Keratin hydrolysate enriched in free amino acids and in free tyrosine, process for obtaining it and use in animal feed and plant nutrition

Abstract

The present invention relates to a keratin hydrolysate comprising at least 88 wt%, preferably at least 90 wt% of free amino acids relative to the total weight of amino acids in the hydrolysate, and comprising from 2 to 4 wt%, preferably from 2.5 to 3.5 wt% of free tyrosine relative to the total weight of free amino acids in the hydrolysate, and to a process for the preparation of said hydrolysate, in particular comprising the steps of acid hydrolysis, pH adjustment and desalination. The present invention also relates to the use of said hydrolysate, in particular for pet food, aquaculture food and plant biostimulants.

Description

[0001] The present invention relates to the field of hydrolysates enriched in free amino acids, and their use in animal feed, especially for felines and canines, aquaculture and agriculture, especially as plant biostimulants.

[0002] Amino acid-based compositions are used in a range of fields, such as nutraceuticals, cosmetics, plants, animals and human nutrition, and for very different and specific applications in each of these fields. These uses include those aimed at making human hair grow and shine, and those aimed at providing free amino acids that constitute a specific protein source in animal feed, especially in aquaculture and in canine and feline nutrition.

[0003] One of the methods for obtaining amino acid-based compositions is to produce hydrolysates of keratin materials.

[0004] Natural keratin materials are essentially composed of high molecular weight polypeptides and have a highly cross-linked structure that makes them resistant to enzymatic attack. This natural keratin material is not easily digestible. However, it is known that hydrolysis of keratin materials into amino acids improves their digestibility.

[0005] Keratin hydrolysates sold, especially as food additives, ingredients for formulating animal nutrition formulas or animal feed raw materials, are generally obtained by very incomplete hydrolysis. These hydrolysates generally have a high molecular weight due to the presence of a large number of so-called "linking" amino acids forming peptides and polypeptides. The molecular weight of commercial compositions is generally between 5000 and 50000 Daltons. These keratin hydrolysates are relatively difficult to digest and contain almost no free amino acids. Indeed, obtaining keratin hydrolysates with very high levels of free amino acids at an industrial level is technically complex and expensive.

[0006] In addition, excessive hydrolysis risks denaturing and destroying amino acids.

[0007] Patent application WO2019 / 043128 outlines a keratin hydrolysate with a high level of free amino acids, obtained by implementing a process in which the hydrolysis step is followed by a tyrosine extraction step. This hydrolysate has the advantages associated with a high level of free amino acids, in particular good digestibility. However, this hydrolysate does not include all the amino acids that are generally present after acid hydrolysis, in particular the content of cysteine and tyrosine is low. Nonetheless, obtaining a hydrolysate that can be used in complete and balanced food without the addition of additional amino acids is a universally sought-after property.

[0008] Moreover, the hydrolysate obtained by chemical hydrolysis, for example acid hydrolysis, is in salt form, whereas most of the uses, especially in animal feed and biostimulation, require a desalted hydrolysate. However, the desalting process generally leads to a significant loss of certain amino acids, in particular those which precipitate at the end of the neutralization stage. Indeed, desalting can only be performed on filtered solutions. Thus, the yield of the hydrolysate obtained according to the prior art methods is not always satisfactory.

[0009] Surprisingly and more advantageously, the inventors of the present application have succeeded in overcoming the problems of the prior art by implementing a process which makes it possible to obtain a desalted keratin hydrolysate having a high level of free amino acids and comprising all the amino acids which are normally present after acid hydrolysis. Moreover, the amino acid profile of the hydrolysate according to the present application is close to that of the original keratin material.

[0010] Thus, the use of the hydrolysate according to the present application makes it possible to dispense with the addition of most of the additional amino acids by those which are present in the original keratin material.

[0011] The free amino acids obtained according to the present application are not destroyed or denatured, in particular the amino acids which are the most difficult to release in free form during the hydrolysis process, such as valine, leucine and isoleucine.

[0012] Moreover, the process according to the present application makes it possible to obtain a good amino acid yield, i.e. a good ratio between the total amount of amino acids of the hydrolysate according to the present application and the original keratin material, since all the phases produced from the various stages of the process are recovered and treated in order to extract the maximum amount, i.e. practically all the amino acids.

[0013] The subject of the present application is a keratin hydrolysate comprising at least 88% by weight, preferably at least 90% by weight of free amino acids relative to the total weight of amino acids in the hydrolysate, said hydrolysate comprising from 2% to 4% by weight, preferably from 2.5% to 3.5% by weight of free tyrosine relative to the total weight of free amino acids in the hydrolysate.

[0014] Advantageously, said hydrolysate comprises at least 90% by weight, preferably at least 93% by weight, preferably at least 95% by weight of cystine in free form relative to the total weight of cystine in the hydrolysate.

[0015] In a preferred embodiment, said hydrolysate is desalted, i.e. it comprises less than 11% by weight, preferably less than 7% by weight of salts relative to the total weight of the hydrolysate, the salts being chosen from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate, preferably sodium chloride.

[0016] The second object of the application relates to a process for the preparation of a keratin hydrolysate according to the application from an animal keratin material, preferably poultry, said process comprising at least the following steps in the order given below:

[0017] - at least one chemical hydrolysis of the keratin material with an acid under conditions enabling to obtain a hydrolysate comprising at least 88% by weight of free amino acids relative to the total weight of amino acids in the hydrolysate, the remaining amino acids of the hydrolysate being in the form of peptides having a molecular weight of less than 800 Dalton or equal to 800 Dalton;

[0018] - a step of adjusting the pH of the hydrolysate to a value comprised between 3 and 5, preferably between 4 and 5, and recovering the precipitate and the liquid phase;

[0019] - separating the precipitate and the liquid phase, preferably by centrifugation;

[0020] - at least one washing of the precipitate with water until obtaining a desalted precipitate comprising less than 1% by weight of salts relative to the total weight of the precipitate, and recovering the desalted precipitate on the one hand and the washing water on the other hand,

[0021] - combining the washing water and the liquid phase to obtain a solution, and desalting this solution by electrodialysis to obtain a desalted solution,

[0022] - adding the desalted precipitate to the desalted solution,

[0023] - recovering the desalted suspension obtained.

[0024] In a preferred variant, the suspension obtained is dried and the solid obtained at the end of the drying is recovered.

[0025] The advantage of the device for implementing the process according to the application is simply that the application can be implemented with equipment using devices commonly used in industry, such as reactors, centrifuges and atomization towers. Several steps can be carried out in the same enclosure, in addition, the collection of the different phases is easy to carry out without significant problems.

[0026] The application also relates to the use of a keratin hydrolysate in animal feed, in particular for felines and canines, in aquaculture or in agriculture. The application more particularly relates to the use of a hydrolysate according to the application or prepared according to the application as a component of a product chosen from the group consisting of a food for animal feed for pets, a food for aquaculture and a plant biostimulant.

[0027] Other aspects, advantages and characteristics of the application will emerge clearly from the description, in particular the examples and the subsequent figures, in a non-exhaustive manner.

[0028] Detailed description

[0029] Advantageously, the hydrolysate is obtained from natural, animal keratin materials, particularly from poultry, and preferably from poultry feathers. Poultry examples include hens, especially laying hens, chickens, turkeys, ducks, geese, etc. Natural keratin materials can also be selected from animal hair, especially pig bristles, animal hooves, and animal nails.

[0030] In particular, the hydrolysate according to the invention is not obtained from human keratin such as hair.

[0031] As already mentioned, the hydrolysate according to the invention contains at least 88% by weight, preferably 90% by weight, of free amino acids relative to the total weight of amino acids in the hydrolysate.

[0032] Advantageously, the total amino acid content of the hydrolysate according to the invention is 40% to 95% by weight, preferably 45% to 93% by weight, relative to the total weight of the hydrolysate, and the hydrolysate also contains minerals and water.

[0033] Furthermore, the hydrolysate based on this invention is characterized by free branched-chain amino acids: undenatured valine, leucine, and isoleucine. However, these branched-chain amino acids are known to be more difficult to release under the same usage conditions.

[0034] As described above, the hydrolysate of the present invention contains at least 90% by weight, preferably at least 93% by weight, and preferably at least 95% by weight of free cystine relative to the total weight of cystine in the hydrolysate.

[0035] In a preferred variant, the hydrolysate according to the invention comprises the following components:

[0036] The free form of aspartic acid is at least 95% by weight relative to the total weight of aspartic acid in the hydrolysate, preferably 100% by weight.

[0037] The free form of threonine is at least 95% by weight relative to the total weight of threonine in the hydrolysate, preferably 100% by weight.

[0038] The free form of serine is at least 95% by weight relative to the total weight of serine in the hydrolysate, preferably 100% by weight.

[0039] The free form of glutamic acid is at least 93% by weight relative to the total weight of glutamic acid in the hydrolysate, preferably 95% by weight or more than 95% by weight;

[0040] The free form of glycine is at least 90% by weight relative to the total weight of glycine in the hydrolysate, preferably 93% by weight or more than 93% by weight;

[0041] The free form of alanine is at least 90% by weight relative to the total weight of alanine in the hydrolysate, preferably 93% by weight or more than 93% by weight;

[0042] at least 90 wt.%, preferably 93 wt.% or higher than 93 wt.% of phenylalanine in free form relative to the total weight of phenylalanine in the hydrolysate; and

[0043] at least 93 wt.%, preferably 95 wt.% or higher than 95 wt.% of proline in free form relative to the total weight of proline in the hydrolysate.

[0044] In addition, at least 90 wt.% of the amino acids in the hydrolysate are characterized by a molecular weight of less than 250 Dalton or equal to 250 Dalton, preferably less than 240 Dalton or equal to 240 Dalton. Thus, the hydrolysate can be used for the preparation of a complete feed for animals with low allergenicity or even no allergenicity.

[0045] The obtained hydrolysate is advantageously desalted, i.e. the obtained hydrolysate comprises less than 11 wt.%, preferably less than 7 wt.% of salts relative to the total weight of the hydrolysate, the salts being selected from the group consisting of sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate, preferably sodium chloride (NaCI).

[0046] The determination of the percentage of salts is within the capabilities of the skilled person. Preferably, the percentage of salts is determined from the anion dosage. In particular, the chloride ions are determined by using 0.1 N silver nitrate, followed by the potential dosage using a combined Ag / AgCI electrode; the phosphate ions are determined by spectrophotometry of the phosphomolybdate complex according to the ISO 6878 standard, the sulfate ions are determined by gravimetric determination with addition of barium salt according to the ISO 2480:1972 standard.

[0047] The anion dosage can also be complemented by the cation dosage, generally the dosage of sodium and potassium is obtained by flame ionization spectrophotometry based on the ISO 9964-2:1993 standard.

[0048] The salt content depends on the washing quality of the precipitate and the duration of the electrodialysis. The skilled person is able to adjust the parameters of these stages, in particular their duration, according to the desired salt content.

[0049] The "dry hydrolysate" or "dried hydrolysate" as defined in the present application means a hydrolysate containing less than 5 wt.% of water. The water weight of the hydrolysate is measured using an infrared thermobalance.

[0050] Preferably, the hydrolysate according to the present application comprises the following amounts of free amino acids relative to the total weight of amino acids in the hydrolysate:

[0051] aspartic acid in an amount of 6.00 wt.% to 10.00 wt.%, preferably 7.00 wt.% to 9.00 wt.%, more preferably 7.83 wt.%;

[0052] threonine in an amount of 3.00 to 7.00 wt.-%, preferably 4.00 to 6.00 wt.-%, more preferably 4.93 wt.-%;

[0053] serine in an amount of 11.00 to 15.00 wt.-%, preferably 12.00 to 14.00 wt.-%, more preferably 12.88 wt.-%;

[0054] glutamic acid in an amount of 8.50 to 12.50 wt.-%, preferably 9.50 to 11.50 wt.-%, more preferably 10.47 wt.-%;

[0055] glycine in an amount of 6.50 to 10.50 wt.-%, preferably 7.50 to 9.50 wt.-%, more preferably 8.56 wt.-%;

[0056] alanine in an amount of 3.00 to 7.00 wt.-%, preferably 4.00 to 6.00 wt.-%, more preferably 5.04 wt.-%;

[0057] valine in an amount of 3.50 to 7.50 wt.-%, preferably 4.50 to 6.50 wt.-%, more preferably 5.61 wt.-%;

[0058] cystine in an amount of 4.00 to 8.00 wt.-%, preferably 5.00 to 7.00 wt.-%, more preferably 5.80 wt.-%;

[0059] methionine in an amount of 0.10 to 2.00 wt.-%, preferably 0.20 to 1.00 wt.-%, more preferably 0.57 wt.-%;

[0060] isoleucine in an amount of 1.50 to 5.50 wt.-%, preferably 2.50 to 4.50 wt.-%, more preferably 3.50 wt.-%;

[0061] leucine in an amount of 6.00 to 10.00 wt.-%, preferably 7.00 to 9.00 wt.-%, more preferably 7.77 wt.-%;

[0062] tyrosine in an amount of 2.50 to 3.50 wt.-%, preferably 3.00 to 3.50 wt.-%, more preferably 3.15 wt.-%;

[0063] phenylalanine in an amount of 3.00 to 7.00 wt.-%, preferably 4.00 to 6.00 wt.-%, more preferably 5.08 wt.-%;

[0064] lysine in an amount of 0.50 to 3.00 wt.-%, preferably 1.00 to 2.00 wt.-%, more preferably 1.66 wt.-%;

[0065] histidine in an amount of 0.10 to 2.00 wt%, preferably 0.20 to 1.00 wt%, more preferably 0.74 wt%;

[0066] arginine in an amount of 4.00 to 8.00 wt%, preferably 5.00 to 7.00 wt%, more preferably 5.82 wt%; and

[0067] proline in an amount of 8.50 to 12.50 wt%, preferably 9.50 to 11.50 wt%, more preferably 10.59 wt%.

[0068] Another advantage of the keratin hydrolysate based on the application is that its amino acid profile is close to that of the original keratin material. Indeed, apart from tryptophan which is destroyed in acid hydrolysis, the 17 amino acids present in the original keratin material are also present in free form in the final hydrolysate.

[0069] Thus, the keratin hydrolysate based on the application comprises 17 amino acids for which the absolute value of the percentage variation between the weight of the free amino acid in the hydrolysate and the weight of this amino acid in the original keratin material is less than 20%, advantageously less than 10% for 15 of these amino acids.

[0070] The percentage variation of an amino acid corresponds to the ratio of the absolute value of the difference between the weight of the amino acid in the keratin material and the weight of the free amino acid in the hydrolysate to the weight of the amino acid in the keratin material multiplied by 100, i.e. the following formula:

[0071] (| weight of the amino acid in the keratin material - weight of the free amino acid in the hydrolysate | / weight of the amino acid in the keratin material) x 100.

[0072] Another advantage of the hydrolysate according to the application is that it is very digestible. Moreover, it is recognized as food grade. The hydrolysate according to the application is characterized by a true digestibility of its protein fraction of at least 98%. This value is very close to the maximum possible value (100%).

[0073] The digestibility is measured in vivo according to the method described in Z.M. Larbier, A.M. Chagneau, and M. Lessire, "Effect of protein intake on true digestibility of amino acids in rapeseed meals for adult roosters force fed with moistened feed." Animal Feed Science and Technology. 34 (1991) 255-260.

[0074] Method

[0075] [ Figure 1 ] is a diagram showing the main steps of the method according to the application and the stages obtained at the end of the different steps. The steps are represented by rectangles and the stages by ovals. According to the diagram in Figure 1 , the method comprises a hydrolysis followed by a pH adjustment step resulting in a liquid phase and a precipitate, the liquid phase and the precipitate are subjected to a solid-liquid separation step. The precipitate is then washed resulting in a desalted precipitate (AA2). The liquid phase and the washing water are combined and the solution (AA1) is subjected to a desalting stage resulting in a desalted solution. The desalted solution and the desalted precipitate (AA2) are then combined and the obtained suspension is subjected to a drying, thereby obtaining a dried desalted hydrolysate.

[0076] Acid hydrolysis

[0077] The method for preparing a keratin hydrolysate according to the application implements at least one chemical hydrolysis by acid under conditions suitable for obtaining a hydrolysate comprising at least 88% by weight of free amino acids relative to the total weight of amino acids in the hydrolysate, the remaining amino acids of the hydrolysate being in the form of small peptides, i.e. having a molecular weight less than or equal to 800 Daltons.

[0078] The percentage of small peptides in the hydrolysate according to the application is generally between 5% and 12% by weight relative to the total weight of the hydrolysate.

[0079] Indeed, the hydrolysis is not complete and the percentage of small peptides in the hydrolysate is not zero, at most 12% by weight.

[0080] The chemical hydrolysis of keratin is performed using an acid, preferably a strong acid chosen from hydrochloric acid, phosphoric acid and sulfuric acid, preferably hydrochloric acid. The concentration of the acid, preferably the concentration of hydrochloric acid, should be between 14% and 34% by weight.

[0081] Preferably, the acid / keratin material weight ratio, in particular the acid / feather weight ratio, is between 2 and 5.

[0082] The chemical hydrolysis is generally carried out at a temperature of 100°C to 115°C for 1 hour to 24 hours, preferably 6 hours to 20 hours.

[0083] Depending on the specific variant, the chemical hydrolysis is carried out in two stages: a first chemical hydrolysis at a temperature of 60°C to 80°C for 4 hours to 5 hours. Then a second chemical hydrolysis at a temperature of 100°C to 115°C for 5 hours to 8 hours.

[0084] In addition, the two hydrolysis can be carried out without an intermediate pause step, or by carrying out an intermediate pause step of 1 hour to 7 days.

[0085] More precisely, the first chemical hydrolysis is carried out at 72°C for 4.5 hours and the second chemical hydrolysis is carried out at 107°C for 6 hours, with an intermediate pause of 24 hours to 80 hours between the two chemical hydrolysis.

[0086] Advantageously, the part floating on the surface of the hydrolysate is removed when the fatty part floats on the surface of the hydrolysate.

[0087] pH adjustment

[0088] The chemical hydrolysis, carried out in one or more stages, is followed by a pH adjustment stage. The pH of the hydrolysate is adjusted to 3 to 5, preferably 4 to 5. This step is carried out by adding a base chosen from sodium hydroxide and potassium hydroxide, preferably sodium hydroxide. This is a typical step, the implementation of which is within the ability of the skilled person.

[0089] This step is also characterized by the effect of precipitation, at least partial precipitation, of the less soluble amino acids, in particular cystine, tyrosine, leucine and isoleucine. These less soluble amino acids form a precipitate, the other amino acids remaining in solution, forming a liquid phase with salts and water.

[0090] Solid-liquid separation - dewatering

[0091] The pH adjustment step is followed by a step of separation of the precipitate from the liquid phase. The separation step can be carried out by implementing any solid-liquid separation technique, in particular by applying a centrifugal force or by pressing, in particular using a filter press. Based on the preferred variant, the separation step is dewatering. The dewatering stage is advantageously carried out by applying a centrifugal force at a rotation speed of approximately 1000 rpm. This method known to the person skilled in the art for carrying out a solid-liquid separation consists in removing the liquid phase by the action of a centrifugal force, while retaining the precipitate (solid part) on the cloth.

[0092] Washing

[0093] The precipitate recovered by centrifugation, preferably on the cloth of the wringer, is then washed with water until a desalinated precipitate is obtained, having a salt content of less than 1% by weight relative to the total weight of the precipitate. The term "salt" means sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate, preferably sodium chloride (NaCI).

[0094] During the washing phase, by means of the water on the fabric, by dissolution, the salts present in the precipitate are taken away, together with some of the amino acids, while leaving the least soluble amino acids in solid form.

[0095] At the end of the washing phase, the water content of the precipitate is between 50% and 60% by weight relative to the total weight of the precipitate.

[0096] The desalinated precipitate mainly comprises the following amino acids: cystine, tyrosine and leucine, also valine, isoleucine and phenylalanine.

[0097] The possibility of carrying out the dehydration and washing within the same enclosure, in particular within the wringer, contributes to simplifying the device for carrying out the method of the present application.

[0098] The washing water is recovered and added to the liquid phase obtained at the end of the centrifugation cycle, to form a solution of salted amino acids.

[0099] Desalination

[0100] The desalination phase is carried out on the solution of salted amino acids by means of electrodialysis. This solution of salted amino acids comprises all the amino acids, but in very small quantities of cystine and tyrosine.

[0101] The desalination phase is aimed at eliminating the salts, in particular the sodium chloride formed during the pH adjustment phase, by adding sodium hydroxide to the hydrochloric acid used in the hydrolysis phase. This desalination step is carried out by means of electrodialysis. Electrodialysis is generally carried out by placing pure water opposite the solution to be desalinated, the two solutions being alternately circulated between an anionic membrane and a cationic membrane to which an electric current is applied. At the end of the desalination phase, the salt content of the solution is less than 1% by weight of salt relative to the total weight of the solution.

[0102] Association

[0103] After the desalination phase, the desalinated precipitate obtained at the end of the water washing step is introduced into the desalinated solution, thus forming a suspension.

[0104] Surprisingly and advantageously, the inventors have shown that all the amino acids produced by acid hydrolysis are present. Furthermore, the amino acid profile of the hydrolysate according to the present application is close to that of the original keratin material.

[0105] Preferably, the obtained suspension is dried and the solid obtained after the drying stage is recovered. The hydrolysate according to the application is preferably in dry form, comprising less than 5% by weight of water, relative to the total weight of the hydrolysate.

[0106] The hydrolysate in dry form comprises less than 11% by weight, preferably less than 7% by weight of salts, relative to the total weight of the hydrolysate.

[0107] The total weight of amino acids recovered in dry form from the hydrolysate is at least equal to 80%, preferably at least 84% of the total weight of amino acids contained in the keratinous material involved in the hydrolysis.

[0108] Use

[0109] As already mentioned, the present application relates to the use of the hydrolysate in animal feed, in particular for felines and canines, in aquaculture or in agriculture.

[0110] The present application more particularly relates to the use of the hydrolysate according to the application or of the hydrolysate prepared according to the application as a component of a product chosen from the group consisting of food for animal feed for pets, food for aquaculture and plant biostimulants.

[0111] According to a first variant, the present application aims at the oral use of a keratin hydrolysate according to the application, or of a keratin hydrolysate obtained by the preparation method according to the application, as a raw material for animal feed.

[0112] The present application also relates to a raw material comprising the hydrolysate according to the application, without added ingredients.

[0113] The term "raw material" means any product of plant or animal origin, in a natural state, fresh or preserved, and obtained from its industrial processing, as well as any organic or inorganic substance, with or without additives, whether or not included, which is intended for direct oral consumption by animals, or after processing for the preparation of compound feed, or as a carrier of premixes (Directive 96 / 25 / EC of the Council of April 29, 1996).

[0114] The raw material according to the application is a mixture of amino acids intended to be incorporated into a complete and balanced food for animals, or as a food supplement for humans. It is therefore intended for oral administration to terrestrial and / or marine animals and / or humans. This raw material does not belong to the therapeutic class.

[0115] The present application more particularly relates to the use of the hydrolysate in animal feed, more particularly to the use of the hydrolysate as a source of raw material of free amino acids, making it possible to dispense with complex and high molecular weight plant and / or animal source food proteins.

[0116] The formulation of the raw material for animal feeding according to the application is carried out according to conventional methods, which form part of the general knowledge of the person skilled in the art.

[0117] As mentioned above, the present application also relates to a complete feed for animal feeding comprising from 0.25% to 40% by weight of the total weight of the complete feed of the composition according to the application or preferably of the hydrolysate according to the application.

[0118] According to the application, the complete feed for animal feeding can be formulated with excipients commonly used in compositions for the oral route, in particular humectants, thickening agents, texturizing agents, flavorings, coating agents, preservatives, antioxidants, colorants, plant extracts, non-protein ingredients such as starches, plant fibers, minerals and vitamins.

[0119] Of course, the person skilled in the art will note the choice of these excipients so as not to modify the properties of the complete feed for animal feeding.

[0120] The complete feed for animal feeding according to the application can be formulated on the basis of one of the following forms: kibbles, dragees, tablets, soft or hard gel capsules, or even suspensions, solutions, gels, dry preparations containing less than 15% by weight of water, or wet preparations containing at least 50% by weight of water and at most 85% by weight of water.

[0121] The formulation of the complete feed for animal feeding according to the application is carried out according to conventional methods, which form part of the general knowledge of the person skilled in the art.

[0122] The present application also relates to the use of the composition according to the application or of the hydrolysate according to the application for the preparation of a raw material or a complete feed for animal feeding.

[0123] According to a second variant, the present application relates to the use of keratin hydrolysates as an ingredient for promoting the palatability of feed for aquaculture, in particular for shrimp farming, in particular in the juvenile stage up to the growing stage.

[0124] According to a third variant, the present application aims to use keratin hydrolysates as plant biostimulants. The hydrolysates according to the application can be used for different parts of the plant: seeds, leaves, flowers and fruits.

[0125] Biostimulants are defined as substances and / or microorganisms, the function of which, when applied to plants or the rhizosphere, is to stimulate natural processes, to promote / enhance the uptake or use of nutrients, tolerance to abiotic stress, the quality or yield of crops, regardless of the presence of nutrients.

[0126] The hydrolysates can also be used with ingredients chosen from products for controlling plant diseases, fertilizers, microorganisms, seaweed extracts, humic and fulvic acids and minerals.

[0127] The following examples are intended to illustrate the application without limiting its scope. Example

[0128] The amino acids shown in Tables 1 to 3 were measured based on a method adapted from the 152 / 2009 EC regulation.

[0129] The amino acids were separated by chromatography with an ion exchange column (HPLC) and analyzed by reaction with ninhydrin and photometric detection at 570 nm.

[0130] Example 1 - Hydrolysate

[0131] Preparation of the hydrolysate

[0132] Hydrolysis

[0133] 9000 kg of poultry feathers containing 50% dry matter were introduced into a 55.000 liter reactor / hydrolyzer. The chemical hydrolysis was performed by adding 18.000 liters of hydrochloric acid (23%) and the hydrolysis was performed at 72°C for 4.5 hours. The resulting product was stored for 48 hours, allowing it to naturally recover to room temperature. Then, without the addition of acid, a second chemical hydrolysis was performed by heating at 107°C for 6 hours. The hydrolysate obtained contained 88% by weight of free amino acids, the remainder of the amino acids of the hydrolysate being in the form of small peptides with a molecular weight less than 800 Dalton or equal to 800 Dalton.

[0134] Purification

[0135] The hydrolysate was then decanted to remove the fat left by the keratin material floating on the surface of the aqueous phase. The excess hydrochloric acid introduced during the hydrolysis step was removed. 8000 kg of concentrate were recovered. Then 4500 kg of water were added, obtaining 12500 kg of diluted concentrate.

[0136] pH adjustment

[0137] 30.5% of sodium hydroxide was added to the hydrolysate to bring the pH to between 4 and 5. When sodium hydroxide is added, the less soluble amino acids, in particular cystine, tyrosine, leucine and isoleucine, at least partially precipitate. The other amino acids are all left in solution in the liquid phase.

[0138] Dehydration

[0139] The suspension was then placed in a wringer to separate the precipitate, which was retained on the canvas, and the liquid phase was recovered in a tank, equivalent to 17.000 kg.

[0140] Washing

[0141] The precipitate remaining on the canvas was then washed by introducing 3000 kg of water directly into the wringer to remove the salt (NaCI). The equivalent of 3440 kg of washing water was sent to the tank already containing the liquid phase resulting from the centrifugation, obtaining 20440 kg of solution called AA1. 1560 kg of washed precipitate were recovered, which corresponded to the product AA2.

[0142] The free amino acid composition of the AA1 solution is shown in Table 1

[0143] [Table 1]

[0144]

[0145] By infrared thermobalance measurement, AA1 was characterized by a dry matter content of 34.07%, a NaCI content of 14.74% and a free amino acid content of 93.4% with respect to the total weight of the amino acids of AA1.

[0146] The 1560 kg of washed precipitate containing less than 1% NaCI were recovered. By using infrared thermobalance measurement, the dry matter content was 44% by weight. The free amino acid composition of the washed and dried precipitate is shown in Table 2.

[0147] [Table 2]

[0148]

[0149] AA2 precipitate was characterized by a free amino acid content of 92.17% by weight with respect to the total weight of the total amino acids of AA2 precipitate.

[0150] Desalination

[0151] The 20.440 kg of AA1 solution (from the liquid phase of the centrifugation drying and washing water of the precipitate) were desalinated by electrodialysis against water, obtaining about 14.300 kg of desalinated solution containing less than 1% of NaCI. The electrodialyzer used to perform the desalination consisted of a double layer of 2 x 600 alternating anionic and cationic membranes, between which the solution was circulated and a direct current was passed.

[0152] Phase combination

[0153] The 1560 kg of washed precipitate and the 14.300 kg of desalinated solution were combined, obtaining 15.860 kg of suspension, which was dried by atomization in a drying tower with an inlet temperature of 172°C and an outlet temperature of 80°C, and sieved at 2500 rpm. About 3860 kg of powder were obtained, the composition of which is shown in Table 3.

[0154] Table 3 shows in the second column: the weight fraction of each FAA (free amino acid) of the hydrolysate with respect to the total FAAs; in the third column: the weight fraction of each AA (amino acid) of the original keratin material with respect to the total AAs; in the fourth column: the absolute value of the percentage variation between the weight of the free amino acid in the hydrolysate and the weight of that amino acid in the keratin material.

[0155] [Table 3]

[0156]

[0157] The hydrolysate obtained has a dry matter content of 98.6% with respect to the total weight of the hydrolysate amino acids, a NaCl content of 4.7% and a free amino acid content of 91.11%.

[0158] The original keratin material contains 93% of total amino acids based on dry matter (4500 kg of dry matter) and the hydrolysate obtained (3860 kg as is) contains 90.6% of total amino acids based on as is, with a yield of total amino acids of 83.6%.

[0159] Furthermore, the amino acid profile of the hydrolysate according to the present application is close to the amino acid profile of the original keratin material. In fact, as shown in the fourth column, for each of the 17 amino acids, the absolute value of the percentage variation between the weight of the free amino acid in the hydrolysate and the weight of that amino acid in the original keratin material is less than 20%. Furthermore, for 15 of them, this weight variation is less than 10%.

[0160] Example 2 - digestibility

[0161] Example 2 - digestibility The true digestibility of the proteins of the hydrolysate according to the present application is very high, since it is equal to 98.99%, thus very close to the maximum possible value (100%). This value was obtained according to the following protocol on cockerels with the caecum removed, which is a reference model for measuring the bioavailability of proteins in the animal kingdom.

[0162] Experimental protocol

[0163] The measurement of digestibility was carried out on adult cockerels with the caecum removed, which were kept in individual cages, fed with standard food outside the test period.

[0164] 2 replicates using 4 cockerels with the caecum removed were used. All the animals were fasted for 24 hours and then ingested an 80 g single meal consisting of 24 g of sample (hydrolysate) mixed with 56 g of sugar.

[0165] In the following 48 hours, all the faeces (= faecal droppings) were collected in two 24-hour periods, including endogenous losses, to avoid their fermentation and possible deterioration.

[0166] These faeces do not contain any contaminant such as feathers, which are carefully removed before freezing (-80°C).

[0167] The faeces are then freeze-dried in an oven, combined together and mixed in 2 pools, corresponding to 2 replicates for 4 animals for each of the 2 hydrolysates. Analyses are performed on these two pools.

[0168] Nutritional analyses (dry matter, crude protein (Dumas method ISO 16634-1 :2008 standard)) are performed on the hydrolysates, on the rooster faeces and on the endogenous losses. This data is used to calculate the true digestibility of the protein.

[0169] For this value of true digestibility of the protein, taking into account the contamination of the bird faeces by urinary nitrogen, the protein nitrogen in the faeces is measured (Terpstra method; Terpstra K.D. and N. De Hart. 1973. "The estimation of urinary nitrogen and faecal nitrogen in poultry excreta" Zeitschrift fur Tierphysiologie und Futtermittelkunde. 32(1-5):306-320).

[0170] Thus, according to the quantitative method, the true protein digestibility, measured in percentage, is calculated from the difference between the amount of hydrolysate ingested and the amount of faeces excreted, corrected for the endogenous losses, based on the following formula.

[0171] True protein digestibility % = (ingested protein hydrolysate - (excreted protein faeces - endogenous excreted protein)) / ingested hydrolysate protein x 100.

[0172] Thus, the hydrolysate according to the application is very easily assimilated by the body.

[0173] Example 3 - raw material for animal feed

[0174] A raw material for animal nutrition is prepared from the hydrolysate, the free amino acid composition of which is listed in Table 3 (column 2), without the addition of additional amino acids, in particular without the addition of L-tyrosine, precursor of melanin, pigment responsible for dark colouring of the coat (brown and black), and without the addition of L-cystine, essential for good health of the skin and keratin component in the coat of the animal.

[0175] The raw material prepared is not allergenic.

[0176] In addition, its palatability for cats and dogs has been observed.​

Claims

1. A keratin hydrolysate, characterized in that... It contains at least 88% by weight of free amino acids relative to the total weight of amino acids in the hydrolysate, and the keratin hydrolysate contains the following free amino acids relative to the total weight of free amino acids in the hydrolysate: Aspartic acid in a content of 6.00% to 10.00% by weight; Threonine in a content of 3.00% to 7.00% by weight; Serine in a content of 11.00% to 15.00% by weight; Glutamic acid in a content of 8.50% to 12.50% by weight; The content of glycine is from 6.50% to 10.50% by weight; Alanine in a content of 3.00% to 7.00% by weight; Valine in a content of 3.50% to 7.50% by weight; Cystine in a content of 4.00% to 8.00% by weight; Methionine in a content of 0.10% to 2.00% by weight; Isoleucine in a content of 1.50% to 5.50% by weight; Leucine in a content of 6.00% to 10.00% by weight; Tyrosine content of 2.50% to 3.50% by weight; Phenylalanine in a content of 3.00% to 7.00% by weight; Lysine in a content of 0.50% to 3.00% by weight; Histidine in a content of 0.10% to 2.00% by weight; Arginine in a content of 4.00% to 8.00% by weight; and The content of proline is 8.50% to 12.50% by weight.

2. The keratin hydrolysate according to claim 1, characterized in that... It contains at least 90% by weight of free cystine relative to the total weight of cystine in the hydrolysate.

3. The keratin hydrolysate according to claim 1, characterized in that... It is desalted, meaning that the keratin hydrolysate contains less than 11% by weight of salt relative to the total weight of the hydrolysate, and the salt is selected from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate.

4. The keratin hydrolysate according to claim 1, characterized in that... It contains the following free amino acids: At least 95% by weight of free aspartic acid relative to the total weight of aspartic acid in the hydrolysate; At least 95% by weight of free threonine relative to the total weight of threonine in the hydrolysate; At least 95% by weight of serine in the free form relative to the total weight of serine in the hydrolysate; At least 93% by weight of free glutamic acid relative to the total weight of glutamic acid in the hydrolysate; At least 90% by weight of glycine in the free form relative to the total weight of glycine in the hydrolysate; At least 90% by weight of alanine in the hydrolysate is in the free form relative to the total weight of alanine in the hydrolysate; At least 90% by weight of free phenylalanine relative to the total weight of phenylalanine in the hydrolysate; and At least 93% by weight of proline in the free form relative to the total weight of proline in the hydrolysate.

5. The keratin hydrolysate according to claim 1, which is obtained from keratin material, contains 17 amino acids, wherein for each amino acid, the absolute value of the percentage change between the weight of the free amino acid in the hydrolysate and the weight of the amino acid in the original keratin material is less than 20%.

6. The keratin hydrolysate according to claim 1, wherein the keratin hydrolysate comprises free amino acids in the following weight relative to the total weight of free amino acids in the hydrolysate: 7.00% to 9.00% by weight of aspartic acid; 4.00% to 6.00% by weight of threonine; 12.00% to 14.00% by weight of serine; 9.50% to 11.50% by weight of glutamic acid; 7.50% to 9.50% by weight of glycine; 4.00% to 6.00% by weight of alanine; 4.50% to 6.50% by weight of valine; Cystine: 5.00% to 7.00% by weight; 0.20% to 1.00% by weight of methionine; 2.50% to 4.50% by weight of isoleucine; 7.00% to 9.00% by weight of leucine; 3.00% to 3.50% by weight of tyrosine; 4.00% to 6.00% by weight of phenylalanine; 1.00% to 2.00% by weight of lysine; 0.20% to 1.00% by weight of histidine; 5.00% to 7.00% by weight of arginine; and 9.50% to 11.50% by weight of proline.

7. A method for preparing the keratin hydrolysate according to claim 1 from animal keratin material, characterized in that... It includes at least the following steps in the order given below: - Under conditions where hydrolysates are available, the keratin material is chemically hydrolyzed at least once with acid, the hydrolysate comprising at least 88% by weight of free amino acids relative to the total weight of amino acids in the hydrolysate, the remaining amino acids in the hydrolysate being peptides with a molecular weight of less than or equal to 800 Daltons. - The steps of adjusting the pH of the hydrolysate to 3 to 5 and recovering the precipitate and liquid phase; - Separate precipitate and liquid phase; - The precipitate is washed with water at least once until a desalinated precipitate is obtained, which contains less than 1% by weight of salt relative to the total weight of the precipitate, and the desalinated precipitate and the wash water are recovered on the one hand. - Combine the wash water and liquid phase to obtain a solution, and desalinate the solution by electrodialysis to obtain a desalinated solution. - Add the desalting precipitate to the desalting solution. -Recover the suspension obtained after desalination.

8. The method for preparing keratin hydrolysate according to claim 7, wherein the chemical hydrolysis is carried out at a temperature of 100°C to 115°C for 1 hour to 24 hours.

9. The method for preparing keratin hydrolysate according to claim 7, wherein the chemical hydrolysis is carried out in two stages: - The first chemical hydrolysis is carried out at a temperature of 60°C to 80°C for 4 to 5 hours, followed by... - A second chemical hydrolysis is carried out at a temperature of 100°C to 115°C for 5 to 8 hours.

10. The method for preparing keratin hydrolysate according to claim 7, wherein the obtained suspension is dried and the solid obtained at the end of drying is recovered.

11. Use of the keratin hydrolysate preparation product according to claim 1, wherein the product is selected from animal feed for pets, feed for aquaculture, and plant biostimulants.

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