Particles containing an active agent and their manufacturing process

Abstract

Particles containing an active agent and their manufacturing process. The invention relates to particles (15) comprising an agglomeration of cohesive grains and having a substantially spherical shape, each grain being made up of a mixture comprising an active agent and a cohesive agent for said grains. The manufacturing process for these particles employs granulation by spraying.

Description

Title of the invention: Particles containing an active agent and method of manufacturing them

[0001] The invention relates to particles containing at least one active agent. The particles are particularly suitable for their incorporation during pelletizing or tablet manufacturing processes for the animal feed, food supplement, pharmaceutical or cosmetics industries.

[0002] In the context of the present invention, the term "pelletization" (or otherwise known as "granulation") means a process consisting of assembling or agglomerating solid particles together so as to form larger elements in various geometric shapes such as spheres or cylinders.

[0003] The invention is particularly described with reference to the animal feed industry, without this limiting its scope. Indeed, the problems described just below inherent to active agents and in connection with the animal feed industry can also arise in the pharmaceutical industry, the industry for manufacturing food supplements for humans or even the cosmetics industry.

[0004] The shaping of animal feed in the form of granules during pelletization is particularly favored compared to that in dry form. Indeed, it has the advantages of being more qualitative, comprising fewer pathogenic agents, generating less dust during manufacturing, being more suitable for handling stages, and finally allowing a more uniform dosage of the active agents contained in animal feed to improve their productivity. These active agents can be, for example, enzymes to improve the digestibility of feed, vitamins, proteins, antimicrobial agents or even pre- or probiotics.

[0005] However, other compounds, said to be "aggressive" towards the active agents, also present in the compositions of animal feed such as acidic or basic compounds which may be organic or inorganic, reducing sugars and hygroscopic substances (in particular choline chloride or sodium chloride) have detrimental effects on these active agents such that they reduce their beneficial activity.

[0006] These detrimental effects may occur during storage of premixes or animal feeds containing these active agents and compounds. For the purposes of the present invention, the term "premix" means a premixture of active agents such as those described above with various other organic or inorganic compounds. (for example, carrier compounds such as limestone or clay) and in particular compounds said to be "aggressive" towards the active agents, said premix being intended to be incorporated into animal feed. Indeed, during storage, oxidation-reduction reactions can occur in the presence of water within the premixes or animal feed, resulting in a significant reduction in the activity of active agents such as enzymes.

[0007] Furthermore, certain active agents (notably enzymes) can cause allergies when handled by operators.

[0008] Furthermore, it is known that process steps for manufacturing animal feeds involving drastic conditions (such as high humidity, high temperatures and high compression forces) can also be detrimental to the activity of the active agents. Indeed, the latter (in particular enzymes) are not always stable when subjected to such conditions, in particular during pelletization for the manufacture of animal feeds.

[0009] Finally, certain active agents are sensitive to humidity, particularly during storage of animal feed which can last several years.

[0010] The solution of storing these so-called "sensitive" active agents outside the premixes is not fully satisfactory for reasons of logistics and therefore of the costs of manufacturing animal feed. Furthermore, this does not resolve the problem mentioned above of the drastic conditions of the animal feed manufacturing process, as well as the risks of allergies among operators.

[0011] This is why it is common in industry, particularly in the animal feed industry but also in the pharmaceutical industry, to formulate these active agents in a protected form so that they can maintain their activity (even if they are subjected to drastic industrial process conditions and incorporated with other compounds said to be "aggressive" towards them) and to minimize the risk of allergies in operators.

[0012] There are different ways to protect active agents. For example, active agents can be incorporated: - in a core made of a porous material (for example made of an inorganic salt such as sodium sulfate) by blocking its pores and therefore remaining relatively on the surface of the core, and / or - within a layer made up of a binder (for example a sugar) which surrounds the core, said core, where appropriate the binder layer, being coated with one or more protective layers (in other words “coating layers”) so as to obtain particles containing perfectly protected active agents. In this regard, the experimental part of patent EP 3 270 893 B1 describes examples of such particles.

[0013] It is important to emphasize that in these embodiments of the state-of-the-art particles, the protective layer is essential to correctly protect the active agent which is present on the surface of the core (namely in a layer surrounding the core and / or in the pores on the surface of said core).

[0014] In order to simplify the manufacture of thermostable particles containing active agents and to limit the costs by reducing, for example, the number of raw materials, it would be advantageous to develop particles which do not require such protective layers while not reducing their capacity to protect the active agents.

[0015] Furthermore, it would be advantageous to have particles in which the protective performance of the active agents is increased compared to that of the particles of the state of the art. Due to better protection of the active agents, this would also have the advantage of being able to lower their quantity within said particles and thus reduce their production costs. In other words, due to better protection of the active agents, an activity of said active agents equivalent to that of the particles of the state of the art could be obtained with particles containing less active agents than the particles of the state of the art contain.

[0016] Thus, there is still a real need to improve the development of thermostable particles containing active agents which remain perfectly protected during contact of these particles with other so-called "aggressive" compounds or during the use of these particles in pelletization or tablet manufacturing processes.

[0017] The inventors of the present invention have discovered, quite surprisingly, particles containing at least one active agent which perfectly fulfill these objectives.

[0018] The invention thus relates to a particle which is characterized in that it comprises an agglomeration of grains which are cohesive with each other and which is of substantially spherical shape, each grain being made up of a mixture which comprises at least: - an active agent and - an agent for the cohesion of said grains between them.

[0019] The cohesion agent allows the grains to be stuck together so as to form an agglomeration of cohesive grains between them. Said agglomeration has a substantially spherical shape.

[0020] The originality of the particles according to the invention lies in the fact that the cohesion agent contained in the mixture of which the grains are made ensures the cohesion of the grains between them so as to obtain an agglomeration of grains of a substantially spherical shape. Thanks to this agglomeration of the grains, the active agent is distributed (or in other words “confined”) in a homogeneous manner within said particles and is thus perfectly protected.

[0021] Unlike the particles of the state of the art mentioned above in which the active agent to be protected can be distributed either in a layer surrounding the core of the particle, or by blocking the pores of a porous material constituting the core of said particle and thus remaining relatively on the surface of said core, in the particles according to the invention, the active agent is distributed, advantageously in a uniform manner, throughout the entire volume (preferably spherical) of said particles.

[0022] In the particles according to the invention, the active agent not being exclusively distributed on the surface of said particles but rather in the entire volume thereof, the protective layer of the active agent is not obligatory. Sufficient protection of the active agent without a protective layer is already obtained with the particles according to the invention thanks to this agglomeration of grains which are made up of the mixture as described above.

[0023] Indeed, and as demonstrated in the experimental part below, the inventors discovered in a completely surprising manner that the particles according to the invention without a protective layer presented protection of the active agent equivalent to that of the particles of the state of the art which have been described above and which comprise at least one protective layer around the core of these particles.

[0024] The particles according to the invention can be thermostable.

[0025] The particles according to the invention can ensure good protection of the active agent against humidity, particularly during storage of products (e.g. animal feed) in which they are incorporated.

[0026] The particle according to the invention may have a median diameter of between 300 pm and 800 pm, preferably between 400 pm and 700 pm, more preferably between 500 pm and 600 pm.

[0027] Preferably, the particles according to the invention have excellent sphericity. For example, the sphericity can be evaluated from the parameters of circularity and / or roundness.

[0028] The circularity of a particle is determined according to the following mathematical formula (1):

[0029] [Math 1]

[0030] circularity = (4 x pi x particle area) / (particle perimeter)2 (1)

[0031] The particle according to the invention has good sphericity when its circularity is of at least 0.85.

[0032] The roundness of a particle is determined according to the following mathematical formula (2):

[0033] [Math 2]

[0034] roundness = (4 x area of ​​the particle) / (pi x length of the major axis of the particle2) (2)

[0035] The particle according to the invention has good sphericity when its roundness is at least 0.85.

[0036] The fact that the particles according to the invention have excellent sphericity is advantageous for being able to coat them with at least one protective layer in a qualitative manner as described below. Indeed, if the particles do not have good sphericity, pores may form on the surface of said protective layer during this coating step. These pores may promote the entry of water into the particles and thus reduce the activity of the active agents if they are sensitive to humidity.

[0037] Advantageously, the particles according to the invention have a narrow particle size distribution, namely with a span less than or equal to 0.5.

[0038] The span is defined according to the mathematical equation (3) below:

[0039] [Math 3]

[0040] span = [D(v,0.9)-D(v,0,l)] / D(v, 0.5)(3)

[0041] in which: - D(v,0.9) represents the particle size for which 90% of the sample volume has a particle size smaller than this size; - D(v,0,l) represents the particle size for which 10% of the sample volume has a particle size smaller than this size, - D(v,0.5) represents the particle size for which 50% of the sample volume has a particle size smaller than this size.

[0042] The lower the span value, the more homogeneous the particle size distribution. A span value less than or equal to about 1 is significant of a very homogeneous particle size distribution and when the span value is less than or equal to about 0.5, the particles have a perfectly homogeneous size distribution.

[0043] The active agent may be chosen from the group consisting of proteins (and in particular enzymes, peptides and polypeptides), antimicrobial agents, amino acids and vitamins, taken alone or as a mixture thereof.

[0044] In preferred embodiments of the invention, the active agent is at least one protein selected from enzymes, peptides and polypeptides.

[0045] Preferably, the active agent is an enzyme. For example, it may be phytase, xylanase (especially endo-1,4-beta-xylanase), beta-glucanase, phosphatase, protease, amylase (especially alpha-, beta- or gluco-amylase), cellulase, lipase, cutinase, oxidase, transferase, reductase, hemicellulase, mannanase, esterase, isomerases, pectinase, lactase, peroxidase, laccase or redox enzymes (such as glucose oxidase, glutamate oxidase, lactate oxidase), taken alone or in a mixture of these.

[0046] In a preferred embodiment of the invention, the enzyme is selected from the group consisting of phytases, xylanases (e.g., endo-1,4-beta-xylanase), phosphatases, proteases, amylases, esterases, redox enzymes, lipases, transferases, cellulases, and beta-glucanases, taken alone or as a mixture thereof. These enzymes are conventionally used in the formulation of animal feed.

[0047] In another preferred embodiment of the invention, the enzyme is selected from the group consisting of proteases, amylases, lipases, hemicellulases, redox enzymes, peroxidases, transferases and cellulases, taken alone or as a mixture thereof. These enzymes are conventionally used in the formulation of food supplements or cosmetic care.

[0048] The particles according to the invention may comprise one or more different active agents (for example up to 3 different active agents).

[0049] In embodiments of the invention, the particles comprise a single enzyme or 2 or 3 different enzymes. For example, the particles may comprise a xylanase (e.g., endo-1,4-beta-xylanase) and a beta-glucanase or a xylanase (e.g., endo-1,4-beta-xylanase), a beta-glucanase, and a phytase.

[0050] The enzymes described above are given as examples and do not limit the scope of the invention. They can be any type of enzyme.

[0051] The active agent may be in solid or liquid form. When it is an enzyme, it may be in solid or liquid form. Preferably, it is in liquid form.

[0052] The quantity of active agents present in the particles according to the invention depends on their application. It is within the reach of those skilled in the art to choose the quantity of active agents according to the application that they envisage for the particles according to the invention.

[0053] The particle according to the invention may comprise, in mass percentages expressed relative to the total mass of said particle, between 10% and 90%, preferably between 15% and 70%, more preferably between 15% and 30%, even more preferably between 15% and 20%, of active agent.

[0054] The particles according to the invention have higher active agent protection performances than those of the particles of the state of the art. Due to this better protection of the active agents, the quantity of active agents contained in the particles according to the invention can be lowered; which makes it possible to reduce their production costs. Indeed, due to this better protection of the active agents, an activity of said active agents equivalent to that of the particles of the state of the art can be obtained with particles according to the invention which contain less active agents than state-of-the-art particles do not contain any.

[0055] The cohesion agent is configured to provide cohesion between the grains.

[0056] It may consist of a hydrophilic polymer or a hydrophobic polymer.

[0057] In an advantageous embodiment of the invention, the cohesion agent is soluble in water.

[0058] In an advantageous embodiment of the invention, the cohesion agent at 15% by mass of dry extract in water has a viscosity at 25°C of between 5 mPa.s and 20 mPa.s.

[0059] When the active agent is an enzyme, the cohesion agent may be chosen to limit the deployment of the enzyme at high temperatures of industrial processes such as those mentioned above. In other words, the cohesion agent may be chosen such that the enzyme maintains its tertiary structure, even if it is subjected to high process temperatures.

[0060] The cohesion agent may be chosen from the group consisting of maltodextrin, lactose, maltose and starch, taken alone or as a mixture thereof.

[0061] Preferably, said cohesion agent is chosen from the group consisting of maltodextrin and starch.

[0062] The particle may comprise, in mass percentages expressed relative to the total mass of said particle, between 10% and 90%, preferably between 15% and 85%, more preferably between 50% and 80%, of said cohesion agent.

[0063] The mixture of which each grain is composed may further comprise at least one additional compound. Advantageously, said additional compound is chosen from the group consisting of sodium sulfate, calcium carbonate and polyols.

[0064] The total mass content of the additional compound(s), expressed relative to the total mass of said particle, may be between 5% and 35%, preferably between 5% and 10%.

[0065] In one embodiment of the invention, the particle further comprises at least one protective layer which surrounds said agglomeration of cohesive grains. The presence of the protective layer(s) makes it possible to improve the protection of the active agents which are confined within the agglomeration of cohesive grains.

[0066] Advantageously, the protective layer makes it possible to protect the active agent against humidity, in particular during storage of the product which contains the particles according to the invention and which can extend over several years.

[0067] In one embodiment of the invention, the particles comprise a plurality of protective layers which may be identical or different from each other.

[0068] For example, the particles may comprise one, two, three or four protective layers which may be the same or different from each other.

[0069] Each protective layer may have a thickness of between 2 μm and 80 μm, preferably between 5 μm and 20 μm.

[0070] Furthermore, in this embodiment of the invention, the particle according to the invention may comprise, in mass percentages expressed relative to the total mass of said particle: - between 5% and 50%, preferably between 15% and 40%, of at least one protective layer, - between 50% and 95%, preferably between 60% and 85%, of said agglomeration of cohesive grains between them.

[0071] The choice of the constituent(s) of the protective layer is perfectly within the reach of those skilled in the art. Indeed, as explained above, it is conventional to coat the core of the particles with at least one protective layer to protect the active agents that said particles contain.

[0072] Thus, within the framework of the present invention, the at least one protective layer may comprise one or more constituents usually used for the implementation of such protective layers.

[0073] As non-limiting examples of the present invention, they may be polymers, sugars, proteins, lipids, oils, fatty acids, inorganic salts or even gums, taken alone or in mixtures thereof.

[0074] Preferably, the constituent(s) of the at least one protective layer has(have) a melting point below 80°C. This is particularly suitable for obtaining qualitative protective layers which best protect the agglomeration of cohesive grains between the particles.

[0075] The polymer may be selected from the group consisting of polysaccharides (e.g. alginates), polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, polyacrylates, polyethylene oxides, polylactic acid, polyvinyl chloride, polyvinyl acetate, polyvinyl pyrrolidones, cellulose ethers, native or modified starches and their substituted derivatives, as well as their hydrolysates and copolymers thereof, hydroxypropyl methyl cellulose, methyl cellulose, carboxymethyl cellulose and ethylcellulose, taken alone or as a mixture thereof. Preferably, it is polyvinyl alcohol, methyl cellulose or hydroxypropyl methyl cellulose.

[0076] As regards starches, these may be native or modified starches of corn, sorghum, rice, wheat, rye, barley, oats, potato, tapioca or cassava.

[0077] As for sugars, these may be sucrose, corn syrup extract, molasses, glucose, fructose and lactose.

[0078] As for proteins, these can be whey powder or concentrate, caseinates, soy protein concentrate, albumin and gelatin.

[0079] As regards lipids, oils and fatty acids, these may be waxes (such as vegetable, mineral and synthetic waxes: for example, camauba, candellila, beeswax, paraffin and microcrystalline waxes), lecithin, fatty acids (for example, stearic, palmitic, linoleic, oleic, butyric and arachidonic acid), as well as their sodium, potassium, calcium and zinc salts, fats and oils, in particular hydrogenated or partially hydrogenated fats and oils (for example, soybean, corn, cottonseed or linseed oil). Preferably, it is lecithin.

[0080] As regards the inorganic salts, these may be sulfate, citrate, chloride, carbonate, sulfite, phosphate, phosphonate salts, as well as bicarbonate salts of sodium, ammonium, potassium, calcium, magnesium and zinc. Preferably, these are salts of magnesium, sodium and ammonium sulfates.

[0081] As for gums, these may be gum arabic, guar, agar, tragacanth, carob, carrageenan and xanthan.

[0082] Preferably, the protective layer comprises at least one constituent chosen from the group consisting of alginates, stearic acid, ethylcellulose, hydroxypropyl methyl cellulose, methyl cellulose, carboxymethyl cellulose and soybean, corn, cottonseed or linseed oil, taken alone or as a mixture thereof.

[0083] The at least one protective layer may further comprise at least one constituent selected from the group consisting of plasticizers, lubricants, pigments and powders such as talc, bentonite, kaolin, magnesium silicate, calcium carbonate and chitosan, taken alone or as a mixture thereof.

[0084] Preferably, the particles according to the invention are incorporated into a premix as defined above.

[0085] Therefore, the invention also relates to a premix comprising at least particles according to the invention as described above. Said premix may comprise, in mass percentages expressed relative to the mass of said premix, between 0.2% and 10%, preferably between 0.1% and 3%, of said particles. For example, if the active agent is an enzyme, the enzymatic activity in the premix may be approximately 500 U / g.

[0086] The premix can be incorporated into an animal feed.

[0087] This is why the invention also relates to an animal feed which comprises at least the premix according to the invention.

[0088] Said animal feed may comprise, in mass percentages expressed relative to the mass of said animal feed, between 0.5% and 6%, preferably between 2% and 5%, of said premix according to the invention.

[0089] In another embodiment of the invention, the particles according to the invention are directly incorporated into an animal feed.

[0090] When the particles according to the invention are incorporated into an animal feed, the quantity of said particles per tonne of said animal feed may be up to approximately 5000 g, and preferably between approximately 25 g and approximately 400 g. Indeed, the quantity of each of the constituents of the animal feed is generally between 0.001 g and 400 g per tonne of said animal feed.

[0091] Thus, the amount of active agent per tonne of said animal feed may be between approximately 0.0001 g and approximately 80 g.

[0092] The invention also relates to a method for manufacturing particles according to the invention which is characterized in that it comprises at least the following steps: a) a mixture is prepared which comprises at least the active agent and the cohesion agent, and optionally at least one solvent so that said mixture can be subjected to granulation by spraying, b) the mixture thus obtained in step a) is subjected to spray granulation so as to obtain said particles according to the invention.

[0093] Advantageously, the manufacturing method further comprises at least one step c) of coating the particles obtained at the end of step b) so as to cover them with at least one protective layer.

[0094] In step a), the mixture may optionally comprise at least one additional compound as described above in the description of the particles according to the invention.

[0095] The active agent, the cohesion agent, any additional compounds, as well as the constituents of the at least one protective layer have been described above in the description of the particles according to the invention.

[0096] In step a), the mixture may optionally comprise at least one solvent.

[0097] Indeed, at least one solvent can be added to the other constituents of the mixture of step a) in order to be able to subject said mixture to spray granulation if this spray granulation is not feasible with only these other constituents of said mixture.

[0098] It is perfectly within the reach of a person skilled in the art to decide whether he must add at least one solvent to the mixture of step a) which comprises at least the active agent and the cohesion agent in order to then be able to carry out step b) of spray granulation. It is also within the reach of a person skilled in the art to determine the quantity of solvent to be added to the mixture in step a). Indeed, the spray granulation technique is perfectly mastered by a person skilled in the art.

[0099] The solvent may be water, preferably demineralized water.

[0100] Preferably, the mixture obtained at the end of step a) has a viscosity at 25°C of between 10 mPa.s (cP) and 50 mPa.s (cP), preferably between 12 mPa.s (cP) and 48 mPa.s (cP). This viscosity of the mixture is particularly suitable for subjecting it to spray granulation during which said mixture will be sprayed through at least one nozzle. In other words, advantageously, the viscosity of the mixture of step a) is compatible with the use of the nozzle through which it is sprayed.

[0101] Advantageously, the mixture is stable over time. This means that it does not exhibit any phase separation or mass gain over time, knowing that spray granulation can last a few hours.

[0102] The choice of all the constituents of the mixture of step a) and their respective quantities is perfectly within the reach of those skilled in the art who will be able to determine them without difficulty depending on the final composition of the particles according to the invention that he wishes to obtain.

[0103] In step a), advantageously, the cohesion agent, as well as any additional compounds, are gently incorporated into the active agent.

[0104] Step a) is preferably carried out at room temperature.

[0105] At the end of step a), the mixture is preferably homogeneous.

[0106] Preferably, in step a), said mixture is prepared with a paddle stirrer, for example at a speed of between 300 rpm and 700 rpm, so as to make the mixture homogeneous.

[0107] In one embodiment of the invention, when carrying out step a), the active agent may be in the form of a concentrate of active agents suspended in an aqueous solution.

[0108] Said concentrate of active agents may advantageously have a dry extract of between 10% and 50%, preferably between 25% and 35%.

[0109] It may be an enzyme concentrate. For example, mention may be made of the enzyme concentrate marketed by the company Danisco under the trade name “Rovabio Advance SD”.

[0110] The cohesion agent may also have a dry extract, for example of 98%, and therefore include a little water.

[0111] In other words, in these embodiments of the invention, in the mixture obtained at the end of step a), a part of the solvent (for example water) which may need to be added so that said mixture is suitable for carrying out granulation by spraying is provided by the concentrate of active agents and / or the cohesion agent.

[0112] The implementation of spray granulation in step b) is perfectly within the reach of those skilled in the art. Step b) of spray granulation can advantageously be carried out with a device as shown schematically in [Fig.l] and described below.

[0113] During step b) of spray granulation, the solvent (for example water, in particular demineralized water) contained in the mixture obtained at the end of step a) is evaporated, preferably until said mixture has a mass content of said solvent which is less than 4%.

[0114] Advantageously, the manufacturing method further comprises at least one step c) of coating the particles obtained at the end of step b) so as to cover them with at least one protective layer.

[0115] The implementation of coating step c) is perfectly within the reach of those skilled in the art. It can, for example, be carried out in a fluidized air bed.

[0116] The invention will be better understood with the help of: - the experimental part carried out with particles according to the invention and comparative particles, - the detailed description which is set out below with reference to the appended drawing representing, by way of non-limiting example, an embodiment of the device used to carry out step b) of granulation by spraying of the process for manufacturing particles according to the invention.

[0117] [Fig. 1] [Fig. 1] is a schematic representation of a device for implementing implements step b) of the process for manufacturing the particles according to the invention.

[0118] EXPERIMENTAL PART:

[0119] Experiments were carried out on particles according to the invention and comparative particles of the state of the art.

[0120] I - Preparation of particles according to the invention without protective layer:

[0121] 4 types of particles according to the invention were prepared according to steps a) to b) of the manufacturing method according to the invention.

[0122] These 4 types of particles included enzymes as the active agent. Specifically, they were endo-l,4-beta-xylanase and beta-glucanase.

[0123] During step a) of the particle manufacturing process, these enzymes were added to the mixture in the form of an enzyme concentrate. This was the enzyme concentrate marketed by the company Danisco under the trade name “Rovabio Advance SD”. It had a dry extract of 28% by mass.

[0124] The cohesion agent ensuring the cohesion of the grains between them was maltodextrin which had a dry extract of 98%.

[0125] During step a), the solvent was demineralized water.

[0126] Step a) of the manufacturing process was carried out at room temperature, using a paddle stirrer set at a speed of approximately 600 rpm, preparing a homogeneous mixture comprising: - the enzyme concentrate described above; - a powder of the aforementioned maltodextrin; - water in such a quantity that the said mixture had a dry extract of 30%.

[0127] The 4 types of particles according to the invention differed from each other only by the quantities of enzymatic concentrate, maltodextrin, as well as demineralized water which were used for the preparation of the mixture of step a) of the process for manufacturing said particles.

[0128] Table 1 below details for each type of particle these quantities of enzyme concentrate, maltodextrin and demineralized water: - 1st type of particles, hereinafter referred to as INV1 particles, - 2nd type of particles, hereinafter referred to as INV2 particles, - 3rd type of particles, hereinafter referred to as INV3 particles, - 4th type of particles, hereinafter referred to as INV4 particles.

[0129] [Tables 1] Constituent: W1 iNV2 1W 3 ;NV4 enzymatic concentrate 2120 kg 2924 kg 2243 kg 1411 kg maltodextrin 1428 kg 3592 kg 3903 kg 4133 kg demineralized water 3118 kg 8151 kg 8854 kg 9457 kg

[0130] From these values ​​of the masses of enzyme concentrate and maltodextrin detailed in Table 1 above, and knowing that the enzyme concentrate had a dry extract of 28% and the maltodextrin a dry extract of 98%, it is possible to calculate the mass contents of dry extract of enzyme concentrate and dry extract of maltodextrin expressed in relation to the sum of the masses of the dry extracts of enzyme concentrate and maltodextrin which were used for each of the 4 types of particles.

[0131] Table 2 below details these mass contents of dry extract of enzyme concentrate and maltodextrin for each type of particle INV1 to INV4.

[0132] [Tables2] Mass content of enzyme concentrate Mass content of maltodextrin (%) 30 70 'SQ 81 14 86 9 91

[0133] Table 2 shows that particles INV1 to INV 4 had varying mass contents of dry extract of enzyme concentrate and maltodextrin.

[0134] Then, the mixture thus obtained at the end of step a) was subjected to granulation by spraying in an apparatus 1 which is shown schematically in [Fig.l].

[0135] This apparatus 1 comprises: - a container 2 configured to contain the mixture 3 obtained at the end of step a) of the manufacturing process and as described above, - a peristaltic pump 5 with a flow rate between 945 and 1000 g / hour, - a 1st means of conveying 4 said mixture 3 from the container 2 to the pump 5, - a nozzle 8 with a needle with an internal opening diameter of 1.2 mm and an atomization pressure of 2.5 bars, said nozzle 8 was arranged substantially vertically on the bottom wall 18 of a tank 7 within which the spray granulation was carried out, - a 2nd means of conveying 6 the mixture 3 from the pump 5 to the nozzle 8, - a 1st air injector (called “process air” because it is used for the implementation of spray granulation) 12 in tank 7, - a 1st air shutter 9a with an opening of +15° to the left and a 2nd air shutter 9b with an opening of +30° to the right, - a 3rd means 19 of conveying the particles formed in the tank 7 to a separator 11, - the separator 11 comprising an elongated portion 20 in the shape of a “zig-zag” configured to implement continuous pneumatic separation, as well as a 1st end 20a and a 2nd end 20b, said 2nd end 20b being in fluid connection on the one hand with said 3rd means 19 for conveying the formed particles in the tank 7 towards the separator 11 and on the other hand with a 4th means of conveying 10 the particles which are too fine from the separator 11 to the tank 7, - a 2nd air injector 13 (called “pressure air” to allow the separation of particles within the separator 11 by continuous pneumatic separation) at the level of the 1st end 20a of the separator 11, - a container 14 for receiving the particles 15 obtained at the end of step b) of granulation by spraying which are sufficiently dense to pass through the separator 11, - an air outlet 21 to avoid overpressure in the tank 7, ensure the renewal of the aforementioned process air and thus evacuate the humid air, - a filtration system 22 configured to retain inside the tank 7 the particles being formed by spray granulation, and so that they do not exit the tank 7 through the aforementioned air outlet 21.

[0136] In [Fig. 1], reference 16 designates the circulation movement of air and particles during manufacture by spray granulation in the tank 7. Reference 23 designates the mixture 3 which is sprayed by the nozzle 8. Reference 17 designates the air flow which is injected into the tank 7 by the Venturi effect.

[0137] Apparatus 1 operated in continuous mode.

[0138] The air temperature at the 1st air injector 12 was between 56°C and 60°C. The air flow rate injected by the 1st air injector 12 into the tank 7 was between 100 and 150 m3 / hour. This was desaturated dry air.

[0139] The 1st and 2nd air flaps 9a, 9b made it possible to obtain a homogeneous air flow in the tank 7.

[0140] The air temperature at the air outlet 21 was between 32°C and 39°C.

[0141] In addition, compressed air at a pressure of 0.8 bar was injected by the 2nd air injector 13 at the separator 11 so that the particles with a median diameter of at least 500 μm passed through the whole of said separator 11, then fell by gravity to be collected in the container 14 and the other particles, which were too small, were pushed upwards and rose in the portion 20 in a “zig-zag” fashion to the 2nd end 20b of the separator 11 to be re-routed into the tank 7 via the 4th conveying means 10 which consisted of a flexible plastic tube. The particles which were too small were again subjected to spray granulation in the tank 7 until they reached a median diameter of at least 500 μm.

[0142] The container 14 made it possible to collect the particles 15 according to the invention which had passed through the entire separator 11.

[0143] State-of-the-art comparative particles

[0144] The comparative particles of the state of the art were particles marketed by the company Adisseo France SAS under the trade name “Rovabio ©Advance T-Flex”.

[0145] They contained in mass percentages expressed in relation to the mass of said comparative particles: - 15% of the same enzymatic concentrate as for the particles according to the invention (namely the Rovabio ® Advance SD enzymatic concentrate), - 20% calcium carbonate, - 15% maltodextrin, and - 50% stearic acid.

[0146] The comparative particles comprised a calcium carbonate core with a diameter of about 285 μm covered by a first layer with a thickness of about 65 μm and which contained a mixture of maltodextrin and the enzyme concentrate. This first layer was itself covered by a protective layer with a thickness of about 55 μm consisting of stearic acid. Thus, in the comparative particles, the enzymes of the enzyme concentrate were protected by the protective layer consisting of stearic acid.

[0147] Measurements carried out on the particles according to the invention and the comparative particles

[0148] The size and size distribution of the particles according to the invention and the comparative particles were measured using a laser granulometer (MasterSizer S model marketed by Malvern Instruments Ltd) in solid state. The result was obtained after counting 5000 particles.

[0149] Table 3 below details for the comparative particles and the 4 types of particles according to the invention (namely INV1, INV2, INV3 and INV4): D(v,0,1), D(v,0,5), D(v,0,9) expressed in pm and the span as defined above.

[0150] [Tables3] Particle type Granulometry DLAl) pm D(vA5| pm Dv.QA span COMPARATIVES 416 632 1070 0.95 iNVl '365 435 513 0.34 JNV2 339 447 585 0.57 ÎNV3 '359 462 596 0.51 iNVT 345 440 566 0.51

[0151] In view of the results detailed in Table 3 above, and in particular in view of the span values, we note that: - the 4 types of particles according to the invention have lower span values ​​than the comparative particles, and this particularly for the INV1, INV3 and INV4 particles: the particles according to the invention thus have a more homogeneous size distribution than the comparative particles, - the 4 types of particles according to the invention have homogeneous size distributions. The INV3 and INV4 particles are of very homogeneous size (span of 0.51) and the INV1 particles have a size distribution showing excellent homogeneity with a span value of only 0.34.

[0152] II - Preparation of particles according to the invention with protective layer:

[0153] Then, the 4 types of particles according to the invention were subjected to an additional coating step, namely optional step c) of the manufacturing process according to the invention.

[0154] Two coating compositions were used.

[0155] The 1st coating composition was obtained by solubilizing at room temperature 73 g of an 8% dry extract ethylcellulose solution in 847 g of ethanol using a paddle stirrer. The dry matter mass content of the 1st coating composition thus obtained was 8%.

[0156] The 2nd coating composition was obtained by melting 133 g of 92% reagent grade stearic acid using a hot plate.

[0157] The coating step with the 1st or 2nd coating composition was carried out on the particles according to the invention in a fluidized air bed.

[0158] More precisely, 3 different coatings described below were carried out on the INV1 particles in order to obtain 3 new types of coated particles according to the invention INV1A, INV1B and INV1C: - the INV1A coated particles were obtained from the INV1 particles which were coated with a single protective layer consisting of the 2nd coating composition in quantities such that the mass percentages expressed relative to the total mass of these INV1A coated particles were: 75% of the INV1 particles and 25% of this protective layer; - the INV1B coated particles were obtained from the INV1 particles which were coated with a single protective layer consisting of the first coating composition in quantities such that the mass percentages expressed in relation to the total mass of these INV1B coated particles were: 90% of the INV1 particles and 10% of this protective layer; - the coated particles INV1C were obtained from the particles INV1 which were coated with a 1st protective layer consisting of the 1st coating composition, then this 1st protective layer was itself covered with a 2nd protective layer of the 2nd coating composition in quantities such that the mass percentages expressed in relation to the total mass of these particles INV1C coated particles were: 77% of INV1 particles, 8% of the 1st protective layer and 15% of the 2nd protective layer.

[0159] 2 different coatings described below were carried out on the particles INV2 in order to obtain 2 new types of coated particles according to the invention INV2A and INV2B: - the INV2A coated particles were obtained from the INV2 particles which were coated with a single protective layer consisting of the 2nd coating composition in quantities such that the mass percentages expressed in relation to the total mass of these INV2A coated particles were: 75% of the INV2 particles and 25% of this protective layer; - the INV2B coated particles were obtained from the INV2 particles which were coated with a single protective layer consisting of the 2nd coating composition in quantities such that the mass percentages expressed in relation to the total mass of these INV2B coated particles were: 50% of the INV2 particles and 50% of this protective layer.

[0160] 2 different coatings described below were carried out on the particles INV3 in order to obtain 2 new types of coated particles according to the invention INV3A and INV3B: - the INV3A coated particles were obtained from the INV3 particles which were coated with a single protective layer consisting of the first coating composition in quantities such that the mass percentages expressed relative to the total mass of these INV3A coated particles were: 90% of the INV3 particles and 10% of this protective layer; - the INV3B coated particles were obtained from the INV3 particles which were coated with a single protective layer consisting of the 2nd coating composition in quantities such that the mass percentages expressed in relation to the total mass of these INV3B coated particles were: 75% of the INV3 particles and 25% of this protective layer;

[0161] 1 coating described below was carried out on the INV4 particles in order to obtain a new type of coated particles according to the INV4A invention: - the INV4A coated particles were obtained from the INV4 particles which were coated with a single protective layer consisting of the first coating composition in quantities such that the mass percentages expressed in relation to the total mass of these INV4A coated particles were: 95% of the INV4 particles and 5% of this protective layer;

[0162] Thus, particles INV1 to INV4 constituted the “cores” of these other particles according to the invention which included one or two protective layers and whose particle size characteristics are detailed in Table 4.

[0163] More precisely, table 4 below details for the particles coated according to the invention INV1A to INV1C, INV2A, INV2B, INV3A, INV3B and INV4A: D(v,0,1), D(v,0,5), D(v,0,9) and the span.

[0164] [Tables4] Core Coated particles Granulometry pm D[v,.0.5' pm pm Span INV1 IN VIA 448 528 631 0.34 INV1B 364 536 812 0.83 iNVlC 514 771 1190 0.87 IW2 INV2A 409 515 550 0.45 INV23 481 589 722 0.41 IW3 INV3A 367 464 581 0.46 INV3B 468 571 704 0.41 INV4 INV4A 360 438 537 0.40

[0165] In view of the results detailed in Table 4 above, and in particular in view of the span values, we note that: - all these particles coated according to the invention have a span value of less than 1: they thus have a homogeneous size distribution, - the particles according to the invention INV1A, INV2B, INV3B and INV4A which comprise protective layers which are different from each other have very low span values: their size distribution is perfectly homogeneous. This demonstrates that the particles according to the invention can have perfectly homogeneous size distributions, and this with very varied protective layer compositions.

[0166] Tables 5 and 6 below detail for particles INV1 to INV4, the coated particles INV1A to INV1C, INV2A, INV2B, INV3A, INV3B and INV4A, as well as the comparative particles: - the enzyme content expressed in mg / g; - endo-l,4-beta-xylanase activity expressed in U / g; - the relative humidity rate measured on 2 g of particles at 105°C; - water activity; - the packed density dlO (g / L); - roundness.

[0167] Endo-l,4-beta-xylanase activity was measured according to a protocol based on the enzymatic hydrolysis of the xylosidic bridges of a soluble solution of wheat arabinoxylan and [3-1,4-xylan polysaccharide substituted with arabinose. Indeed, endo-1,4-beta-xylanase hydrolyzes the xylosidic bridges of xylan. The enzymatic activity is thus proportional to the reduction in the viscosity of this soluble solution in the presence of the enzyme to be measured.

[0168] The relative humidity level was determined using an infrared balance marketed under the trade name HS 153 by Mettler Toledo. Approximately 2 g of the sample of tested particles were placed in a previously tared aluminum cup, then once the balance was closed, it heated the sample to 105°C to remove as much moisture as possible from it in order to determine this relative humidity level.

[0169] Water activity was determined at room temperature using the Aw-meter device marketed under the trade name Labswift-aw by the company Novasina.

[0170] The tapped density dlO was determined as follows: a sample of test particles was placed in a plastic graduated cylinder, and then the density dO was calculated by dividing the mass by the volume multiplied by 100. The cylinder was then placed in an automated tapped density analyzer marketed under the trade name Autotap by the company Anto Paar, which tapped the cylinder 10 times. The density dlO was obtained by dividing the mass by the volume of the tapped sample, then multiplying by 100.

[0171] Microscopic observations were carried out using a microscope from Techsystems equipped with an optical camera from Navitar. The particle sample was placed on a slide. Archimed software enabled images to be taken at different magnifications. The images obtained by microscopy were used in ImageJ software to obtain the roundness of the tested particles. This parameter was determined by drawing the outline of the particles.

[0172] Roundness was determined according to the mathematical formula (2) detailed above.

[0173] [Tables5] Enzyme content Fendo 1,4-beta-xylanase activity Relative humidity (¾ Water activity Packed density dlO ]g / L) Sounder 300 123 300 3.99 0.58' 792 0.900 ±0.057 20G 72990 3.51 0.57 7592 0.832 ±0.081 INV3 150 58563 3.65% 0.51 733 0.809 ±0.073 100 45449 3.58% 0.52 759 0.904 ±0.043

[0174] In view of the results detailed in Table 5 above, we note that: - these particles according to the invention have excellent sphericity because their roundness is always greater than 0.8; - the activity of endo-l,4-beta-xylanase) increases with increasing enzyme content; - the relative humidity is very low (between 3.5% and 4%) and of a completely acceptable value to avoid the degradation of enzymes; - water activity is also very low and of a value that is also quite acceptable to avoid the degradation of enzymes. Enzyme content Endo-1,4-beta xyianase activity (0 / g) Relative humidity Water activity Packed density 610 Roundness COMPARATIVES 150 48 280 0.22 943 0.724 ±0.077 iNVIA 220 90 318 0.79 0.55 667 0.843 ±0.126 iNVIB 268 110 118 0.58 0.58 .Not determined 0.890 ±0.078 BW1C 223 91744 0.95 0.48 625 0.901 ±0.036 INV2A 150 52 247 0.85% 0.21 653 0.884 ±0.075 1W2B 100 38254 0.79% 0.19 630 0.911 ±0.022 ^V3A 135 50 178 1.2.5% 0.28 781. 0.860 ±0.052 W3B 113 39 409 0.91% 0.34 667 0.875 ±0.059 IW4Â 95 43971 1.85% 0.31 719 0.918 ±0.041

[0176] In view of the results detailed in Table 6 above, we note that:

[0177] - these particles coated according to the invention have excellent sphericity because their roundness is always greater than a value of 0.8. They have better sphericity than comparative particles; - the relative humidity of these particles according to the invention is lower than that of the comparative particles.

[0178] Then, the thermostability to pelletization of particles INV1 to INV4, INV1A to INV1C, INV2A, INV2B, INV3A, INV3B, INV4A and comparatives were tested.

[0179] All these particles were introduced at a rate of 200 g / tonne into a poultry premix, then extruded in a press at 90°C to obtain granules.

[0180] The pelletizing conditions were as follows: - press output: 44 to 46 kg / hour; - temperature at the conditioner outlet: 90°C; - die temperature: 85°C; - steam flow rate: 2.6 kg / hour; - steam pressure: 1.6 bars; - die size: 4 mm x 24 mm.

[0181] The rate of maintenance of enzymatic activity is the ratio of the activity of the enzymes contained in the granules to the activity of said enzymes before the incorporation of the particles into the livestock feed. It is determined according to the following mathematical formula (4):

[0182] [Math.4] Enzyme activity maintenance rate = B (|) / 1000 (4)

[0183] in which: - A is the activity of the enzymes contained in the animal feed pellets (i.e. after pelletization) expressed in U / kg, - B is the activity of the enzymes before incorporation of the particles into the animal feed expressed in U / g, - C is the quantity of particles incorporated into the food expressed in g / T.

[0184] Table 7 below details the rates of maintenance of enzymatic activity obtained for the particles according to the invention INV1 to INV4 and the comparative particles.

[0185] [Tables?] Maintenance rate of enzymatic activity mvi iNV2 ms INV4 COMPARATIVES 58 51 70 52 between 47 and 67

[0186] In view of the results detailed in Table 7 above, it is noted that all these particles according to the invention have a rate of maintenance of the enzymatic activity comparable to those of the comparative particles for which the rate of maintenance of the enzymatic activity can be between 47% and 67%. Thus, the particles according to the invention, although devoid of any protective layer, protect the enzymes with an efficiency almost equivalent to that of the comparative particles of the state of the art which comprise two protective layers.

[0187] Table 8 below details the rates of maintenance of enzymatic activity obtained for the particles coated according to the invention INV1A to INV1C, INV2A, INV2B, INV3A, INV3B and INV4A and the comparative particles.

[0188] [Tables8] Enzyme activity retention rate (%) 1W1A iNVIB INV1C INV2A INV2B iNV3A ÎNV3B INV4A COMPARATIVES 84 86 92 71 73 78 92 83: between 47 and 67

[0189] In view of the results detailed in Table 8 above, we note that: - these particles coated according to the invention all have an excellent rate of maintenance of enzymatic activity which is much higher than that of the comparative particles; - INV1C and INV3B particles show the best rate of maintenance of enzymatic activity.

[0190] By comparing the results in Table 7 with those in Table 8, it can be seen that the addition of one or more protective layers improves the rate of maintenance of the enzymatic activity of the particles according to the invention.

Claims

Claims

1. Particle (15), characterized in that it comprises an agglomeration of grains which are cohesive with each other and which is of substantially spherical shape, each grain being made up of a mixture which comprises at least: - an active agent and - an agent for cohesion of said grains with each other.

2. Particle (15) according to claim 1, characterized in that it has a median diameter of between 300 pm and 800 pm, preferably between 400 pm and 700 pm, more preferably between 500 pm and 600 pm.

3. Particle (15) according to claim 1 or 2, characterized in that the active agent is chosen from the group consisting of proteins, antimicrobial agents, amino acids and vitamins, taken alone or as a mixture thereof.

4. Particle (15) according to claim 3, characterized in that the active agent is at least one protein chosen from enzymes, peptides and polypeptides.

5. Particle (15) according to any one of claims 1 to 4, characterized in that it comprises, in mass percentages expressed relative to the total mass of said particle, between 10% and 90%, preferably between 15% and 70%, more preferably between 15% and 30%, of active agent.

6. Particle (15) according to any one of claims 1 to 5, characterized in that the cohesion agent is chosen from the group consisting of maltodextrin, lactose, maltose and starch, taken alone or as a mixture thereof.

7. Particle (15) according to any one of claims 1 to 6, characterized in that it comprises, in mass percentages expressed relative to the total mass of said particle, between 10% and 90%, preferably between 15% and 85%, more preferably between 50% and 80%, of said cohesion agent.

8. Particle according to any one of claims 1 to 7, characterized in that it further comprises at least one protective layer which surrounds said agglomeration of cohesive grains between them.

9. Particle according to claim 8, characterized in that the protective layer comprises at least one constituent chosen from the group consisting of alginates, stearic acid, ethylcellulose, hydroxypropyl methyl cellulose, methyl cellulose, carboxymethyl cellulose and soybean, corn, cottonseed or linseed oil, taken alone or in a mixture thereof.

10. Premix, characterized in that it comprises at least particles according to any one of claims 1 to 9.

11. Animal feed, characterized in that it comprises at least the premix according to claim 10.

12. Method for manufacturing particles (15) according to any one of claims 1 to 7, characterized in that it comprises at least the following steps: a) a mixture is prepared which comprises at least the active agent and the cohesion agent, and optionally at least one solvent so that said mixture can be subjected to spray granulation, b) the mixture thus obtained in step a) is subjected to spray granulation so as to obtain said particles.

13. Manufacturing method according to claim 12 of particles according to claims 8 or 9, characterized in that it further comprises at least one step c) of coating the particles (15) obtained at the end of step b) so as to cover them with at least one protective layer.

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