EP4117447A1

EP4117447A1 - Process for producing compounds of interest from at least one mineralized connective tissue, and compounds of interest derived from this production process

Info

Publication number: EP4117447A1
Application number: EP21710294.6A
Authority: EP
Inventors: Vincenza FERRARO
Original assignee: Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Current assignee: Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
Priority date: 2020-03-11
Filing date: 2021-03-10
Publication date: 2023-01-18
Also published as: US20230110281A1; WO2021180806A1; FR3108116A1; FR3108116B1

Abstract

The present invention relates to a process for producing compounds of interest from at least one mineralized connective tissue, and to said compounds of interest comprising at least collagen. The production process comprises a step of solid/liquid extraction in which said at least one mineralized connective tissue is mixed with an eluent suitable for extracting said compounds of interest from said at least one mineralized connective tissue to said eluent. This extraction step comprises successive operations of adjusting the pH of said eluent, separated by a time interval; which operations of adjusting the pH each comprise an addition of an acid to the mineralized connective tissue/eluent mixture in order to reduce the pH of said eluent to achieve a pH value of less than or equal to 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4.

Description

Process for the manufacture of compounds of interest from at least one mineralized connective tissue, and compounds of interest from this manufacturing process

Technical field of the invention

The present invention relates to the general field of the valorization of the co-products which are generated by the animal food industries.

It relates in particular to processes for the manufacture of compounds of interest from at least one mineralized connective tissue, said compounds of interest comprising at least collagen.

It also relates to products of biological interest, comprising the compounds of interest resulting from these manufacturing processes.

State of the art

Animal sectors generate many by-products and co-products, which are rich in compounds of industrial interest.

This is particularly the case with biopolymers which have potential in many applications, including biomaterials and technical materials, biomedicine or textiles.

Among these biopolymers, collagen is particularly interesting because of its physical and biological properties.

There is thus a definite interest in extracting such compounds of interest, in particular collagen, from these by-products.

Certain processes thus envisage a solid / liquid extraction of compounds of interest from mineralized connective tissues which come from the animal food industry.

Solid / liquid extraction is a fundamental unitary operation which aims to extract, separate, dissolve, either by immersion or by percolation, one or more components (liquid or solid) mixed with a solid.

This material transfer or exchange operation is thus carried out between, on the one hand, a solid phase which contains the material to be extracted and, on the other hand, a liquid phase, the extraction solvent (or eluent).

The methods reported in the prior art use an acid, often 0.5 M acetic acid, in combination with enzymes. These processes are carried out at a refrigeration temperature of 4 ° C and with variable pH (towards increase) throughout the extraction. The volumes of eluent used are variable, generally 5 to 30 times the mass of the treated tissue.

For example, document US2007 / 0219128 describes such a solid / liquid extraction process which is carried out from fish bones immersed in water or an acid solution. In this prior art, the acid solution is added at the start of the extraction step, with a view to immersion for 2 or 3 days, without regulation of the pH thereafter. However, the current manufacturing processes are not completely satisfactory quantitatively (in particular in terms of extraction yield) and / or qualitatively (the collagen obtained is often denatured or hydrolyzed).

Presentation of the invention

In order to remedy the aforementioned drawback of the state of the art, the present invention proposes a new process for the manufacture of compounds of interest from at least one mineralized connective tissue, said compounds of interest comprising at least collagen.

More particularly, the present invention relates to a manufacturing process which comprises the following steps:

- a step of supplying said at least one mineralized connective tissue,

- a solid / liquid extraction step, advantageously by a single cycle, in which said at least one mineralized connective tissue is mixed with an eluent suitable for extracting said compounds of interest from said at least one mineralized connective tissue to said eluent,

- a step of separating the eluent, containing said compounds of interest, from said minus one mineralized connective tissue,

- an optional step of drying said compounds of interest.

And according to the invention, said extraction step comprises successive operations of adjusting the pH of said eluent, spaced by a time interval.

The pH adjustment operations each comprise an addition of an acid in the mineralized connective tissue / eluent mixture, to decrease the pH of said eluent until reaching a pH value less than or equal to 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4.

Indeed, the inventors have found that, without intervention on the pH, the value of this parameter tends to increase throughout the extraction step.

Now, the inventors then demonstrate that, surprisingly and unexpectedly, the adjustment of the pH (via successive adjustment operations) confers a significantly improved extraction yield, in the most natural way possible, compared to the extraction processes. known (devoid of such pH adjustment operations).

In addition, the collagen obtained is qualitatively very interesting: the collagen obtained is non-denatured and / or non-hydrolyzed.

In addition, the process according to the invention has the advantage of being able to be used on bones obtained from adult bovine animals (over 5 years of age). The methods of the state of the art are in fact preferably applicable only on the bones of young animals (less than 2 years old at slaughter) for the greater ease of extraction compared to an adult bone whose collagen is more crosslinked (the chemical availability of collagen being reduced due to age). Other non-limiting and advantageous characteristics of the process in accordance with the invention, taken individually or in any technically possible combination, are as follows:

- The pH adjustment operations are configured to maintain the pH of said eluent at a pH value less than or equal to 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4;

- the time interval between two pH adjustment operations is one to five hours, preferably every 1h30 to 2h30;

- the solid / liquid extraction step is carried out in the form of a single cycle in which said at least one mineralized connective tissue is subjected to a single solid / liquid extraction step, which at least one mineralized connective tissue is mixed with a determined quantity of eluent which is fully introduced at the start of the solid / liquid extraction step, which solid / liquid extraction step is advantageously carried out in a batch reactor;

- during each pH adjustment operation, the pH of the eluent is measured, and, following each measurement, a suitable quantity of acid is added so as to reduce the pH of the eluent to the value predetermined target pH;

- during the solid / liquid extraction step, the temperature of the eluent is maintained at a value corresponding to the physiological temperature of the animal from which said at least one mineralized connective tissue is derived, for example 37 ° C, plus or minus 5 ° C;

- The pH adjustment operations each comprise an addition of an acid chosen from inorganic acids, for example hydrochloric acid;

- the solid / liquid extraction step is carried out over a period of time of at least 5 days, for example ranging from 5 to 10 days;

- during the solid / liquid extraction step, the mass ratio of mineralized connective tissue / eluent is within a range ranging from 1/5 to 1/40 p / p, preferably from 1/15 to 1/25 p / p;

- said at least one mineralized connective tissue is chosen from mineralized connective tissues from mammals or fish, which mineralized connective tissues from mammals are advantageously chosen from bones of bovines or pigs, in particular adult bovines with more than 5 years, which mineralized connective tissues obtained from fish are advantageously chosen from bones and scales;

- the solid / liquid extraction step is carried out under pseudo-static conditions or stirring conditions;

- said eluent is devoid of enzymes, in particular proteolytic enzymes, and / or calcium chelating agents;

- the supply step comprises an operation of physicochemical preparation of said at least one mineralized connective tissue, which preparation operation comprises an operation mechanical treatment ensuring a powder reduction of said at least one mineralized connective tissue, which powder advantageously has a particle size ranging from 0.5 to 2 mm;

- initially, the eluent has a NaCl concentration of less than 60 g / L, preferably a NaCl concentration of less than 10 g / L, more preferably a value equal to 9 g / L;

- the separation step is chosen from decantation, centrifugation or filtration;

- the optional step of drying said compounds of interest is chosen from lyophilization or atomization.

The present invention also relates to the compounds of interest resulting from the manufacturing process according to the invention, said compounds of interest comprising collagen and optionally proteoglycans and / or minerals and / or non-collagen proteins.

The present invention also relates to a product of biological interest, comprising the compounds of interest resulting from the manufacturing process according to the invention, said compounds of interest comprising collagen and optionally proteoglycans and / or minerals, preferably from calcium, phosphorus, and magnesium, and / or non-collagenous protein.

The present invention also relates to this product of biological interest for its use as a medicament.

The present invention also relates to the use of a product of biological interest according to the invention, in an application chosen from:

- dietary supplement,

- materials and biomaterials,

- soil fertilization.

Of course, the different characteristics, variants and embodiments of the invention can be associated with each other in various combinations insofar as they are not incompatible or mutually exclusive.

Detailed description of the invention

In addition, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate non-limiting embodiments of the invention and in which:

[Fig. 1] is a block diagram which illustrates the main steps of the manufacturing process according to the invention;

[Fig. 2] is a block diagram illustrating one embodiment of the supply step;

[Fig. 3] is a graph representing the kinetics of collagen extraction by multicycle extraction (mean values and standard deviation bars), expressed as an amount of collagen (mg) over time (day);

[Fig. 4] is a graph representing the evolution of total mass yields by extraction of multicycle (1/5 w / w) and unicycle (1/20 w / w) collagen (average values and bars standard deviation), expressed as mass yield (mg of collagen per gram of bone) over time (day);

[Fig. 5] is a diagram representing the mass yield of collagen (mg of collagen per gram of bone) at the end of the extraction (mean values and standard deviation bars), for the ratios 1/5 p / p and 1 / 20 p / p;

[Fig. 6] is a diagram illustrating the mass yield of collagen (mg of collagen per gram of bone) as a function of the stirring regime (static - without stirring / pseudo-static - with stirring), at the end of the extraction ( mean values and standard deviation bars);

[Fig. 7] is a curve showing the kinetics of collagen extraction (mean values and standard deviation bars) in mg of collagen / g bone, over time (days);

[Fig. 8] is a graph that illustrates the effect of temperature on collagen extraction efficiency (mg collagen / g bone) over time (day);

[Fig. 9] is a diagram showing the effect of pH on collagen yield (mg collagen / g bone), with two pH management by temperature;

[Fig. 10] is a diagram illustrating the glucose content (mg), with and without added NaCl (NaCl concentration w / w);

[Fig. 11] is a diagram illustrating the effect of pH and temperature on glucose solubilization, expressed in mg with two different pHs (normal and 2.5 constant) for each temperature;

[Fig. 12] is a diagram illustrating the effect of pH and temperature on dry matter, showing percent dry matter with two pH management by temperature.

It should be noted that, in these figures, the structural and / or functional elements common to the different variants may have the same references.

The present invention thus relates to a process for the manufacture of compounds of interest from at least one mineralized connective tissue, and the compounds of interest resulting from this process.

By "compounds of interest" is meant at least collagen.

Collagen especially includes fibrillar collagens (FACIT), network forming collagens, anchor fibrils, transmembrane collagens, basement membrane collagens and others with unique functions.

The members of the collagen family advantageously have one characteristic in common: a straight triple helix composed of three a (alpha) chains. These can be formed by three identical chains (homotrimers), or by two or more different chains (heterotrimers), which are characteristic of humans and terrestrial animals.

The collagen is also advantageously chosen from type I fibrillar collagen (characteristic of mineralized animal tissues of which it represents more than 90% of all the collagens). The collagen can also optionally be chosen from type II and V fibrillar collagen, basement membrane collagen (type IV), anchoring collagen (type VII), transmembrane collagen (type XVII) and FACIT collagen (Fibril Associated Collagen with Interrupted Triple helixes) (type XXII).

These different types of collagen are further described in the document Gelse et al., 2003. “Collagens - structure, functions and biosynthesis”, Advanced Drug Delivery Reviews, 55, 1531-1546.

The collagen (resulting from the process according to the invention) advantageously comprises at least one type I collagen.

By "collagen" is thus meant a single type of collagen or a mixture of at least two types of collagen.

The term "compounds of interest" also advantageously encompasses proteoglycans and / or minerals and / or non-collagen proteins.

Proteoglycans result from the combination of a protein and a glycosaminoglycan (GAG).

The proteoglycans advantageously consist of proteoglycans of the extra-cellular bone matrix, advantageously chosen from SLRPs (small leucine-rich proteoglycans) such as biglycan, decorin, mimecane, osteomodulin, and PRELPs (roline / arginine- rich repeat and leucine-rich repeat proteins).

The minerals are preferably chosen from calcium, phosphorus and magnesium.

The non-collagenous proteins preferably include osteocalcin.

Manufacturing process

For the manufacture of such compounds of interest, the process according to the invention comprises the following succession of steps (Figure 1):

- a supply step (A) of said at least one mineralized connective tissue,

- a solid / liquid extraction step (B) (also called “extraction step (B)”) during which said at least one mineralized connective tissue is mixed with an eluent suitable for extracting said at least one compound d 'interest,

- a separation step (C) of the eluent, containing said compounds of interest, from said minus one mineralized connective tissue,

- an optional drying step (D) of the compounds of interest.

Supply stage

The supply step (A) therefore consists in supplying said at least one mineralized connective tissue (raw material) with a view to its treatment by the solid / liquid extraction step (B).

By “mineralized connective tissue” is meant in particular bone tissue, including reticular bone tissue, lamellar bone tissue and spongy bone tissue. This mineralized connective tissue is advantageously chosen from mineralized connective tissues obtained from mammals or fish.

From mammals, the mineralized connective tissues are advantageously chosen from the bones of cattle or pigs, in particular from adult animals over 5 years old.

The bones used are then preferably long bones, and more preferably the diaphysis part of these long bones (obtained by removing the ends of the bone - epiphysis and metaphysis).

The long bones advantageously comprise the bones chosen from: femur, clavicle, humerus, radius, ulna, metacarpals, phalanges, tibia, fibula (or fibula).

Cattle include in particular bulls, oxen and cows.

Starting from fish, the mineralized connective tissues are advantageously chosen from edges and scales.

For the purpose of the extraction step (B), said at least one mineralized connective tissue is preferably subjected to at least one physicochemical preparation operation.

This preparation operation preferably comprises a mechanical treatment operation to ensure a powder reduction of said at least one mineralized connective tissue (also called “crushing and / or grinding”).

The mineralized connective tissue powder advantageously has a particle size ranging from 0.5 to 2 mm (advantageously measured by a sieving method).

This preparation operation can also comprise, before grinding, operations chosen from:

- a cleaning / drying operation, for example by immersion in at least one alcohol bath (for example 70% ethanol),

- an operation to remove the bone marrow.

By way of example only, FIG. 2 illustrates a particular embodiment of this supply step (A) starting from long bone.

This particular embodiment comprises the following successive operations:

A1. an operation to remove the ends of the bone (epiphysis and metaphysis),

A2. a cutting operation of the diaphysis (middle part of a long bone),

A3 an operation of washing the slices (in a 70% ethanol bath) and drying (at temperature ranging from 40 ° C to 50 ° C), then

A4 a bone marrow removal operation, crushing and grinding of slices.

This embodiment thus generates a bone powder (in particular of the shaft), suitable for the following extraction step (B).

Extraction step

The extraction step (B) consists of a solid / liquid extraction technique, also called “washing” or “leaching”, advantageously of the “maceration” type. During this extraction step (B), said at least one mineralized connective tissue (solid phase) is mixed with an eluent (also called “solvent”, “extraction solvent” or “liquid phase”) which is suitable. extracting the compounds of interest from said at least one mineralized connective tissue to said eluent.

This extraction step (B) is advantageously carried out in the form of a single cycle (also called "unicycle extraction").

In such a "single cycle", said at least one mineralized connective tissue is then subjected to a single extraction step.

Said at least one mineralized connective tissue is then mixed with (immersed in) a determined amount of eluent which is fully introduced at the start of the extraction step (B).

This eluent, containing said at least one compound of interest, is recovered at the end of the extraction step (B).

The eluent is advantageously of food grade / grade (usable for human consumption).

The eluent advantageously consists of an aqueous solution which has an acidic pH and which optionally contains NaCl.

More precisely, the eluent preferably consists of an acidic aqueous solution (also called “acidic medium”), the (initial) pH value of which is less than or equal to 5.5, preferably less than or equal to 4, preferably. ranging from 2 to 4.

This aqueous solution is for example chosen from one of the following acids: formic acid, acetic acid, propionic acid, malonic acid, butyric acid, succinic acid, malic acid, citric acid, tartaric acid, lactic acid, phosphoric acid, hydrochloric acid , sulfuric acid, nitric acid.

Initially, the eluent has a NaCl concentration of less than 60 g / L, more preferably a NaCl concentration of less than 10 g / L, more preferably a value equal to 9 g / L (including a value approximately equal to 9 g / L, for example 8.5-9.5 g / L).

The eluent is also advantageously devoid of enzymes and / or calcium chelating agents.

By “enzymes” is meant in particular enzymes capable of degrading collagen, in particular proteolytic enzymes, namely for example metalloproteinases (collagenases, gelatinases), serine proteinases (elastase, cathepsin G), cysteine proteinase (papain, calpains, cathepsins B, H, L, N and S), aspartic proteinases (pepsin, chymosin, cathepsins D and E).

Calcium chelating agents include, for example, EDTA (ethylenediaminetetraacetic acid).

Preferably, the mineralized connective tissue / eluent mass ratio is within a range from 1/5 to 1/40 w / w (weight / weight), preferably from 1/15 to 1/25 w / w (weight / weight). Furthermore, during this extraction step (B), the pH of the eluent is subjected to successive pH adjustment operations, which operations are spaced by a time interval (two pH adjustment operations successive are thus separated from each other by a lapse of time).

Specifically, each pH adjustment operation involves adding an acid to the mineralized connective tissue / eluent mixture.

Each addition is adjusted so as to obtain a decrease in the pH of the eluent until a pH value (also known as the “predetermined target value”) of less than or equal to 5.5, preferably less than or equal to 4, is reached. more preferably ranging from 2 to 4.

In other words, the pH of the eluent is measured, advantageously regularly; and, following each measurement, a suitable amount of acid is added so as to decrease the pH of the eluent to the predetermined target value.

More preferably, the pH adjustment operations are configured to maintain (advantageously continuously) the pH of the eluent at a pH value which is less than or equal to 5.5, preferably less than or equal to 4, of preferably ranging from 2 to 4.

In other words, these pH adjustment operations are adjusted so that, throughout the extraction step (B), the pH of the eluent has a value which is less than or equal to a value maximum threshold of 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4.

This preferred mode of operation (maintaining the pH) is also referred to as “constant pH” (as opposed to a variable pH in the absence of regular control of the pH of the mixture during the extraction step (B)). .

In this case, the time interval between two pH adjustment operations is advantageously from one to five hours, preferably from 1h30 to 2h30 (1h30 to 2h30).

Each pH adjustment operation advantageously comprises adding an acid to the mixture, advantageously a 0.5 M acidic aqueous solution.

The acid is advantageously chosen from inorganic acids, for example: hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid.

As illustrated in the example below, such successive pH adjustment operations allow significantly improved extraction yield compared to known extraction methods (without successive pH adjustment operations).

Also during this solid / liquid extraction step (B), the temperature of the eluent is advantageously maintained at a constant value, corresponding to the physiological temperature of the animal from which said at least one mineralized connective tissue is derived.

In practice, this temperature is advantageously adjusted to a value of 37 ° C, plus or minus 5 ° C. Such a temperature ensures an interesting extraction yield, without risk of denaturing the extracted collagen.

The solid / liquid extraction step (B) is also advantageously carried out under conditions which are chosen from:

- pseudo-static conditions, for example with a mixture of 15 to 20 minutes every 1 to 3 hours, or

- stirring conditions, with a mixture maintained throughout the extraction step.

Furthermore, the extraction step (B) advantageously lasts over several days, preferably a period of at least 5 days, for example ranging from 5 to 10 days.

In practice, the extraction step (B) is advantageously carried out in a batch reactor (also called "batch").

At the end of this extraction step (B), the eluent thus forms a collagen solution, optionally still containing proteoglycans and / or minerals and / or non-collagen proteins.

Separation step

Following the extraction step (B), the separation step (C) consists of separating the eluent (containing the compounds of interest) from the bone.

This separation step is advantageously chosen from techniques conventional per se, for example decantation, centrifugation or filtration.

The term “decantation” is intended in particular for passive sedimentation following which the supernatant is recovered.

Drying step

The optional drying step (D) consists in drying the compounds of interest, advantageously by eluting in a solution of low acidity (for example, pH of approximately 5.5), to obtain a soluble powder.

This drying operation (D) is advantageously aimed at eliminating the eluent by a phenomenon of evaporation and / or sublimation, so as to recover the compounds of interest.

This optional drying step (D) is for example chosen from lyophilization or atomization.

Lyophilization (low temperature operation) is carried out after freezing of the eluent (for example at -30 ° C), followed by sublimation of the same eluent at a pressure ranging from 0.1 to 5 mbar and at a temperature ranging from from -30 ° C to 15 ° C, for a period ranging from 1.5 to 3 days.

This operation makes it possible, for example, to obtain a powder containing 90 to 92% dry matter, 5 to 7% water and 1.5 to 4% acid.

The powder thus obtained advantageously exhibits an assembly in the form of microparticles of small sizes (ranging from 0.5 to 20 micrometers in diameter) according to observation under a scanning electron microscope (magnification 7000X). The atomization (drying at high temperature) is carried out on the eluent in its liquid state which is heated, for example to a temperature of 70 ° C, before passing through an addition which pulverizes it and which introduces it into a drying chamber, the temperature of which is set for example at 150 ° C.

The powder is then collected in a tank at room temperature. It advantageously has 95 to 96% dry matter, 3 to 3.5% water, 1 to 2% acid.

The powder thus obtained has for example an assembly in the form of fibers, the diameter of which is advantageously of the same order of dimension as the diameter of the collagen fibers in the bone (ranging from 0.05 to 2 micrometers) according to an observation at scanning electron microscope (magnification 7000X).

Product of biological interest and applications

At the end of the manufacturing process according to the invention, a product of biological interest is advantageously obtained which comprises the aforementioned compounds of interest.

This product of biological interest thus comprises at least collagen, and optionally proteoglycans and / or minerals and / or non-collagen proteins.

The collagen obtained advantageously consists of a natural collagen, which is advantageously neither fragmented nor denatured, thus retaining its biological and mechanical properties.

This collagen can be used to produce, for example, biomaterials and technical materials, or as a source of nutrients for soils.

In addition to significantly increasing the yield of collagen (3 times higher than the method of the prior art), this method according to the invention has the advantage of promoting the co-extraction of other molecules of biological interest, such as proteoglycans, and essential macroelements such as calcium, phosphorus and magnesium, to preserve the primary structure (amino acid sequence of the alpha-helix) and telopeptides characteristic of bone collagen (unlike conventional methods which use enzymes and which results in loss of telopeptides and partial hydrolysis of the protein chain).

Such a product of biological interest can also be envisaged in a therapeutic use, as a medicament, for example to relieve inflammation due to osteoarthritis or rheumatoid arthritis, as a hemostatic, as an agent for regenerating connective tissues (bone , skin, tendons, ligaments), as an agent for healing wounds, as an antioxidant (collagen peptides, less than 3000 Dalton in molecular weight), or even as an agent for combating type II diabetes, in particular thanks to the presence of the non-collagen protein (osteocalcin).

This product of biological interest can also be used in a non-therapeutic application, advantageously chosen from the following applications:

- nutritious (food supplement), - cosmetics,

- materials and biomaterials,

- soil fertilization.

The product of biological interest according to the invention can thus be incorporated into a cosmetic, pharmaceutical, food or biomaterial composition.

The product of biological interest can be processed to form various oral preparations as medicaments or health foods via methods well known per se. Examples of specific preparations include: tablets (including coated tablets, regular tablets, gastric suspension tablets, mouth tablets, effervescent tablets and chewable tablets), capsules (including soft capsules and microcapsules), granules, powdered preparations for infusion, effervescent granules, powders (including lyophilized powders), tablets (drops), controlled release tablets (including enteric coated tablets, prolonged release tablets), capsules controlled release (including enteric coated capsules and prolonged release tablets), fruit flavored preparations, oral solutions, syrups, suspensions, aerosol preparations, solutions (including broth), gels, emulsions, suspensions, drops, etc. .

In a pharmaceutical composition, the collagen of the product of biological interest according to the invention is advantageously used for the production of microparticles, injectable dispersions, ophthalmic solutions, drug delivery systems, etc.

This application in the pharmaceutical and biomedical field is due to its characteristics such as low antigenicity, cell attachment capacity, biodegradability and biocompatibility.

The collagen of the product of biological interest according to the invention can also be used in the field of tissue creation. It can be used as a basic matrix for cell culture systems and in tissue engineering such as injectable matrices, scaffolds for bone regeneration, etc.

Of course, various other modifications can be made to the invention within the scope of the appended claims.

Examples

Solid-liquid extraction of collagen from bovine bone

I. Material and method

1. Bovine bone

Bovine bones, of 5 different ages at slaughter, were used for collagen extraction. The cattle were all adult animals (over 5 years of age).

The product was ground and then stored at -20 ° C until use. The first step was the study of the particle size distribution of bone powders for the characterization of the particle size.

For each age, the powder was sieved through six different meshes, with dimension of 0.2mm, 0.4mm, 0.8mm, 1.0mm, 1.4mm and 2mm. Then the particles of different sizes and ages studied were mixed with an identical mass ratio.

2. Extraction of collagen

The collagen was extracted by solubilization in an acidic medium. A 0.5 M acetic acid solution, food grade, was used.

The extraction was studied under different agitation regimes, as described below.

In addition, the extraction was carried out at variable pH (increase in pH due to the solubilization of collagen) and at constant pH by the addition of acid.

2.1. Collagen extraction through several cycles (multicycle extraction)

The collagen extraction was carried out at a S / L (Solid / Liquid) ratio of 1/5 w / w (w = weight), i.e. 80 g of bone powder / 400 g of eluent, acetic acid 0, 5 M.

The tests were carried out over a period of time allowing a plateau to be reached (no variation in concentration with increasing time).

During the extraction process, samples of the supernatant were taken every 2 hours in order to determine the change in the concentration of collagen over time.

At the end of each extraction cycle, the collagen solution (the supernatant) is collected; 0.5 M acetic acid solution is added to the residual bone to start a new extraction cycle of the same duration as the previous one, and with the same S / L ratio. Four consecutive cycles were carried out.

2.2. Single cycle collagen extraction (unicycle extraction)

Single-cycle collagen extraction was performed to compare the yield versus multicycle extraction.

Collagen extraction is carried out at a S / L ratio of 1/20 w / w, i.e. 80 g of bone powder / 1600 g of 0.5 M acetic acid (i.e. the total mass eluent which was used for the four cycles of the multicycle extraction).

Samples of the supernatant were taken every 2 h in order to determine the concentration of collagen over time.

Several parameters were taken into account in order to identify the conditions having an effect on the extraction of collagen: the stirring regime, the temperature, the pH, the duration of extraction, the addition of salt (sodium chloride , NaCl).

Influence of the agitation regime

To study the influence of this parameter, the extraction was carried out in different regimes:

- static regime, stirring the solution for 30 seconds every 2 hours, and just before taking the samples, and - pseudo-static regime, stirring the solution every 30 minutes for 30 seconds.

Influence of the extraction time

In order to study the influence of the collagen extraction time, the collagen concentration was determined every 2 hours over several days and until the maximum plateau was reached (no variation in concentration over time).

Influence of temperature

The extraction was carried out at different temperatures: in refrigeration at 4 ° C, at room temperature of 20 ° C and at physiological bone temperature of 37 ° C.

Influence of pH

The effect of pH on the yield of collagen was studied by a comparative experiment:

- at normal pH, i.e. the pH of the supernatant during extraction (without intervention on the value of this pH), and

- at constant acidic pH (constant pH 2.5, using a 37% mass HCl solution) throughout the process.

Influence of N a Cl

The effect of NaCl on the solubility of collagen was studied by two parallel experiments: NaCl 0% and 0.9% (w / w) (i.e. the physiological concentration in humans), added to the solution of 0.5 M acetic acid used for the extraction.

3. Quantification of compounds

The collagen was quantified by UV-VIS spectrophotometry (ultraviolet / visible) and was characterized by infrared spectroscopy, through which it is possible to verify the presence and assembly of collagen, proteoglycans and minerals.

The collagen concentration was determined by the BCA (BicinChoninic acid Assay) method, based on bicinchonic acid and on the Biuret reaction, calibrated on type I collagen from mouse bone (analytical control).

The eluent obtained at the end of the extraction was also analyzed via high performance liquid chromatography (HPLC) in order to determine the level of amino acids and the concentration of collagen, by the presence in particular of hydroxyproline (single amino acid of collagen ).

The different types of collagen, proteoglycans and non-collagen proteins were demonstrated by mass spectrometry (LC-MS / MS).

Total glucose (expressing the level of glycosaminoglycans in proteoglycans) was quantified by the sulfuric acid method, implemented by Du Bois et al. (see Ferraro et al., (2017), Collagen type I from bovine bone. Effect of animal age, bone anatomy and drying methodology on extraction yield, self-assembly, thermal behavior and electrokinetic potential, International Journal of Biological Macromolecules, 97, 55-66). The dry matter was estimated by drying in an oven at 105 ° C for 16 h, according to the official method AOAC 968.08. For this, 20 g of Fontainebleu sand were placed in a ceramic crucible and 20 g of the supernatant at the end of the extraction was immediately added. After drying, the crucibles were placed in a desiccator to cool before weighing. The percentage of dry matter was calculated by dividing the amount of material recovered after drying by the amount of material before drying and multiplying by 100.

The minerals in the eluent were quantified by ion chromatography, and by X-ray energy dispersion spectroscopy (also called X-ray microanalysis) in the powders obtained after drying.

II. Results and discussion

The results obtained showed that the extraction of collagen is influenced by most of the parameters studied. But, among these parameters, surprisingly and unexpectedly, the management of the pH value is decisive in the extraction yield.

1. Extraction of collagen from bone

From the particle size study, the results showed that the weight average particle size was 1.2 mm; there is also a similarity in size between bone powders of different ages.

For each particle size, the powders of different ages were mixed in equal proportions before extracting the collagen.

1.1. Effect of S / L ratio (Solid / Liquid) and multicycle / unicycle effect

The use of two different S / L ratios (1/5 and 1/20 w / w) demonstrated the impact of the amount of solvent on the solid matter.

The result for the multicycle extraction, performed at the S / L ratio set at 1/5, is shown in Figure 3.

After four successive cycles, it was noticed an absence of growth in the yield of collagen over time; the curve of the kinetics of the 4th cycle in fact exhibits a slope close to zero and the additional quantity of collagen obtained at the end of the extraction is close to zero.

On the other hand, as illustrated by Figures 4 and 5, the solid / liquid ratio of 1/20 p / pa allowed an extraction of a higher quantity of collagen, this with a single cycle whose duration is identical to the 'one of the cycles of the multicycle extraction (that is to say 5 days).

Figures 4 and 5 illustrate the collagen yield by extraction, unicycle and multicycle.

Without being limited by any theory, by increasing the solid / liquid ratio, the saturation of the eluent is decreased and therefore the amount of collagen in solution is increased which is maintained in equilibrium with the collagen in the bone powder. The 1/20 (w / w) ratio was therefore maintained in the following steps.

1.2. Effect of agitation

The results relating to the effect of agitation on collagen solubilization are shown in Figure 6.

The pseudo-static diet extracted more collagen (11.53 mg collagen / g bone) than the static diet (9.66 mg collagen / g bone) at normal pH.

Without being limited by any theory, mechanical agitation of the particles in the solvent allows homogenization of the medium, constant access of the solvent to the solid, and also provides an amount of energy which drives the transfer of the material to the interface. solid-liquid.

Therefore, the pseudo-static regime was retained for the following experiments.

1.3. Effect of extraction time

The change in the collagen extraction yield over time is shown in Figure 7.

To study the effect of time on the extraction step (residence time), a bone powder / solvent ratio of 1/20 (w / w) in a pseudo-static regime was used and the extraction continued for 10 days.

In theory, the extraction time corresponds to the time necessary for the total recovery of the collagen contained in the initial product.

The results show that the collagen extraction efficiency increases over time, but from the 8th day onwards, a maximum plateau is reached.

Therefore, the duration of 8 days was chosen for the following experiments.

1.4. Temperature effect

The effects of temperature (at 4 ° C, 20 ° C and 37 ° C) were studied with a bone powder / solvent ratio 1/20 (w / w) in a pseudo-static regime, over a period of 8 days.

The results, shown in Figure 8, demonstrate that the yield increases with temperature.

Temperatures above 37 ° C, which is the physiological temperature, were not used to avoid a risk of denaturing the extracted collagen.

1.5. Effect of pH

The management of the pH is found to confer a significant / determining effect on the solubilization of collagen. The yields obtained, as a function of the pH, are shown in Figure 9.

The results show that the yield is higher at “constant” pH 2.5, whatever the experimental temperature.

The yield thus changes from approximately 18 mg of collagen / g bone to 30.11 mg of collagen / g bone when the temperature varies from 4 ° C to 37 ° C at constant pH 2.5.

However, at “normal” pH (without intervention on the pH value), this yield changes from about 9.9 mg of collagen / g bone to 15.11 mg of collagen / g bone. Thanks to the “constant” pH, advantageously in combination with a suitable physiological temperature, a level of collagen extracted is therefore obtained of 30.11 mg collagen / g bone at pH 2.5 and 37 ° C, against only 9.9 mg / g os at normal pH and 4 ° C.

1.6. Effect of NaCl concentration

The addition of NaCl (at a physiological concentration of 0.9% w / w) to the extraction solvent has no effect on the yield of collagen.

2. Extraction of glucose and dry matter

2.1. Glucose (expressing the level of glycosaminoglycans in proteoglycans)

The results show that the amount of solubilized glucose is dependent on the salt level, the pH and the temperature (Figures 10 and 11).

As with collagen, the level of glucose increases with, on the one hand, an increase in temperature and, on the other hand, a decrease in pH (Figure 11).

NaCl here has a positive effect on glucose solubilization (Figure 10).

The quantity of glucose obtained is higher at constant pH 2.5, for each of the experimental temperatures (FIG. 11).

2.2. Dry matter rate

The results show that the amount of dry matter recovered at the end of the extraction varies with temperature and pH (Figure 12).

This variation is in agreement with the results observed for collagen.

The results confirm that the solubilization yield is higher at pH 2.5 for each of the experimental temperatures.

An increase in temperature promotes the extraction of material.

The mineral matter represents from 60 to 65% of the dry matter, with 30 to 35% of calcium, 8 to 15% of phosphorus, 3 to 6% of magnesium, the remainder being represented by mineral oxygen (bound to calcium and phosphorus).

Sodium (without addition of NaCl), chloride (without addition of NaCl), potassium and sulfur are present in the form of traces (less than 0.05%).

III. Conclusion

The influence of various parameters (pH management, extraction temperature, time, agitation, etc.) has been studied on the extraction of collagen from animal bone.

The collagen extraction yield increases significantly by maintaining the acidic pH of the S / L mixture, advantageously in combination with the increase in the temperature of the mixture and the duration of the extraction process.

Claims

[Claim 1] A process for the manufacture of compounds of interest from at least one mineralized connective tissue, said compounds of interest comprising at least collagen, which manufacturing process comprises:

- a step of supplying said at least one mineralized connective tissue,

- a solid / liquid extraction step, in which said at least one mineralized connective tissue is mixed with an eluent suitable for extracting said compounds of interest from said at least one mineralized connective tissue to said eluent,

- an optional step of drying said compounds of interest, characterized in that said step of solid / liquid extraction comprises successive operations of adjusting the pH of said eluent, spaced apart by a time interval, which operations of adjusting the pH each comprise an addition of an acid in the mineralized connective tissue / eluent mixture, to decrease the pH of said eluent until reaching a pH value less than or equal to 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4.

[Claim 2] The manufacturing method according to claim 1, characterized in that the pH adjustment operations are configured to maintain the pH of said eluent at a pH value less than or equal to 5.5, preferably less than or equal to 4, preferably ranging from 2 to 4.

[Claim 3] The manufacturing method according to any one of claims 1 or 2, characterized in that the time interval between two pH adjustment operations is from one to five hours, preferably every 1h30 to 2h30.

[Claim 4] The manufacturing method according to any one of claims 1 to 3, characterized in that the solid / liquid extraction step is carried out in the form of a single cycle in which said at least one mineralized connective tissue is subjected to a single solid / liquid extraction step, which at least one mineralized connective tissue is mixed with a determined amount of eluent which is fully introduced at the start of the solid / liquid extraction step, which step of solid / liquid extraction is advantageously carried out in a batch reactor.

[Claim 5] The manufacturing method according to any one of claims 1 to 4, characterized in that, during each pH adjustment operation, the pH of the eluent is measured, and, following each measurement, a A suitable amount of acid is added so as to decrease the pH of the eluent to the predetermined target pH value.

[Claim 6] The manufacturing method according to any one of claims 1 to 5, characterized in that, during the solid / liquid extraction step, the temperature of the eluent is maintained at a value corresponding to the physiological temperature of the animal from which said at least one mineralized connective tissue is derived, for example 37 ° C, plus or minus 5 ° C.

[Claim 7] A method of making any one of claims 1 to 6, characterized in that the pH adjustment operations each comprise an addition of an acid selected from inorganic acids, for example hydrochloric acid.

[Claim 8] The manufacturing method according to any one of claims 1 to 7, characterized in that the solid / liquid extraction step is carried out over a period of time of at least 5 days, for example ranging from 5 to 10 days.

[Claim 9] The manufacturing method according to any one of claims 1 to 8, characterized in that, during the solid / liquid extraction step, the mineralized connective tissue / eluent mass ratio is within a range of from 1/5 to 1/40 w / w, preferably from 1/15 to 1/25 w / w.

[Claim 10] The manufacturing method according to any one of claims 1 to 9, characterized in that said at least one mineralized connective tissue is chosen from mineralized connective tissues from mammals or fish, which mineralized connective tissues from mammals are advantageously chosen from the bones of bovines or pigs, preferably adult bovines over 5 years old, which mineralized connective tissues obtained from fish are advantageously chosen from bones and scales.

[Claim 11] The manufacturing method according to any one of claims 1 to 10, characterized in that the solid / liquid extraction step is carried out under pseudo-static conditions or under agitation conditions.

[Claim 12] The manufacturing method according to any one of claims 1 to 11, characterized in that the step of providing comprises an operation of physicochemical preparation of said at least one mineralized connective tissue, which operation of preparation comprises an operation of mechanical treatment ensuring a reduction to powder of said at least one mineralized connective tissue, which powder advantageously has a particle size ranging from 0.5 to 2 mm.

[Claim 13] Manufacturing process according to any one of claims 1 to 12, characterized in that, initially, the eluent has a NaCl concentration of less than 60 g / L, preferably a NaCl concentration of less than 10 g / L, more preferably a value equal to 9 g / L.

[Claim 14] Compounds of interest resulting from the manufacturing process according to any one of claims 1 to 13, said compounds of interest comprising collagen and optionally proteoglycans and / or minerals and / or non-collagen proteins.

[Claim 15] Product of biological interest, comprising the compounds of interest resulting from the manufacturing process according to any one of claims 1 to 13, said compounds of interest comprising collagen and optionally proteoglycans and / or minerals and / or non-collagenous proteins.