Valorization of Marine Animal By-products

Abstract

The present invention relates to methods and processes for the industrial-scale extraction of nutritional and cosmetic products from marine animal by-products, including proteins and oils for nutritional purposes (e.g., animal food), and collagen for medical, nutritional and cosmetic purposes.

Description

Technical Field

[0001] The present invention relates to methods and processes for extracting nutritional and beauty products from marine animal by-products.

Background Art

[0002] Introduction A large amount of waste and by-products are generated in the food production chain that are suitable for further use. These animal by-products (ABP) are defined as animal carcasses, parts of carcasses and other products of animal origin that are not intended for human consumption. This includes both cooked and uncooked fish, shellfish and other aquatic products that are not for human consumption. Animal by-products can pose a threat to animal and human health if not properly handled or disposed of. Animal by-products are a significant part of biological waste vapors and there are strict regulations applicable to the disposal of these wastes.

[0003] Therefore, strict standards exist in the EU for the management of the collection, transport, storage, handling, processing and use or disposal of all animal by-products (EU Regulation 1069 / 2009).

[0004] The infrastructure of the waste industry in the Western world and the associated waste costs vary between countries, but the option of landfilling ABP is becoming increasingly infeasible in many countries. In some cases, landfilling of ABP is even prohibited, in other cases taxes are introduced or, as in the UK, there is also the possibility of fines for exceeding the allocated limit capacity, and the costs are increasing. There are many technologies for the treatment of ABP, including rendering and incineration, and for certain categories of ABP, composting and biogas production. One technology for ABP recycling is in-vessel composting, which has the advantage of producing a safe and valuable compost for use as a soil improver and fertilizer while avoiding the need for landfilling. The industrial-scale production of fish protein meal as animal feed has the potential to valorize marine ABP (WO 2005 / 115176). However, all of these methods leave valuable biological resources of ABP unutilized. Furthermore, especially with regard to marine ABP, the residual salt content and other undesirable impurities of the low-level products produced by these methods continue to be a problem.

[0005] Collagen is the main component of connective tissue, the most abundant fibrous protein in vertebrates, and constitutes approximately 30% of the total vertebrate protein. Collagen has a wide range of applications in leather, the film industry, pharmaceuticals, cosmetics and biomedical materials, and food. Collagen has been widely used in tissue engineering biomaterials in the last 20 years due to its properties that mimic human proteins and can be used in the treatment of pain associated with hypertension, urinary incontinence and osteoarthritis, in tissue engineering for human grafts, and in the inhibition of angiopathic diseases such as diabetic complications, obesity and arthritis.

[0006] Approximately 30% of fish waste is skin and bone, which are very rich in collagen. Annually, more than 100 million tons of fish are caught worldwide, and one third of the total catch is used for fish meal and animal feed. Therefore, the extraction of collagen from fish bones is a promising means of obtaining a value-added product and reducing environmental pollution.

[0007] EP1016347 relates to a process for preparing fish gelatin from the skin of raw fresh thawed fish by acid extraction. However, this process is not suitable for the processing of more complex ABP that contains fish bones, meat, cartilage and other fractions of ABP, and only gelatin is produced.

[0008] Small-scale studies for extracting collagen from fish by-products have shown that these starting materials are promising sources of valuable collagen (Jeong, H.-S. et al. Biotechnology and Bioprocess Engineering 18: 1185-1191 (2013); Matmaroth, K. Food Chem. 112: 1179-1186 (2011); Schmidt, M.M. et al. Int. Food. Red. J. 23(3): 913-922 (2016)). However, these laboratory-scale methods cannot be scaled up in an economical way, require less complex source materials (skin), and do not utilize other valuable components of the starting product. Therefore, these methods are not yet mature for the industrial valorization of usually complex marine ABP containing skin, meat, fish bones, fins and cartilage. SUMMARY OF THE INVENTION

[0009] Accordingly, there is still a need for an industrial method of utilizing marine ABP, for example to prevent the need for landfill or disposal of these products, and to produce high-quality native products derived from marine ABP that can be used in the beauty industry. Furthermore, there is still a need for a method that can economically produce collagen and other products that can utilize complex ABP starting materials and are pure enough for various further applications.

[0010] One object of the present invention is to provide large-scale valorization methods and processes that start from marine animal by-products and produce relatively high-quality products for human and animal use. A particular object of the present invention is to provide high-quality raw products, especially collagen, for use in the beauty industry.

[0011] The present invention meets this need by providing methods for preventing the presence of unwanted quality-degrading components in products resulting from marine ABP. These methods are capable of handling large-scale and complex starting materials.

[0012] Summary of the Invention Products manufactured from marine by-products play an increasingly important role not only in providing nutritious dietary components for pigs, poultry, and aquaculture but also in the growing pet food market, where high-quality and high-value ingredients are used to manufacture pellets, pouches, and treats. The freshness of the raw materials is important because it affects the quality of the protein in the final product. The spoilage of fish and its products occurs mainly due to enzymatic and bacterial action and begins immediately after the fish dies. Hydrolyzed collagen and protein products manufactured by prior art methods often contain high levels of monovalent ions and biogenic amines, which are toxic at increasing concentrations and produce an unpleasant taste and odor in the final product and are thus problematic. The present invention is based on the discovery that, in the process of manufacturing collagen hydrolysates and / or protein hydrolysates, the use of specific filtration steps results in a very high-quality final product even when starting from lower-quality starting materials. The present invention achieves these goals by providing large-scale methods for valorizing industrial quantities of marine raw materials.

[0013] In a first aspect, the present invention is a method for extracting collagen from marine animal by-products, comprising: (a) providing pieces of marine animal by-products preferably having a size of less than 5 cm, more preferably between 1 and 2 cm, said pieces of by-products containing at least two components selected from the meat, skin, fish bones, fins and cartilage of marine animals; (b) separating the pieces of animal by-products into at least two fractions, including a protein-rich fraction and a fraction containing skin and cartilage, wherein the skin and cartilage fraction is essentially free of meat; (c) optionally, comminuting the fraction containing skin and cartilage so as to produce a particle size preferably between 0.5 mm and 1 cm, more preferably between 1 mm and 4 mm; (d) enzymatically hydrolyzing the fraction containing skin and cartilage to produce a raw hydrolysate of collagen, preferably carrying out the hydrolysis at a pH between 5 and 9, more preferably between 6 and 8; (e) separating the raw hydrolysate of collagen into a lipid fraction, an aqueous collagen hydrolysate fraction free of solids, and a fraction containing solids, preferably by three-phase decanter centrifugation; (f) subjecting the aqueous collagen hydrolysate fraction to at least one filtration step using a membrane with a pore size of 2000 to 100,000 Da, preferably 4000 to 50,000 Da, more preferably about 10,000 Da, to produce a first permeate containing collagen hydrolysate and a retentate; (g) using a membrane having a pore size of less than 1000 Da, preferably between about 150 and 300 Da, to demineralize the first permeate by nanofiltration and / or dialysis to produce a purified collagen solution. The present invention further relates to a method as described above, further comprising deodorizing the purified collagen solution in step (g), preferably by using activated carbon and / or dialysis for deodorization.The present invention further relates to a method as described above, which comprises the steps of concentrating and sterilizing a purified collagen solution or a deodorized purified collagen solution, preferably by evaporation such as direct steam injection. The present invention further relates to a method as described above, which comprises the step of drying a purified collagen solution, a purified deodorized collagen solution, or a concentrate thereof to produce a collagen powder, preferably by spray drying, freeze drying, lyophilization, or a nozzle atomizer, and most preferably by continuous horizontal spray drying.

[0014] In another embodiment, the present invention is a method for producing a protein hydrolysate from marine animal by-products, comprising: (a) providing pieces of marine animal by-products, wherein the by-product pieces comprise at least two components selected from the meat, skin, fish bones, fins and cartilage of marine animals; (b) separating the by-product pieces into at least two fractions, including a protein-rich fraction and a fraction containing skin and cartilage; (c) mixing the protein-rich fraction with water and separating a lipid fraction, an aqueous fraction and a protein-rich solid fraction, preferably by three-phase decanter centrifugation; (d) enzymatically hydrolyzing the protein-rich solid fraction to yield a protein hydrolysate solution; (e) separating the protein hydrolysate solution into a second lipid fraction, an aqueous protein hydrolysate fraction and a second solid fraction, preferably by three-phase decanter centrifugation; (f) collecting the aqueous protein hydrolysate; and (g) further comprising demineralizing the aqueous protein hydrolysate of step (f) by filtration and / or dialysis using a membrane having a pore size of less than 1000 Da, preferably between 100 and 500 Da or 150 and 300 Da. The present invention further relates to a method as described above, which further comprises concentrating and sterilizing the aqueous protein hydrolysate or the demineralized aqueous protein hydrolysate, preferably by evaporation such as direct steam injection. The present invention further relates to a method as described above, which further comprises drying the aqueous protein hydrolysate, the demineralized aqueous protein hydrolysate, or their respective concentrates to produce a protein powder, preferably by spray drying, freeze drying, lyophilization, or nozzle sprayer, and most preferably by continuous horizontal spray drying.The present invention further relates to a method as described above, further comprising: (c’) collecting the aqueous fraction of step (c) and subjecting this fraction to filtration and / or dialysis using a membrane having a pore size of less than 1000 Da, preferably between 100 and 500 Da; and (c”) adding the retentate of step (c’) to the protein-rich solid fraction prior to step (d).

[0015] The present invention further relates to an integrated process for upcycling or valorizing marine animal by-products by combining the methods described above. In one embodiment of this aspect, the integrated process comprises: (a) providing pieces of marine animal by-products, wherein the by-product pieces comprise at least two components selected from the meat, skin, fish bones, fins and cartilage of marine animals; (b) separating the by-product pieces into at least two fractions comprising a protein-rich fraction and a fraction comprising skin and cartilage, wherein the skin and cartilage fraction is essentially free of meat; (c1) subjecting the protein-rich fraction to steps (c) and subsequent steps of the method for producing the protein hydrolysate described above; and (c2) subjecting the fraction comprising skin and cartilage to steps (c) and subsequent steps of the method for extracting collagen as described above. Further variations and refinements of the method for producing the protein hydrolysate and the method for extracting collagen can be fully applied in the context of the integrated process of this aspect of the present invention.

[0016] In any of the methods described above, the marine animal by-products used as starting materials may be fish by-products, such as heads, viscera, fish bones, scraps and / or skin. In a preferred embodiment, the marine by-products are by-products of tuna.

[0017] The above method manufactures a collagen solution and / or a collagen powder. The collagen produced by the above method has been found to be of very high quality. Accordingly, the present invention relates to a purified collagen solution or collagen powder produced by the method detailed above. In certain embodiments, the collagen solution and / or the collagen powder comprises at least one matrikine. The at least one matrikine of the collagen solution and / or powder may be any one or more of arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lumican, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endostatin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatins 7, neostatins 14, palmitoyl pentapeptide-4, the collagen hexapeptide GFOGER, and CLAC. In particular, the present invention is a collagen solution or powder produced by the method of the present invention, wherein at least 50% of the peptides of the collagen hydrolysate or powder are <2 kDa, preferably at least about 60% of the peptides are <2 kDa, more preferably at least about 65%, 66%, 67%, 68%, 69%, 70%, ···, 79%, 80%, 81%, 82%, 83%, 84% or 85% of the peptides are <2 kDa. In one embodiment, the purified collagen solution or powder of the present invention is characterized in that the residual salt content is less than 4% by dry weight, preferably less than 2%, 1% or 0.5% by dry weight.

[0018] The present invention also relates to a protein hydrolysate or a protein powder produced by the method of the present invention. In one embodiment, the protein hydrolysate or protein powder contains at least 3% (by weight) of omega-3 lipids and at least 1% (by weight) of phospholipids, preferably the hydrolysate or powder contains at least 2% (by weight) of monounsaturated lipids, at least 3% (by weight) of polyunsaturated lipids and at least 2.5% (by weight) of omega-3 lipids. In one embodiment, the purified protein hydrolysate or powder is characterized in that the residual salt content is less than 20% by dry weight, preferably less than 15%, 10% or 5%, the lipid content is less than 20% by dry weight, preferably less than 15%, 12% or 10%, and the protein content is more than 60% by dry weight, preferably more than 70%, 75% or 80%.

[0019] The collagen solution and collagen powder of the present invention can be used for pharmaceutical, nutritional and cosmetic purposes.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] In the method of the present invention, when steps (a) ··· (x) are described, the steps must be carried out in the order of appearance. Preferably, the method does not include other procedural steps than those explicitly specified. However, the different steps may be interrupted by non-procedural steps of transportation, cooling / heating and / or storage. However, further processing steps are not excluded from the recited method. The methods described herein are industrial methods, i.e., large-scale methods. These methods are carried out with at least several hundred kilograms of starting material and can be carried out to process several tons of starting material. One skilled in the art will recognize that operations on this scale require their respective equipment and that the methods of the present invention are not necessarily scaled down to small-scale (laboratory-scale) methods and, as is evident, small-scale methods cannot simply be scaled up to industrial scale (see, for example, Crater & Lievense, FEMS Microbiol Lett. 2018 Jul; 365(13): fny138). Very often, laboratory methods are either completely non-scaleable or non-scaleable in an economically meaningful way.

[0022] In a first aspect, the present invention relates to a method for a method of extracting collagen from marine animal by-products on an industrial scale.

[0023] The term "marine animal by-products" relates to by-products in the form of heads, viscera, skeletons, skin and other things such as tails, fins, scales, minced meat, blood, etc. resulting from the processing of fish for human consumption. This material can constitute up to 70% of the processed fish and shellfish, and the yield of fish fillets is species-dependent and often in the range of 30 - 50% of the fish. The waste generated after processing is actually a valuable raw material from which fish meal and fish oil can be manufactured, and this material is not yet fully utilized. The main sources of by-products are finfish and whitefish trimmings (pollock, cod, hake, haddock, etc.) as well as salmon (wild and farmed), tuna, herring, mackerel, and can be obtained from the processing of wild or farmed fish. The quality of the raw material often has to be in the region of 50 mg N / 100 g of raw material, measured by the total volatile basic nitrogen content. Preferred sources of marine animal by-products are fish-derived, especially tuna-derived, for example from Thunnus albacares or Katsuwonus pelamis.

[0024] The method of the present invention for extracting collagen comprises: (a) providing pieces of marine animal by-products; (b) separating the pieces of animal by-products into at least two fractions including a protein-rich fraction and a fraction containing skin and cartilage; (c) optionally, grinding the fraction containing skin and cartilage; (d) enzymatically hydrolyzing the fraction containing skin and cartilage to produce a tropocollagen hydrolysate; (e) separating the tropocollagen hydrolysate into a lipid fraction, an aqueous collagen hydrolysate fraction free of solids, and a fraction containing solids; (f) subjecting the aqueous collagen hydrolysate fraction to at least one filtration step using a membrane with a pore size of 2000 - 100,000 Da to produce a first permeate and a retentate containing collagen hydrolysate; (g) using a membrane with a pore size of less than 1000 Da to demineralize the first permeate by nanofiltration and / or dialysis to produce a purified collagen solution.

[0025] The by-product pieces in step (a) may have a size of less than 5 cm, more preferably a size of 1-2 cm. The production of pieces of this size from the supplied raw material may be carried out by known techniques, for example using an industrial macerator or other means for shredding this material. Notably, this size of the pieces does not have to be exact as shown, and the shredded or macerated material may exhibit pieces with a diverse size distribution. That is, a size of less than X cm refers to a macerated raw material where most of the pieces are smaller than X and no pieces larger than 5 cm are shown. A size between X cm and Y cm indicates that the average particle size must be within this range, although there may still be larger and smaller pieces.

[0026] Marine by-products and the pieces produced therefrom will typically contain at least two components selected from the meat, skin, fish bones, fins and cartilage of marine animals. However, with respect to the method of producing collagen, it is sufficient if the material contains skin and / or fish bones and / or fins.

[0027] In the separation step (b), the separation may be carried out with any suitable separator. Exemplary separators include separation devices such as RSBF (model 04 or 04), RSTC series, RSDD04, RSTD06, Beehive S02, SD and Beluga series (models 208, 210, 310, 620, 820, 1830, 2050, 3060, 4100, 5100, 6100), SM and Barracuda series (models 208, 210, 310, 620, 820, 1830, 2050, 3060, 4100, 5100, 6100), Modernpack Sepamatic (models 410, 1400, 1800, 2000, 3000, 4000) or AM2C Barracuda Beluga 1830 separator. These separators can be operated in a routine with parameters (pressure, grill / mesh size) that produce a skin and cartilage fraction that is essentially meat-free. Parameters such as the porosity of the screen, the pressure applied to the screen and the flow rate of the raw material are well described by the manufacturer depending on the type of separator used.

[0028] A product that "essentially contains no" other components or essentially contains no plurality of other components in the sense of the present invention refers to a product that contains less than 20% of other component(s), preferably less than 15% or 10%, in particular less than 5, 4, 3, 2, or 1% of other component(s). That is, a fraction that is essentially meat-free contains less than 20% meat, preferably less than 15% or 10%, in particular less than 5, 4, 3, 2, or 1% meat. A protein-rich fraction that is essentially free of bone and cartilage refers to a composition that contains less than 20% bone and cartilage, preferably less than 15% or 10%, in particular less than 5, 4, 3, 2, or 1% bone and cartilage.

[0029] "Consisting essentially of" in the meaning of the present invention represents a product containing more than 80%, preferably more than 85 or 90%, particularly more than 95, 96, 97, 98 or 99% of the specified components. It is to be understood that a protein-rich fraction that is essentially free of bone and cartilage can be a fraction consisting essentially of meat.

[0030] Thus, in step (b), the separator is operated according to the manufacturer's specification with parameters such that two fractions are obtained: a protein-rich fraction (which can be further used as described hereinafter in this specification) and a fraction consisting essentially of components such as skin, cartilage, fish bones, fins, etc. containing collagen. This separation step is important for the present invention because the quality of the collagen product produced is improved when the fraction containing skin and cartilage is essentially free of meat. Further, a further washing step (with associated loss of material) of the fraction containing skin and cartilage can be avoided.

[0031] In step (c) according to the method of the present invention, the particle size of the fraction of skin and cartilage can be further reduced so that a particle size between 0.5 mm and 1 cm, preferably between about 1 mm and about 5 mm, or between about 1 mm and about 4 or 3 mm is obtained. As shown above in connection with step (a), a size between X and Y mm indicates that the average particle size must be within that range, although larger and smaller pieces may be present. The advantage of further reducing the particle size of the cartilage and skin particles is that the subsequent hydrolysis step (d) requires less time until completion and the yield of the collagen hydrolysate is improved.

[0032] Step (d) of enzymatically hydrolyzing the fraction containing skin and cartilage (optionally triturated) yields tropocollagen hydrolysate. The hydrolysate contains hydrolyzed collagen, lipids, as well as other organic materials and ash (solids). The enzyme used in this step is a protease that can be used alone or in a mixture. Essentially, the protease used has a cleavage motif present in the amino acid sequence of collagen (types I, II, III, IV and / or V) and cleaves collagen into smaller peptides, as in the case of bacterial alkaline protease. Preferred enzyme types are serine endopeptidases, exopeptidases, di- and tripeptidases and carboxypeptidases, as well as metalloproteases.

[0033] The source of protease is not critical and plant proteases as well as bacterial proteases or proteases of animal origin may be used. Suitable enzymes include, but are not limited to, papain plant protease, bromelain plant protease, Protease Plus (Dupont / Dyadic), Sumizyme BNP L, Sumizyme LP, Sumizyme FLG (Shin Nihon), Alcalase 2.4 and 2.5 L, Neutrase, Protamex, Esperase, Savinase (Novozymes), Corolase 7089 (AB enzyme), papain, bromelain, Promod 950L (Biocatalysts).

[0034] Generally, hydrolysis is carried out at a pH between 5 and 10, more preferably between 6 and 9, or between 6 and 8. However, each enzyme has its own pH range in which it can act. Therefore, the enzyme hydrolysis conditions follow the recommendations of the enzyme supplier. By way of example, Protease Plus (Dyadic) is a bacterial alkaline protease produced by the controlled fermentation of Bacillus licheniformis. This enzyme is an endopeptidase capable of hydrolyzing internal peptide bonds of protein molecules with broad substrate specificity; this enzyme has pH stability between 6.0 and 10.0, can operate effectively in the pH range of 7.0 - 10.0, and has an optimal pH range of 9.0 - 10.0. Papain has an optimal pH for activity between 6.0 and 7.0. That is, in step (d), when Protease Plus is used, the process is operated at a pH between 9 and 10, and when papain is used, the step is operated at a pH between 6 and 7.

[0035] The temperature of enzymatic hydrolysis, like the pH conditions, depends on the enzyme or enzyme mixture used. Generally, the temperature will be from about 50 °C to about 70 °C, preferably up to about 65 °C. By way of example, Protease Plus has temperature stability up to 70 °C, the effective temperature range is up to 65 °C, and the optimal activity is obtained at 60 °C. Papain has a temperature optimum for activity at 65 °C.

[0036] The exact amount of enzyme added depends on the process and protein substrate concentration, the degree of protein hydrolysis desired, pH, temperature, and time. Dosage is calculated as % per weight of protein to be hydrolyzed. Typical dosage levels are 0.2% - 2% or 0.3% - 1% of the kg of protein contained in the material to be treated. The dosage may naturally be adjusted as appropriate to meet economic considerations according to the type of enzyme(s) used (e.g., increasing the enzyme amount to reduce processing time, or reducing the enzyme amount if processing time is not a concern). By way of example, for Promod 950 L (Biocatalyst), the amount of enzyme added to the skin and cartilage-containing fraction is from about 0.3% to about 0.7%, preferably about 0.4% - 0.6% or about 0.5% ± 0.05%. Those skilled in the art can readily control and adjust the parameters of the hydrolysis step according to the nature of the protease(s) and / or starting material used. If the progress of hydrolysis is too slow, the enzyme may be replenished as necessary.

[0037] The duration of enzymatic hydrolysis depends on the activity and amount of enzyme used. Maintaining a given temperature for hydrolysis requires a significant amount of energy, so the hydrolysis time is selected not to exceed 10 hours. To achieve good extraction, the hydrolysis time must also be no shorter than about 1 hour. Generally, as shown above, providing smaller particles for the skin and cartilage fractions allows for a shorter hydrolysis time. For example, when using Protease Plus for well-macerated material, hydrolysis is carried out within about 3 - 4 hours, and when using Promod 950 L (Biocatalyst), the hydrolysis time is about 5 hours.

[0038] Therefore, the hydrolysis time ranges between 1 hour and 10 hours, preferably between 2 hours and 8 hours, more preferably between 3 hours and 6 hours, or any value between these times.

[0039] To control the efficiency and progress of hydrolysis, the solubility of the protein may be measured with a refractometer (brix). Further, the hydrolysis rate may be determined by analytical methods (e.g., Sorensen method and also pH-stat method (Eric Rothenbuhler & John E. Kinsella; J. Agric. Food Chem. 1985, 33, 3, 433-438)). Other methods of controlling and determining protein hydrolysis are known to those skilled in the art (see the review on pages 263-271 of Silvestre, Food Chemistry, Volume 60, Issue 2, October 1997). These methods are capable of controlling the cleavage of amino acids, which affects the pH and, if necessary, allows the adjustment of the pH with NaOH or acid to operate the hydrolysis at the optimal pH.

[0040] Enzymatic hydrolysis results in a liquid phase, a lipid phase and a solid phase rich in peptides derived from collagen hydrolysis. These phases are separated in step (e) of the method of the invention. Any suitable technique or combination of techniques may be used to separate the lipid phase, the aqueous phase and the solid phase. In a preferred embodiment, the separation is carried out in a decanter, for example a three-phase decanter. There are many suitable (three-phase) decanters available from different suppliers, and exemplary devices include, but are not limited to, the Flottweg Z series (e.g., Z3E, Z5E, Z6E, Z4 or Z35), the three-phase decanters of GEA (e.g., ACE 345 or Alfa Laval). The main parameters, such as temperature, rotation speed (acceleration > 3500g), flow, adjustment of the extraction pipe (no value), screw type (angle, D / L of the bowl), depend on the device used and are operated according to the operating method as suggested by the manufacturer of the device.

[0041] The separation step (e) results in three fractions: (i) one lipid fraction (fish oil) that can be collected and used for various purposes, (ii) a solid fraction (mineral fraction, ash, which may be ground and dried to a white powder) that can be used as a source of minerals for the dietary supplement market, and (iii) an aqueous phase containing hydrolyzed collagen.

[0042] The aqueous fraction is subjected to at least one filtration step (f) using a membrane having a pore size of 2000 - 100,000 Da, preferably 4000 - 50,000 Da, to yield a first permeate and a retentate containing collagen hydrolysate. Particularly preferred pore size ranges are between 5000 Da and 25,000 Da, or between 6000 Da and 15,000 Da. In particular, the membrane may have a pore size of 7000 - 12,000 Da, for example 10,000 Da (10 kDa). This pore size of the ultrafiltration step enables the filtration of collagen hydrolysate with respect to non-hydrolyzed macromolecules, as well as residual fats and lipid vesicles. The fraction containing hydrolyzed collagen (collagen peptides) is the permeate.

[0043] Filtration may be carried out by tangential flow filtration of cross-flow filtration. The nature of the membrane is not essential and may be cellulose-based, polymeric material or ceramic-derived. Exemplary membranes may be polyethersulfone (PES), cellulose acetate, polyacrylonitrile, polyvinyl fluoride, as well as other fluorinated polymer membranes and ceramic filters made from zirconium oxide or titanium oxide.

[0044] Exemplary membranes that can be used include, but are not limited to, GE, KOCH, SYNDER, TIA, PALL, TAMI, MICRODYN NADIR (NADIR, SPIRA-CEL (spiral module), SEPRODYN (module for fine filtration), MICRODYN (tubular and capillary modules), ULTRADYN (hollow fiber module for ultrafiltration).

[0045] Suitable equipment includes the Alfa Laval Pilot Unit Multi and a 1x5m supplied by TIA with a permeate capacity of 200 - 500 l / hour 2 It includes an ultrafiltration pilot equipped with Organique UF and OI.

[0046] An exemplary 10 kDa ultrafiltration using Kock HKF 131 UF was used on chondroitin hydrolysate to produce collagen and remove high molecular weight impurities: The retentate fraction containing large proteins and fat residues was collected and may be recycled together with other protein hydrolysates (scrap, protein dough, charged water). The collagen hydrolysate is found in the permeate and is a clear, slightly colored (yellow to brownish) liquid.

[0047] The flow rate and ultrafiltration pressure are not critical to the process as long as ultrafiltration is effective, depending on the membrane selected and the equipment used; fouling can be prevented, for example, by adding water to the clarified collagen raw hydrolysate (fraction (iii) of step (e)) before or during ultrafiltration.

[0048] The collagen hydrolyzate (first permeate) resulting from step (f) is then subjected to a nanofiltration step (g). Alternatively, dialysis may be performed. The collagen hydrolyzate solution may be used as it is, or it may be further concentrated if necessary (for example, a concentrated first permeate is preferred for dialysis). The nanofiltration / dialysis step demineralizes the first permeate by nanofiltration and / or dialysis using a membrane with a pore size of less than 1000 Da to yield a purified collagen solution. Preferably, the membrane used for nanofiltration or dialysis has a pore size of less than about 500 Da, preferably about 100 - 400 Da, and particularly preferably 150 - 350 Da. By way of example, Lenntech organic nanofiltration membranes with a pore size of 150 - 300 Da, Snyder NFX, and NFW series, NF 90 (Filmtec), NF 270 (Filmtec), NF 2 (Sepro), and NF PES 10 (Microdyn-Nadir), Filmtec membranes (Dupont) can be used.

[0049] The pore size used in the nanofiltration step (g) must retain peptides and low molecular weight proteins while allowing aqueous monovalent ions (such as sodium, chloride ions) and optionally divalent ions (such as magnesium, nitrate) and other small molecules (such as biogenic amines, heavy metals, etc.) to pass through the membrane.

[0050] Depending on the transparency, color, and odor of the collagen hydrolyzate (permeate) in step (g), the resulting collagen solution may be subjected to further steps of deodorization and / or dialysis if necessary. Therefore, the present invention further relates to the above-described method which further includes the step of deodorizing the purified collagen solution in step (h) using activated carbon and / or dialysis. When using activated carbon, residual small molecules that may cause a slight odor in the collagen hydrolyzate solution are effectively removed, and thus the quality of the final product is further improved. When using dialysis, the effect of repeatedly adding water also has the effect of washing the collagen solution, further reducing unwanted components. This means also improves the removal of salts from the collagen solution. Dialysis may be performed with or without subsequent filtration of the retained material (collagen solution) on the activated carbon. Alternatively, the collagen solution may first be filtered through activated carbon and then subjected to dialysis.

[0051] The present invention further relates to the above-described method which further includes the steps of concentrating and sterilizing the purified collagen solution or deodorized purified collagen solution by evaporation, such as direct steam injection using suitable equipment (e.g., external or internal regulated direct contact steam heater). The sterilization may be UHT sterilization, i.e., injecting direct steam into the product to reach 140°C and then subjecting the product to a vacuum system for a very short time to lower the temperature to 60°C. By way of example, purified collagen hydrolyzate is directly injected into high-pressure steam (higher than 5 bar, e.g., 7 - 10 bar) for several seconds and the pressure is reduced to vacuum (e.g., 250 mmbar) at 60°C to form and collect particles. The present invention further relates to the above-described method which further includes the step of drying the purified collagen solution or purified deodorized collagen solution, or a concentrate thereof, to produce collagen powder. The drying step is performed by spray drying using a nozzle atomizer, freeze drying, or lyophilization, and preferably, the drying is performed by continuous horizontal spray drying. In particular, concentration and sterilization can be performed simultaneously with drying using a suitably integrated device.

[0052] The inventors have observed that nanofiltration and dialysis have an excellent effect on the deodorization of collagen. Therefore, by using these further steps, a better collagen solution / powder can be produced.

[0053] In a second aspect, the present invention relates to a method for producing a protein hydrolysate from marine animal by-products, comprising: (a) providing pieces of marine animal by-products as described in the method for producing the collagen hydrolysate as described above; (b) separating the by-product pieces into at least two fractions including a protein-rich fraction and a fraction containing skin and cartilage as described above; (c) mixing the protein-rich fraction with water and separating a lipid fraction, an aqueous fraction, and a protein-rich solid fraction; (d) enzymatically hydrolyzing the protein-rich solid fraction to yield a protein hydrolysate solution; (e) separating the protein hydrolysate solution into a second lipid fraction, an aqueous protein hydrolysate fraction, and a second solid fraction; and (f) collecting the aqueous protein hydrolysate.

[0054] In the context of the present invention, the expression "protein-rich" refers to proteins derived from meat in the context of the present invention. Further, this expression indicates that the protein content of the fraction that is "protein-rich" contains only trace amounts (less than about 15%), preferably minimal amounts (less than 5%) of bone and cartilage. Ideally, the protein-rich fraction is substantially free of bone and cartilage (less than 1.5%).

[0055] Step (c) of separating the protein-rich fraction into a lipid fraction, an aqueous fraction, and a protein-rich solid fraction may be performed using the methods and apparatus of the routine. Preferably, the separation is performed by three-phase decanter centrifugation as described above for the method of producing a collagen hydrolysate. In particular, the parameters for operating the three-phase decanter are adapted for the protein-rich fraction as needed, which are described in the manufacturer's operating instructions for each device used. The protein-rich solid fraction is then subjected to an enzymatic hydrolysis step (d).

[0056] The enzymatic hydrolysis (d) of the protein-rich solid fraction yields a protein hydrolysate solution. The hydrolysis conditions such as time, temperature, and pH depend on the choice of enzyme as described above for the method of producing a collagen hydrolysate. However, the proteins in the protein-rich fraction are more accessible to hydrolysis. Therefore, the incubation time for hydrolysis is significantly shorter. By way of example, when Protease Plus is used as the enzyme, the hydrolysis is completed in about 1 to 2 hours at a pH of 8 (about 7.5 to about 9) and a temperature of about 60 °C (about 52 °C to about 65 °C).

[0057] After hydrolysis, the protein crude hydrolysate is subjected to a separation step (e) of separating it into (i) a (second) lipid fraction, (ii) an aqueous protein hydrolysate fraction, and (iii) a (second) solid fraction. As shown above, any suitable means for achieving this separation may be used. Preferably, the parameters of the decanter are adapted to the softer type of the supplied material, and the separation is performed by further three-phase decanter centrifugation as described above.

[0058] (f) The step of collecting the aqueous protein hydrolyzate then collects it (step (f)) for storage or further processing as described below. One (optional) further step to improve the quality of the protein hydrolyzate is to perform a demineralization (desalting) step (g). As described above for collagen hydrolyzates, demineralization may be performed by nanofiltration and / or dialysis using a membrane with a pore size of less than 1000 Da, preferably between 100 and 500 Da (or between 150 and 300 Da). The conditions (e.g., membrane type) applied to the nanofiltration of collagen hydrolyzates are fully applicable to the demineralization of protein hydrolyzates.

[0059] The present invention further relates to a method as described above, which further comprises the step of concentrating and sterilizing the aqueous protein hydrolyzate or the demineralized aqueous protein hydrolyzate, preferably by evaporation, for example by direct steam injection as described above. Further, the method for producing a protein hydrolyzate may further comprise the step of drying the aqueous protein hydrolyzate, the demineralized aqueous protein hydrolyzate or their respective concentrates to produce a protein hydrolyzate powder. In one embodiment, the drying is performed by spray drying, freeze drying, lyophilization or using a nozzle atomizer as described above. Most preferably, the drying is performed by continuous horizontal spray drying as described in the method for producing collagen hydrolyzates. The present invention further relates to a method for producing a protein hydrolyzate as described above, which further comprises the step of collecting the aqueous fraction of step (c') and subjecting this fraction to filtration and / or dialysis using a membrane having a pore size of less than 1000 Da, preferably between 100 and 500 Da (most preferably between 150 and 300 Da), and the step of adding the retentate of step (c') to the protein-rich solid fraction before step (d). These further steps improve the yield of the protein hydrolyzate.

[0060] In a third aspect, the present invention further relates to an integrated process for upcycling or valorizing marine animal by-products, which combines the above-described methods for producing collagen hydrolysates and protein hydrolysates. The details described above with respect to the two methods are fully applicable to the integrated process. In one embodiment of this aspect, the integrated process comprises: (a) providing pieces of marine animal by-products, wherein the by-product pieces comprise at least two components selected from the meat, skin, fish bones, fins and cartilage of marine animals; (b) separating the by-product pieces into at least two fractions, including a protein-rich fraction and a fraction containing skin and cartilage, wherein the skin and cartilage fraction is essentially free of meat; (c1) subjecting the protein-rich fraction to steps (c) and subsequent steps of the method for producing the protein hydrolysate described above; and (c2) subjecting the fraction containing skin and cartilage to steps (c) and subsequent steps of the method for producing the collagen hydrolysate as described in the first aspect of the present invention above. Further variations and refinements of the method for producing the protein hydrolysate and the method for extracting the collagen can be fully applied in the context of the integrated process of this aspect of the present invention.

[0061] In any of the above methods of any of the three aspects of the present invention, the marine animal by-products used as starting materials may be fish by-products, such as heads, viscera, fish bones, scraps and / or skin. In a preferred embodiment, the marine by-products are by-products of tuna.

[0062] The above method yields a collagen (hydrolysate) solution and / or a collagen (hydrolysate) powder. The collagen hydrolysate produced by the above method has been found to be of very high quality. Accordingly, the present invention relates to a purified collagen (hydrolysate) solution or a collagen (hydrolysate) powder produced by the method of the present invention. In one embodiment, the collagen (hydrolysate) solution and / or the collagen (hydrolysate) powder contains at least one matrikine. At least one matrikine of the collagen (hydrolysate) solution and / or the powder may be any one or more of arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lumican, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endostatin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatins 7, neostatins 14, palmitoyl pentapeptide-4, the collagen hexapeptide GFOGER, and CLAC. However, the collagen (hydrolysate) may further contain additional matrikines, as well as more than one, more than two, or more than three matrikines.

[0063] In particular, the present invention relates to a collagen (hydrolysate) solution or powder produced by the method of the present invention, wherein at least 50% of the peptides of the collagen hydrolysate or powder are <2 kDa, preferably at least about 60% of the peptides are <2 kDa, and more preferably at least about 65%, 66%, 67%, 68%, 69%, 70%, ···, 79%, 80%, 81%, 82%, 83%, 84% or 85% of the peptides are <2 kDa. In one embodiment, the purified collagen (hydrolysate) solution or powder of the present invention is characterized in that the residual salt content is less than 4% by dry weight, preferably less than 2%, 1% or 0.5% by dry weight.

[0064] The present invention also relates to a protein hydrolysate or protein powder produced by the method of the present invention. This product may be referred to as FPH fish protein hydrolysate. In one embodiment, the protein hydrolysate or protein powder contains at least 3% (by weight) of omega-3 lipids and at least 1% (by weight) of phospholipids, preferably the hydrolysate or powder contains at least 2% (by weight) of monounsaturated lipids, at least 3% (by weight) of polyunsaturated lipids and at least 2.5% (by weight) of omega-3 lipids. In one embodiment, the purified protein hydrolysate or powder is characterized in that the residual salt content is less than 15% by dry weight, preferably 10%, 7%, 6%, 5% or less than 4%, the lipid content is less than 20% by dry weight, preferably 15%, 12% or less than 10%, and the protein content is more than 60% by dry weight, preferably more than 70%, 75% or 80%. In particular, the final product of the protein hydrolysate contains about 17 - 18% lipids in the hydrolysate in the powder, together with about 3.7% phospholipids and about 2% omega-3 lipids.

[0065] A typical protein powder final product produced by the method of the present invention has a residual moisture content of about 2 - 4%, a protein between 70 - 80%, a total lipid content of about 11 - 19% (about 2 - 5% phospholipids) and a total mineral (ash) content of about 6 - 10% (about 2 - 4% chlorine). The product may be referred to as fish protein hydrolysate powder or FPHP. By way of example, the FPHP final product has about 73 - 76% total protein, 13 - 16% total lipids (2.5% - 3.5% phospholipids), 7 - 9% total mineral content (about 2.5% - 3.5% chlorine). All % values in this paragraph are by weight of the final product.

[0066] The peptide profile of the collagen hydrolysate shows a typical profile of amino acids dominated by the collagen protein sequence, namely three specific amino acids; glycine (about 18 - 20%), hydroxyproline (about 7 - 8%) and proline (about 10 - 12%) are prominent.

[0067] The collagen hydrolysate and protein hydrolysate produced by the method of the present invention exhibit a specific and unique peptide size distribution as described below.

[0068] In particular, the collagen hydrolysate of the present invention contains peptides with a size of about 4 - 10 kDa accounting for about 12 - 18%, peptides with a size of about 1 - 4 kDa accounting for about 60 - 70%, peptides with a size of about 0.5 - 1 kDa accounting for about 12 - 18%, and peptides with a size of less than 0.5 kDa accounting for about 3 - 7%. The collagen hydrolysate of the present invention contains less than 1% of peptides larger than 35 kDa, preferably essentially does not contain them, contains less than 5%, preferably less than 2%, and more preferably essentially does not contain peptides larger than 10 kDa. The high content of relatively small-sized peptides has the advantages of being reproducible, having enhanced biological properties, and higher digestibility.

[0069] The heavy metal and PCB / dioxin content is below the threshold defined in the European Union, and thus the collagen hydrolysate is suitable for human and animal nutrition and / or cosmetics.

[0070] The protein hydrolysate of the present invention contains peptides with a size of about 4 - 10 kDa accounting for about 25 - 35%, peptides with a size of about 1 - 4 kDa accounting for about 45 - 60%, peptides with a size of about 0.5 - 1 kDa accounting for about 8 - 14%, and peptides with a size of less than 0.5 kDa accounting for about 3 - 7%. The protein hydrolysate of the present invention contains less than 4%, preferably less than 3 or 2%, of peptides larger than 35 kDa, and less than 5%, preferably less than 4%, and more preferably less than 3%, of peptides larger than 10 kDa. The high content of relatively small-sized peptides has the advantages of high reproducibility, excellent uniformity, and higher digestibility. Due to the small peptide size, the hydrolysate has excellent biological availability and thus improved biological properties.

[0071] The fatty acid profile of the protein hydrolysate of the present invention is as follows:

[0072]

Table A

[0073] The histamine content of the protein hydrolysate is less than 300 ppm, preferably less than 250 ppm, particularly less than 200 ppm. The total amount of biogenic amines is less than 1800 ppm, preferably less than 1400 ppm, particularly less than 1000 ppm. The heavy metal and PCB / dioxin contents are below the thresholds defined in the European Union, and thus the protein hydrolysate is suitable for human and animal nutrition.

[0074] In all of the above steps of any one of the three embodiments of the present invention, additional components, such as stabilizers, foam control agents, or antioxidants, may be supplied to the reaction in order to regulate, protect, or improve each reaction. Suitable antioxidants include, but are not limited to, Roseen, Paramega (Kemin), and Hybrilox (Kemin). Those skilled in the art will understand that many other antioxidant products are available and suitable for use in the present invention.

[0075] Usefulness and industrial applicability

[0076] The collagen solution and collagen powder of the present invention can be used for pharmaceutical, nutritional, and cosmetic purposes.

[0077] Collagen injection can improve the contour of the skin. Cosmetically using a filler containing collagen can remove lines and wrinkles from the face. Collagen can also improve scars and assist in wound healing by attracting new skin cells to the wound site. Collagen promotes healing and provides a scaffold for new tissue growth. Collagen dressings can be used for chronic wounds that do not respond to other treatments, wounds in tissues that release body fluids such as urine or sweat, granulating wounds, necrotic or septic wounds, partial-thickness and full-thickness wounds, second-degree burns, and areas of skin grafts and skin flaps. Collagen-based membranes have also been used in periodontal and transplantation therapies. In oral surgery, the collagen barrier prevents rapidly proliferating cells from migrating to the tooth wound, thereby maintaining a space for the tooth cells to have a chance to regenerate. Donor-derived collagen tissue grafts have been used for peripheral nerve regeneration, artificial blood vessels, and arterial reconstruction. Collagen adjuvants or formulations can be used for the treatment of osteoarthritis. Collagen can reduce pain symptoms and improve joint function in patients with osteoarthritis. For cosmetic use, collagen can be used for skin reactivation.

[0078] Due to the fact that collagen hydrolysate mainly contains smaller peptides, it can be digested in the form of a food supplement or a functional food or beverage to assist joint and bone health and enhance skin health. Hydrolyzed collagen, such as that resulting from the method of the present invention, has a much smaller molecular weight compared to native collagen or gelatin. More than 90% of the hydrolyzed collagen is digested within one hour and is available as small molecule peptides in the bloodstream. The peptides are transported from the blood to target tissues (such as skin, bone, and cartilage), where the peptides act as building blocks for local cells and assist in boosting the production of new collagen fibers.

[0079] Definition

[0080] The terms "hydrolysis" or "hydrolysing" in the context of the present invention relate to the treatment of by-product fractions with protease enzymes. Other types of hydrolysis, such as chemical hydrolysis (e.g., thermal acidic hydrolysis), are clearly not within the scope of the present invention. The term "hydrolysate" refers to the product of enzymatic hydrolysis.

[0081] "Solids" are components that cause sedimentation in each solution and / or can be removed from the solution by filtration through a sieve with a mesh size of 0.5 mm or by centrifugation at a g-force of up to 20 xg.

[0082] The term "ultrafiltration" is used to relate to filtration through a semi-permeable membrane where high molecular weight suspended solids and solutes are retained in the so-called retentate while water and low molecular weight solutes pass through the membrane in the permeate (filtrate). Ultrafiltration will retain molecules with molecular weights between 2 kDa and 100 kDa.

[0083] The term "nanofiltration" is used in a similar way to microfiltration, but nanofiltration retains molecules with molecular weights between 100 Da and 1000 Da and requires a higher pressure inside the membrane, which is different.

[0084] The terms "pore size" related to ultrafiltration and nanofiltration describe the molecular weight cut-off of each membrane or filter. For example, a pore size of 300 Da retains peptides and other solutes with an MW > 300 Da, while peptides, amino acids, and other solutes with an MW < 300 Da pass through the membrane.

[0085] "Filtration membrane" refers to a sheet of membrane that achieves the function of filtration as well as other configurations of materials (filter / membrane stack, ceramic cylinder, etc.).

[0086] "Permeate" is the solution that exits or passes through a given filter or membrane in the filtration step. Accordingly, "retentate" is the fraction that does not pass through a given filtration membrane at the end of filtration, and the retentate may be a solution or a solid fraction. When the retentate is a solid fraction, it may or may not be redissolved in a further solution of the same or different hydrophobicity as the solution from which the retentate is derived.

[0087] The terms "demineralizing" and "desalting" refer to the removal of salts (e.g., but not limited to NaCl) from a composition or solution.

[0088] The terms "dialysing" and "dialysis" refer to the process of removing excess salts, toxins, and other small molecular components by diffusion using a semi-permeable membrane, by adding water and pushing out the excess compounds through the membrane, thereby "washing" the remaining components. Except for the driving force used (diffusion versus pressure), the effect of dialysis is similar to nanofiltration. The solution processed by dialysis is also diluted, while the solution subjected to nanofiltration is concentrated.

[0089] The terms "purified" and "purification" in the broadest sense mean enriching or isolating one of components A, B, or C, or from a mixture of different components A, B, C, ···.

[0090] The term "deodorizing" in the context of the present invention refers to the process of removing the components that cause the odor of the composition.

[0091] The term "concentrate" in the context of the present invention refers to an increase in the dry matter of a solution by the removal of water.

[0092] The term "dry" describes a composition with a water content of less than 10%, preferably less than 5%, more preferably less than 4% or 3%. Correspondingly, the process of "drying" refers to the removal of water from a solution to the extent that the resulting composition has a water content of less than 10%, preferably less than 5%, more preferably less than 4% or 3%.

[0093] An "aqueous" solution either does not contain an organic solvent or contains less than 10% or less than 5% of an organic solvent, i.e., a solution essentially based on water.

[0094] Extracellular proteins and glycosaminoglycans (GAGs) can, through limited enzymatic cleavage, sometimes result in the release of fragments that exhibit biological activities different from those of the full-length molecules (Richard-Blum & Salza, Experimental Dermatology, (2014), Vol. 23, p. 457-463). These bioactive fragments are referred to herein as matricines. These fragments control many pathophysiological processes, including angiogenesis, cancer, fibrosis, inflammation, neurodegenerative diseases, and wound healing. Exemplary matricines include arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lumican, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endostatin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatins 7 and 14, palmitoyl pentapeptide-4, the collagen hexapeptide GFOGER, and CLAC.

[0095] The expression "about" immediately preceding any value presented herein indicates a variation of about 10%, preferably about 5% of the value.

Examples

[0096] The following examples illustrate specific embodiments of the invention and should not be construed as limiting the scope of the invention as defined in the appended claims.

[0097] This example shows the valorization of four different available by-products: cooking scraps (from canneries), protein dough, filling water, and cartilage and skin residues (from the oil recovery process from tuna heads).

[0098] Raw materials Tests were conducted using all tuna heads from the Seychelles and the Indian Ocean. The heads were packaged in cardboard pallets and frozen in a cryogenic storage before processing. The tuna heads from the Seychelles are derived from tuna that were not stored in brine and not frozen, resulting in a significant difference in composition and thus salt content in the composition.

[0099] The heads were then processed on an AM2C Barracuda Beluga1830 separator to yield protein dough and cartilage residues.

[0100] Volume used: Received 19 pallets with a total weight of 7211 kg. Two types of tuna were provided: (i) Yellowfin tuna (Thunnus albacares): 2 T (ii) Long-finned tuna (Thunnus alalunga): 5.2 T (also known as albacore or thon germon)

[0101] Raw material characteristics: Upon receipt, there were also unfrozen heads, and most were bigeye tuna. Random temperature management yielded values between -15°C and 2.7°C. The yellowfin tuna heads were frozen and adhered together, and a mallet or bar had to be used to conduct crushing tests and quality control sampling. The bigeye tuna heads were mostly frozen at the outer edge of the pallet, and the internal temperature was -3.5 to 2.7°C. Blood was flowing out from one pallet. According to the supplier, all the heads had been placed in the freezer. The bigeye tuna heads were smaller, sedimentation occurred, and the cold air did not seem to diffuse sufficiently to ensure rapid and complete freezing. When using the heads, some heads showed browning and some even showed obvious signs of deterioration. The blood had flowed to the bottom of the container.

[0102]

Table 1

[0103] Histamine standard according to Regulation (EC) No 2073 / 2005 and its applicable amendments: for fishery products: the average content of 9 samples of fish must not exceed 100 mg / kg; 2 samples may contain an amount more than 100 mg but not exceeding 200 mg / kg; no sample must exceed 200 mg / kg. For products matured enzymatically or in brine: multiply the upper limit by 2. TCB-N = Total volatile basic nitrogen.

[0104] When reduced to a dry matter content of 12%, the heavy metal content complies with the regulatory threshold for animal feed.

[0105] However, the levels of IP, ABVT, and biogenic amines were high, indicating that the freezing was too slow. Therefore, the quality of these raw materials is relatively low compared to materials available from other sources. This example thus demonstrates the excellent effect of the method of the present invention in that a very highly purified product of high quality can be obtained even from suboptimal starting materials.

[0106] Process Figure 1 shows a simplified material balance for the main steps of the method for producing protein hydrolysates (Figure 1A) and collagen hydrolysates (Figure 1B). The expected products are: Demineralized collagen powder Demineralized protein hydrolysate (dough) Mineral residue respectively.

[0107] The balance described in Figure 1 is shown per ton of raw material (Figure 1A is the balance for the protein hydrolysate process and Figure 1B is the balance for the collagen hydrolysate process).

[0108] Equipment used Shredder: The raw materials were processed into pieces of marine by-products using a Pallmann shredder to shred frozen fish plates to obtain 1 or 2 cm chips. The dimensions of the cutting chamber of the PALLMANN grinder are 350 mm (thickness) × 750 mm (width). Separator: AM2C supplied with a 3 mm sieve was used as a separator to produce a protein-rich fraction and a fraction containing skin and cartilage using a Barracuda Beluga 1830 separator.

[0109] Grinder (optional): A BIRO crusher AFM G52 type was used with a 12 - 14 mm grid for the cartilage and skin from the separator to finely grind the cartilage to facilitate hydrolysis.

[0110] A 5 m stirred reactor equipped with a variable speed drive and a half-shell heating system was used to achieve enzymatic hydrolysis. This apparatus includes a manual bottom valve coupled with a pneumatic discharge valve. At the outlet of this reactor, a sieve screw is used to recover the cartilage residue. A minimum volume of 700 liters is required to start stirring. Promod 950L was used as the enzyme. 3 ​

[0111] The GEA ACD 345 was used as a three-phase decanter and operated according to the manufacturer's recommendations to separate the lipid phase, the hydrolyzate, and the residual solid phase.

[0112] 1 Skid ultrafiltration (UF) / nanofiltration (NF): The UF / nano skid may be equipped with a variety of different porous membranes. In this example, two different membranes were used: (a) a Food & Dairy UF of the HFK-131 type with a surface element of 4.2 m 2 and a porosity of 10,000 Daltons, and (b) a DK3840C-50D type nanofiltration GE Waters and Process Technologies membrane with a surface area of 5.4 m 2 and a porosity of 150 - 300 Daltons.

[0113] Alternatively, a Food & Dairy UF element of the HFK-328 type with a surface area of 4.3 m 2 and a porosity of 5,000 Daltons may be used. The filtration device is equipped with a 100-liter feed tank, which limits the volume to be processed, but an external tank can be provided to increase the processable volume. The filtration device enables the production and purification of collagen hydrolyzate (1 kDa), demineralizes the collagen with a nano membrane (150 - 300 Da), and performs pre-concentration and demineralization of the hydrolyzate (with a nano 150 - 300 Da membrane).

[0114] In this example, a filter press with activated carbon was used. The filter press was used to remove specific coloring or odor particles. 3% coal and Smellrite (deodorant) of each dry substance were mixed with the hydrolyzate. Filtration was then carried out with a filter plate containing the adjuvant type Pall K200, retaining the coal and Smellrite in the press cake.

[0115] As an evaporator, a single-effect evaporator equipped with thermocompression was used. This plate evaporator had a capacity of approximately 1 T / hour and increased the solution concentration between 35 - 40% of the dry weight content. A minimum volume of 300 liters was required.

[0116] Drying: For the final drying step, a nozzle atomizer with a 200 bar HP pump and an 800 μ pre-filter was used, and the nozzle atomizer was adapted to dry soluble protein and fish protein products. Other atomizers may be used that allow the injection of an anti-caking agent in the pneumatic transport pipe to facilitate the extraction of the moving powder. Nozzle selection is to obtain sufficient spray pressure in the chamber considering the viscosity and dry matter content of the product.

[0117] Packaging: Using a vacuum packaging device (Bernhardt), a small vacuum bag was created to protect the product against oxidation and the final product was packaged.

[0118] Timeline of the method Tests were conducted on two similar batches of 3 - 4 tons of raw materials (heads). Each test took a total of 2 days, and the second batch was allowed optimization compared to the first batch.

[0119] Day 1 Management of raw materials

[0120] Day 2: First batch Grinding of yellowfin tuna heads / Pallman (approx. 1.5 T) Separation of protein paste / cartilage from bigeye tuna heads (1.8 T), then grinding of yellowfin tuna heads Decantation after hydrolysis of bigeye tuna head protein paste (1.9 T) Nanofiltration / hydrolysate sample protein paste after addition of salt Atomization of the hydrolysate of the protein paste (batch 1)

[0121] Day 3: First and Second Batches Cartilage Hydrolysis (Batch 1) Ultrafiltration of Cartilage Hydrolysate (Hydrolysate 1) Deodorization and Decolorization of Collagen Hydrolysate Grinding of Yellowfin Tuna Head / Pallman (approx. 0.5T) Separation of Protein Paste / Cartilage from Mash of Yellowfin Tuna Head and Bigeye Tuna Head (3.4T) Decantation after Hydrolysis of Yellowfin Tuna Head Dough (1.4T)

[0122] Day 4: Second Batch Concentration and Micronization of Hydrolysate Protein Paste in 2 Batches Using Different Antioxidants (Batch 2 and 3) Addition of Salt after Nanofiltration of Collagen Hydrolysate Sample (Batch 1) Cartilage Hydrolysis (Batch 2)

[0123] Day 5: Second Batch Ultrafiltration of Cartilage Hydrolysate (Hydrolysate 2) Deodorization and Decolorization of Collagen Hydrolysate (Batch 2) Vacuum Packaging of Hydrolysate Sample

[0124] Day 6: Collagen Concentration

[0125] Day 7: Collagen Drying (Tecoma Dryer) The antioxidants Paramega and Hybrilox (both Kemin) were used to stabilize the products as follows: Batch 1: Paramega was used at 500 ppm / paste for the protein paste, and Paramega was used at 5,000 ppm / dry matter or 900 ppm (18.1% dry matter) for the product before drying. Batch 2: Hybrilox was used for the paste, equivalent to 2000 ppm / paste at the start of hydrolysis; after concentration and before drying, Hybrylox was used at 7,500 ppm / dry matter for batch 2 and Paramega was used at 2,000 ppm / dry matter for batch 3. Six samples from each test were conditioned under vacuum to allow comparison of vacuum-induced oxidation over several months. All samples showed no signs of decomposition and showed satisfactory storage times over 12 months.

[0126] Separation In two batches, based on 7.2 tonnes of heads (raw material) processed as described above, the tuna heads were separated into a skin and cartilage-containing fraction on the one hand and a protein and meat-rich fraction on the other hand:

[0127] Batch 1: Albacore tuna head separation: From 1845 kg of head, approximately 250 L of water was added during separation to obtain approximately 1900 kg of protein rich fraction (paste) or approximately 90% paste compared to the head weight (not counting the added water). Separation of pre-ground yellowfin tuna heads: from 1342 kg of heads (+250 L water) 1164 kg of protein-rich paste (about 68% of the raw material) is obtained. Overall, for the head weight used, 20% skin / cartilage A fraction was obtained (about 650 kg).

[0128] Because the heads were still partially frozen, the mechanical separation was suboptimal and the resulting skin / cartilage fraction still contained some meat. For batch 2, the process was optimized as described below.

[0129] Batch 2 Separation of pre-ground yellowfin tuna heads: From 638 kg of heads (+ 150 L of water), 675 kg of protein paste (about 82% of the raw material) was obtained. Separation of bigeye tuna heads: From 3277 kg of heads (+ 250 L of water), about 3300 kg of protein paste was obtained. For Batch 2, about 7% skin / cartilage (283 kg) was obtained compared to the head weight used.

[0130] The difference in the yield of the skin / cartilage fraction results from the fact that more yellowfin tuna was processed on the second day. The difference can also be explained by the improvement in separation obtained by differences in settings (increase in pressure and temperature) that allow for better separation. The skin / cartilage fraction of Batch 2 is less "red" and contains less meat compared to Batch 1.

[0131] Over the two batches, this corresponds to an average of 13% skin / cartilage relative to the head weight.

[0132] In a further series, from the raw material, a protein paste (PP) to cartilage (C) ratio of 61% PP (5.8 T) to 39% C (3.7 T) was obtained.

[0133] Composition of the protein-rich fraction: [Table 2] Table 2: Composition of the protein-rich fraction

[0134] The mineral and protein contents are very similar for both species, while the fat content is higher in the protein fraction of bigeye tuna.

[0135] Composition of the skin / cartilage fraction:

[0136] [Table 3] Table 3: Composition of Skin / Cartilage Fraction

[0137] The mineral content is higher (exceeding 50%) in the cartilage obtained from the head of bluefin tuna compared to 40% for yellowfin tuna.

[0138] Mincing of cartilage and skin To optimize the hydrolysis of collagen contained in the cartilage, fine grinding of the cartilage is important (the performance is even better when the grinding is finer). At the outlet of the separator, the cartilage was ground on a BIRO grinder with a 20 mm grid.

[0139] Enzymatic hydrolysis (protein-rich fraction) For the enzymatic hydrolysis of the protein paste (protein-rich fraction), the following conditions were used:

[0140]

Table 4

[0141] The composition of the extracted fat phase (batch 2) was: dry matter: 98.5%, mineral: 0%, protein: 0.7%. Thus, the process yields oil of relatively high purity.

[0142] Enzymatic hydrolysis (skin / cartilage fraction; collagen) For the enzymatic hydrolysis of the cartilage (skin and cartilage fraction containing collagen), the following conditions were used.

[0143]

Table 5

[0144] Hydrolysis was carried out without particular problems, and for both batches, the discharge from the reactor was achieved with good control by "pushing out" with a small amount of water.

[0145] The resulting collagen hydrolysate was fairly transparent. It was possible to obtain a fairly white fraction (tailing) of solid cartilage and bone, which could be finely ground into a white powder, forming an interesting fraction as a source of minerals for the nutrition market.

[0146] Filtration Hydrolysates from protein paste, nanofiltration (desalting) of protein hydrolysates Before filtration, the protein hydrolysate had a mineral content of approximately 15% in the dry matter. For the nanofiltration of the protein hydrolysate, an organic nanofiltration membrane (150 - 300 Da) was used.

[0147] The initial volume of the hydrolysate was 73 liters; during the process, water was added to maintain the flow rate and improve desalting. The total volume of the hydrolysate processed was 110 liters. The fractions resulting from the nanofiltration of the protein hydrolysate were treated as follows: Permeate was stored in a plastic container (a fraction to be discarded later, containing a high concentration of salts and very little protein), Retentate was recycled into the pilot feed tank (the noble protein fraction). After the retentate concentration step, distilled water was added to extract more salts (dialysis). Volume after concentration = 61 liters; total volume of water added: 102 liters (52 + 16 + 34).

[0148] Regular monitoring of the resistance of the dry extract, as well as the retentate and permeate fractions, was carried out at the outlet. The resistance (R / ES) ratio to the dry matter is an excellent indicator of the soluble mineral content. During recycling, the process was continued for 12 hours to monitor the membrane performance over time.

[0149] During nanofiltration, samples were taken regularly. Some of these samples were analyzed (dry matter, protein, minerals), and the conductivity / dry matter ratio was monitored thereby. In the retentate, the ratio decreased by a factor of 5 (from over 260 to a final volume of about 51).

[0150] Flow rate: The membrane surface area used was 5.39 m 2 It was. The permeate flow rate varied from 15 l / h / m 2 after 15 - 18 hours to 5 - 6 l / h / m 2 which is excellent and very classical for this type of membrane.

[0151] The following characteristics of the protein hydrolysate fraction resulting from NF were obtained:

[0152]

Table 6

[0153] Protein hydrolysate fraction, retentate. After nanofiltration, the mineral content corresponded to only 2.92% of the dry matter, which is very satisfactory and below the standard of prior art hydrolysates. Approximately 90% of the nitrogenous substances are found in the retentate. The loss of amino acids or small - molecule peptides is very weak. Only 10% of the ash initially present in the hydrolysate is found in the retentate. Thus, demineralization was very effective.

[0154] Hydrolysate from skin / cartilage fraction, filtration (desalting) of collagen hydrolysate After hydrolysis of cartilage and skin, the procollagen hydrolysate obtained after separation is purified by two consecutive filtration steps:

[0155] 1) Ultrafiltration UF 10 kDa: Kock HKF 131 UF for cartilage hydrolysate to produce collagen and remove "impurities": The retentate fraction containing large proteins and fat residues may be recycled together with other protein hydrolysates (scrap, protein paste, loading water). Collagen hydrolysate is found in the permeate.

[0156] 2) Nanofiltration NF 300 Da for the permeate from UF. This step enables the demineralization of collagen hydrolysate: Valuable fraction = retentate. Permeate (= "brine"). The membrane used was a thin-film nanofiltration membrane characterized by an approximate molecular weight cut-off of 150 - 300 Daltons for uncharged organic molecules (GE Dairy Processing Sanitary Nanofiltration D series).

[0157] Ultrafiltration (batch 1) The collagen hydrolysate was processed by continuous UF without recycling or dialysis (without recirculation or addition of water). Ultrafiltration was started at a volume of 80 liters (tank capacity). This volume decreases by a volume fraction corresponding to the permeate volume. Then, to continue ultrafiltration, the volume is completed.

[0158] The cartilage hydrolysate was processed in 2 batches:

[0159] Lot 1: 366 liters of processed hydrolysate Lot 2: 280 liters of processed hydrolysate

[0160] The resulting permeate contained approximately 10% minerals and 90% protein. The flow rate was approximately 50 l / h / m at the start of filtration 2 and 30 l / h / m 2 at the end.

[0161] The following characteristics of the collagen hydrolysate fraction from UF were obtained:

[0162]

Table 7

[0163] In this balance, the permeate composition is the average of different analyses weighted by the volume obtained at the time of analysis. The composition of the retentate was reproducible. More than half of the protein was in the permeate (collagen hydrolysate).

[0164] A total of 646 liters of cartilage hydrolysate was processed by UF at 2 h 41. The flow rate decreased over time: at the start of UF, it was between 216 L / h and 266 L / h of permeate per hour compared to 160 L / h at the end of UF (1 hour later); i.e., 49 L / h / m at the start 2 and at the end, it was 30 L / h / m 2 in terms of the flow rate.

[0165] Considering the obtained collagen yield (>50%), dialysis (without water addition) was necessary to optimize the collagen yield. Thus, the 10 kDa membrane enabled direct ultrafiltration without dialysis, which also means water conservation and indicates that the smaller surface area of the attached membrane can be operated efficiently to reduce costs.

[0166] Ultrafiltration (batch 2) To ensure excellent stirring, due to the amount of water added for hydrolysis, the dry matter content of the hydrolysate of batch 2 was quite low (about 5%). Therefore, before ultrafiltration, the hydrolysate was concentrated to be closer to the material balance value (14 - 15%). A value close to 11% / dry weight was obtained.

[0167] Processing volume: 263 liters concentrated at 10.69% The conditions and the membrane were the same as those for Batch 1. The permeate contained approximately 10% minerals and 90% protein, as for the first batch. The protein content of the retentate was stable over the processing time. Here, the flow rate was lower than during the ultrafiltration of the first batch of cartilage hydrolysate: the flow rate was approximately 23 L / h / m 2 at the start and 7 L / h / m 2 at the end. This difference was due to differences in hydrolysate concentration and viscosity because the second hydrolysate was pre-concentrated.

[0168] The following characteristics of the collagen hydrolysate fraction from UF were obtained.

[0169]

Table 8

[0170] The composition of the hydrolysate feed was estimated to be the same as that of the first batch. On the other hand, its dry matter was higher because the hydrolysate was pre-concentrated.

[0171] The ratio of protein in the permeate (collagen hydrolysate) is greater than 70%. This yield is better than that for the first batch. This can be explained by the fact that meat / cartilage separation may have been more effective in Batch 2.

[0172] Nanofiltration of collagen hydrolysate (batch 1) After the above ultrafiltration, nanofiltration is performed on the permeate obtained from ultrafiltration to remove the minerals present.

[0173] At the end of nanofiltration, the retentate is recycled to the feed tank to allow the liquid to pass through the membrane several times. Thereby, the retentate is concentrated while the permeate (salt) is continuously removed. After concentration, water is added and dialysis is performed to further improve the removal of soluble minerals. This process was carried out in a closed circuit for 12 hours to monitor the performance of the membrane over time.

[0174] To obtain a mineral content equivalent to that of collagen obtained from the head preserved in saline, this test was conducted on a sample of the first batch of collagen hydrolysate after adding salt.

[0175] The volume of the first collagen hydrolysate was 119 liters, which could be reduced to a volume of 40 liters by concentration / desalting (before dialysis). For dialysis, a total of 100 liters of water was added in different volumes (40 + 20 + 20 + 20L).

[0176] The protein content in the permeate was low, and the permeate mainly contained minerals (80%). The protein content increased slightly over time. The higher content at the end was due to long - term filtration (12 hours), which was intended to check the fouling rate of the membrane.

[0177] The mineral content in the retentate (collagen hydrolysate) decreased rapidly: it became half in 15 minutes. After 3 hours, it reached a dry mineral content of about 3%.

[0178] The initial flow rate was 78 l / h / m 2 It was. After 3 - fold concentration (FVC3), this increased to 42 l / h / m 2 After water addition, the flow rate returned to 74 l / h / m 2 In the closed - loop circuit, the flow rate after 12 hours was 38 l / h / m 2 The following characteristics of the permeate and retentate were observed:

[0179]

Table 9

[0180] The composition of the retentate corresponds to the final retentate to be analyzed.

[0181] The permeate composition is the average of the permeate composition during filtration, weighted by the associated volume. The mineral content of the permeate is probably overestimated. The protein content in the retentate is also probably overestimated. The total amount of protein and ash in the retentate and permeate is more than the initial amount in the solution before nanofiltration.

[0182] After correction, it was observed that only 10% of the protein was removed in the permeate. Less than 20% of the ash is in the retentate. Thus, nanofiltration made it possible to effectively separate the minerals from the collagen hydrolysate, and the demineralization was very effective. In the collagen hydrolysate, a mineral content of 3.8% was achieved, which is in line with market standards. It was also observed that nanofiltration and dialysis have a beneficial effect on the deodorization of collagen.

[0183] Deodorization and decolorization on a plate filter press After nanofiltration was completed, a part of the collagen hydrolysate was subjected to decolorization and deodorization using a plate filter press (activated carbon) as described above. It should be noted that the collagen obtained based on the raw materials of the present invention used remained more colored than the collagen fraction obtained from lighter fish skin. However, the color and odor were satisfactory and improved compared to the initial tests.

[0184] Concentration and drying Protein hydrolysate Various drying operations were performed on the spray drying tower. The spray drying device consists of a conical bottom and a tower equipped with a nozzle for spraying and a high-pressure pump. This spray drying device was constructed by TGE and has an evaporation capacity of 250 l / h.

[0185] The first batch of the protein hydrolyzate was dried directly (i.e., no further concentration was performed for the separation step). Due to the low dry matter content (15%), it was impossible to obtain sufficient pressure from the HP pump (high pressure) because of the nozzle arrangement and low viscosity. Since the first batch of the protein hydrolyzate had a high fat content (24% lipid content), the spray in the drying chamber was poor and became sticky in the tower. This problem could be solved by using an anti-caking agent.

[0186] The second batch of the protein hydrolyzate was pre-concentrated until evaporation and had a dry matter content of 33%. By driving decanter centrifugation at high temperature and hydrolyzing for a longer time, it became possible to extract a part of the oil in the form of fluid oil. Finally, the initial content of the skipjack tuna protein paste (used for this test batch) was lower than that of the bigeye tuna. Therefore, the drying process was better than that of the first batch, and very little sticking was observed on the tower.

[0187] The addition of the anti-caking agent improves the fluidity in the pneumatic conveyor and various rotary valves, leading to an improvement in the drying process of the protein hydrolyzate.

[0188] Collagen hydrolysate Due to the smaller volume, the deodorized collagen hydrolyzate was concentrated and dried on an Alfa Laval vacuum evaporator. Batch 1: 70 L of 25 / 30% brix (dry soluble matter), Batch 2: 53 L of 30% brix + 7 L of 10% brix. The concentrated collagen solution was dried on a Tecoma Gatedryer. The following amounts were obtained: Batch 1: 19 kg, Batch 2: 14 kg. The resulting powder was a powder ranging from bright white to ivory color.

[0189] Composition of the obtained hydrolysate

[0190] Properties of the protein (powder) hydrolysate

[0191] Nutritional composition

Table 10

[0192] The only differences between batches 2 and 3 are the type of antioxidant and the drying level (dry matter content) only. Hydrolysates 2 and 3 contain 72.6% protein and 12.3% fat in the dry matter. Its mineral content (12.8%) is higher than that in the hydrolysate of batch 1 (5%). There is no particular reason other than using the protein paste derived from the head of bluefin tuna for the first batch and yellowfin tuna for the second batch. It should be noted that only batch 1 was subjected to nanofiltration. The dried and analyzed samples of batches 2 and 3 were not demineralized and the raw materials were not frozen in brine, so the salt content was lower, but the final mineral content was considered relatively high. Therefore, the implementation of the nanofiltration step is recommended to reduce the overall mineral content of the protein hydrolysate.

[0193] Quality The quality of different batches of protein hydrolysate was tested.

[0194]

Table 11

[0195] An assay for biogenic amines, PCBs and heavy metals was initiated for the hydrolysate of batch 2.

[0196]

Table 12

[0197] The heavy metal levels complied with the regulations and only the mercury content was close to the limit.

[0198]

Table 13

[0199] The levels of PCB and dioxin comply with the European regulation threshold (2017 / 644).

[0200] Biogenic amines (mg / kg) The biogenic amine content was determined as follows (mg / kg of dry matter):

[0201]

Table 14

[0202] The biogenic amine levels were quite high. This was due to the fact that the raw materials were not very fresh.

[0203]

Table 15

[0204] The main amino acids in the protein hydrolysate are glutamic acid (13%), followed by glycine (9.6%) and aspartic acid (9%).

[0205] Fatty acid profile

Table 16

[0206] 31% of the fatty acids (11.2%) are omega 3. There are 4.1 grams of polyunsaturated fatty acids per 100 g of protein hydrolysate. The detailed fatty acid profile is provided in a separate table (batch 2).

[0207] Properties of the collagen hydrolysate (powder) Nutritional composition

[0208]

Table 17

[0209] The resulting collagen hydrolyzate contains 88% protein and 12% minerals on a dry basis.

[0210] After nanofiltration, the mineral content decreased to about 2 - 3% of the dry matter, and the protein content was expected to be >97% as this was obtained on the filtered sample.

[0211] Amino acid profile [Table 18]

[0212] The resulting hydroxyproline content was slightly lower than the average of marine collagen (8 - 8.5%). The glycine content was in line with the standard, and the proline content was slightly lower. Therefore, the type of raw material and the parameters of meat / cartilage separation are important points regarding collagen purity.

[0213] Quality [Table 19] [Table 20]

[0214] Molecular weight profile The protein size of the sample was estimated by HPLC analysis. The sample was diluted in an appropriate buffer and analyzed through a filtration gel column that enabled the molecules to be sorted by size: during the analysis, the largest molecules came out of the column first, and the smallest ones last. A detection device set at 214 nm was used to detect protein compounds. The column can be calibrated by using standard proteins. The following table shows the correspondence between the retention time in the column and the size of the protein / peptide of the collagen hydrolysate and protein hydrolysate produced.

[0215]

Table 21

[0216] The hydrolyzed collagen contains about 67% peptides <2 kDa. The hydrolyzed protein batches (CPSP1 - CPSP3) contain about 66 - 70% peptides <2 kDa. The profiles are very similar for the two batches, and batch 1 appears to be slightly more hydrolyzed.

[0217] Conclusion The above examples show that protein hydrolysates and collagen hydrolysates can be efficiently produced and purified by including ultrafiltration and nanofiltration steps to achieve a well - defined peptide distribution, low salt concentration, and otherwise excellent sensory properties. Due to the fact that the initial raw material was slightly degraded by inadequate or incomplete storage, a relatively high content of biogenic amines occurred. This problem would not occur with properly stored raw materials. Nevertheless, even based on sub - optimal raw materials, the composition of the hydrolysates was satisfactory.

[0218] This example shows that desalting by nanofiltration was effective with an acceptable permeate flow. Furthermore, despite the low quality of the raw material, it was possible to obtain collagen with little odor and partially decolorized.

[0219] Matricain Unconstrained by theory, MMP (matrix metalloproteinase) is very important for generating matricines from ECM (extracellular matrix) collagen components. Therefore, the collagen products of the present invention are expected to contain these valuable components. Exemplary matricines are arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, ramstatin, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endostatin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatins 7, neostatins 14, palmitoyl pentapeptide-4, the collagen hexapeptide GFOGER, and CLAC.

[0220] Separate table

[0221] [Table 22]

[0222] [Table 23]

[0223] [Table 24] <Appendix> Item 1 A method for extracting collagen from marine animal by-products, comprising: a) providing pieces of marine animal by-products, said by-product pieces comprising at least two components selected from the meat, skin, fish bones, fins and cartilage of said marine animal; b) separating said animal by-product pieces into at least two fractions, including a protein-rich fraction and a fraction containing skin and cartilage, said skin and cartilage fraction being essentially free of meat; c) optionally, comminuting the fraction containing skin and cartilage so as to produce a particle size preferably between 0.5 mm and 1 cm, more preferably between 1 mm and 4 mm; d) enzymatically hydrolyzing the fraction containing skin and cartilage to produce a procollagen hydrolysate, preferably performing said hydrolysis at a pH between 5 and 9; e) separating said procollagen hydrolysate into a lipid fraction, an aqueous collagen hydrolysate fraction free of solids, and a fraction containing solids, preferably performing said separation by three-phase decanter centrifugation; f) subjecting said aqueous collagen hydrolysate fraction to at least one filtration step using a membrane with a pore size of 2000 to 100,000 Da, preferably 4000 to 50,000 Da, more preferably about 10,000 Da, to produce a first permeate containing collagen hydrolysate and a retentate; g) demineralizing said first permeate by nanofiltration and / or dialysis using a membrane having a pore size of less than 1000 Da, preferably between about 150 and 300 Da, to produce a purified collagen solution; h) deodorizing the purified collagen solution of step g), preferably performing said deodorization... Item 2 A step of deodorizing the purified collagen solution in step g), preferably... ... ... ... ... ... 、 ... ... ... A method as described above. Item 2 A step of deodorizing the purified collagen solution in step g), preferably... The method according to claim 1, further comprising a step of deodorization using activated carbon and / or dialysis. Claim 3 A step of concentrating and sterilizing the purified collagen solution or the deodorized purified collagen solution, preferably, the concentrating and sterilizing are performed by evaporation such as direct steam injection The method according to claim 1 or 2, further comprising the step. Claim 4 A step of drying the purified collagen solution, the purified deodorized collagen solution, or a concentrate thereof to produce a collagen powder, preferably, the drying is performed by spray drying, freeze drying, lyophilization, or a nozzle sprayer, and most preferably, the drying is performed by continuous horizontal spray drying. The method according to any one of claims 1 to 3, further comprising the step. Claim 5 A method for producing a protein hydrolyzate from marine animal by-products, comprising: a) providing pieces of marine animal by-products, wherein the by-product pieces comprise at least two components selected from meat, skin, fish bones, fins, and cartilage of the marine animal; b) separating the by-product pieces into at least two fractions including a protein-rich fraction and a fraction containing skin and cartilage; c) mixing the protein-rich fraction with water and separating a lipid fraction, an aqueous fraction, and a protein-rich solid fraction, preferably, the separation is performed by three-phase decanter centrifugation; d) enzymatically hydrolyzing the protein-rich solid fraction to produce a protein hydrolyzate solution; e) separating the protein hydrolyzate solution into a second lipid fraction, an aqueous protein hydrolyzate fraction, and a second solid fraction, preferably, by three-phase decanter centrifugation; f) collecting the aqueous protein hydrolyzate. Claim 6 g) further comprising a step of demineralizing the aqueous protein hydrolyzate of step f) by filtration and / or dialysis using a membrane having a pore size of less than 1000 Da, preferably between 100 and 500 Da. The method according to claim 5. Claim 7 A step of concentrating and sterilizing the aqueous protein hydrolyzate or the demineralized aqueous protein hydrolyzate, preferably, the concentrating and sterilizing are performed by evaporation such as direct steam injection. The method according to claim 5 or 6, further comprising the step. Claim 8 ​ ​ ​ ​ 、 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The aqueous protein hydrolyzate, the demineralized aqueous protein hydrolyzate, or a step of drying the respective concentrates thereof to produce a protein powder, preferably, the drying is carried out by spray drying, freeze drying, lyophilization, or a nozzle sprayer and most preferably, the drying is carried out by continuous horizontal spray drying The method according to any one of items 5 to 7, further comprising. Item 9 c’) Collecting the aqueous fraction of step c), and subjecting this fraction to filtration and / or dialysis using a membrane having pore sizes of less than 1000 Da, preferably between 100 and 500 Da; c”) Before step d), adding the retentate of step c’) to the protein-rich solid fraction; The method according to any one of items 5 to 8, further comprising. Adding the retentate of step c’) to the protein-rich solid fraction before step d). The method according to any one of items 5 to 8, further comprising. Item 10 An integrated process for upcycling marine animal by-products, a) providing pieces of marine animal by-products, said by-product pieces comprising at least two components selected from the meat, skin, fish bones, fins and cartilage of said marine animal; b) separating the by-product pieces into at least two fractions comprising a protein-rich fraction and a fraction comprising skin and cartilage, said skin and cartilage fraction being essentially free of meat; c1) subjecting the protein-rich fraction to the steps of step c) and subsequent steps of any one of items 5 to 9; c2) subjecting the fraction comprising skin and cartilage to the steps of step c) and subsequent steps of any one of items 1 to 4; The process comprising. The protein-rich fraction is subjected to the steps of step c) of any one of items 5 to 9 and subsequent steps. The fraction containing skin and cartilage is subjected to the steps of step c) of any one of items 1 to 4 and subsequent steps. The process comprising. Item 11 The marine animal by-products are fish by-products, such as heads, viscera, fish bones, scraps and / or skins, and preferably, the marine by-products are tuna by-products. The method according to any one of items 1 to 10. Item 12 A purified collagen solution or collagen powder produced by the method according to any one of items 1 to 4, 10 or 11, said collagen solution or collagen powder containing at least one matrikine. Item 13 The at least one matrikine is arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lumstatin, Hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endotrophin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2 , restin 3, restin 4, endostatin, neostatine 7, neostatine 14, palmitoyl pentapeptide-4, collagen hexapeptide GFOGER, and CLAC , a collagen solution or powder according to claim 12, selected from the group consisting of . The collagen solution or powder according to claim 12, selected from the group consisting of hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endotrophin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatine 7, neostatine 14, palmitoyl pentapeptide-4, collagen hexapeptide GFOGER, and CLAC Claim 14 At least 55% of the peptides of the hydrolyzate or powder are <2 kDa, preferably at least about 60% of the peptides are <2 kDa, more preferably at least about 65% of the peptides Are <2 kDa, the collagen solution or powder according to claim 12 or 13 . Claim 15 A protein hydrolyzate or protein powder produced by the method according to any one of claims 5 to 11, wherein the hydrolyzate or powder contains at least 3% (by weight) of omega-3 lipids and at least 1% (by weight) of phospholipids, preferably the hydrolyzate or powder contains at least 2% (by weight) of monounsaturated lipids, at least 3% (by weight) of polyunsaturated lipids and at least 2.5% (by weight) of omega-3 lipids, a protein hydrolyzate or protein powder

Claims

1. A method for extracting collagen from marine animal by-products, comprising: a) providing pieces of marine animal by-products, said pieces of by-products comprising at least two components selected from the meat, skin, fish bones, fins and cartilage of said marine animal; b) separating said pieces of animal by-products into at least two fractions, including a protein-rich fraction and a fraction containing skin and cartilage, said fraction containing skin and cartilage containing less than 20% meat; d) enzymatically hydrolyzing said fraction containing skin and cartilage to produce a tropocollagen hydrolysate; e) separating said tropocollagen hydrolysate into a lipid fraction, an aqueous collagen hydrolysate fraction free of solids, and a fraction containing solids; f) subjecting said aqueous collagen hydrolysate fraction to at least one filtration step using a membrane with a pore size of 2,000 to 100,000 Da to produce a first permeate containing collagen hydrolysate and a retentate; g) demineralizing said first permeate by nanofiltration and / or dialysis using a membrane having a pore size of less than 1,000 Da to produce a purified collagen solution. A method as described above.

2. The method according to claim 1, further comprising deodorizing the purified collagen solution in step g).

3. The method according to claim 1 or 2, further comprising concentrating and sterilizing said purified collagen solution or said deodorized purified collagen solution.

4. The method according to any one of claims 1 to 3, further comprising drying said purified collagen solution or said purified deodorized collagen solution, or a concentrate thereof, to produce a collagen powder.

5. Between steps b) and d), c) A step of grinding the fraction containing the skin and cartilage so that a particle size between 0.5 mm and 1 cm is produced The method according to any one of claims 1 to 4, further comprising.

6. In step d), the method according to any one of claims 1 to 5, wherein the hydrolysis is carried out at a pH between 5 and 9.

7. In step e), the method according to any one of claims 1 to 6, wherein the separation is carried out by three-phase decanter centrifugation.

8. In step f), the method according to any one of claims 1 to 7, wherein the membrane has a pore size of 4000 to 50,000 Da.

9. In step g), the method according to any one of claims 1 to 8, wherein the membrane has a pore size of 150 to 350 Da.

10. An integrated process for upcycling marine animal by-products, a) A step of providing marine animal by-product pieces, wherein the by-product pieces contain at least two components selected from the meat, skin, fish bones, fins and cartilage of the marine animal, and the step, b) A step of separating the by-product pieces into at least two fractions including a protein-rich fraction and a fraction containing skin and cartilage, wherein the fraction containing skin and cartilage contains less than 20% meat, and the step, c) A step of subjecting the fraction containing skin and cartilage to the method according to any one of claims 1 to 9, and including, (i) A step of mixing the protein-rich fraction and water and separating a lipid fraction, an aqueous fraction, and a protein-rich solid fraction, (ii) A step of enzymatically hydrolyzing the protein-rich solid fraction to produce a protein hydrolysate solution, (iii) A step of separating the protein hydrolysate solution into a second lipid fraction, an aqueous protein hydrolysate fraction, and a second solid fraction, (iv) collecting the aqueous protein hydrolysate; A process further comprising.

11. (v) using a membrane having a pore size between less than 1000 Da, and demineralizing the aqueous protein hydrolysate of step (iv) by filtration and / or dialysis The process according to claim 10, further comprising.

12. The process according to claim 10 or 11, further comprising concentrating and sterilizing the aqueous protein hydrolysate or the demineralized aqueous protein hydrolysate.

13. The process according to any one of claims 10 to 12, further comprising drying the aqueous protein hydrolysate, the demineralized aqueous protein hydrolysate, or their respective concentrates to produce a protein powder.

14. The process according to any one of claims 10 to 13, wherein in step (i), the separation is carried out by three-phase decanter centrifugation.

15. The process according to any one of claims 10 to 14, wherein in step (iii), the separation is carried out by three-phase decanter centrifugation.

16. The method or process according to any one of claims 1 to 15, wherein the marine animal by-product is a fish by-product.

17. A collagen solution or collagen powder produced by the method or process according to any one of claims 1 to 16, at least 50% of the peptides of the hydrolysate or powder are <2 kDa, the collagen hydrolysate contains less than 1% of peptides larger than 35 kDa and less than 5% of peptides larger than 10 kDa, and the residual salt content is less than 4% by dry weight, a collagen solution or powder.

18. A protein hydrolyzate or protein powder produced by the process according to any one of claims 10 to 16, wherein the hydrolyzate or powder contains at least 3% (by weight) of omega-3 lipids and at least 1% (by weight) of phospholipids, the residual salt content is less than 10% by dry weight, the total lipid content is less than 20% by dry weight, and the protein content is more than 60% by dry weight, the protein powder is a protein hydrolyzate or protein powder having a total mineral content of 6 to 10% and a chlorine content of 2 to 4% at a residual moisture content of 2 to 4%.

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