Patent Application
20240032554
The present invention relates to a new method for preparing an isolate of cationic whey proteins of high purity lactoferrin.
The applicant has developed a method for obtaining an isolate of whey proteins whose lactoferrin protein purity is higher than 90%; this method allows the control of the vitamin B12 (cobalamin) content in the lactoferrin isolate.
This method is characterised on the one hand by the use of previously concentrated milk material (such as concentrated skimmed milk or concentrated whey) by a membrane technology (such as reverse osmosis, nanofiltration, or ultrafiltration) and on the other hand by a selective extraction using strong cation exchange resins of the sulphopropyl (SP) type packed in a radial chromatography column. The eluted lactoferrin pure fraction is concentrated and demineralised by ultrafiltration to obtain an isolate of cationic whey proteins whose lactoferrin purity higher than at least 90% and preferably 95%. This liquid isolate obtained is debacterised or sterilised by a microfiltration and optionally dried by spray drying or freeze drying to obtain the powder isolate.
The method for preparing an isolate of cationic whey proteins of high purity lactoferrin comprises the following steps a) to f):
It should be noted that the pasteurisation and microfiltration treatments are not essential for the method.
The passage step on cation exchange resins serves to bind cationic proteins present in the starting raw material while allowing major constituents of skimmed milk such as lactose, minerals, acidic proteins such as caseins, β-lactoglobulin, α-lactoglobulin, serum albumin and most immunoglobulins to pass. The first elution step serves to selectively extract specific cationic proteins by keeping the majority of lactoferrin, the major protein of milk cationic proteins, bound to the resins. The pure bovine lactoferrin fraction is therefore eluted in the second eluate.
Advantageously, the use of a mammalian milk material (e.g. pasteurised skimmed cow's milk, cheese whey from pasteurised goat's milk) concentrated by UF/NF/RO membrane allows for a reduction in the flow passage speed for the equivalent amount of protein present for a passage through an extraction column. Due to this extended contact time with the strong cation exchange resins of the SP type, the extraction efficiency of the cationic proteins is significantly improved.
In addition, the use of a radial flow column (e.g. Albert Handtmann Armaturenfabrick GmbH), due to its trapezoidal geometry, allows to sustainably withstand the pressure generated by the passage of a concentrated mammalian milk material through packed resins.
This combination of a concentrated milk material and a radial flow column is essential to execute a stable and regular industrial production.
Another advantage of the method according to the invention is that it can be carried out effectively over a wide temperature range; in particular, while resin manufacturers recommend an implementation at temperatures between 30 and 50° C., the applicant has succeeded in developing a method that is effective at cold temperatures, i.e. at temperatures below 15° C., preferably below 10° C.
The present invention thus relates to an isolate of cationic whey proteins enriched in lactoferrin obtained or obtainable by the method according to the invention, such that the proportion of protein on dry material is greater than or equal to 90% by weight and whose proportion of lactoferrin on the total proteins is greater than 90% by weight, preferably greater than 95% by weight, still more preferably greater than 98% by weight.
The present invention also relates to an isolate of cationic whey proteins enriched with lactoferrin from milk or whey from cow's milk or goat's milk obtained or obtainable by the method according to the invention, whose protein proportion on dry material is greater than or equal to 90% by weight, the proportion of lactoferrin on the total proteins is greater than 95% by weight (w/w), preferably greater than 98% by weight, and containing cobalamin in complex form with transcobalamin at a concentration of less than or equal to 5 μg/g of proteins, in particular, the concentration of cobalamin in complex form with transcobalamin is between 1 and 5 μg/g of proteins.
The present invention also relates to an isolate of cationic whey proteins enriched with lactoferrin from milk or whey from cow's milk or goat's milk obtained or obtainable by the method according to the invention, whose protein proportion on dry material is greater than or equal to 90% by weight, the proportion of lactoferrin on the total proteins is greater than 90% (w/w), preferably greater than 95% by weight, containing cobalamin in complex form with transcobalamin at a concentration greater than or equal to 5 μg/g, preferably greater than or equal to 8 μg/g, still more preferably greater than or equal to 10 μg/g of protein.
The isolates according to the invention may be in liquid form (step f not implemented) or in powder form (step f implemented). If it is in liquid form, it has the same characteristics as the powder in terms of composition relative to the dry material and generally comprises between 5 and 25%, preferably between 10 and 20%, by weight of water.
According to another object, the present invention relates to a food product for human or animal consumption, a human or animal medicine or a food supplement containing an isolate of cationic proteins according to the invention.
Preferably, the isolates according to the invention have a microbial load such that the count of aerobic mesophilic flora is less than 1000, preferably less than 100 or 10 and even more preferably less than 1 cfu/g of powder of isolate according to the invention or less than 100, preferably less than 10, even more preferably less than 1 cfu/ml of liquid isolate. The combination of the use of a concentrated milk material and a radial flow column allows the stable and efficient production of two kinds of isolates of high purity lactoferrin by cation exchange chromatography under the appropriate conditions:
Such isolates according to the invention are of particular interest for the preparation of infant formulas (infant milks or follow-on milks) based on cow's or goat's milk.
This isolate may have a nutritional benefit for a food supplement for vegetarians or for a nutritional preparation for people deficient in the absorption of vitamin B12, such the people who has undergone gastrectomy or people chronically treated with PPI (proton pump inhibitors). Indeed, this isolate can provide, in addition to the benefits of lactoferrin, an important source of vitamin B12 with high bioavailability even in the absence of the intrinsic factor secreted by the stomach.
The present invention thus also relates to dietary supplements comprising the isolate according to the invention enriched in vitamin B12, i.e. an isolate whose lactoferrin protein purity is >90% or 95% and whose vitamin B12 content is 5 μg/g protein, preferably 8 μg/g protein, still more preferably greater than or equal to 10 μg/g protein.
The isolate content according to the invention in the food supplement will be chosen according to the profile of the population to be supplemented, thus the dose of vitamin B12 to be administered, and the vitamin B12 content of the isolate. For example, a daily dose of 150 to 1000 mg protein of the isolate according to the invention enriched with vitamin B12 at 6 μg/g protein can provide 0.9 to 6.0 μg vitamin B12 in complex form with transcobalamin. Also, 100 to 600 mg of protein of the isolate according to the invention enriched with vitamin B12 at 10 μg/g protein can provide 1.0 to 6.0 μg of vitamin B12 in complex form with transcobalamin. Thus, such a food supplement can cover the needs of each population group shown in the table below even if intestinal absorption of vitamin B12 is disturbed.
Nutritional references for vitamin B12 (μg/d) according to ANSES 2016
The present invention further relates to an isolate whose lactoferrin protein purity is >90% or 95% and whose vitamin B12 content is 5 μg/g protein, preferably 8 μg/g protein, more preferably 10 μg/g protein for preventing and/or treating a vitamin B12 deficient absorption, e.g. in patients who have undergone gastrectomy or chronically treated with PPI (proton pump inhibitors).
Cobalamin (vitamin B12) is present in milk in complex form with a binding protein. In cow's milk, it is present in complex form with transcobalamin, which is a cationic protein of 43 kDa (S. N. Fedosov, T. E. Petersen, E. Nex∅, Transcobalamin from cow milk: isolation and physico-chemical properties, Biochimica et Biophysica Acta—Protein Structure and Molecular Enzymology. 1292 (1996) 113-119). The nutritional interest of this cobalamin-transcobalamin complex is important, as it is thought to be responsible for the bioavailability of vitamin B12 (S. N. Fedosov, Ebba Nexo, Christian W. Heegaard, Vitamin B12 and its binding proteins in milk from cow and buffalo in relation to bioavailability of B12, Journal of Dairy Science. American Dairy Science Association. 102 (2019) 4891-4905).
Although the behaviour of this complex during the treatment by cation exchange chromatography is close to that of lactoferrin, it is possible to vary the vitamin B12 content in the eluate obtained by the method according to the invention depending on the conditions used.
Furthermore, despite its nutritional interest, for certain applications (e.g. infant formulas, i.e. infant formulas/milks and/or follow-on formulas/milks) in certain specific cases (high incorporation dose), there may be an interest in limiting this vitamin B12 content in a pure lactoferrin fraction ingredient. In this context, the method according to the invention, which allows to adjust the final vitamin B12 content, is of great interest.
The invention therefore relates to food products for human or animal consumption comprising an isolate according to the invention; in the context of infant formulas, i.e. infant formulas/milks and/or follow-on formulas/milks, an isolate whose lactoferrin protein purity is >95% and whose vitamin B12 content is 5 μg/g protein is preferably used. The incorporation rate of the isolate according to the invention is 50 to 1000 mg of protein in one litre of a ready-to-eat formula.
The present invention also relates to non-food products, such as hygiene products and cosmetic products comprising an isolate according to the invention.
Also included in the present invention are the products for oral hygiene, such as toothpastes in gel or paste form, mouthwashes, chewing gums, comprising an isolate according to the invention. The incorporation rate of the isolate according to the invention is 1 to 100 mg of protein in one gram of a product.
1) The skimmed cow's milk whose DM is 92 g/L was pasteurised at 73° C. for 20 seconds, then cooled to 6° C. The concentration of bovine lactoferrin in this pasteurised skimmed milk was measured by HPLC SCX (High Performance Liquid Chromatography by strong cation exchange; column Propac SCX, 20 mM phosphate buffer in NaCl gradient, detection at 280 nm);
2) The pasteurized skimmed milk was passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a linear speed of 400 cm/hr with a variable volume of pasteurized skimmed milk;
3) After rinsing with 5 BV of osmosis water, the bound proteins are eluted with 6 BV of a 10% (w/v) NaCl solution at 20° C.;
4) The bovine lactoferrin content of each eluate was measured by RP HPLC (Reversed-Phase High-Performance Liquid Chromatography; C18 column 300 Å, 0.1% TFA/CH3CN gradient, detection at 280 nm). Thus, the amount of bovine lactoferrin in each eluate was obtained.
The conditions and the results of the assays are shown in Table 1:
1) The cow's milk was skimmed, then pasteurised at 73° C. for 20 seconds, then cooled to 6° C. This pasteurised skimmed cow's milk whose DM is 92 g/L was concentrated by a reverse osmosis to 130 g/L DM at 6° C. The concentration of bovine lactoferrin in this concentrated pasteurized skimmed milk was measured by HPLC SCX (column Propac SCX, 20 mM phosphate buffer in NaCl gradient, detection at 280 nm);
2) The concentrated pasteurized skimmed milk (130 g/L DM) is passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a variable linear speed with variable volume of pasteurized skimmed milk;
3) After rinsing with 5 BV of osmosis water, bound proteins are eluted with 5 BV of a 10% (w/v) NaCl solution at 20° C.;
4) The bovine lactoferrin content of each eluate was measured by RP HPLC (C18 column 300 Å, 0.1% TFA/CH3CN gradient, detection at 280 nm). Thus, the amount of bovine lactoferrin in each eluate was obtained.
The conditions and the results of the assays are presented in Table 2:
The comparison of Tables 1 and 2 shows that the yield of bovine lactoferrin obtained is much higher (>20%) with previously concentrated pasteurised skimmed milk using the comparable binding conditions (e.g. the passage of ˜300 BV equivalent of pasteurised skimmed milk with the DM flow rate 37-39 g/cm2/h).
1) The cow's milk was skimmed, then pasteurised at 73° C. for 20 seconds, then cooled to 6° C. This pasteurised skimmed cow's milk whose DM is 92 g/L was concentrated by a reverse osmosis to 130 g/L DM at 6° C. The concentration of bovine lactoferrin in this concentrated pasteurized skimmed milk was measured by HPLC SCX (column Propac SCX, 20 mM phosphate buffer in NaCl gradient, detection at 280 nm);
2) The concentrated pasteurized skimmed milk (130 g/L DM) is passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a variable linear speed with variable volume of pasteurized skimmed milk;
3) After rinsing with 5 BV of osmosis water, bound proteins are partially eluted with 6 BV of a 2.6% (w/v) NaCl solution at 38 mS/cm at 20° C. Lactoperoxidase, ribonucleases and other basic proteins were recovered from this eluate;
4) Still bound proteins are eluted with 5 BV of a 10% (w/v) NaCl solution at 20° C. The bovine lactoferrin was recovered from this eluate;
5) The proportion of lactoferrin in the total protein in this 2nd eluate was determined by RP HPLC (C18 column 300 Å, 0.1% TEA in H20/CH3CN gradient, detection at 280 nm) as the relative area of the peak of the bovine lactoferrin.
The cobalamin (vitamin B12) content in this 2nd eluate was also measured by the AOAC method. Thus, its total protein content was obtained.
The conditions and the results of 2 series of assays are shown in Table 3:
These results show that using appropriate conditions, two kinds of fraction of high lactoferrin purity (e.g. >95% on total proteins) can be obtained by cation exchange chromatography with the passage of concentrated pasteurised skimmed milk:
Although the pasteurised skimmed milk concentrated to ˜130 g/L can be passed through an axial column at bench scale, it is difficult to envisage a stable production over time at industrial scale with a passage of a complex matrix such as milk material, especially concentrated, due to both high pressure loss and filter surface clogging.
We checked the pressure loss behaviour through an industrial radial flow column by passing the skimmed milk of different DM and at different flow rates.
1) An industrial radial flow column of 260 L (Albert Handtmann Armaturenfabrick GmbH) was prepared with 280 L of SP Sepharose Big Beads Food Grade resins. It was regenerated with 10% NaCl, then saturated with 1 N NaOH and finally rinsed with osmosis water;
2) The cow's milk was skimmed, then pasteurised at 73° C. for 20 seconds, then cooled to 6° C. A portion of pasteurised skimmed cow's milk was concentrated by a reverse osmosis at 6° C. The compositions of these unconcentrated and concentrated pasteurised skimmed milks are as follows:
3) After preparing the concentration level of pasteurised skimmed milk by in-line mixing, it is passed at different flow rates through the previously prepared radial flow column at a temperature of 10° C.
The observed skimmed milk compositions, flow rates and pressures are shown in Table 5. The pressure losses (i.e. pressure) generated by the radial flow column increased as a function of flow rates and mobile phase material concentrations (
1) The cow's milk was skimmed, then pasteurised at 73° C. for 20 seconds, then cooled to 6° C., then concentrated by a reverse osmosis to 128 g/L DM at 6° C.;
2) 80 m3 of this concentrated pasteurised skimmed milk was passed through an industrial radial flow column of 260 L (Albert Handtmann Armaturenfabrick GmbH) packed with 280 L of SP Sepharose Big Beads Food Grade resins at a flow rate of 2.6 m/h;
3) After rinsing with 5 BV of osmosis water, bound proteins are partially eluted by 6 BV of a 38 mS/cm NaCl solution at 20° C. Lactoperoxidase, ribonucleases and other basic proteins were recovered from this eluate;
4) Still bound proteins are eluted with 4 BV of a 10% (w/v) NaCl solution at 20° C. This eluate containing the bovine lactoferrin was cooled and stored at 6° C.; Steps 2-4 were repeated 10 times;
6) 11.2 m3 of 2nd pooled eluate was concentrated on an ultrafiltration (organic spiral membrane with MWCO of 20 kDa), then diafiltered on UF (MWCO 20 kDa) with osmosis water down to 1 mS/cm, and finally microfiltered on a 1.4 μm ceramic membrane in a double layer (Membrarox®, Pall Corporation);
7) The isolate of whey proteins enriched with bovine lactoferrin obtained in the form of the micro-filtrate has undergone a spray drying and 40 kg of powder was obtained (Ingredient 1);
8) The ingredient 1 was analysed; in particular the proportion of lactoferrin in the total protein was determined by RP HPLC (C18 column 300 Å, 0.1% TFA in H20/CH3CN gradient, detection at 280 nm) as the relative peak area of the bovine lactoferrin (
1) The cow's milk was skimmed, then pasteurised at 73° C. for 20 seconds, then cooled to 6° C., then concentrated by a reverse osmosis to 120 g/L DM at 6° C.;
2) 60 m3 of this concentrated pasteurised skimmed milk was passed through a 260 L radial flow industrial column (Albert Handtmann Armaturenfabrick GmbH) packed with 280 L of SP Sepharose Big Beads Food Grade resins at a flow rate of 2.6 m/h;
3) After rinsing with 5 BV of osmosis water, bound proteins are partially eluted by 6 BV of a 36 mS/cm NaCl solution at 20° C. Lactoperoxidase, ribonucleases and other basic proteins were recovered from this eluate;
4) Still bound proteins are eluted with 4 BV of a 10% (w/v) NaCl solution at 20° C.
This eluate containing the bovine lactoferrin was cooled and stored at 6° C.;
5) Steps 2-4 were repeated 15 times;
6) 116.8 m 3 of 2nd pooled eluate were concentrated on an ultrafiltration (organic spiral membrane with cutoff threshold (MWCO) 20 kDa), then diafiltered on UF (MWCO 20 kDa) with osmosis water down to 1 mS/cm, and finally microfiltered on a 0.8 μm ceramic membrane in a double-layer (Membrarox®, Pall Corporation);
7) The isolate of whey proteins enriched with bovine lactoferrin obtained in the form of the micro-filtrate has undergone the following additional treatments to ensure the stability of this following protein fraction:
8) The ingredients 2 and 3 were analysed; in particular the proportion of lactoferrin in the total protein in this 2nd eluate was determined by RP HPLC (C18 column 300 Å, 0.1% TFA in H20/CH3CN gradient, detection at 280 nm) as the relative peak area of the bovine lactoferrin (
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2) 3000 L of pasteurized skimmed goat's milk, after being held at 50° C. for 30 minutes, were passed through a 0.1 μm ceramic microfiltration (Membrarox®, Pall Corporation) to obtain a whey as micro-filtrate of goat's milk free of fat and casein micelles;
3) 2000 L of whey from goat's milk was concentrated on an ultrafiltration (organic spiral membrane with a cutoff threshold (MWCO) of 10 kDa). The compositions of the resulting retentate (450 L) were in Table 7 below:
The concentration of goat β-Lactoglobulin and goat α-Lactalbumin in this concentrated whey was measured by SEC HPLC (TSK G3000PWxl column, CH3CN/H20/TFA, detection at 210 nm). The concentration of goat lactoferrin in this concentrated whey was measured by HPLC SCX (Propac SCX column, 20 mM phosphate buffer in NaCl gradient, detection at 280 nm).
4) 3 L of concentrated goat whey is passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a linear speed of 200 and 300 cm/h;
After rinsing with 5 BV of osmosis water, bound proteins are partially eluted with 6 BV of a 2.2% (w/v) NaCl solution at 20° C. Cationic proteins other than lactoferrin such as lactoperoxidase were recovered from this eluate;
6) Still bound proteins are eluted with 5 BV of a 10% (w/v) NaCl solution at 20° C. The goat lactoferrin was recovered from this eluate. The goat lactoferrin content in the eluate was measured by RP HPLC (C18 column 300 Å, 0.1% TFA in H20/CH3CN gradient, detection at 280 nm).
As shown in Table 8, a goat lactoferrin fraction whose very high protein purity was extracted very efficiently from concentrated whey from goat's milk.
1) 240 L of cheese whey from pasteurised goat's milk (at 74° C. for 30 seconds) was concentrated on an ultrafiltration (organic spiral membrane with MWCO of 10 kDa). The compositions of the retentate obtained (60 L) were in the table below:
The concentration of goat (β-Lactoglobulin and goat α-Lactalbumin in this concentrated whey was measured by SEC HPLC (TSK G3000PWxl column, CH3CN/H20/TFA, detection at 210 nm). The concentration of goat lactoferrin in this concentrated whey was measured by HPLC SCX (Propac SCX column, 20 mM NaPB/NaCl gradient, detection at 280 nm).
2) 3 L of concentrated goat whey is passed through an axial flow column (1.6 cm diameter) containing 20 mL (BV) of SP Sepharose Big Beads at a linear speed of 200 and 300 cm/h;
3) After rinsing with 6 BV of osmosis water, bound proteins are partially eluted with 6 BV of 2.2% (w/v) NaCl solution at 20° C. Cationic proteins other than lactoferrin such as lactoperoxidase were recovered from this eluate;
4) Still bound proteins are eluted with 5 BV of a 10% (w/v) NaCl solution at 20° C. The goat lactoferrin was recovered from this eluate. The goat lactoferrin content in the eluate was measured by RP HPLC (C18 column 300 Å, 0.1% TFA in H20/CH3CN gradient, detection at 280 nm).
As shown in Table 10, a goat lactoferrin fraction with high protein purity was extracted very efficiently from concentrated cheese whey from goat's milk.
1) An infant milk powder based on cow's milk has been prepared by a standard manufacturing method by formulating with skimmed cow's milk, lactose, maltodextrins, oleic sunflower oil, anhydrous milk fat, demineralised whey, soluble protein, galacto-oligosaccharides, sunflower oil, rapeseed oil, soy lecithin, sunflower lecithin, calcium phosphate, fish oil, potassium phosphate, Mortierella alpina oil, choline bitartrate, calcium chloride, potassium citrate, magnesium citrate, sodium chloride, fructo-oligosaccharides, vitamin C, ferric pyrophosphate, calcium carbonate, taurine, potassium hydroxide, potassium chloride, inositol, nucleotides, L-phenylalanine, tocopherol rich extract, L-ascorbyl palmitate, zinc sulphate, L-tryptophan, vitamin E, potassium iodide, L-carnitine, nicotinamide, sodium selenite, calcium pantothenate, copper sulphate, thiamine, vitamin A, vitamin B6, manganese sulphate, folic acid, vitamin K, biotin, vitamin D, riboflavin, vitamin B12.
2) The infant milk powder has been mixed with Ingredient 1 at an incorporation rate of 82 mg/100 g.
1) A powder infant milk (food for special medical purposes, i.e. FSMP) based on cow's milk has been prepared by a standard manufacturing method by formulating with skimmed milk, vegetable oils (palm, rapeseed, copra, sunflower), demineralised soluble protein, lactose, starch, locust bean flour, lecithin, calcium citrate, fish oil, Mortierella alpina oil, calcium carbonate, vitamin C, calcium phosphate, potassium citrate, sodium citrate, calcium hydroxide, choline chloride, taurine, vitamin E, inositol, ferrous sulphate, L-tryptophan, potassium chloride, calcium chloride, tocopherol rich extract, L-ascorbyl palmitate, L-carnitine, magnesium sulphate, nucleotides, zinc sulphate, vitamin A, nicotinamide, vitamin K, vitamin D, calcium pantothenate, copper sulphate, thiamine, vitamin B6, riboflavin, manganese sulphate, folic acid, potassium iodide, sodium selenite, biotin.
2) The FSMP infant milk powder has been mixed with Ingredient 2 at an incorporation rate of 400 mg/100 g. An intake of 3.1 μg of vitamin B12 in complex form with transcobalamin per 100 g of the powdered formulation was achieved by this incorporation of Ingredient 2.
1) A liquid infant milk (foods for special medical purposes, FSMP) based on cow's milk has been prepared by a standard manufacturing method by formulating with Skimmed milk, demineralised soluble protein, vegetable oils (palm, palm kernel, rapeseed, sunflower), lactose, soybean lecithin, sunflower lecithin, sodium citrate, calcium phosphate, potassium citrate, calcium chloride, calcium carbonate, vitamin C, Mortierella alpina oil, fish oil, calcium hydroxide, potassium chloride, vitamin E, choline chloride, taurine, ferrous sulphate, tocopherol-rich extract, L-ascorbyl palmitate, inositol, zinc sulphate, nucleotides, L-carnitine, nicotinamide, vitamin A, magnesium sulphate, vitamin K, vitamin D, calcium pantothenate, copper sulphate, thiamine, vitamin B6, riboflavin, manganese sulphate, folic acid, potassium iodide, sodium selenite, biotin.
2) The FSMP infant milk in liquid form was sterilised by an ultra-high temperature (UHT) thermal treatment and then mixed with Ingredient 3 at an incorporation rate of 410 mg/100 g (on the dry material). An intake of 0.43 μg of vitamin B12 in complex form with transcobalamin per 100 mL of the liquid formulation was achieved by this incorporation of Ingredient 3.
A capsule food supplement was prepared from the mixture of Ingredient 2 of Example 5 (99.5% of the mixture) and colloidal silica (0.5% of the mixture). Each capsule contains 200 mg of protein.
The content of vitamin B12 in complex form with transcobalamin is 1.6 μg/capsule. The recommended daily dose for each population group is as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2013945 | Dec 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/087268 | 12/22/2021 | WO |
