Compositions containing chitin and digestible proteins

Abstract

The present invention relates to compositions containing chitin and digestible proteins. The present invention relates to a composition comprising at least 67% by weight of crude protein, at least 5% by weight of chitin, the weight percentages relating to the total weight of the composition, and 85% by weight relative to the total weight of crude protein being digestible protein. The invention also relates to a method for preparing the composition and to the use thereof, in particular in human and animal nutrition.

Description

[0001] This application is a divisional application of Chinese Patent Application 201580077227.9, filed on December 30, 2015, entitled "Composition containing chitosan and digestible protein". Technical Field

[0002] This invention relates to compositions comprising proteins and chitosans. It also relates to methods for preparing the compositions and their use in human or animal nutrition, and more particularly in fish feed. Background Technology

[0003] Aquaculture is now one of the most dynamic sectors in the food industry. High demand for fish has led to a significant increase in the expected price of feed used in fish farming.

[0004] One of the most commonly used products in fish feed is fishmeal. In fact, fishmeal is one of the main sources of protein in aquaculture feed. It is a meal rich in easily digestible animal protein (rich in amino acids such as lysine and methionine). The increasing demand accompanying limited supply has led to a significant increase in its price, posing a risk to the sustainable growth of aquaculture. Therefore, there is a high demand for high-quality, and preferably renewable, alternative sources of protein for aquaculture feed.

[0005] Insect meal provides a natural alternative protein source, and the possibility of mass production with a minimal ecological footprint. In particular, certain insects such as mealworms ( Tenebrio molitor It has the advantage of being suitable for intensive mass production.

[0006] However, test results for replacing fishmeal with various insect meal yielded mixed results. Where substitution was proven possible, it generally did not exceed 50%; exceeding this level resulted in adverse effects on fish growth. Summary of the Invention

[0007] The inventors’ work has enabled the demonstration that certain compositions can be advantageously used as a substitute for fishmeal in aquaculture feeds.

[0008] The present invention therefore relates to a composition comprising at least 67% crude protein by weight, at least 5% chitosan by weight, the percentages by weight being given relative to the total weight of the composition, and 85% digestible protein by weight relative to the total weight of the crude protein.

[0009] It should be noted that, in the context of this application, and unless otherwise specified, the range of values ​​indicated should be understood as inclusive.

[0010] The quantification of crude protein is well known to those skilled in the art. For example, the Dumas method or the Kjeldahl method may be mentioned. Preferably, the Dumas method corresponding to the standard NF EN ISO 16634-1 (2008) is used.

[0011] When no date is specified for a regulation, standard, or directive throughout the application process, it refers to the regulation, standard, or directive that is in effect on the date of submission.

[0012] Preferably, the composition comprises 68% crude protein by weight, more preferably 70% crude protein by weight, the percentage by weight being given relative to the total weight of the composition.

[0013] "Digestible protein" means a protein that can be digested by means of pepsin digestibility. Quantification of digestible protein is preferably performed by the method described in Directive 72 / 199 / EC.

[0014] Preferably, the composition comprises 86% by weight, more preferably 88% digestible protein relative to the total weight of crude protein.

[0015] According to the present invention, "chitosan" means any type of chitosan derivative, that is, any type of polysaccharide derivative containing N-acetylglucosamine units and D-glucosamine units, especially chitosan-peptide copolymers (sometimes referred to as "chitosan-peptide complexes"). These copolymers can also be combined with pigments that are typically melanin-type.

[0016] Chitosan is the second most abundant synthetic polymer in the biological world after cellulose. In fact, chitosan is synthesized by numerous species: it forms the exoskeleton of crustaceans and insects, as well as the sidewalls that surround and protect fungi. More specifically, in insects, chitosan constitutes 3 to 60% of their exoskeleton.

[0017] The chitosan content was determined by extraction. This method can be the method described in Example 2, AOAC991.43, and is the preferred method for this determination.

[0018] Preferably, the composition comprises 5 to 16% chitosan by weight, more preferably 8 to 14% chitosan by weight, the percentage by weight being given relative to the total weight of the composition.

[0019] Prior art compositions containing both protein and chitosan are generally derived from insects and / or crustaceans. However, the high crude protein and digestible protein content in the compositions according to the invention can only be obtained through insect and / or crustacean processing methods that include a hydrolysis step. The hydrolysis step has the effect of reducing the chitosan content to an order of magnitude of 5% relative to the total weight of the composition, for example, less than 5% by weight.

[0020] Chitosan is currently generally considered an anti-nutritional factor because it is difficult to digest. This explains why, in agri-food applications, insect-based compositions are dechisandized, i.e., a chitosan removal step is performed. The inventors' work has also enabled the demonstration that, contrary to commonly held belief, chitosan has no effect on the growth of fish fed with compositions according to the invention containing a significant chitosan content (see Example 4 below). On the contrary, compositions according to the invention can advantageously replace not only some but all of the fishmeal in aquaculture feed. In fact, compositions according to the invention enable the improvement of the growth of animals fed with such compositions.

[0021] Furthermore, the introduction of the composition according to the invention also has certain advantages in the feed manufacturing process: reducing the loss of water-soluble vitamins during optional heat treatment, and reducing the energy required during optional extrusion steps.

[0022] Preferably, the composition according to the invention has a residual moisture content of between 2% and 15%, more preferably between 5% and 10%, and more preferably between 4% and 8%. This moisture content can be determined, for example, according to the method (103°C / 4 hours) of EC Regulation 152 / 2009 derived from 27-01-2009.

[0023] Advantageously, the composition according to the invention has an ash content of less than or equal to 4% by weight relative to the total weight of the composition, and even more advantageously less than or equal to 3.5%.

[0024] The ash content is a residue generated from the combustion of the composition according to the invention.

[0025] Methods for determining ash content are well known to those skilled in the art. Preferably, the ash content is determined according to the method specified in EC Regulation 152 / 2009 of 27-01-2009.

[0026] The fat content of the composition according to the invention is preferably between 5% and 20% by weight relative to the total weight of the composition, more preferably between 9% and 17%.

[0027] The methods used to determine fat content are well known to those skilled in the art. By way of example and in a preferred manner, the content will be determined according to the method of EC Regulation 152 / 2009.

[0028] As shown above, the compositions according to the present invention can be obtained from insects.

[0029] "Obtained from insects" more specifically means a composition obtained solely from insects and optionally water. The composition is produced by mechanical or thermal treatment, excluding any chemical treatment of the insects (other than water).

[0030] More specifically, the composition is insect powder. “Insect powder” means powder having a particle size acceptable to human or animal nutrition. “Particle size acceptable to human or animal nutrition” means a particle size between 100 μm and 1.5 mm, preferably between 300 μm and 1 mm, and more preferably between 500 μm and 800 μm.

[0031] Preferred insects for preparing this powder are, for example, beetles (Coleoptera), Diptera, Lepidoptera, Isoptera, Orthoptera, Hymenoptera, Blattoptera, Hemiptera, Heteroptera, Ephemeroptera, and Mecoptera), with beetles, Diptera, Orthoptera, Lepidoptera, or mixtures thereof being more preferred.

[0032] Preferably, the insect is selected from mealworms ( Tenebrio molitor Black soldier fly ( Hermetia illucens ), Galleria mellonella ( Galleria mellonella ), black fungus insect ( Alphitobius diaperinus ), barleyworm ( Zophobas morio ), Blattera fusca Red grain beetle ( Tribolium castaneum ), Red-brown weevil ( Rhynchophorus ferrugineus ), houseflies ( Musca domestica ), big-headed golden fly ( Chrysomya megacephala ), locusts Locusta migratoria Desert locusts Schistocerca gregaria ), house cricket ( Acheta domesticus ), Ailanthus altissima ( Samia ricini (or a mixture thereof, and even more preferably, mealworms.)

[0033] Advantageously, the composition according to the invention comprises 35 to 65% by weight of soluble protein relative to the total weight of crude protein, and at least 50% of the soluble protein has a size of less than or equal to 12,400 g / mol.

[0034] "Soluble protein" means those crude proteins that are soluble in an aqueous solution having a pH between 6 and 8, advantageously between 7.2 and 7.6.

[0035] Preferably, the aqueous solution is a buffer solution with a pH between 6 and 8, advantageously between 7.2 and 7.6. Preferably, the buffer solution is a NaCl phosphate buffer solution with a pH of 7.4 + / - 0.2.

[0036] The digestibility of proteins in humans and animals is highly dependent on protein size. In animal nutrition, protein size is typically reduced to facilitate digestion. This reduction in protein size is generally achieved through hydrolysis methods (e.g., enzymatic hydrolysis), which are particularly expensive to implement.

[0037] The compositions according to the invention, obtained through a process that does not involve hydrolysis, contain a large amount of soluble protein, the size of which is sufficiently reduced to facilitate digestion in animals. The compositions according to the invention also have the advantage of being able to be prepared at a lower cost.

[0038] Advantageously, the composition according to the invention comprises soluble protein at a weight of 30 to 60%, preferably 35 to 55%, relative to the total weight of crude protein.

[0039] Preferably, at least 60%, more preferably at least 70%, of the soluble protein has a size of less than or equal to 12,400 g / mol.

[0040] More specifically, soluble proteins have sizes ranging from 6,500 to 12,400 g / mol.

[0041] Advantageously, less than 10%, preferably less than 8%, more preferably less than 5%, and even more preferably 0% of soluble protein has a size greater than or equal to 66,000 g / mol.

[0042] This distribution of soluble proteins was confirmed in Example 6.

[0043] The present invention also discloses a method for preparing the composition according to the present invention.

[0044] A method for preparing the composition according to the invention includes the step of pressing insects.

[0045] The purpose of pressing is to remove the oil from the insects and thus obtain a cake with an oil (or fat) content of less than or equal to 20%, preferably less than or equal to 17%, by weight relative to the dry weight of the cake.

[0046] The pressing step is described more fully in step 2 of the preparation method detailed below.

[0047] Specifically, it is capable of hot or cold pressing. Preferably, a single-screw press is used.

[0048] More specifically, the preparation method according to the present invention includes the following steps: i) Kill insects, ii) Pressing insects to obtain press cake, and iii) Grinding and pressing the cake.

[0049] Insects can be killed by scalding or blanching, as described more fully in step 1 of the detailed method below.

[0050] Similarly, grinding is described more fully in step 4 of the detailed method.

[0051] Finally, the preparation method according to the present invention may further include the step of drying the press cake.

[0052] The drying step is advantageously carried out after the pressing step and before the grinding step.

[0053] The drying process is described in more detail in step 3 of the method described in detail.

[0054] Detailed method for preparing the composition according to the invention ● Step 1: Kill the insects The killing step 1 can advantageously be carried out by scalding or blanching. This step 1 enables the killing of insects by reducing the microbial load (reducing the risk of spoilage and health risks) and by inactivating the insect's internal enzymes, which can trigger autolysis and thus trigger rapid browning.

[0055] For scalding, insects, preferably larvae, are scalded with hot water for 2 to 20 minutes, preferably 5 to 15 minutes. Preferably, the water is at a temperature between 95 and 105°C, preferably 100°C.

[0056] The amount of water introduced during the scalding process is determined as follows: the ratio of water volume in mL to insect weight in g is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, and even more preferably about 1.

[0057] For blanching, insects, preferably larvae, are blanched using steam (steam nozzles or steam beds) at a temperature between 80 and 130°C, preferably between 90 and 120°C, more preferably between 95 and 105°C, and most preferably 98°C; or water at a temperature between 95 and 105°C, preferably 100°C (through a nozzle); or a mixture of water and steam at a temperature between 80 and 130°C, preferably between 90 and 120°C, more preferably between 95 and 105°C, and most preferably 98°C. The residence time in the blanching chamber is between 1 and 15 minutes, preferably between 3 and 7 minutes.

[0058] ● (Optional) Step: Grinding The insects are removed from the scalding tank or scalding chamber, then sieved (or drained) and placed in a grinder such as a knife grinder, which allows the insects to be crushed into granules.

[0059] To facilitate grinding, a certain amount of water may be added. This amount of water is similar to the amount of water introduced during the blanching in step 1: the ratio of water volume in mL to insect weight in g is preferably between 0.3 and 10, more preferably between 0.5 and 5, even more preferably between 0.7 and 3, and even more preferably about 1. It is also possible to retain the blanching water and / or the water generated during rinsing for this step.

[0060] Preferably, at the end of grinding, the insect particles are smaller than 1 cm (the maximum particle size observable under a microscope), and more preferably less than 0.5 cm. Preferably, the particle size is between 300 μm and 3 mm, and even more preferably between 500 μm and 1 mm. There is no need to excessively reduce the particle size, for example, to less than 250 μm.

[0061] ● Step 2: Pressing The killed insects from step 1 or the wet paste from an optional grinding step are then placed in a press according to a procedure that allows for pressing and separation of the juice containing both fat and protein fractions.

[0062] Preferably, the pressing step enables the production of a cake containing less than or equal to 20%, preferably less than or equal to 17%, and more preferably less than or equal to 15% oil by weight relative to the dry weight of the cake.

[0063] Similarly, the pressing step enables the production of a cake with a dry matter content between 30% and 60%, preferably between 40% and 55%, and more preferably between 45% and 55%.

[0064] Any pressing system can be used to perform the pressing step, such as a single-screw or twin-screw press (Angel-type twin-screw press), a filter press (Choquenet-type filter press), a flat-press extruder, etc. These systems are well known to those skilled in the art, who are able to determine the pressing conditions to obtain the aforementioned oil and / or water content.

[0065] In particular, hot or cold pressing is possible. Advantageously, pressing is carried out hot, which allows for increased oil removal from the press cake. In particular, hot pressing makes it possible to obtain a press cake containing less than or equal to 17% oil content, preferably less than or equal to 15% oil by weight relative to the dry weight of the press cake.

[0066] ● Step 3: Drying The press cake is then dried using standard techniques known to those skilled in the art. Drying can be direct or indirect (using a blister dryer, paddle dryer, tubular dryer, disc dryer, etc.) at a temperature between 60°C and 200°C for a duration of 15 minutes to 24 hours. For example, the press cake can be arranged and dried in ventilated / stirred air at a temperature between 80°C and 100°C, preferably 90°C, for a duration between 3 and 7 hours, preferably 5 hours.

[0067] The purpose of this drying step is to obtain a press cake with a moisture content of between 2% and 15%, preferably between 5% and 10%, and even more preferably between 4% and 8%.

[0068] ● Step 4: Final grinding The dried press cake is then placed in a grinder, such as a hammer mill, so that the press cake can be crushed into granules.

[0069] Advantageously, at the completion of this final grinding, the size of the insect particles is less than 0.5 cm (the maximum particle size observable under a microscope), preferably around 1 mm. More particularly, the particle size is contained between 300 μm and 1 mm, and even more preferably between 500 and 800 μm.

[0070] This series of four steps enables the preparation of the composition of the present invention, which contains high levels of crude protein and digestible protein, while maintaining a chitosan content of at least about 5% by weight relative to the total weight of the composition.

[0071] As shown above, the pressing step can be performed cold or hot.

[0072] As an example of a method for obtaining the composition according to the invention, cold pressing is involved: For example, mealworm larvae are introduced into a beaker containing 200 mL of pre-boiled water and killed by scalding in a water bath at 100°C. After 5 minutes, the beaker is removed from the water bath, the larvae are drained, and then mixed with 200 mL of water. The resulting liquid is then transferred to a twin-screw press. The resulting press cake is dried in an oven at 70°C for 24 hours and then ground to 250 μm.

[0073] As an example of a method for obtaining the composition according to the invention, hot pressing is involved: For example, mealworm larvae are introduced into a blanching chamber and blanched in steam at 100°C for 5 minutes. The blanched larvae are then introduced into a "drying" type press suitable for products containing water. The resulting press cake is dried in an oven at 90°C for 5 hours and then ground to 1 mm in a hammer mill.

[0074] Preferably, the method for preparing the composition according to the invention comprises the following steps: i) Kill insects, ii) Pressing insects to obtain press cake, iii) Dry the press cake, and iv) Grinding and pressing the cake.

[0075] According to a first embodiment of the method according to the invention, the pressing step is preceded by a grinding step of the insects.

[0076] The present invention therefore relates to a method for preparing a composition according to the invention, comprising the following steps: i) Kill insects, ii) Pressing insects to obtain press cake, iii) Dry the press cake, and iv) Grinding and pressing the cake, The pressing step is preceded by the grinding of insects.

[0077] The advantages of the step of grinding the insects before pressing are described more fully in Example 5.

[0078] According to a second embodiment of the method according to the invention, the step of pressing the insects is carried out hot.

[0079] The present invention therefore relates to a method for preparing a composition according to the invention, comprising the following steps: i) Kill insects, ii) Pressing insects to obtain press cake, iii) Dry the press cake, and iv) Grinding and pressing the cake, The pressing step is performed hot.

[0080] As shown above, hot pressing enables the production of a press cake containing an oil content of less than or equal to 17%, preferably less than or equal to 15%, by weight relative to the dry weight of the press cake.

[0081] According to a third embodiment of the method according to the invention, the step of grinding the press cake is carried out to a particle size of 300 μm to 1 mm, preferably between 500 and 800 μm.

[0082] The present invention therefore relates to a method for preparing a composition according to the invention, comprising the following steps: i) Kill insects, ii) Pressing insects to obtain press cake, iii) Dry the press cake, and iv) Grinding and pressing the cake, The grinding and pressing of the cake is carried out to a particle size between 300 μm and 1 mm.

[0083] More specifically, in this third embodiment of the method according to the invention, the step of pressing the insects can be performed hot. Alternatively, the pressing step can be preceded by a step of grinding the insects.

[0084] Finally, this invention relates to the use of the compositions according to the invention in human or animal nutrition.

[0085] Advantageously, the compositions according to the invention can be used in the feed of pets such as dogs, cats, birds, fish, reptiles and rodents.

[0086] More specifically, the compositions according to the invention can be used in aquaculture (fish, crustaceans, mollusks, shellfish), as feed for poultry (chickens, turkeys, birds such as quails, pheasants, bustards), pigs, ruminants (cattle, sheep, goats, horses) and mink.

[0087] Finally, the compositions according to the invention can be advantageously used as a substitute for protein powder.

[0088] Protein powder more specifically refers to fish meal, milk powder or lactose serum, concentrated soy protein (“CSP”), and meat meal such as poultry meal.

[0089] The substitution can be partial or complete.

[0090] Preferably, the composition according to the invention is used to partially or completely replace fishmeal, for example, 50% or 100% replacement.

[0091] Other features and advantages of the invention will become apparent from the following embodiments, which are given by way of example. Attached Figure Description

[0092] - Figure 1 It is a graph showing the changes in water temperature and dissolved oxygen levels in a tank in which trout fed with different doses of the composition according to the invention were raised. - Figure 2A-2B This includes showing the final weight of the trout ( Figure 2A ) and feed conversion ( Figure 2B Two graphs showing the effects of feeding the trout with different doses of the composition according to the invention. - Figure 3 The distribution of insect-derived lipids found in juice and press cake obtained by a method comprising a pressing step or a grinding step followed by pressing is shown. - Figure 4This is a graph representing the size exclusion chromatography analysis of the protein size of the composition according to the present invention. Detailed Implementation

[0093] Example 1: Method for preparing the composition according to the invention The compositions according to the invention are prepared from mealworm larvae. Upon receipt of the larvae, they can be stored in their rearing tanks at 4°C for 0 to 15 days without significant degradation, prior to being killed. The weight (age) of the larvae used is variable, and therefore their composition can vary, as shown in Table 1 below: Biomass (insects) mg 23 35 58 80 108 154 dry matter %* 34 34 34.2 37.9 39.6 39.5 Ash %* 1.59 1.52 1.6 1.75 1.67 1.43 crude protein %* 22.6 22.2 22 23.2 23.1 23.2 lipids %* 6.62 6.88 7.98 10.3 10.9 11.7 *% represents the dry weight relative to the wet weight of the larvae. Table 1 Biochemical composition of mealworm larvae based on their weight.

[0094] ● Step 1: Blanching the insects Live larvae (+4°C to +25°C) are conveyed to the scalding chamber on a perforated conveyor belt (1 mm) in a layer with a thickness between 2 and 10 cm. The insects are thus scalded in steam (steam nozzles or steam beds) at 98°C, or in water (nozzles) at 100°C, or in a mixed mode (water + steam). The residence time in the scalding chamber ranges from 1 to 15 minutes, ideally 5 minutes.

[0095] The temperature of the larvae after scalding is between 75°C and 98°C.

[0096] ● Step 2: Pressing Once blanched, the larvae are fed into the feed hopper of a continuous single-screw press. While inside the press, the larvae are maintained at a temperature above 70°C to increase oil removal yield. Oil removal is achieved by pressurizing the material within a cylindrical cage using an arrangement of screws and rings on a central shaft. The cage is lined with strips distributed throughout its sections and separated by spaces of varying thicknesses depending on the working area. This arrangement allows for the flow of the oil / fat fraction while restricting the passage of the so-called "dry" protein fraction (referred to as the "press cake"), thus involving pressurization.

[0097] The obtained crushing yield ranges from 48% to 55%. Y 饼 = (mass) 饼 / quality 汁 + quality 饼 ).

[0098] The resulting press cake contains 35-40% dry matter, 67% to 75% protein and 13% to 17% fat, the percentages by weight relative to the dry weight of the press cake.

[0099] ● Step 3: Drying The press cake is then laid out in a thin layer (about 2 cm) on a tray and dried in ventilated / stirred air at 90°C for 5 hours to obtain a press cake with a dry matter content of more than 92%.

[0100] This step ensures that the animal is free from any contamination that may occur after the kill.

[0101] The water activity (Aw) after drying was 0.35. Microbiological results showed the absence of Salmonella spp. (Method: IRIS Salmonella BKR 23 / 07-10 / 11) and Enterobacteriaceae values ​​less than 10 CFU / g (Method: NF ISO 2128-2, December 2004, 30°C and 37°C).

[0102] ● Step 4: Grinding Finally, a continuous hammer mill (6 reversible moving parts - 8 mm thickness) was used to grind the dried press cake, which consisted mainly of protein. The mill was fed by a hopper with flow rate control vanes (180 kg / h). A perforated grid of 0.8 mm was used for controlling the output particle size measurement. The engine speed was 3,000 rpm. (Electric, power absorption 4 kW (5.5 CV)) .

[0103] Example 2: Characterization of the composition according to the present invention Characterize the composition prepared in Example 1.

[0104] 1. analyze 1.1 Determination of moisture content Moisture content was determined according to the method (103°C / 4 hours) derived from the EU Regulation 27-01-2009.

[0105] 1.2 Determination of crude protein content Crude protein was determined according to a method known as Dumas and in accordance with standard NF EN ISO 16634-1 (2008).

[0106] 1.3 Determination of Chitosan Content The dietary fiber in insect powder is primarily composed of chitosan, which was therefore determined according to method AOAC 991.43. Consequently, the obtained value is slightly overestimated.

[0107] 1.4 Determination of Fat Content Fat was determined according to the method of EC Regulation 152 / 2009.

[0108] 1.5 Determination of Ash Content The crude ash content was determined according to the method specified in EC Regulation 152 / 2009 dated 27-01-2009.

[0109] 1.6 Determination of Phosphorus Content Phosphorus is determined by ICP (“Inductively Coupled Plasma”) and internal calibration.

[0110] 1.7 Energy Measurement The energy value is obtained using the coefficients of EU Regulation 1169 / 201.

[0111] 1.8 Determination of amino acid and fatty acid content The determination was performed by gas chromatography after the hydrolysis and derivatization of amino acids and fatty acids, respectively.

[0112] 1.9 Determination of pepsin digestibility Pepsin digestibility is measured by the method described in Directive 72 / 199 / EC.

[0113] 2. result The composition of the compositions according to the present invention is detailed in Table 2 below. Table 2 :composition In addition, a pepsin digestibility of 90+ / -2% was obtained.

[0114] Example 3: Alternative method for preparing the composition according to the invention 200 g of mealworm larvae were introduced into a beaker placed in a water bath at 100°C containing 200 mL of pre-boiled water. After 5 minutes, the beaker was removed from the water bath, the larvae were drained, and then mixed with 200 mL of water. The resulting liquid was then fed into a twin-screw press. The resulting press cake was dried in an oven at 70°C for 24 hours and then ground to 250 μm. The composition according to the invention was thus obtained.

[0115] Example 4: Introducing the composition according to the invention into fish feed In this embodiment, the effects of including the composition according to the invention in the feed on the growth, feed intake, feed conversion, body composition, and apparent digestibility of nutrients in rainbow trout were investigated.

[0116] 1. Materials and Methods 1.1. The composition according to the invention The composition used in this embodiment is the composition obtained according to Example 1 and described more fully in Example 2.

[0117] 1.2. Experimental Diet Fishmeal-based diets (CTRL) are formulated with convenient ingredients to meet the known nutritional requirements of rainbow trout juveniles. This CTRL diet consists of 25% fishmeal, 8% other marine-derived protein sources (squid meal and krill meal), and the remaining protein sources are soy protein concentrate, wheat gluten, and corn gluten. Based on this formulation, four test diets (Y5, Y7.5, Y15, and Y25) were formulated in which fishmeal was replaced with compositions according to the invention at contents of 20%, 30%, 60%, and 100%, respectively (see Table 3 below). *Dry matter percentage relative to the total weight of the composition **% of the total weight of dry matter, calculated on a dry weight basis** 1 Peruvian fishmeal LT70: 71% crude protein (CP), 11% crude fat (CF), EXALMAR, Peru; 2 Krill meal: 61% CP, 19% CF, Aker BioMarine Antarctic AS, Norway; 3 Super Prime (excluding offal): 82% CP, 3.5% CF, Sopropêche, France; 4 Soycomil P: 62% CP, 0.7% CF, ADM, Netherlands; 5 VITEN: 84.7% CP, 1.3% CF, ROQUETTE, France; 6 Corn gluten meal: 61% CP, 6% CF, COPAM, Portugal; 7PREMIX Lda, Portugal. Vitamins (IU or mg / kg diet): DL-α-tocopherol acetate, 100 mg; sodium methylnaphthoquinone bisulfite, 25 mg; retinyl acetate, 20,000 IU; DL-cholecalciferol, 2,000 IU; thiamine, 30 mg; riboflavin, 30 mg; pyridoxine, 20 mg; cyanocobalamin, 0.1 mg; niacin, 200 mg; folic acid, 15 mg; ascorbic acid, 1,000 mg; inositol, 500 mg; biotin, 3 mg; calcium pantothenate, 100 mg; choline chloride, 1,000 mg; betaine, 500 mg. Minerals (g or mg / kg): Cobalt carbonate, 0.65 mg; Copper sulfate, 9 mg; Ferric sulfate, 6 mg; Potassium iodide, 0.5 mg; Manganese oxide, 9.6 mg; Sodium selenite, 0.01 mg; Zinc sulfate, 7.5 mg; Sodium chloride, 400 mg; Calcium carbonate, 1.86 g; Excipient: Wheat; 8 Yttrium oxide is incorporated into only a portion of the feed used for digestibility measurements. Table 3 The formula and composition of the experimental diet.

[0118] The levels of squid and krill were kept constant across all diets to ensure high palatability. Minor adjustments were made to the tested diets to maintain isonitrogenous (crude protein, 48.5% DM), islipinous (22.7% DM), and isoenergetic (crude energy, 23.2 MJ / kg DM) conditions. The levels of methionine and monocalcium phosphate supplementation in the tested diets were adjusted to correspond to those found in CTRL feeds.

[0119] Using a 55.5 mm screw diameter and a temperature range of 119 to 123 °C, diets were produced on a pilot scale using a CLETRALT BC45 twin-screw extruder (particle sizes: 1.2 and 2.0 mm). During extrusion, all batches of the extruded feed were dried in a vibrating fluidized bed dryer (model DR100, TGC Extrusion, France). After cooling the pellets, oil was added by vacuum coating (model PG-10VCLAB, Dinnisen, Netherlands). Throughout the testing period, the experimental feeds were stored at ambient temperature, but in a cool, well-ventilated place. Samples representative of each diet were obtained for analysis (Tables 4-5). Content is indicated as a percentage by weight relative to the total weight of the granules before drying. Table 4 Overview of amino acids in the experimental diet. Content is indicated as a percentage by weight relative to the total weight of the granules before drying. Table 5 Synthesis of fatty acid profiles from experimental diets.

[0120] 1.3. Growth Performance Test Thirty-five rainbow trout with an initial body weight (IBW) of 5.01 ± 0.1 g were selected. Oncorhynchus mykiss Triple groups were fed one of five experimental diets for 90 days. The fish were grown in circular fiberglass tanks (volume: 250 L) supplied with a constant flow of fresh water at temperatures between 14.1 ± 0.3 °C and dissolved oxygen levels above 7.4 mg / L (see [reference]). Figure 1 The fish were subjected to summer conditions with natural photoperiod variations (May–July). They were manually fed to apparent satiety three times daily during the week (9:00, 14:00, and 18:00) and twice daily at the weekend (10:00 and 16:00) to minimize feed waste. Feed allocations were quantified throughout the study. Anesthetized fish were individually weighed at the start and end of the study, and groups were weighed on days 28 and 60. At the start, 15 fish from the same initial population were sampled and stored at -20°C for subsequent analysis of whole-body composition. After 90 days of experimental feeding, 6 fish from each tank were sampled for the same purpose.

[0121] 1.4. Apparent digestibility measurement At the end of the growth test, and after all relevant sampling, 12 fish (weight: 45 g) from each replicate tank were used to determine the apparent digestibility of dry matter, protein, lipids, energy, and phosphorus via an indirect method using an equivalent diet containing yttrium oxide (200 mg / kg) as an inert tracer. The fish were stored in cylindrical-conical tanks (volume: 60 L; flow rate: 3.7 L / min; dissolved oxygen level >6.4 mg / L) at a constant water temperature of 14°C. The fish were acclimatized to the rearing conditions and experimental diet for more than 10 days. The fish were then manually fed once daily (10 h00) to a slightly overfeed. After deep cleaning of the rearing tanks to remove all feed residue, feces were collected daily for the next 8 days using a continuous effluent filtration system (Choubert-INRA system). After daily collection, the feces were frozen at -20°C. The mixed feces from each group of fish were freeze-dried prior to analysis. Triples of each diet were tested.

[0122] The apparent digestibility coefficients (ADCs) of nutrients and feed energy in the experimental diet were calculated using the following formula: ADC (%) = 100 - [% concentration of Y2O3 in feed / % concentration of Y2O3 in feces × % energy or nutrient in feces / % energy or nutrient in feed].

[0123] 1.5. Analytical Methods Test components, diet, and freeze-dried feces were ground prior to analysis. Whole-body samples were chopped, mixed, and representative samples were freeze-dried and homogenized using a laboratory grinder prior to analysis. Chemical composition analysis of components, diet, feces, and whole fish was performed using the following procedures: dry matter dried at 105°C for 24 hours; ash obtained by combustion at 550°C for 12 hours; crude protein (N x 6.25) obtained by flash combustion followed by gas chromatography separation and thermal conductivity determination (LECO FP428); fat obtained by extraction with dichloromethane (Soxhlet); total phosphorus obtained using vanadomolybdic reagent according to ISO / DIS 6491 method; and crude energy in an adiabatic bomb calorimeter. Yttrium oxide in feed and feces was determined by ICP-AES.

[0124] For the analysis of total amino acids, the test components and test diet were hydrolyzed (in a nitrogen-washed glass flask, 6M HCl at 116°C for 22 hours), and then derivatized with AccQ (6-aminoquinolinyl-N-hydroxysuccinimide) fluorine reagent (Waters, USA) according to the AccQ-Tag method. Analysis was performed by high-performance liquid chromatography (HPLC) in a reverse-phase amino acid analysis system, using valine as an internal standard. Tryptophan was not determined because it was partially destroyed by acid hydrolysis. The obtained peaks were analyzed using EMPOWER software (Waters, USA). For the analysis of fatty acids, lipids were extracted according to the method of Folch et al. (1957), and the fatty acid composition of the fish fillets was subsequently determined by gas chromatography analysis of methyl esters according to the Lepage and Roy procedure (1986).

[0125] 1.6. Standards for assessing growth and nutrient use IBW (g): Initial body weight.

[0126] FBW (g): Final body weight.

[0127] Specific growth rate, SGR (% / day): (Ln FBW – Ln IBW) x 100 / day.

[0128] Feed conversion ratio, FCR: Total feed ration / weight gain.

[0129] Voluntary feed intake, VFI (% BW / day): (Total feed ration / (IBW + FBW) / 2 / day) x 100.

[0130] Protein efficiency ratio (PER): wet weight gain / crude protein intake.

[0131] Retention (% of intake): 100 x (FBW x final nutrient content of cadaver - IBW x initial nutrient content of cadaver) / nutrient intake.

[0132] 1.7. Statistical Analysis Data are presented as mean ± standard deviation over three replicates. Data underwent one-way ANOVA. Values ​​expressed as a percentage (%) were subjected to a square root arcsine transformation prior to ANOVA. Statistical significance was tested at a probability level of 0.05. All statistical tests were performed using IBM SPSS V21 software.

[0133] 2. result 2.1. Growth performance Data on growth performance, feed conversion, and protein efficiency of rainbow trout fed the experimental diet for 28, 60, and 90 days are reported in Tables 6-8 and 7-8. Figure 2A-2B No deaths occurred during the test. The value is the mean ± standard deviation (n = 3). Values ​​with different indices in the same row are significantly different (P<0.05). Table 6 Growth performance on day 28.

[0134] After 28 days of experimental feeding (Table 5), the fish increased more than threefold compared to their initial body weight. Feed intake was high (3.22–3.31% BWM / day) and was unaffected by escalating doses of the composition incorporated according to the invention (P>0.05). This observation suggests that the composition according to the invention has no negative effect on palatability and may even compensate for the overall loss of fishmeal without impairing feed intake. Growth rate ranged from 4.19 to 4.50% / day. Compared to the CTRL treatment, while the Y5 and Y7.5 diets did not affect FBW and SGR, the Y15 and Y25 diets resulted in a significant increase in FBW and SGR (P<0.05). The feed conversion ratio values ​​varied between 0.81 and 0.87. Compared to CTRL, the composition according to the invention, including 5% and 7.5% (Y5 and Y7.5% diets), did not affect FCR. However, including high levels of the composition according to the invention (Y15 and Y25 diets) resulted in a significant decrease in FCR (P<0.05). Protein efficiency ratio (PER) varied between 2.55 and 2.72. Fish fed the Y25 diet showed a significant increase in PER compared to fish fed the CTRL, Y5, and Y7.5 diets. The value is the mean ± standard deviation (n = 3). Values ​​with different indices in the same row are significantly different (P<0.05). Table 7 Growth performance at day 60.

[0135] After 60 days of experimental feeding (Table 6), fish receiving the most effective treatment showed an 8-fold increase in initial body weight. Growth rate ranged from 3.00 to 3.57% / day. All diets with the composition according to the invention showed a significant increase in SGR compared to CTRL treatment (P<0.05). FCR values ​​varied between 0.85 and 1.10, and compared to CTRL, including the composition according to the invention at all tested doses resulted in a significant decrease in FCR (P<0.05). Protein efficiency ratio (PER) varied between 2.01 and 2.56. The lowest PER values ​​were found in fish fed the CTRL diet, while improvements in PER were closely associated with escalating doses of the composition according to the invention. The value is the mean ± standard deviation (n = 3). Values ​​with different indices in the same row are significantly different (P<0.05). Table 8 Growth performance at day 90.

[0136] At the end of the 90-day experimental feeding test (Table 8), the fish that received the most effective treatment showed an 11-fold increase in initial body weight. Fish fed with an insect-rich diet showed a significant increase in final body weight compared to CTRL-treated fish (P<0.05). This increase was dose-related, with a moderate increase for the Y5 diet, moderate increases for Y7.5 and Y15, and the highest increase for Y25. Specific growth rate (SGR) varied between 2.39 and 2.67% / day, with the minimum observed in fish fed the CTRL diet, while fish fed with diets containing the composition according to the invention showed significantly higher SGR values ​​(p<0.05). The composition according to the invention resulted in a significant decrease in FCR regardless of the incorporation level (P<0.05). All diets containing insect meal resulted in a significant increase in PER values ​​compared to the CTRL treatment (P<0.05).

[0137] 2.2. Composition of the whole body Data on the overall composition of the trout at the end of the test are presented in Table 9. Feeding treatment had no effect on the whole fish's moisture, protein, lipid, ash, phosphorus, and energy levels (P>0.05). The percentage is a percentage by weight relative to the total weight of the fish. The value is the mean ± standard deviation (n = 3). Initial fish composition: moisture 75.0%; protein 14.1%; fat 8.7%; ash 2.2%; phosphorus 0.4%; energy 6.7 kJ / g. Table 9 The whole body composition of trout fed with various feed treatments.

[0138] 2.3. Nutrient Retention Nutrient and energy retention values ​​(expressed as a percentage of intake) are presented in Table 10. Compared with the CTRL treatment, fish fed a diet rich in the composition according to the invention showed a significant increase in protein and energy retention (P<0.05). Similarly, the Y7.5, Y15, and Y25 diets showed significantly higher P retention than the CTRL diet (P<0.05). Fat retention was not affected by diet (P>0.05). The value is the mean ± standard deviation (n = 3). Values ​​with different indices in the same row are significantly different (P<0.05). Table 10 Nutrient and energy retention in trout fed with various diets.

[0139] 2.4. Apparent digestibility The composition of feces collected from trout fed with various feed treatments is shown in Table 11. * Percentage by weight relative to the total weight of dry feces. The value is the mean ± standard deviation (n = 3). Table 11 Composition of feces from trout fed with various diets.

[0140] The apparent digestibility coefficients (ADC %) for different nutrients and energy are presented in Table 12. Increasing the dosage of the composition according to the invention had no significant effect on the apparent digestibility of dry matter, protein, fat, phosphorus and energy (P>0.05). The value is the mean ± standard deviation (n = 3). Table 12 Apparent digestibility of nutrients and energy in trout.

[0141] 3. in conclusion At the end of the 90-day experimental feeding period, overall growth performance was considered very satisfactory, and in the high range for juvenile rainbow trout, with SGR values ​​varying between 2.4 and 2.7% / day over the total duration of the test. In the most effective treatment, the fish showed an 11-fold increase in their initial body weight. The feed conversion ratio varied between 0.79 and 0.93 in the treatments, suggesting good nutritional adequacy of the diet and good feeding practices.

[0142] The experimental data generated in this embodiment enables confirmation that: ▪ The incorporation of the composition according to the invention at gradually increasing doses (5%, 7.5%, 15%, and 25%) with decreasing fishmeal content is increasingly associated with a significant increase in fish weight. ▪ All diets containing the compositions according to the present invention showed significant improvements in SGR, FCR and PER. ▪ The gradually increasing dosage of the composition according to the invention has no effect on the overall composition of the trout. ▪ The gradually increasing doses of the composition according to the invention incorporated had no effect on the apparent digestibility of dry matter, protein, lipids, phosphorus and energy in different experimental diets. Protein, phosphorus, and energy retention are enhanced in trout fed with a diet containing the composition according to the invention.

[0143] Generally speaking, the composition according to the invention used in this embodiment can effectively replace 100% fishmeal in the diet of rainbow trout juveniles, and has a positive effect on FCR and overall growth performance.

[0144] Example 5: Methods with or without grinding before pressing Using only pressing method 200 g of mealworm larvae were introduced into a beaker placed in a water bath at 100°C containing 200 mL of pre-boiled water. After 5 minutes, the beaker was removed from the water bath, the larvae were drained, and the beaker was then transferred to a twin-screw press. Pressed cake was thus obtained.

[0145] The method involves grinding followed by pressing. 200 g of mealworm larvae were introduced into a beaker placed in a water bath at 100°C containing 200 mL of pre-boiled water. After 5 minutes, the beaker was removed from the water bath, the larvae were drained, and then mixed with a predetermined volume of 200 mL of water. The resulting liquid was then fed into a twin-screw press. Pressed cake was thus obtained.

[0146] Measurement of lipid content Place 2 g of sample in a beaker, add 0.2 g of Na₂SO₄ and 15 mL of CHCl₃ / MeOH (2 / 1 v / v). Stir the mixture magnetically for 20 minutes, then filter the solution. Place the residue back into a beaker containing 10 mL of CHCl₃ / MeOH (2 / 1 v / v). Stir the mixture magnetically for 15 minutes, then filter the solution. Combine the solvent phases and evaporate to constant weight. Lipid content is determined as a percentage by weight relative to the initial weight (2 g) of the sample after extraction-evaporation.

[0147] in conclusion The significance of grinding upstream of pressing was studied. Figure 3 Therefore, it is clear that when pre-grinding is performed, the lipid distribution between the press cake and the press juice is much more effective, 12.9 vs. 87.1, and 42.7 vs. 57.3.

[0148] Example 6: Analysis of the size of soluble proteins in the composition according to the present invention.

[0149] 100 mg of the composition prepared in Example 1 was placed in 10 mL of NaCl phosphate buffer (pH 7.4, 0.137 mM). The sample was stirred for 1 minute (vortexed) and then centrifuged at 900 g for 1 minute. After centrifugation, the sample was filtered through a 0.45 μm membrane. Analysis of soluble protein size was performed using a size exclusion chromatography system with a Nucleogel GFC-300 column. NaCl phosphate buffer (pH 7.4, 137 mM) was used as the eluent. The flow rate was 1.0 mL / min. Detection was performed at 280 nm using a UV detector.

[0150] The analysis results are presented in Figure 4 The details are summarized in Table 13 below. Protein size (kg / mol) Relative abundance (%) 6.5 to 12.4 74.4 12.4 to 29 20.5 29 to 66 5.1 Table 13 Distribution of soluble protein size in the composition prepared in Example 1.

[0151] The results showed that approximately 74.4% of the soluble protein present in the composition according to the invention had a molar mass of less than 12,400 g / mol (or Da, Daltons).

Claims

1. Beetle powder, comprising at least 67% crude protein by weight, at least 5% chitosan by weight, fat by weight in an amount between 9% and 17% by weight, the percentages by weight being given relative to the total weight of the beetle powder, 85% digestible protein by weight relative to the total weight of the crude protein, and a residual water content between 2% and 15%.

2. The beetle powder according to claim 1, comprising ash in an amount of less than or equal to 4% by weight relative to the total weight of the beetle powder.

3. The beetle powder according to any one of claims 1 to 2, comprising 30 to 60% by weight of soluble protein relative to the total weight of crude protein, wherein at least 50% of said soluble protein has a size of less than or equal to 12,400 g / mol.

4. The beetle powder of claim 3, wherein at least 70% of the soluble protein has a size of less than or equal to 12,400 g / mol.

5. The beetle powder according to claim 3 or 4, wherein less than 10% of the soluble protein has a size greater than or equal to 66,000 g / mol.

6. A method for preparing beetle powder according to any one of claims 1 to 5, comprising the following steps: i) Kill the beetle, ii) Press the beetle to obtain a press cake, and iii) Grind the pressed cake.

7. The method of claim 6, further comprising the step of drying the press cake.

8. The method of claim 7, comprising the following steps: i) Kill the beetle, ii) Press the beetle to obtain a press cake. iii) Dry the pressed cake, and iv) Grind the pressed cake, The pressing step is preceded by the grinding step of the beetle.

9. The method of claim 7, comprising the following steps: i) Kill the beetle, ii) Press the beetle to obtain a press cake. iii) Dry the pressed cake, and iv) Grind the pressed cake, The pressing step is performed hot.

10. The method of claim 7, comprising the following steps: i) Kill the beetle, ii) Press the beetle to obtain a press cake. iii) Dry the pressed cake, and iv) Grind the pressed cake, The step of grinding the pressed cake is carried out until the particle size is between 300 μm and 1 mm.

11. Use of the beetle powder according to any one of claims 1 to 5 in human or animal nutrition.

12. The use according to claim 11, wherein the beetle powder is used as a substitute for protein powder.

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