Method for producing keratin hydrolyzate

Abstract

The present invention relates to a keratin hydrolysate containing 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, and 2-4% by weight, preferably 2.5-3.5% by weight, of free tyrosine relative to the total weight of free amino acids in the hydrolysate, and a method for preparing the hydrolysate, in particular comprising the steps of acid hydrolysis, pH adjustment and desalting.The present invention also relates to the use of the hydrolysate, in particular for pet food, aquaculture feed and as a biostimulant for plants.

Description

Technical Field

[0001] The present invention relates to the field of hydrolyzates with high free amino acid content, and their use in animal feed, particularly for cats and dogs, in aquaculture and agriculture, especially as plant biostimulants.

Background Art

[0002] Amino acid-based compositions are used in various fields such as nutritional supplements, cosmetics, plant, animal and human nutrition, and for very different specific uses in each of these fields. Among these, there are uses aimed at the growth and gloss of human hair, and uses aimed at supplying free amino acids that constitute specific protein sources in animal feed, particularly in aquaculture and feeding of dogs and cats.

[0003] One way to obtain amino acid-based compositions is to produce hydrolyzates of keratin materials.

[0004] Natural keratin materials consist essentially of high molecular weight polypeptides and have a highly cross-linked structure, so they are not easily accessible to enzymes. This natural keratin material cannot be easily digested. However, it is known that the digestibility is improved by hydrolyzing the keratin material into amino acids.

[0005] Keratin hydrolysates provided for sale, in particular 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 high molecular weights due to the presence of high levels of so-called "linked" amino acids that form peptides and polypeptides. The molecular weights of commercially available compositions typically range from 5,000 to 50,000 Daltons. These keratin hydrolysates are relatively indigestible and contain few free amino acids. In fact, obtaining keratin hydrolysates with very high levels of free amino acids at the industrial level is technically complex and expensive. Furthermore, excessive hydrolysis poses a risk of amino acid denaturation and destruction.

[0006] Patent application WO2019 / 043128 outlines a keratin hydrolysate with high levels of free amino acids obtained by implementing a process in which a tyrosine extraction step follows the hydrolysis step. This hydrolysate has the advantage of high levels of free amino acids and particularly good digestibility. However, this hydrolysate does not contain all the amino acids that are generally present after acid hydrolysis, especially cysteine and tyrosine are at low levels. Nevertheless, obtaining a hydrolysate that can be used in complete and balanced foods without adding amino acids is a generally desired property.

[0007] Furthermore, hydrolysates obtained by chemical hydrolysis such as acid hydrolysis are in a salt-containing state, but for most applications, especially in animal feed and biostimulants, desalted hydrolysates are required. However, in the desalting process, generally, certain amino acids, especially those that precipitate at the end of the neutralization process, are lost in large quantities. In fact, desalting can only be carried out on the filtered solution. Therefore, the yield of hydrolysates obtained according to prior art methods is not necessarily 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 enables the obtaining of a desalted keratin hydrolyzate having a high level of free amino acids and containing all the amino acids generally present after acid hydrolysis. Furthermore, the amino acid profile of the hydrolyzate according to the present invention is close to the amino acid profile of the original keratin material. Therefore, the use of the hydrolyzate according to the present invention makes it possible to omit most of the addition of additional amino acids among the amino acids present in the original keratin material.

[0009] The free amino acids obtained according to the present invention, in particular the amino acids that are most difficult to be released in free form during the hydrolysis process, such as valine, leucine and isoleucine, are not damaged or denatured.

[0010] Furthermore, in the process according to the present invention, all the phases resulting from the various stages of the process are recovered and processed to extract maximally, i.e., substantially all the amino acids, so that a good yield of amino acids, i.e., a good ratio between the total of the amino acids of the hydrolyzate according to the present invention and the starting point of the keratin material, can be obtained.

[0011] The subject of the present invention is a keratin hydrolyzate containing at least 88% by weight, preferably at least 90% by weight of free amino acids based on the total weight of the amino acids of the hydrolyzate, and the hydrolyzate consists of free tyrosine in a content ranging from 2 to 4% by weight, preferably from 2.5 to 3.5% by weight, based on the total weight of the free amino acids of the hydrolyzate.

[0012] Advantageously, the hydrolyzate 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 hydrolyzate.

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

[0014] A second object of the present invention relates to a process for preparing a keratin hydrolyzate according to the present invention from keratin materials of animals, preferably poultry, and comprises at least the following steps in the order schematically shown below: - subjecting the keratin material to at least one chemical hydrolysis with an acid under conditions such that a hydrolyzate can be obtained that contains at least 88% by weight of free amino acids relative to the total weight of the amino acids of the hydrolyzate, where the remainder of the amino acids of the hydrolyzate is in the form of peptides having a molecular weight of 800 daltons or less; - subjecting the hydrolyzate to a step of adjusting it to a value in the range of pH 3 to 5, preferably in the range of 4 to 5, and recovering the precipitate and the liquid phase; - separating the precipitate and the liquid phase, preferably by centrifugation (essorage); - subjecting the precipitate to at least one washing with water until a desalted precipitate is obtained that contains less than 1% by weight of salts relative to the total weight of the precipitate, recovering the desalted precipitate on the one hand and the washing water on the other hand; - collecting the washing water and the liquid phase to obtain a solution, and further desalting this solution by electrodialysis to obtain a desalted solution; - adding the desalted precipitate to the desalted solution; - recovering the obtained desalted suspension.

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

[0016] The means for implementing the process according to the invention has the advantage of being simple: the invention can be implemented with an apparatus using means commonly used in industries such as reactors, centrifugal dryers, and spray drying towers. Several steps can be carried out in the same apparatus, and furthermore, the collection of different steps can be easily carried out without significant problems.

[0017] The invention further relates to the use of keratin hydrolysates in animal feed, particularly in cat, dog, aquaculture or agricultural applications. More particularly, the invention relates to the use of hydrolysates according to or prepared according to the invention as a component of products selected from pet food, aquaculture feed, and plant biostimulants.

[0018] This specification, particularly the examples, and the drawings that follow do not exhaust the invention, but other aspects, advantages, and characteristics of the invention are made clear by these disclosures.

Brief Description of the Drawings

[0019]

Figure 1

Modes for Carrying Out the Invention

[0020] Preferably, this hydrolysate is obtained from natural, animal, particularly poultry keratin materials, preferably from poultry feathers. Examples of poultry include chickens, particularly laying hens, domestic chickens, turkeys, ducks, geese, etc. The natural keratin material may also be selected from animal hair, particularly pig bristles, animal hooves, and 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 hydrolyzate 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 the amino acids of the hydrolyzate.

[0023] Advantageously, the total amino acid content of the hydrolyzate according to the invention ranges from 40% to 95%, preferably from 45% to 93%, relative to the total weight of the hydrolyzate, and the hydrolyzate further contains minerals and water.

[0024] Furthermore, the hydrolyzate according to the invention is characterized by free branched-chain amino acids: unmodified valine, leucine and isoleucine. However, it is known that these branched-chain amino acids are more difficult to release under the same use conditions.

[0025] As already mentioned, the hydrolyzate according to the invention contains cystine in a free form of at least 90%, preferably at least 93%, more preferably at least 95%, relative to the total weight of cystine in the hydrolyzate.

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

[0027] Furthermore, at least 90% by weight of the amino acids in the hydrolyzate is characterized by a molecular weight of 250 Daltons or less, preferably 240 Daltons or less. As a result, a complete feed for animals with low allergenicity or non-allergenicity can be prepared using this hydrolyzate.

[0028] The obtained hydrolyzate is preferably desalted, that is, it consists of less than 11% by weight, preferably less than 7% by weight, of salts based on the total weight of the hydrolyzate. The salts are selected from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, and potassium phosphate, and preferably sodium chloride (NaCl).

[0029] Determining the proportion of salts can be carried out within the capabilities of those skilled in the art. Preferably, the proportion of salts is determined by the input amount of anions. Specifically, chloride ions are measured by potentiometric titration using 0.1N silver nitrate at this time, and then an Ag / AgCl composite electrode is used; phosphate ions are quantified by a spectrophotometer of a phosphomolybdic acid complex conforming to the ISO 6878 standard, and furthermore, sulfates are measured by weight by the addition of barium salts conforming to ISO 2480:1972.

[0030] The input amount of anions can be further supplemented by the input of cations. Generally, the input amounts of sodium and potassium are measured by spectrophotometry using flame ionization based on the ISO 9964-2:1993 standard.

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

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

[0033] Preferably, the hydrolyzate according to the present invention contains the following free amino acids, by weight relative to the total weight of the free amino acids of the hydrolyzate: Aspartic acid in a content ranging from 6.00 to 10.00% by weight, preferably from 7.00 to 9.00% by weight, more preferably from 7.83% by weight; Threonine in a content ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, more preferably from 4.93% by weight; Serine in a content ranging from 11.00 to 15.00% by weight, preferably from 12.00 to 14.00% by weight, more preferably from 12.88% by weight; Glutamic acid in a content ranging from 8.50 to 12.50% by weight, preferably from 9.50 to 11.50% by weight, more preferably from 10.47% by weight; Glycine in a content ranging from 6.50 to 10.50% by weight, preferably from 7.50 to 9.50% by weight, more preferably from 8.56% by weight; Alanine in a content ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, more preferably from 5.04% by weight; Valine in a content ranging from 3.50 to 7.50% by weight, preferably from 4.50 to 6.50% by weight, more preferably from 5.61% by weight; Cystine in a content ranging from 4.00 to 8.00% by weight, preferably from 5.00 to 7.00% by weight, more preferably from 5.80% by weight; Methionine in a content ranging from 0.10 to 2.00% by weight, preferably from 0.20 to 1.00% by weight, more preferably from 0.57% by weight; Isoleucine in an amount 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 in an amount ranging from 6.00 to 10.00% by weight, preferably from 7.00 to 9.00% by weight, more preferably 7.77% by weight; Tyrosine in an amount 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 in an amount ranging from 3.00 to 7.00% by weight, preferably from 4.00 to 6.00% by weight, more preferably 5.08% by weight; Lysine in an amount ranging from 0.50 to 3.00% by weight, preferably from 1.00 to 2.00% by weight, more preferably 1.66% by weight; Histidine in an amount ranging from 0.10 to 2.00% by weight, preferably from 0.20 to 1.00% by weight, more preferably 0.74% by weight; Arginine in an amount ranging from 4.00 to 8.00% by weight, preferably from 5.00 to 7.00% by weight, more preferably 5.82% by weight; Proline in an amount ranging from 8.50 to 12.50%, preferably from 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, except for tryptophan which is destroyed by acid hydrolysis, the 17 amino acids present in the original keratin material also appear in free form in the final hydrolysate.

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

[0036] The percent variation for 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 hydrolyzate, multiplied by 100, to the weight of the amino acid in the keratin material, i.e., it corresponds to the following formula: (|weight of amino acid in keratin material - weight of free amino acid in hydrolyzate| / weight of amino acid in keratin material) × 100.

[0037] Another advantage of the hydrolyzate according to the present invention is that it is very easy to digest. Also, it is recognized as food grade. The hydrolyzate according to the present invention is characterized in that the true digestibility of its protein fraction is at least 98%. This value is very close to the maximum possible value (100%).

[0038] Digestibility is measured in vivo according to the method shown by Z.M. Larbier, A.M. 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 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 figure of Figure 1, the process, after hydrolysis, undergoes a pH adjustment step to produce a liquid phase and a precipitate, which is then subjected to a solid-liquid separation step. Thereafter, the precipitate is 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 process to obtain a desalted solution. Next, the desalted solution and the desalted precipitate (AA2) are combined, and the resulting suspension is dried to obtain a dried desalted hydrolyzate.

[0041] Hydrolysis with an acid

[0042] The process for preparing a keratin hydrolyzate according to the invention involves performing at least one chemical hydrolysis with an acid under conditions suitable for obtaining a hydrolyzate containing at least 88% by weight of free amino acids with respect to the total weight of the amino acids of the hydrolyzate, and the remaining amino acids of the hydrolyzate are small peptides, i.e., those having a molecular weight of 800 daltons or less.

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

[0044] In fact, since the hydrolysis is not complete, the proportion of small peptides in the hydrolyzate is not zero, but on the other hand, it 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, in a specific case the acid / feather weight ratio, is in the range of 2 to 5.

[0047] 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 hour to 24 hours, preferably in the range of 6 to 20 hours.

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

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

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

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

[0052] pH adjustment

[0053] After chemical hydrolysis carried out in one or more steps, a pH adjustment step follows. The hydrolyzate is adjusted to a pH with a value in the range of 3 to 5, preferably 4 to 5. This step is carried out by adding a base selected from sodium hydroxide and potassium hydroxide, preferably sodium hydroxide. This step is classical and its implementation is within the capabilities of those skilled in the art.

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

[0055] Solid-liquid separation - dehydration

[0056] After the pH adjustment step, a step of separating the precipitate from the liquid phase is performed. The separation step can be carried out by implementing any solid-liquid separation technique, particularly by applying centrifugal force or particularly by pressing using a filter press. According to a preferred variant, the separation stage is carried out by dehydration. The dehydration stage 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 performing solid-liquid separation and consists of eliminating the liquid phase by the effect of centrifugal force while maintaining the precipitate (solid fraction) on the cloth.

[0057] Washing

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

[0059] During the washing step, the water passing over the precipitate at a predetermined position on the cloth incorporates, by solubilization, the salts and some amino acids present in the precipitate, while leaving the most insoluble amino acids in solid form.

[0060] At the end of the washing step, the water content of the precipitate is in the range of 50 to 60% by weight with respect to the total weight of the precipitate.

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

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

[0063] The washing water is recovered and added to the liquid phase obtained at the end of the centrifugation cycle to form a solution of amino acids in the form of salts.

[0064] Desalting

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

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

[0067] Phase combination

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

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

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

[0071] The hydrolyzate in dry form comprises less than 11% by weight, preferably less than 7% by weight, of salts based on the total weight of the hydrolyzate.

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

[0073] Use

[0074] As already mentioned, the present invention relates to the use of hydrolyzates in animal nutrition, particularly in cat, dog, aquaculture, or agriculture.

[0075] More particularly, the present invention relates to the use of hydrolyzates according to or prepared according to the present invention as a component of products selected from pet food, aquaculture feed, and plant biostimulants.

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

[0077] The present invention also relates to a raw material consisting of the hydrolyzate according to the present invention without the addition of components.

[0078] The term "raw material" means natural, fresh or preserved, substances derived from plants or animals, and those obtained from their industrial processing, and organic or inorganic substances, whether or not they contain additives, which are intended to be used orally, as such or after processing, for the preparation of compound feeds or as carriers for premixes in animal feed (Directive 96 / 25 / EC of the Council of April 29, 1996).

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

[0080] The present invention relates to the use of a hydrolyzate as a source of raw material for free amino acids in animal feed, thereby making it possible to omit complex molecular structures and high molecular weight plant and / or animal-derived food proteins.

[0081] Formulating the raw material for animal feed according to the present invention can be carried out by conventional processes, which may form part of the general skills of those skilled in the art.

[0082] As already mentioned, the present invention further relates to a complete feed for animal feeding, comprising 0.25 to 40% by weight of the composition according to the present invention or preferably a hydrolyzate, based on the total weight of the complete feed.

[0083] The complete feed for animal feeding according to the present invention can be formulated with excipients commonly used in compositions intended for the oral route, in particular non-protein components such as humectants, thickeners, texture improvers, flavoring agents, coating agents, preservatives, antioxidants, coloring agents, plant extracts, starch, etc., plant fibers, minerals, and vitamins.

[0084] Of course, those skilled in the art will take care to select these excipients so as not to change the properties of the complete feed for animal feeding.

[0085] The complete feed for animal feeding according to the present invention can be formulated based on one of the following presentations: kibble, dragee, tablet, soft capsule or hard capsule, or suspension, solution, gel, dry preparation containing less than 15% by weight of water, or wet preparation containing at least 50% by weight and up to 85% by weight of water.

[0086] The formulation of the complete feed for animal feeding according to the present invention may be carried out by conventional processes, which may form part of the general skills of those skilled in the art.

[0087] The present invention also relates to the use of the composition according to the invention or the hydrolyzate according to the invention for preparing raw materials or complete feeds for animal feeding.

[0088] According to a second variant, the present invention relates to the use of keratin hydrolyzate as a component for promoting palatability in feeds for aquaculture, in particular for shrimp aquaculture, especially up to the larval and growth stages.

[0089] According to a third variant, the present invention is directed to the use of keratin hydrolyzate as a biostimulant for plants. The hydrolyzate according to the invention can be used on various parts of plants: seeds, leaves, flowers, and fruits.

[0090] A biostimulant is defined as a substance and / or microorganism that, when applied to plants or the rhizosphere, has the function of stimulating natural processes that promote / enhance nutrient uptake or use, tolerance to abiotic stress, crop quality, or yield, regardless of the presence of nutrients.

[0091] The hydrolyzate can further be used in combination with components selected from pesticides (produits phytosanitaires), fertilizers, microorganisms, seaweed extracts, humic acids, and fulvic acids, and also with minerals.

[0092] The following examples are for illustrative purposes and do not limit the scope of the present invention.

[0093] Examples

[0094] The amino acids shown in Tables 1 to 3 are measured by a method adapted to Regulation (EC) No 152 / 2009.

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

[0096] Example 1 - Hydrolysate

[0097] Preparation of Hydrolysate

[0098] Hydrolysis

[0099] 9000 kg of chicken feathers, with a dry weight accounting for 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 resulting product is left for 48 hours to allow the temperature to change naturally to room temperature. Next, a second hydrolysis is carried out by heating at 107 °C for 6 hours without adding acid. The resulting hydrolysate consists of 88 wt% free amino acids, and the remaining amino acids in the hydrolysate are in the form of small peptides with a molecular weight of 800 daltons or less.

[0100] Purification

[0101] Next, the hydrolysate is 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 concentrated solution is recovered. Then, 4500 kg of water is added to obtain 12500 kg of diluted concentrated solution.

[0102] pH Adjustment

[0103] 30.5% sodium hydroxide is added to the hydrolysate to bring the pH to a value between 4 and 5. When sodium hydroxide is added, poorly soluble amino acids, especially cystine, tyrosine, leucine, and isoleucine, precipitate at least partially. The other amino acids remain completely dissolved in the liquid phase.

[0104] Dehydration

[0105] The suspension is passed through a filter press to separate the precipitate, and 17,000 kg of the liquid phase is recovered in a tank.

[0106] Washing

[0107] The precipitate remaining on the canvas is washed by introducing 3000 kg of water directly into the filter press to remove salt (NaCl). The washing water corresponding to 3440 kg is sent to the tank that already contains the liquid phase generated from centrifugation, and 20440 kg of a solution called AA1 is obtained. 1560 kg of the washed precipitate is 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 in that the dry matter content measured by an infrared thermobalance is 34.07% by weight, the NaCl content is 14.74% by weight, and the content of free amino acids relative to the total weight of AA1 amino acids is 93.4% by weight.

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

[0112]

Table 2

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

[0114] Desalting

[0115] 20,440 kg of AA1 solution (the liquid phase obtained by combining the water generated by centrifugal drying and the water used to wash the precipitate) is desalted by electrodialysis with respect to water to obtain approximately 14,300 kg of desalted solution (containing less than 1% NaCl). The electrodialysis device is composed of 2 × 600 anion membranes and cation membranes stacked alternately, with the solution circulating between them and a direct current flowing through.

[0116] Phase combination

[0117] Combine the washed precipitate corresponding to 1560 kg and the desalted solution corresponding to 14,300 kg to obtain 15,860 kg of suspension. This is atomized and dried in a drying tower equipped with a sieve device at an inlet temperature of 172 °C, an outlet temperature of 80 °C, and 2500 rpm, and then sieved. Approximately 3860 kg of powder is obtained, and its composition is as shown in Table 3.

[0118] Table 3 shows in the second column: the weight ratio of each FAA in the hydrolyzate to the total FAA (free amino acid); in the third column: the weight ratio of each AA in the original keratin material to the total AA (amino acid); in the fourth column: the variation in the weight of the free amino acid in the hydrolyzate and the weight of this amino acid in the keratin material is shown as an absolute value in percentage.

[0119]

Table 3

[0120] The obtained hydrolyzate has a dry matter content of 98.6%, an NaCl content of 4.7%, and a free amino acid content of 91.11% by weight based on the total weight of the hydrolyzate's total amino acids.

[0121] The starting keratin material contains 93% total amino acids with respect to the dry matter (4500 kg of dry matter), and the obtained hydrolyzate (3860 kg as it is) contains 90.6% total amino acids as it is. Therefore, the yield of total amino acids was 83.6%.

[0122] Moreover, the amino acid profile of the hydrolyzate according to the present invention is close to that of the original keratin material. In fact, as shown in column 4, for each of the 17 amino acids, the coefficient of variation between the weight of the free amino acids in the hydrolyzate and the weight of this amino acid in the starting keratin material is less than 20% in absolute value. Furthermore, for 15 of them, this weight variation is less than 10%.

[0123] Example 2 - Digestibility

[0124] The true digestibility of the protein of the hydrolyzate according to the present invention is very high, equal to 98.99%, and thus very close to the maximum possible value (100%). This value was obtained in accordance with the following protocol for castrated male chickens (coqs caecectomises), which is a reference model for measuring the bioavailability of proteins in the animal kingdom.

[0125] Experimental protocol

[0126] The digestibility measurement is carried out on castrated adult male chickens (coqs adultes caecectomises) housed in individual cages and given standard feed outside the test period.

[0127] Four castrated male chickens (coqs caecectomises) are used in two repetitions. All animals are fasted for 24 hours and fed 80 g of a single feed consisting of 24 g of the sample (hydrolyzate) mixed with 56 g of sugar.

[0128] All excreta (= feces), including endogenous losses, are collected twice at 24 - hour intervals over the next 48 hours to avoid fermentation and putrefaction as much as possible.

[0129] These feces are carefully removed to remove various contaminants such as feathers and then frozen (-80°C).

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

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

[0132] For this true protein digestibility value, the protein nitrogen in the feces is measured considering the contamination of bird feces with urea nitrogen (Terpstra method; Terpstra K. D. 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] Therefore, the true protein digestibility measured as a percentage is calculated according to a quantitative method by correcting for endogenous losses based on the following formula by the difference between the amount of hydrolysate ingested and the amount of feces excreted.

[0134] [True protein digestibility (%)] = ([Protein hydrolysate ingested] - ([Protein feces excreted] - [Endogenous excreted protein])) / [Hydrolysate protein ingested] × 100.

[0135] Therefore, the hydrolysate according to the present invention is very easily absorbed by the body.

[0136] Example 3 - Raw material for animal feed

[0137] Raw materials for animal feed are prepared from this hydrolyzate. The composition of its free amino acids does not require additional amino acids as shown in Table 3 (second column). In particular, it is not necessary to add L-tyrosine, which is a precursor of the pigment melanin that causes darkening of the coat (brown and black), and it is also not necessary to add L-cystine, which is essential for skin health and constitutes the keratin of the animal's coat.

[0138] The prepared raw materials do not cause allergies.

[0139] Also, the palatability for dogs and cats has been confirmed.

Claims

1. A method for producing a keratin hydrolyzate from an animal keratin material, wherein the hydrolyzate contains at least 88% by weight of free amino acids relative to the total weight of the amino acids of the hydrolyzate, and the hydrolyzate contains 2-4% by weight of free tyrosine relative to the total weight of the free amino acids, the method includes at least the following steps in the order shown below, - subjecting the keratin material to at least one chemical hydrolysis with an acid under conditions such that at least 88% by weight of the free amino acids relative to the total weight of the amino acids of the hydrolyzate are contained in the hydrolyzate, where the remainder of the amino acids of the hydrolyzate are in the form of peptides having a molecular weight of 800 daltons or less; - subjecting the hydrolyzate to a step of adjusting the pH to a value in the range of 3 to 5, and recovering a precipitate and a liquid phase; - separating the precipitate and the liquid phase; - subjecting the precipitate to at least one washing with water until a desalted precipitate containing less than 1% by weight of salt relative to the total weight of the precipitate is obtained, recovering the desalted precipitate on the one hand and washing water on the other hand; - combining the washing water and the liquid phase to obtain a solution, and subjecting the solution to desalting by electrodialysis to obtain a desalted solution; - adding the desalted precipitate to the desalted solution; - recovering the desalted suspension obtained by desalting, wherein the chemical hydrolysis - is carried out in two stages: a first hydrolysis carried out at a temperature in the range of 60-80°C for 4-5 hours, followed by - a second hydrolysis carried out at a temperature in the range of 100-115°C for 5-8 hours, A method for producing a keratin hydrolyzate.

2. including at least 90% by weight of cystine in free form relative to the total weight of cystine in the hydrolyzate, The method for producing a keratin hydrolyzate according to claim 1.

3. containing less than 11% by weight of salt relative to the total weight of the hydrolyzate, and the salt is selected from sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate and potassium phosphate, The method for producing a keratin hydrolyzate according to claim 1 or 2.

4. at least 95% by weight of aspartic acid in free form relative to the total weight of aspartic acid in the hydrolyzate; at least 95% by weight of threonine in free form relative to the total weight of threonine in the hydrolyzate; At least 95% by weight of serine in free form, based on the total weight of serine in the hydrolyzate At least 93% by weight of glutamic acid in free form, based on the total weight of glutamic acid in the hydrolyzate; At least 90% by weight or more of glycine in free form, based on the total weight of glycine in the hydrolyzate; At least 90% by weight or more of alanine in free form, based on the total weight of alanine in the hydrolyzate; At least 90% by weight or more of phenylalanine in free form, based on the total weight of phenylalanine in the hydrolyzate; and At least 93% by weight or more of proline in free form, based on the total weight of proline in the hydrolyzate, containing free amino acids A method for producing the keratin hydrolyzate according to any one of claims 1 to 3.

5. A method for producing a keratin hydrolyzate obtained from a keratin material containing 17 amino acids of 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 value of the variation in the ratio between the weight of the free amino acid in the hydrolyzate and the weight of these amino acids in the starting keratin material is less than 20%. A method for producing the keratin hydrolyzate according to any one of claims 1 to 4.

6. In terms of weight ratio to the total weight of the free amino acids of the hydrolyzate, 6.00 to 10.00% by weight of aspartic acid; 3.00 to 7.00% by weight of threonine; 11.00 to 15.00% by weight of 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; and 8.50 to 12.50% by weight of proline, containing free amino acids A method for producing a keratin hydrolyzate according to any one of claims 1 to 5.

7. By weight ratio to the total weight of the free amino acids of the hydrolyzate, 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; 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; and 9.50 to 11.50% by weight of proline, containing free amino acids, A method for producing a keratin hydrolyzate according to claim 6.

8. A method for producing a keratin hydrolyzate according to any one of claims 1 to 7, wherein the obtained suspension is dried and the solid obtained at the end of drying is recovered.

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