Keratin hydrolysate with high contents of free amino acid and free tyrosine

Abstract

To provide hydrolysates for use in pet foods, aquaculture feeds, and plant biostimulants.SOLUTION: A keratin hydrolysate includes at least 88 wt.% of free amino acids relative to the total weight of the amino acids of the hydrolysate, where the hydrolysate contains free tyrosine of 2 to 4 wt.% relative to the total weight of the free amino acids, and includes a specific amino acid composition relative to the total weight of the free amino acids of the hydrolysate.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of hydrolysates with a high content of free amino acids and their use in animal feed, especially for cats and dogs, in aquaculture and agriculture, especially as biostimulants for plants. [Background technology]

[0002] Compositions based on amino acids are used in various fields such as dietary supplements, cosmetics, botanical, animal and human nutrition, and for very different specific applications within each of these fields, such as for the growth and shine of human hair, or to provide free amino acids constituting a specific protein source in animal feed, especially in aquaculture and in the feeding of dogs and cats.

[0003] One method of obtaining amino acid-based compositions is to prepare hydrolysates of keratin materials.

[0004] Natural keratin materials are essentially composed of high molecular weight polypeptides and have a highly cross-linked structure, which makes them inaccessible to enzymes. These natural keratin materials are not easily digestible. However, it is known that hydrolysis of keratin materials into amino acids can improve digestibility.

[0005] Keratin hydrolysates offered for sale, especially as food supplements, ingredients for formulating animal nutrition recipes or raw materials for animal feed, are generally obtained by very partial hydrolysis. These hydrolysates are generally characterized by a high molecular weight due to the presence of high levels of so-called "linking" amino acids that form peptides and polypeptides. The molecular weight of commercial compositions typically ranges from 5.000 to 50.000 daltons. These keratin hydrolysates are relatively indigestible and contain very few free amino acids. In fact, obtaining keratin hydrolysates with very high levels of free amino acids at industrial levels is technically complex and expensive. Moreover, excessive hydrolysis presents a risk of denaturation and destruction of amino acids.

[0006] Patent application WO2019 / 043128 outlines a keratin hydrolysate with a high level of free amino acids, obtained by carrying out a process in which a hydrolysis step is followed by a tyrosine extraction step. This hydrolysate has the advantage of a high level of free amino acids, in particular good digestibility. However, this hydrolysate does not contain all the amino acids typically present after acid hydrolysis, in particular low levels of cysteine ​​and tyrosine. Nevertheless, obtaining a hydrolysate that can be used in a complete and balanced food product without the need for additional amino acids is a commonly sought property.

[0007] Moreover, the hydrolysate obtained by chemical hydrolysis, such as acid hydrolysis, is salt-containing, whereas desalted hydrolysates are required for most applications, especially animal feed and biostimulants. However, the desalting process generally results in a large loss of certain amino acids, especially those that precipitate at the end of the neutralization process. In fact, desalting can only be performed on the filtered solution. Therefore, the yield of hydrolysates obtained according to the prior art methods is not always satisfactory. Summary of the Invention

[0008] Surprisingly, and as a further advantage, the inventors of the present invention have succeeded in overcoming the problems of the prior art by implementing a process that allows obtaining desalted keratin hydrolysates with a high level of acid-free amino acids and containing all the amino acids typically present after acid hydrolysis.Moreover, the amino acid profile of the hydrolysates according to the present invention is close to that of the original keratin material.The use of the hydrolysates according to the present invention therefore allows the omission of most of the addition of additional amino acids among those present in the original keratin material.

[0009] The free amino acids obtained by the present invention, especially those most difficult to release in free form during the hydrolysis process such as valine, leucine and isoleucine, are not damaged or denatured.

[0010] Furthermore, the process according to the invention makes it possible to obtain a good yield of amino acids, i.e. a good ratio between the sum of the amino acids of the hydrolysate according to the invention and the starting keratin material, since all the phases resulting from the various stages of the process are recovered and treated so as to extract a maximum, i.e. substantially all, of the amino acids.

[0011] The subject of the present invention 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 of the hydrolysate, said hydrolysate consisting of free tyrosine with a content ranging from 2 to 4% by weight, preferably from 2.5 to 3.5% by weight, relative to the total weight of free amino acids of the hydrolysate.

[0012] Advantageously, said hydrolysate contains at least 90%, preferably at least 93%, more preferably at least 95% of cystine in free form relative to the total weight of cystine in the hydrolysate.

[0013] In a preferred embodiment, the hydrolysate is desalted, i.e. contains less than 11% by weight, preferably less than 7% by weight, of salts relative to the total weight of the hydrolysate, the salts being selected from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate, preferably sodium chloride.

[0014] A second object of the invention relates to a process for preparing a keratin hydrolysate according to the invention from animal keratin material, preferably from poultry, comprising at least the following steps in the order outlined below: - subjecting the keratin material to at least one chemical hydrolysis with an acid under conditions allowing to obtain a hydrolysate containing at least 88% by weight of free amino acids relative to the total weight of the amino acids of the hydrolysate, wherein the remainder of the amino acids of the hydrolysate are in the form of peptides having a molecular weight of less than or equal to 800 Daltons; - subjecting the hydrolysate to a pH value ranging from 3 to 5, preferably from 4 to 5, and recovering the precipitate and the liquid phase; - Separating the precipitate and the liquid phase, preferably by centrifugation; - subjecting the precipitate to at least one washing with water, recovering on the one hand the desalted precipitate and on the other hand the washing water, until a desalted precipitate is obtained having less than 1% by weight of salts relative to the total weight of the precipitate; - collecting the wash water and the liquid phase to obtain a solution, which is further desalted by electrodialysis to obtain a desalted solution; - adding the desalted precipitate to said desalted liquid; - The resulting desalted suspension is recovered.

[0015] In a preferred variant, the suspension obtained is dried and the solid obtained at the end of drying is recovered.

[0016] The means for carrying out the process according to the invention have the advantage of being simple: it is possible to carry out the invention in equipment using the means commonly used in industry, such as reactors, centrifugal dryers and spray drying towers. Several stages can be carried out in the same equipment, and furthermore, the collection of the different stages is easily carried out without significant problems.

[0017] The invention further relates to the use of the keratin hydrolysate in animal feed, in particular for cats, dogs, aquaculture or agriculture, and more particularly to the use of the hydrolysate according to the invention or prepared according to the invention as an ingredient of a product selected from pet food, aquaculture food and plant biostimulants.

[0018] The present specification, particularly the examples and drawings which follow, are not exhaustive, but other aspects, advantages and characteristics of the present invention will become apparent from the disclosure thereof. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the main steps of the process according to the invention and the phases obtained at the end of these different steps. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Preferably, the hydrolysate is obtained from natural animal, in particular poultry, keratinous material, preferably from poultry feathers, such as chickens, in particular laying hens, chickens, turkeys, ducks, geese, etc. Natural keratinous material may also be selected from animal hair, in particular pig bristles, animal hooves, animal claws.

[0021] In particular, the hydrolysate according to the invention is not obtained from human keratin such as hair.

[0022] As already mentioned, the hydrolysate according to the invention consists of at least 88% by weight, preferably 90% by weight, of free amino acids relative to the total weight of amino acids of the hydrolysate.

[0023] Advantageously, the total amino acid content of the hydrolysate according to the invention ranges from 40% to 95%, preferably from 45% to 93%, relative to the total weight of the hydrolysate, which further comprises mineral matter and water.

[0024] Furthermore, the hydrolysates according to the invention are characterized by the free branched amino acids: unmodified valine, leucine and isoleucine, which are however known to be more difficult to release under the same conditions of use.

[0025] As already mentioned, the hydrolysate according to the invention is one which contains at least 90%, preferably at least 93%, more preferably at least 95% of cystine in free form, based on the total weight of cystine in the hydrolysate.

[0026] In a preferred variant, the hydrolysate according to the invention comprises: at least 95% by weight, preferably 100% by weight, of aspartic acid in free form, based on the total weight of aspartic acid in the hydrolysate; at least 95% by weight, preferably 100% by weight, of threonine in free form, based on the total weight of threonine in the hydrolysate; at least 95% by weight, preferably 100% by weight, of serine in free form, relative to the total weight of serine in the hydrolysate; at least 93% by weight, preferably 95% by weight or more, of glutamic acid in free form, based on the total weight of glutamic acid in the hydrolysate; at least 90% by weight, preferably 93% by weight or more, of glycine in free form, based on the total weight of glycine in the hydrolysate; at least 90% by weight, preferably 93% by weight or more, of alanine in free form, based on the total weight of alanine in the hydrolysate; at least 90% by weight, preferably 93% by weight or more, of phenylalanine in free form, based on the total weight of phenylalanine in the hydrolysate; At least 93% by weight, preferably 95% by weight or more of proline in free form, based on the total weight of proline in the hydrolysate.

[0027] Furthermore, at least 90% by weight of the amino acids of the hydrolysate are characterized by a molecular weight of less than 250 Daltons, preferably less than 240 Daltons, so that the hydrolysate can be used to prepare a hypoallergenic or non-allergenic complete animal feed.

[0028] The hydrolysate obtained is advantageously 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 (NaCl).

[0029] Determining the salt ratio is within the capabilities of a person skilled in the art. Preferably, the salt ratio is determined by the amount of anion added. Specifically, chloride ions are measured by potentiometric titration using 0.1N silver nitrate and then using a Ag / AgCl composite electrode; phosphate ions are quantified by spectrophotometric determination of phosphomolybdic acid complexes according to ISO 6878 standard, and sulfates are measured gravimetrically by addition of barium salts according to ISO 2480: 1972.

[0030] The anion dosage can be further supplemented by the dosage of cations, typically sodium and potassium dosages being determined spectrophotometrically by flame ionisation according to the ISO 9964-2:1993 standard.

[0031] The salt(s) content depends on the quality of the washing of the precipitate and on the extent to which the electrodialysis is continued. It is within the capabilities of the person skilled in the art to adjust the parameters of these steps, in particular their duration, based on the required salt(s) content.

[0032] "Dried hydrolysate" or "dry hydrolysate" as defined in the present invention means a hydrolysate containing less than 5% by weight of water. The weight of water in the hydrolysate is measured using an infrared thermobalance.

[0033] Preferably, the hydrolysate according to the invention comprises the following free amino acids, by weight relative to the total weight of free amino acids of the hydrolysate: aspartic acid with a content ranging from 6.00 to 10.00% by weight, preferably from 7.00 to 9.00% by weight, more preferably 7.83% by weight; Threonine with a content ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, and more preferably 4.93% by weight; serine with a content ranging from 11.00 to 15.00% by weight, preferably from 12.00 to 14.00% by weight, and more preferably 12.88% by weight; glutamic acid with a content ranging from 8.50 to 12.50% by weight, preferably from 9.50 to 11.50% by weight, more preferably 10.47% by weight; glycine with a content ranging from 6.50 to 10.50% by weight, preferably from 7.50 to 9.50% by weight, more preferably 8.56% by weight; alanine with a content ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, and even more preferably from 5.04% by weight; valine with a content ranging from 3.50 to 7.50% by weight, preferably from 4.50 to 6.50% by weight, more preferably 5.61% by weight; cystine with a content ranging from 4.00 to 8.00% by weight, preferably from 5.00 to 7.00% by weight, more preferably 5.80% by weight; methionine with a content ranging from 0.10 to 2.00% by weight, preferably from 0.20 to 1.00% by weight, more preferably 0.57% by weight; isoleucine with a content ranging from 1.50 to 5.50% by weight, preferably from 2.50 to 4.50% by weight, more preferably 3.50% by weight; leucine with a content ranging from 6.00 to 10.00% by weight, preferably from 7.00 to 9.00% by weight, and even more preferably from 7.77% by weight; tyrosine with a content ranging from 2.50 to 3.50% by weight, preferably from 3.00 to 3.50% by weight, more preferably 3.15% by weight; phenylalanine with a content ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, and even more preferably from 5.08% by weight; lysine with a content in the range of 0.50 to 3.00% by weight, preferably 1.00 to 2.00% by weight, more preferably 1.66% by weight; histidine with a content in the range of 0.10 to 2.00% by weight, preferably 0.20 to 1.00% by weight, and more preferably 0.74% by weight; arginine with a content ranging from 4.00 to 8.00% by weight, preferably from 5.00 to 7.00% by weight, and even more preferably from 5.82% by weight; Proline content in the range of 8.50 to 12.50%, preferably 9.50 to 11.50%, more preferably 10.59% by weight.

[0034] Another advantage of the keratin hydrolysate according to the invention is that its amino acid profile is close to that of the original keratin material: in fact, with the exception of tryptophan, which is destroyed during the acid hydrolysis, the 17 amino acids present in the original keratin material are also present in free form in the final hydrolysate.

[0035] The keratin hydrolysate according to the invention therefore consists of 17 amino acids, for each of which the percentage variation between the weight of the free amino acid in the hydrolysate and the weight of this amino acid at the starting keratin material is less than 20% in absolute value, preferably for 15 of these amino acids said variation is less than 10% in absolute value.

[0036] For amino acids, the percent variation 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 multiplied by 100 to the weight of the amino acid in the keratin material, i.e., the following formula: (|weight of amino acid in keratin material-weight of free amino acid in hydrolysate| / weight of amino acid in keratin material) x 100.

[0037] Another advantage of the hydrolysate according to the invention is that it is highly digestible and is recognized as food grade. The hydrolysate according to the invention is characterized by a true digestibility of its protein fraction of at least 98%, which is very close to the maximum possible (100%).

[0038] Digestibility is measured in vivo according to the method presented by ZM Larbier, AM Chagneau, and M. Lessire in “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.

[0039] process

[0040] Figure 1 shows the main steps of the process according to the invention, as described below, and the phases obtained at the end of these different steps. The steps are shown as rectangles and the phases as ellipses. According to the diagram in Figure 1, the process proceeds through a pH adjustment step after hydrolysis, producing a liquid phase and a precipitate, which is subjected to a solid-liquid separation step. The precipitate is then washed to obtain a desalted precipitate (AA2). The liquid phase and the washing water are combined and this solution (AA1) is subjected to a desalting step to obtain a desalted solution. The desalted solution and the desalted precipitate (AA2) are then combined and the resulting suspension is dried, thereby obtaining a dry desalted hydrolysate.

[0041] Acid hydrolysis

[0042] The process for preparing a keratin hydrolysate according to the invention comprises carrying out at least one chemical hydrolysis with an acid under conditions suitable for obtaining a hydrolysate containing at least 88% by weight of free amino acids relative to the total weight of amino acids of the hydrolysate, the remainder of the amino acids of the hydrolysate being small peptides, i.e. having a molecular weight of less than 800 Daltons.

[0043] The proportion of small peptides in the hydrolysate according to the invention generally ranges from 5 to 12% by weight relative to the total weight of the hydrolysate.

[0044] In fact, the hydrolysis is not complete, so that the proportion of small peptides in the hydrolysate is not zero, but is at most 12% by weight.

[0045] The chemical hydrolysis of keratin is carried out using an acid, preferably a strong acid selected from hydrochloric acid, phosphoric acid and sulfuric acid, preferably hydrochloric acid. The concentration of the acid, preferably hydrochloric acid, is preferably in the range of 14 to 34% by weight.

[0046] Preferably, the acid / keratin material weight ratio, and in certain cases the acid / feather weight ratio, is in the range of 2-5.

[0047] The chemical hydrolysis is generally carried out at a temperature in the range of 100 to 115° C. for a time in the range of 1 to 24 hours, preferably in the range of 6 to 20 hours.

[0048] In certain variations, the chemical hydrolysis is carried out in two stages: there is a first chemical hydrolysis carried out at a temperature in the range of 60 to 80° C. for a period of time in the range of 4 to 5 hours, followed by a second chemical hydrolysis carried out at a temperature in the range of 100 to 115° C. for a period of time in the range of 5 to 8 hours.

[0049] Furthermore, the two hydrolysis steps can be carried out without an intermediate resting step or with an intermediate resting step of between 1 hour and 7 days.

[0050] More specifically, the first chemical hydrolysis can be carried out at 72° C. for 4.5 hours and the second chemical hydrolysis can be carried out at 107° C. for 6 hours, with an intermediate rest period of 24 to 80 hours between the two chemical hydrolysis.

[0051] Preferably, if fat floats on the surface of the hydrolysate, this supernatant is removed.

[0052] Adjusting pH

[0053] The chemical hydrolysis, carried out in one or more stages, is followed by a pH adjustment stage. The hydrolysate is adjusted to a pH value ranging from 3 to 5, preferably from 4 to 5. This stage is carried out by adding a base chosen from sodium hydroxide and potassium hydroxide, preferably sodium hydroxide. This step is classical and its implementation is within the capabilities of the person skilled in the art.

[0054] This step is also characterized by the effect of at least partially precipitating the less soluble amino acids, in particular cystine, tyrosine, leucine and isoleucine, which form a precipitate while the other amino acids remain in solution and form a liquid phase with the salts formed and with water.

[0055] Solid-liquid separation-dehydration

[0056] After the pH adjustment step, a step of separating the precipitate from the liquid phase is carried out. The separation step can be carried out by implementing any solid-liquid separation technique, in particular by applying centrifugal force, or by pressing, in particular with a filter press. According to a preferred variant, the separation step is carried out by dewatering. The dewatering step is preferably carried out by applying centrifugal force by rotation at about 1000 rpm. This method is known to those skilled in the art for carrying out solid-liquid separation and consists in eliminating the liquid phase by the effect of centrifugal force, while keeping the precipitate (solid fraction) on the fabric.

[0057] Washing

[0058] The essore and collected precipitate, preferably on the fabric of the centrifugal dryer, is washed with water until a desalted precipitate is obtained having a salt content of less than 1% by weight relative to the total weight of the precipitate. The term "salts" means sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate, preferably sodium chloride (NaCl).

[0059] During the washing stage, the water passing over the precipitate in place on the fabric takes up, by solubilization, the salts and some of the amino acids present in said precipitate, while leaving the most insoluble amino acids in solid form.

[0060] At the end of the washing stage, the water content of the precipitate is in the range of 50-60% by weight relative to the total weight of the precipitate.

[0061] The desalted precipitate consists mainly of the following amino acids: cystine, tyrosine and leucine, but also valine, isoleucine and phenylalanine.

[0062] It is also possible for the centrifugal drying and washing to take place in the same housing, in particular in a centrifugal dryer (essoreuse), which contributes to simplifying the means for carrying out the process according to the invention.

[0063] The wash water is collected and added to the liquid phase obtained at the end of the centrifugation cycle to form a solution of salified amino acids.

[0064] Desalination

[0065] The solution of amino acids in salts is subjected to a desalting step by electrodialysis. This solution of amino acids in salts contains all the amino acids, except for cystine and tyrosine, which are present therein in very small amounts.

[0066] The desalination stage aims to remove salts formed in the pH adjustment stage, specifically sodium chloride, by adding sodium hydroxide to the hydrochloric acid used in the hydrolysis stage. This desalination step is carried out by electrodialysis. Conventionally, electrodialysis is carried out by pairing pure water with the solution to be desalted, and circulating the two solutions alternately and separately between anion and cation membranes through which an electric current is passed. The salt concentration of the solution at the end of the desalination process is 1% by weight or less based on the total weight of the solution.

[0067] Phase Combination

[0068] Following the desalting step, the suspension is formed by introducing the desalted precipitate obtained at the end of the water washing step into a desalting solution.

[0069] Surprisingly and favourably, the inventors have shown that all amino acids resulting from acid hydrolysis are present. Moreover, the amino acid profile of the hydrolysates according to the invention is close to that of the original keratin material.

[0070] Preferably, the suspension obtained is dried and the solid obtained is recovered following the drying step. The hydrolysate according to the invention is preferably in a dry form, which consists of less than 5% by weight of water relative to the total weight of the hydrolysate.

[0071] 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.

[0072] The weight of the total amino acids recovered from the hydrolysate in dry form is at least equal to 80% and preferably at least 84% of the weight of the total amino acids contained in the keratin material involved in the hydrolysis.

[0073] Purpose

[0074] As already mentioned, the present invention relates to the use of the hydrolysates in animal nutrition, in particular in cats, dogs, aquaculture or agriculture.

[0075] The invention more particularly relates to the use of a hydrolysate according to the invention or prepared according to the invention as an ingredient of a product selected from food for feeding pet animals, feed for aquaculture, and biostimulants for plants.

[0076] According to a first variant, the present invention aims at the oral use of a keratin hydrolysate according to the invention or obtained by a preparation process according to the invention, as a raw material for animal feed.

[0077] The present invention also relates to a raw material consisting of a hydrolysate according to the invention without added ingredients.

[0078] The term "raw materials" refers to natural, fresh or preserved, substances of vegetable or animal origin and those resulting from their industrial processing, as well as organic or inorganic substances, whether or not they contain additives, intended, as such or after processing, for the preparation of compound feed or as carriers for premixes, or orally in animal feed (Directive 96 / 25 / EC of the Council of April 29, 1996).

[0079] The material according to the invention is a mixture of amino acids intended to be incorporated into a complete and balanced feed in animals or to be used as a food supplement in humans. It is therefore intended for oral administration in terrestrial and / or marine animals and / or humans. This material does not fall into the category of therapeutic drugs.

[0080] The present invention relates to the use of hydrolysates in animal feed, more particularly as a raw source of free amino acids, which makes it possible to dispense with food proteins of complex molecular structure and high molecular weight of vegetable and / or animal origin.

[0081] The formulation of animal feed ingredients according to the present invention may be carried out by conventional processes which may form part of the general skill of the person skilled in the art.

[0082] As already mentioned, the present invention further relates to a complete feed for feeding animals, which comprises 0.25 to 40% by weight of the composition or preferably the hydrolysate according to the invention, relative to the total weight of said complete feed.

[0083] The complete feed for feeding animals according to the invention may contain excipients typically used in compositions intended for the oral route, in particular humectants, thickeners, texture improvers, flavourings, coating agents, preservatives, antioxidants, colouring agents, plant extracts, non-protein ingredients such as starch, plant fibres, minerals and vitamins.

[0084] Of course, the skilled artisan will take care to select these excipients so as not to alter the characteristics of the complete feed for feeding the animals.

[0085] The complete feed for feeding animals according to the invention can be formulated according to one of the following presentations: kibbles, dragees, tablets, soft or hard capsules, or alternatively suspensions, solutions, gels, dry formulations containing less than 15% by weight of water or wet formulations containing at least 50% by weight of water and at most 85% by weight of water.

[0086] The formulation of a complete feed for feeding animals according to the invention may be carried out by conventional processes which may form part of the general skill of the person skilled in the art.

[0087] The present invention also relates to the use of the composition according to the invention or the hydrolysate according to the invention for preparing an ingredient or a complete feed for feeding animals.

[0088] According to a second variant, the invention relates to the use of keratin hydrolysates as a palatability-promoting ingredient in feed for aquaculture, in particular for shrimp farming, in particular for the larval and adult stages.

[0089] According to a third variant, the present invention is directed to the use of keratin hydrolysates as biostimulants in plants. The hydrolysates according to the invention can be used on different parts of the plant: seeds, leaves, flowers and fruits.

[0090] Biostimulants are defined as substances and / or microorganisms that, when applied to a plant or the rhizosphere, have the function of stimulating natural processes that promote / enhance nutrient uptake or utilization, resistance to abiotic stresses, crop quality or yield, independent of the presence of nutrients.

[0091] The hydrolysates can furthermore be used together with ingredients selected from pesticides (phytosanitary products), fertilisers, microorganisms, seaweed extracts, humic and fulvic acids, and also minerals.

[0092] The following examples are intended to illustrate the invention but not to limit its scope.

[0093] Working Example

[0094] The amino acids shown in Tables 1 to 3 are measured by methods complying with EC Regulation 152 / 2009.

[0095] The amino acids are separated by chromatography on an ion-exchange column (HPLC) and assayed by reaction with ninhydrin and photometric detection at 570 nm.

[0096] Example 1 - Hydrolysate

[0097] Preparation of hydrolysates

[0098] Hydrolysis

[0099] 9000 kg of chicken feathers, with a dry weight of 50%, are introduced into a 55,000 liter reactor / hydrolyzer. Chemical hydrolysis is carried out by adding 18,000 liters of hydrochloric acid (23%) and the hydrolysis is carried out at 72 °C for 4.5 hours. The product obtained is left for 48 hours to naturally change temperature to room temperature. A second hydrolysis is then carried out by heating at 107 °C for 6 hours without the addition of acid. The hydrolysate obtained consists of 88% by weight of free amino acids, the remaining amino acids of the hydrolysate are in the form of small peptides with a molecular weight below 800 daltons.

[0100] purification

[0101] The hydrolysate is then decanted to remove the fat, which is a residue of keratin material floating on the surface of the aqueous phase. The hydrochloric acid introduced in excess during the hydrolysis step is removed. 8000 kg of concentrate are recovered. 4500 kg of water are then added, obtaining 12500 kg of diluted concentrate.

[0102] pH adjustment

[0103] 30.5% sodium hydroxide is added to the hydrolysate to bring the pH to a value between 4 and 5. Upon addition of sodium hydroxide, the less soluble amino acids, especially cystine, tyrosine, leucine and isoleucine, are at least partially precipitated, while the other amino acids remain completely dissolved in the liquid phase.

[0104] dehydration

[0105] The suspension is passed through a squeezer to separate the sediment, and 17,000 kg of the liquid phase is collected in a tank.

[0106] Washing

[0107] The sediment remaining on the canvas is washed to remove the salt (NaCl) by introducing 3000 kg of water directly into the wringer. Wash water amounting to 3440 kg is sent to a tank already containing the liquid phase coming from the centrifuge, obtaining 20440 kg of solution called AA1. 1560 kg of washed sediment are recovered, which corresponds to product AA2.

[0108] The free amino acid composition of the AA1 solution is shown in Table 1.

[0109] [Table 1]

[0110] AA1 is characterized by a dry matter content of 34.07% by weight, measured by infrared thermobalance, a NaCl content of 14.74% by weight and a content of free amino acids of 93.4% by weight relative to the total weight of AA1 amino acids.

[0111] 1560 kg of washed precipitate containing less than 1% NaCl is recovered. The dry matter content is 44% by weight as determined by infrared thermobalance. The free amino acid composition of the washed and dried precipitate is given in Table 2.

[0112] [Table 2]

[0113] The AA2 precipitate is characterized by a content of free amino acids of 92.17% by weight relative to the total weight of all amino acids in the AA2 precipitate.

[0114] Desalination

[0115] 20,440 kg of AA1 solution (liquid phase consisting of the water resulting from the centrifugal drying and the water used to wash the precipitate) is desalted by electrodialysis against water to obtain about 14,300 kg of desalted solution (containing less than 1% NaCl). The electrodialysis apparatus consists of 2 x 600 alternating layers of anion and cation membranes, between which the solution circulates and a direct current flows.

[0116] Phase Combination

[0117] The washed precipitate, amounting to 1560 kg, and the desalted solution, amounting to 14,300 kg, give a suspension of 15,860 kg, which is dried by atomization in a drying tower equipped with a sieving device at 2500 rpm, with an inlet temperature of 172°C and an outlet temperature of 80°C, and then sieved. Approximately 3860 kg of powder is obtained, the composition of which is shown in Table 3.

[0118] Table 3 shows, in column 2, the weight ratio of each FAA (free amino acid) of the hydrolysate to the total FAA; in column 3, the weight ratio of each AA (amino acid) of the original keratin material to the total AA; in column 4, the variation in absolute values ​​as percentages between the weight of the free amino acid in the hydrolysate and the weight of this amino acid in the keratin material.

[0119] [Table 3]

[0120] The obtained hydrolysate had a dry matter content of 98.6%, a NaCl content of 4.7% and a free amino acid content of 91.11% by weight based on the total weight of all amino acids in the hydrolysate.

[0121] The starting keratin material contained 93% total amino acids based on the dry matter (4500 kg dry matter), and the resulting hydrolysate (3860 kg as is) contained 90.6% total amino acids as is, resulting in a total amino acid yield of 83.6%.

[0122] Moreover, the amino acid profile of the hydrolysates according to the invention is close to that of the original keratin material. Indeed, as shown in column 4, for each of the 17 amino acids, the percentage variation between the weight of the free amino acid in the hydrolysate and the weight of this amino acid in the starting keratin material is less than 20% in absolute terms. Moreover, for 15 of them, this weight varies by less than 10%.

[0123] Example 2 - Digestibility

[0124] The true digestibility of the proteins of the hydrolysates according to the invention is very high, being equal to 98.99%, and therefore very close to the maximum possible (100%). This value was obtained according to the following protocol on castrated roosters (coqs caecectomises), a reference model for measuring the bioavailability of proteins in the animal kingdom:

[0125] Experimental Protocol

[0126] Digestibility measurements were performed on castrated adult male chickens (coqs adultes caecectomises) housed in individual cages and fed a standard diet outside the trial period.

[0127] Four castrated roosters (coqs caecectomises) are used in two replicates. All animals are fasted for 24 h and fed a single diet of 80 g consisting of 24 g of sample (hydrolysate) mixed with 56 g of sugar.

[0128] All excreta (faeces), including endogenous losses, are collected twice in 24-hour periods during the following 48 hours in order to avoid as much as possible fermentation and putrefaction.

[0129] These faeces are carefully cleaned of various contaminants such as feathers before being frozen (-80°C).

[0130] The feces are freeze-dried in an oven and pooled in two pools, corresponding to the two replicates of four animals used for each of the two hydrolysates, and mixed. The two pools are then analyzed.

[0131] Nutritional analysis (dry matter, crude protein (Dumas method ISO 16634-1:2008 standard)) is performed on the hydrolysate, chicken feces and endogenous losses. These data are used to calculate the true protein digestibility.

[0132] To determine the true protein digestibility, the protein nitrogen in the feces is measured, taking into account the presence of urea nitrogen in the feces (Terpstra method; Terpstra KD and N. De Hart. 1973. "The estimation of urinary nitrogen and faecal nitrogen in poultry excreta" Zeitschrift fur Tierphysiologie Tierernahrung und Futtermittelkunde. 32 (1-5): 306-320).

[0133] The true protein digestibility, measured as a percentage, is therefore calculated according to the quantitative method by the difference between the amount of hydrolysate ingested and the amount of faeces excreted, corrected for endogenous losses according to the following formula:

[0134] [True protein digestibility (%)] = ([ingested protein hydrolysate] - ([excreted protein feces] - [endogenous excreted protein])) / [ingested hydrolysate protein] x 100.

[0135] The hydrolysate according to the invention is therefore very easily absorbed by the body.

[0136] Example 3 - Ingredients for animal feed

[0137] From this hydrolysate a raw material for animal feed is prepared, the composition of free amino acids of which, as shown in Table 3 (column 2), does not require the addition of any additional amino acids, in particular L-tyrosine, which is a precursor of the pigment melanin responsible for the darkening of the coat (brown and black), nor L-cystine, which is essential for skin health and constitutes the keratin in animal hair.

[0138] The ingredients used do not cause allergies.

[0139] It has also been confirmed that they have a preference for dogs and cats.

Claims

1. A keratin hydrolysate, The hydrolysate contains at least 88% by weight of free amino acids based on the total weight of amino acids, the hydrolysate contains 2-4% by weight of free tyrosine based on the total weight of the free amino acids; Relative to the total weight of the free amino acids in the hydrolysate, 6.00 to 10.00% by weight of aspartic acid, 3.00 to 7.00% by weight of threonine, 11.00 to 15.00 wt. % serine, 8.50 to 12.50% by weight of glutamic acid, 6.50 to 10.50% by weight of glycine, 3.00 to 7.00% by weight of alanine, 3.50 to 7.50% by weight of valine, 4.00 to 8.00% by weight of cystine, 0.10 to 2.00% by weight of methionine, 1.50 to 5.50% by weight of isoleucine, 6.00 to 10.00% by weight of leucine, 2.50 to 3.50% by weight of tyrosine, 3.00 to 7.00% by weight of phenylalanine, 0.50 to 3.00% by weight of lysine, 0.10 to 2.00% by weight of histidine, 4.00 to 8.00% by weight of arginine, 8.50 to 12.50 wt. % proline, Hydrolysates.

2. Contains at least 90% by weight of cystine in free form, based on the total weight of cystine in the hydrolysate; The hydrolysate according to claim 1.

3. being desalted, i.e. containing less than 11% by weight of salts relative to the total weight of the hydrolysate, the salts being selected from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate; The hydrolysate according to claim 1 or 2.

4. at least 95% by weight of aspartic acid in free form, based on the total weight of aspartic acid in the hydrolysate; at least 95% by weight of threonine in free form, based on the total weight of threonine in the hydrolysate; At least 95% by weight of serine in free form, based on the total weight of serine in the hydrolysate. at least 93% by weight of glutamic acid in free form, based on the total weight of glutamic acid in the hydrolysate; at least 90% by weight of glycine in free form, based on the total weight of glycine in the hydrolysate; at least 90% by weight of alanine in free form, based on the total weight of alanine in the hydrolysate; at least 90% by weight of phenylalanine in free form, based on the total weight of phenylalanine in the hydrolysate; At least 93% by weight of proline in free form, based on the total weight of proline in the hydrolysate; The hydrolysate according to any one of claims 1 to 3.

5. It is derived from a keratin material containing 17 amino acids: aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline; For each of the amino acids, the absolute percentage variation between the weight of the free amine in the hydrolysate and the weight of the amino acid in the starting keratin material is less than 20%. The hydrolysate according to any one of claims 1 to 4.

6. Relative to the total weight of the 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 wt. % 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, 5.00 to 7.00% by weight of cystine, 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, 9.50 to 11.50% by weight of proline, The hydrolysate according to any one of claims 1 to 5.

7. The hydrolysate is used as an ingredient in a product selected from pet food and / or aquaculture food. The hydrolysate according to any one of claims 1 to 6.

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