 1, but using a smaller container, 10 mL and a quantity of paste of the order of 5 g. The CO2 flow rate is set at 3 g/min.

The residual amount of acetone is measured by gas chromatography using the headspace method. Approximately 2 g of sample is filled into a 10 ml vial with a septum and sealed. The vial is kept in a 60 °C bath for 60 minutes until phase equilibrium is reached. A 0.5 ml sample is introduced into the chromatograph from the gas phase using a syringe. Each sample is punctured twice and the average of the areas obtained is calculated to minimize error. A gas chromatograph equipped with a trace ion detector (GC-FID SHIMAZDU GC-2010 PLUS) is used. The column is a capillary silica column "Zebron- ZB-1-HT Inferno” (length 20 m, diameter 0.18 mm, wall thickness 0.18 ^m). The operating conditions are: oven temperature 40 °C, analysis time 6 min, injector temperature 180 °C, SPLIT mode with a ratio of 19.5, pressure 40.9 kPa, nitrogen flow 7 ml/min and FID temperature 280 °C. Acetone-water liquid samples are used for calibration.

A summary of the results obtained is shown in Table 3. The amount of CO2 used in each sample and the humidity of the collagen obtained, the native collagen content according to the ELISA test and the residual acetone are indicated. All samples have zero counts in dilutions up to six orders of magnitude in both the aerobic and anaerobic counts, therefore, they could be considered sterile.

Table 3. Properties of native collagen obtained from chicken sternum paste pretreated with acetone or ethanol prior to processing with supercritical CO<2> at 150 bar. Samples 6 to 9 were pretreated with acetone, samples 10 and 11 with ethanol.

The pretreatment with acetone prior to CO2 extraction reduces the water content of the paste to approximately 35%. Therefore, when operating with CO2 at a much lower pressure than in the previous tests in example 1 (150 vs. 350), a similar amount of CO2 is needed to obtain a defatted, sterile collagen with the desired humidity < 10%. The acetone that soaks the collagen could have acted as a modifier (co-solvent) of the CO2, improving the solubility of water and fats.

After this procedure the residual acetone content is >150 ppm in samples obtained with CO2 ratios lower than 230 kg CO2/kg paste (samples 6, 7 and 8). According to the recommendations of the US Federal Drug Administration [18], the maximum residual amount of acetone in pharmaceutical products should be <5000 ppm; while in foods it should be 50 ppm [19]. To adjust the amount of residual acetone, CO2 can be passed only for a longer time as was done in sample 9. By increasing the CO2 ratio to 299 kg CO2/kg paste, the residual acetone concentration is 13 ppm.

Regarding the ethanol pretreatment, after the vacuum filtration operation, a mass loss of around 40% is achieved. Then, with a ratio of 300 g CO2/g paste, a product is obtained that meets humidity criteria at 150 bar and 38 °C (Sample 11). In this last sample, the native collagen content according to ELISA tests is 66%.

Example 6.

The obtaining of native collagen from chicken sternal cartilage with supercritical CO2 modified with ethanol is described.

Essentially, it is the same procedure as in the previous examples, with the addition of an ethanol supply line, so that CO2 and ethanol are fed in parallel by pumps at the desired operating pressure. The CO2 flow rate is set at 3 g/min and the ethanol flow rate is adjusted to give the desired percentage (3 or 10% by mass with respect to the main fluid, CO2). Both fluids are mixed and the obtained stream is then preheated to the operating temperature. The fluid mixture thus enters the high-pressure vessel where it passes through the ground chicken sternum cartilage paste (approx. 3.5 g). When the operation ends (= when the desired amount of CO2 passes through the loaded paste mass), the pumps are turned off and depressurization begins slowly. In these tests, it is possible to carry out this at an average speed of 6 bar/min. The product obtained is discharged from the extractor for the relevant subsequent analyses. A summary of the samples obtained is shown in Table 4. The deviation in the humidity measurement is high, in the order of ± 5% due to the retention of some ethanol in the samples treated with a higher proportion. To eliminate it, supercritical CO2 can be additionally passed through.

Table 4. Properties of native collagen obtained from chicken sternum paste after treatment with ethanol-modified CO<2> at 150 bar and 40°C. The moisture content of the paste is 75%.

When 3% ethanol is used, just over 350 g CO2/g collagen are needed to achieve a product with 15% moisture. When 10% ethanol is added, the amount of CO2 needed to achieve 13% moisture is 200 g CO2/g paste, and to achieve a moisture content of 5%, 350 g CO2/g paste. Compared to the tests with CO2 alone shown in example 3, the pressure is reduced by using ethanol as co-solvent, as is the operating time. This is important because it greatly increases the process capacity. In exchange, 20 g of ethanol must be introduced per g of paste.

Example 7.

This example refers to the equipment used to obtain native collagen from chicken sternal cartilage.

Figure 4 shows a schematic of this at pilot scale. The vessel where the paste is loaded has a capacity of 11 litres (R1). The CO2 supply line uses a refrigerant (E1) to sub-cool it avoiding cavitation before compressing it in a pump (P1), and a heat exchanger (E2) to adjust its temperature. There is a parallel supply line of co-solvent. At this scale, the CO2 is recirculated, so after contact with the paste, the pressure of the CO2 is reduced in two consecutive separators (S1, S2) where what it carries is precipitated and extracted. In this specific installation, in the first separator, the pressure is 75 bar and the temperature 35°C. In the second separator, the conditions are 35 bar and 10°C. The CO2 is then cleaned of impurity residues in an adsorbent bed filled with activated alumina (A1) and liquefied in a condenser (C1) before being returned to the process. The CO2 lost in the depressurization during the collagen discharge and in the collection of the precipitated residues in the separators is compensated with fresh CO2 from an external storage tank.

The tests are carried out on batches of between 600 g and 2 kg of pasta, drained in a sieve to remove excess free water (around 4%). In this way, the pasta has an initial water content of 73%. To avoid compaction of the pasta bed and channelling problems, redistributors are placed at various heights. The geometry of the bed is similar to that used in the other examples, i.e. length to diameter ratios of 6 to 1 or higher, up to 8 to 1.

First, the CO2 flow rate is varied between 30 and 60 kg CO2/h/kg of pulp, operating at 350 bar and 40°C. The results are shown in Figure 5. For comparison, the data from Example 3 on a laboratory scale at 16 kg CO2/h/kg of pulp are also included. Increasing the CO2 flow rate increases the process efficiency. For example, at 60 kg CO2/h/kg of pulp, it takes 6 h to obtain the native collagen product with the required moisture content <10%. At 30, it takes twice as long. Up to 22 hours would be needed at low CO2 flow rates to achieve a similar mass loss result. On the other hand, and in general, the maximum reduction in mass of the pulp during the treatment is around 70%; that is, the collagen yield of the process is 30%. This loss of mass corresponds to the elimination of water (approx. 60%) and other substances (approx. 10%), including fat.

Figure 6 shows the impact of temperature on the process speed, again measured as the loss of mass of the paste. It is greater at 43°C than at 40°C. In example 3 of the laboratory scale tests, the heating of the container where the paste is housed is done by surrounding it with a heating jacket. In the pilot plant in Figure 4, CO2 is heated in-line before entering, but the container where the paste is housed is only thermally insulated. This latter way of operating surely makes the collagen paste at a more homogeneous temperature inside the container without hot zones on the walls. This is why, in the laboratory tests of example 3, collagen degradation is observed at temperatures above 40°C, while, in the pilot tests, degradation is not observed when operating at 43°C. Figure 7 shows TEM images of both samples. In both cases, the fibrils were defined, with the typical banding, without signs of degradation.

A slight improvement in process speed is also obtained when operating at higher pressures, as already detected in Example 3 performed at laboratory scale. For example, the average rate of mass loss of the paste increases by 10% when raising the pressure from 300 to 400 bar, operating at 40°C.

In all samples of product discharged from the container, the reading on aerobic and anaerobic counting plates is zero up to dilutions of 6 orders of magnitude and the native collagen level was maintained at 60% on average.

Claims (8)

1. Integrated process for obtaining type II or native collagen from cartilage of animal origin by contact with CO2, which includes: - putting said cartilage of animal origin into contact with subcritical or supercritical CO2 under pressure conditions of 50 to 500 bar, a temperature of 20 to 100°C, a CO2 flow rate of between 5 and 120 kg CO2/h/kg collagen, for a time of between 1 and 24 h. - remove the remaining CO2 from the previous stage by depressurization at a rate less than 20 bar/min. - recover the removed CO2 and recirculate it to the process - recover the resulting degreased and sterile collagen. where the animal collagen used as raw material has a humidity of 60-80% and is partially dehydrated to a humidity of 20-30% before being put into contact with CO2. 2. Integrated process, according to claim 1, where the partial dehydration is carried out by drying at a moderate temperature in a vacuum dryer or in an intermittent dryer. 3. Integrated process, according to claim 1, where the partial dehydration is carried out by treatment with solvent such as acetone or ethanol. 4. Integrated process, according to claim 1, where the contact with CO2 is carried out in the presence of a co-solvent. 5. Integrated process, according to claim 4, wherein the co-solvent is ethanol, ethyl acetate, ethyl lactate or acetone. 6. Integrated process, according to claim 1, wherein the cartilage of animal origin is chicken sternum cartilage. 7. Integrated process, according to claim 6, where the raw material is put in contact with CO2 at a temperature between 38 and 43°C and a pressure between 150 and 350 bar. 8. Process, according to claim 7, where the depressurization rate of the remaining CO2 is less than 10 bar/min.