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 cosmetic products from marine animal by-products. [Background technology]

[0002] Introduction Large amounts of waste and by-products suitable for further use are generated in the food production chain. These animal by-products (ABP) are defined as animal carcasses, carcass parts and other products of animal origin that are not intended for human consumption. This includes fish, shellfish and other aquatic products, both cooked and uncooked, 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 streams and there are strict regulations that apply to the disposal of these wastes.

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

[0004] The waste industry base and associated disposal costs in the Western world vary between countries, but the option of landfilling ABP is becoming more unfeasible in many countries. In some cases, landfilling ABP is even prohibited, and in others, taxes have been introduced or even possible fines for exceeding quota limits, as in the UK, increasing costs. Many techniques exist for the treatment of ABP, including rendering and incineration, and for certain categories of ABP, composting and biogas production. One technique for ABP recycling is composting in bins, which has the advantage of avoiding the need for landfilling while producing safe and valuable compost for use as a soil conditioner and fertilizer. 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 the valuable biological resource of ABP unutilized. Furthermore, the residual salt content and other undesirable impurities of the low-level products these methods produce continue to be problematic, especially with regard to marine ABP.

[0005] Collagen is the main component of connective tissue and the most abundant fibrous protein in vertebrates, constituting about 30% of the total protein in vertebrates. Collagen has a wide range of applications in leather, film industry, medicine, cosmetic and biopharmaceutical materials, and food. Collagen has been widely used in tissue engineering biomaterials in the last two decades due to its properties that mimic human proteins, and can be used in treating pain associated with hypertension, urinary incontinence and osteoarthritis, in tissue engineering for implants in humans, and in inhibiting vasculogenic diseases such as diabetic complications, obesity and arthritis.

[0006] About 30% of fish waste is skin and bones, which are very rich in collagen. Over 100 million tons of fish are caught worldwide annually, and one-third of the total catch is used for fishmeal and animal feed. Therefore, extraction of collagen from fish bones is a promising means to obtain value-added products and reduce environmental pollution.

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

[0008] Small-scale studies for collagen extraction 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, MM et al. Int. Food. Red. J. 23(3): 913-922 (2016)). However, these small-scale laboratory methods cannot be scaled up in an economical manner, require less complex source materials (skins), and do not utilize other valuable components of the starting product. Thus, these methods have not matured for industrial-scale valorization of the typically complex marine ABPs, which contain skins, meat, fish bones, fins, and cartilage. Summary of the Invention

[0009] Thus, there remains a need for industrial methods that utilize marine ABP to produce high quality raw products from marine ABP that can be used in the cosmetic industry, for example, to prevent the need to landfill or dispose of these products.Furthermore, there remains a need for methods that can utilize complex ABP starting materials and economically produce collagen and other products that are sufficiently pure for a variety of further applications.

[0010] It is one object of the present invention to provide a large scale valorization method and process starting from marine animal by-products to produce relatively high quality products for human and animal use. It is a particular object of the present invention to provide high quality raw products, in particular collagen, for use in the cosmetic industry.

[0011] The present invention fulfills this need by providing methods to prevent the presence of undesirable spoilage components in the resulting products obtained from marine ABPs. These methods are capable of handling complex starting materials on a large scale.

[0012] Summary of the Invention Products made from marine by-products play an increasingly important role not only in providing nutritious dietary ingredients for pigs, poultry and aquaculture, but also in the expanding pet food market, where superior quality and high value ingredients are used to produce pellets, pouches and treats. Freshness of the raw materials is important as it affects the protein quality in the final product. Deterioration of fish and its products occurs mainly due to enzymatic and bacterial action and begins as soon as the fish die. Hydrolyzed collagen and protein products produced by prior art methods often contain high levels of monovalent ions and biogenic amines, which are toxic in increasing concentrations and produce bad tastes and odors in the final product and are therefore problematic. The present invention is based on the discovery that the use of a specific filtration step in the process of producing collagen hydrolysate and / or protein hydrolysate results in a very superior quality final product, even when starting from lower quality starting materials. The present invention achieves these goals by providing a large-scale method for valorizing industrial quantities of marine raw materials.

[0013] In a first aspect, the present invention provides a method for extracting collagen from marine animal by-products, comprising the steps of: (a) providing marine animal by-product pieces, preferably less than 5 cm in size, more preferably between 1-2 cm in size, the by-product pieces comprising at least two components selected from marine animal meat, skin, fish bones, fins and cartilage; (b) separating the animal by-product pieces into at least two fractions comprising a protein rich fraction and a fraction comprising skin and cartilage, the skin and cartilage fraction being essentially meat free; (c) optionally grinding the fraction comprising skin and cartilage to produce a particle size preferably between 0.5 mm and 1 cm, more preferably between 1 mm and 4 mm; and (d) enzymatically hydrolyzing the fraction comprising skin and cartilage to produce a raw collagen hydrolysate (raw collagen hydrolysate). (e) separating the raw collagen hydrolysate into a lipid fraction, a solid-free aqueous collagen hydrolysate fraction and a solid-containing fraction, 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 2000-100,000 Da, preferably 4000-50,000 Da, more preferably about 10,000 Da to produce a first permeate and a retentate containing collagen hydrolysate; and (g) demineralizing the first permeate by nanofiltration and / or dialysis using a membrane with a pore size of less than 1000 Da, preferably between about 150-300 Da to produce a purified collagen solution. The present invention further relates to a method as described above, further comprising the step of deodorizing the purified collagen solution of step (g), preferably by means of activated carbon and / or dialysis.The present invention further relates to a method as described above, further comprising the steps of concentrating and sterilizing the purified collagen solution or the deodorized purified collagen solution, preferably wherein the concentration and sterilization are performed by evaporation, such as direct steam injection.The present invention further relates to a method as described above, further comprising the steps of drying the purified collagen solution or the purified deodorized collagen solution or a concentrate thereof to produce a collagen powder, preferably wherein the drying is performed by spray drying, freeze drying, lyophilization, nozzle atomizer, most preferably wherein the drying is performed by continuous horizontal spray drying.

[0014] In another embodiment, the present invention relates to a method for producing a protein hydrolysate from a marine animal by-product comprising: (a) providing marine animal by-product pieces, the by-product pieces comprising at least two components selected from marine animal meat, skin, fish bones, fins and cartilage; (b) separating the by-product pieces into at least two fractions comprising a protein rich fraction and a fraction comprising 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 produce 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; and (f) collecting the aqueous protein hydrolysate. The present invention further relates to a method as described above, further comprising the step of (g) 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 100-500 Da or 150-300 Da. The present invention further relates to a method as described above, further comprising the step of concentrating and sterilizing the aqueous protein hydrolysate or the demineralized aqueous protein hydrolysate, preferably wherein the concentration and sterilization are performed by evaporation, such as direct steam injection. The present invention further relates to a method as described above, further comprising the step of drying the aqueous protein hydrolysate, the demineralized aqueous protein hydrolysate, or the respective concentrates thereof to produce a protein powder, preferably wherein the drying is performed by spray drying, freeze drying, lyophilization, nozzle atomizer, most preferably wherein the drying is performed by continuous horizontal spray drying.The present invention further relates to a method as described above, further comprising the steps of: (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 above-mentioned methods. In one embodiment of this aspect, the integrated process includes the steps of: (a) providing marine animal by-product pieces, the by-product pieces comprising at least two components selected from marine animal meat, skin, fish bones, fins and cartilage; (b) separating the by-product pieces into at least two fractions comprising a protein-rich fraction and a fraction comprising skin and cartilage, the fraction comprising skin and cartilage being essentially meat-free; (c1) subjecting the protein-rich fraction to step (c) and subsequent steps of the method for producing a protein hydrolysate as described above; and (c2) subjecting the fraction comprising skin and cartilage to step (c) and subsequent steps of the method for extracting collagen as described above. Further variations and refinements of the method for producing a protein hydrolysate and the method for extracting collagen may be fully applicable in the context of the integrated process of this aspect of the present invention.

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

[0017] The above-mentioned method produces a collagen solution and / or collagen powder. It has been found that the collagen produced by the above-mentioned method is of very high quality. Thus, the present invention relates to a purified collagen solution or collagen powder produced by the method detailed above. In a particular embodiment, the collagen solution and / or 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, lamstatin, hexastatin 1, hexastatin 2, NC1 domain of collagen IV or XIX, endotropin, ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin 7, neostatin 14, palmitoyl pentapeptide-4, collagen hexapeptide GFOGER, and CLAC. In particular, the invention relates to a collagen solution or powder produced by the method of the 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 invention is characterized by a residual salt content of 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 protein powder produced by the method of the present invention.In one embodiment, the protein hydrolysate or protein powder comprises at least 3% (by weight) omega-3 lipids and at least 1% (by weight) phospholipids, preferably the hydrolysate or powder comprises at least 2% (by weight) monosaturated lipids, at least 3% (by weight) polysaturated lipids and at least 2.5% (by weight) omega-3 lipids.In one embodiment, the purified protein hydrolysate or powder is characterized by a residual salt content of less than 20% by dry weight, preferably less than 15%, 10%, or 5%, a lipid content of less than 20% by dry weight, preferably less than 15%, 12%, or 10%, and a protein content of 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 medicinal, nutritional and cosmetic purposes. [Brief description of the drawings]

[0020] [Figure 1A] FIG. 1 shows the balance of protein and cartilage fractions obtained from raw material (tuna heads) from different batches of raw material. [Figure 1B] FIG. 1 shows the balance of protein and cartilage fractions obtained from raw material (tuna heads) from different batches of raw material. [Diagram 2] A: HPLC analysis of the produced collagen hydrolysate (batch 1), 214 nm spectrogram using a Biosep SEC 2000 gel filtration column (phosphate buffer 0.1M + 0.5% SDS, analytical flow = 1 mL.min-1). B: Peak analysis table. C: Correspondence table between retention time and molecular weight. [Diagram 3]A: HPLC analysis of the produced collagen hydrolysate (batch 2), 214 nm spectrogram using a Biosep SEC 2000 gel filtration column (phosphate buffer 0.1M + 0.5% SDS, analytical flow = 1 mL.min-1). B: Peak analysis table. C: Correspondence table between retention time and molecular weight. [Figure 4] A: HPLC analysis of produced protein hydrolysate (CPSP) (batch 2), 214 nm spectrogram using Biosep SEC 2000 gel filtration column (phosphate buffer 0.1 M + 0.5% SDS, analytical flow = 1 mL.min-1). B: Peak analysis table. C: Correspondence table between retention time and molecular weight. [Diagram 5] FIG. 1 shows a flow diagram of the integration of the processing of different by-products to produce protein hydrolysates and collagen hydrolysates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In describing the steps (a)...(x) in the methods of the invention, the steps must be performed in the order of appearance. Preferably, the methods do not include other procedural steps than those explicitly stated. 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 listed methods. The methods described herein are industrial methods, i.e. large-scale methods. These methods are carried out on at least hundreds of kilograms of starting material and can be carried out to process several tons of starting material. The skilled person will recognize that operations of this scale require respective instrumentation and the methods of the invention are not necessarily scaled down to small-scale (laboratory-scale) methods, which, as is self-evident, 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 not scalable at all, or are not scalable in a way that makes economic sense.

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

[0023] The term "marine animal by-products" refers to by-products in the form of heads, viscera, skeletons, skins and other such as tails, fins, scales, mince, blood etc. resulting from the processing of fish for human consumption. This material may constitute up to 70% of the fish and shellfish after processing, with the yield of fish fillets being species dependent and often ranging from 30-50% of the fish. The waste material resulting from processing is in fact a valuable raw material from which fishmeal and fish oil can be produced, a material that is still underutilized. The main sources of by-products are from finfish and whitefish trimmings (pollock, cod, hake, haddock etc.) as well as salmon (wild and farmed), tuna, herring, mackerel and may be obtained from wild or farmed fish processing. The quality of the raw material is often measured by the total volatile base nitrogen content, which must be in the region of 50 mg N / 100 g raw material. The preferred source of marine animal by-products is from fish, especially tuna, such as yellowfin tuna (Thunnus albacares) or skipjack tuna (Katsuwonus pelamis).

[0024] The method of the present invention for extracting collagen includes the steps of: (a) providing marine animal by-product pieces; (b) separating the animal by-product pieces 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 collagen raw hydrolysate; (e) separating the collagen raw hydrolysate into a lipid fraction, a solids-free aqueous collagen hydrolysate fraction, and a solids-containing fraction; (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; and (g) demineralizing the first permeate by nanofiltration and / or dialysis using a membrane having a pore size of less than 1000 Da 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 the material. Notably, this size of the pieces does not have to be exactly as indicated, and the hashed or macerated material may exhibit pieces of a diverse size distribution. That is, a size less than X cm refers to a macerated raw material in which the majority of the pieces are smaller than X and which does not exhibit pieces larger than 5 cm. A size between X cm and Y cm indicates that the average particle size must be within this range, but still larger and smaller pieces may be present.

[0026] Marine by-products and pieces produced therefrom will typically include at least two components selected from marine animal flesh, skin, fish bones, fins and cartilage, however, for the method of producing collagen, it is sufficient that the material contains skin and / or fish bones and / or fins.

[0027] In the separation step (b), the separation may be carried out in any suitable separator. Exemplary separators include separation equipment 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 routinely operated with parameters (pressure, grill / mesh size) such as to produce an essentially meat-free skin and cartilage fraction. Parameters such as screen porosity, pressure applied to the screen and feedstock flow rate depend on the type of separator used and are well described by the manufacturer.

[0028] A product "essentially free" of other ingredients or essentially free of other ingredients in the sense of the present invention describes a product that contains less than 20% of other ingredient(s), preferably less than 15% or 10%, in particular less than 5, 4, 3, 2, or 1% of other ingredient(s). That is to say, a fraction essentially free of meat contains less than 20% meat, preferably less than 15% or 10%, in particular less than 5, 4, 3, 2, or 1% of meat. A protein-rich fraction 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% of bone and cartilage.

[0029] "Consisting essentially of" in the sense of the present invention describes a product that contains more than 80%, preferably more than 85 or 90%, in particular more than 95, 96, 97, 98 or 99% of the specified ingredients. It shall be understood that the protein-rich fraction that is essentially free of bone and cartilage may be a fraction that consists essentially of meat.

[0030] Thus, in step (b), the separator is operated according to the manufacturer's specifications with parameters that result in two fractions, a protein-rich fraction (which may be further used as described herein below) and a fraction that essentially contains only collagen-containing components such as skin, cartilage, fish bones, fins, etc. This separation step is important to the present invention, since the quality of the collagen product produced is improved when the skin- and cartilage-containing fraction is essentially meat-free. Furthermore, an additional washing step of the skin- and cartilage-containing fraction (with associated loss of material) can be avoided.

[0031] In optional step (c) of the method of the invention, the particle size of the hide and cartilage fraction may be further reduced to yield particle sizes 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. As indicated above in relation to 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 still be present. The advantage of further reducing the size of the cartilage and hide particles is that the subsequent hydrolysis step (d) requires less time to complete and the yield of collagen hydrolysate is improved.

[0032] Step (d) of enzymatic hydrolysis of the (optionally ground) fraction containing the skin and cartilage results in a collagen raw hydrolysate. The raw hydrolysate contains hydrolyzed collagen, lipids, and other organic materials and ash (solids). The enzymes used in this step are proteases that can be used alone or in mixtures. Essentially, the proteases used have cleavage motifs present in the amino acid sequence of collagen (types I, II, III, IV and / or V) and cleave the collagen into smaller peptides, as is the case for example with bacterial alkaline proteases. Preferred enzyme classes are serine endopeptidases, exopeptidases, di- and tripeptidases and carboxypeptidases, as well as metalloproteases.

[0033] The source of protease is not important, and plant proteases as well as bacterial or animal-derived proteases 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] Usually, hydrolysis is carried out at a pH between 5 and 10, more preferably at a pH between 6 and 9, or at a pH between 6 and 8. However, each enzyme has its own pH range in which it can operate. Therefore, the enzymatic hydrolysis conditions are followed according to the recommendations of the enzyme supplier. By way of example, Protease Plus (Dyadic) is a bacterial alkaline protease produced by 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 a pH stability between 6.0 and 10.0, can operate effectively in a pH range of pH 7.0 to 10.0, and has an optimum pH range of pH 9.0 to 10.0. Papain has an optimum pH for activity of 6.0 to 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 the enzymatic hydrolysis depends on the enzyme or enzyme mixture used as well as the pH conditions. Usually 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 a temperature stability up to 70°C, an effective temperature range up to 65°C, with optimal activity being obtained at 60°C. Papain has a temperature optimum for activity of 65°C.

[0036] The exact amount of enzyme to be added depends on the process and protein substrate concentration, the degree of protein hydrolysis desired, pH, temperature and time. The dosage is calculated as % per weight of protein to be hydrolyzed. Typical dosage levels are 0.2% to 2%, or 0.3% to 1% of kg of protein contained in the material to be treated. The dosage may naturally be adjusted accordingly to meet economical considerations (e.g. increasing the amount of enzyme to decrease the processing time, or decreasing the amount of enzyme if processing time is not an issue) depending on the type of enzyme (mixture) used. By way of example, for Promod 950 L (Biocatalyst), the amount of enzyme added to the skin and cartilage containing fraction is about 0.3% to about 0.7%, preferably about 0.4% to 0.6% or about 0.5% ± 0.05%. Those skilled in the art can easily control and adjust the parameters of the hydrolysis step depending on the nature of the protease (mixture) used and / or the starting material. If hydrolysis proceeds too slowly, enzyme may be replenished as necessary.

[0037] The duration of the enzymatic hydrolysis depends on the activity and amount of enzyme used. Since maintaining a given temperature for hydrolysis requires a significant amount of energy, the hydrolysis time is selected not to exceed 10 hours. To achieve good extraction, the hydrolysis time should also not be shorter than about 1 hour. In general, the provision of smaller particles for the skin and cartilage fractions allows shorter hydrolysis times, as shown above. For example, when using Protease Plus for well-macerated material, hydrolysis takes place within about 3 to 4 hours, and when using Promod 950 L (Biocatalyst), the hydrolysis time is about 5 hours.

[0038] Thus, 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 by refractometer (brix). In addition, the rate of hydrolysis may be determined by analytical methods, such as the Sorensen method and also the 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 review in Silvestre, Food Chemistry, Volume 60, Issue 2, October 1997, pages 263-271). These methods allow the control of the cleavage of amino acids, which affects the pH, allowing, if necessary, the adjustment of the pH with NaOH or acid to operate the hydrolysis at the optimum pH.

[0040] The enzymatic hydrolysis produces a liquid phase, a lipid phase and a solid phase, which are enriched in peptides derived from collagen hydrolysis. These phases are separated in step (e) of the method of the present invention. Any suitable technique or combination of techniques may be used to separate the lipid, aqueous and solid phases. 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, exemplary devices include, but are not limited to, Flottweg Z series (for example Z3E, Z5E, Z6E, Z4 or Z35), GEA three-phase decanters (for example 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 bowl) depend on the device used and are operated according to the operating method as suggested by the device manufacturer.

[0041] The separation step (e) results in three fractions: (i) a lipid fraction (fish oil) that can be collected and used for a variety of 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 with a pore size of 2000 to 100,000 Da, preferably 4000 to 50,000 Da, resulting in a first permeate and a retentate containing the 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 to 12,000 Da, for example 10,000 Da (10 kDa). The pore size of this ultrafiltration step makes it possible to filter the collagen hydrolysate with respect to large proteins that have not undergone hydrolysis, as well as residual fat and lipid vesicles. The fraction containing the hydrolyzed collagen (collagen peptides) is the permeate.

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

[0044] Exemplary membranes that may be used include, but are not limited to, GE, KOCH, SYNDER, TIA, PALL, TAMI, MICRODYN NADIR (NADIR, SPIRA-CEL (spiral wound 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 1x5m supplied by TIA, with a permeate capacity of 200-500 l / hour. 2 Includes ultrafiltration pilot with Organique UF and OI.

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

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

[0048] The collagen hydrolysate (first permeate) resulting from step (f) is then subjected to a nanofiltration step (g). Alternatively, dialysis may be performed. The collagen hydrolysate solution may be used as is or, if necessary, may be further concentrated (e.g. for dialysis, the concentrated first permeate is preferred). The nanofiltration / dialysis step demineralizes the first permeate by nanofiltration and / or dialysis using membranes with a pore size of less than 1000 Da to yield a purified collagen solution. Preferably, the membranes used for nanofiltration or dialysis have a pore size of less than about 500 Da, preferably between about 100 and 400 Da, particularly preferably between 150 and 350 Da. By way of example, Lenntech organic nanofiltration membranes 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 small proteins while allowing aqueous monovalent ions (e.g. sodium, chloride) and optionally divalent ions (e.g. magnesium, nitrate) as well as other small molecules (e.g. biogenic amines, heavy metals, etc.) to pass through the membrane.

[0050] Depending on the clarity, color and odor of the collagen hydrolysate (permeate) of step (g), the resulting collagen solution may be subjected to a further optional step of deodorization and / or dialysis. Thus, the present invention further relates to a method as described above, further comprising a step of deodorizing the purified collagen solution of step (h) using activated carbon and / or dialysis. The use of activated carbon effectively removes residual small molecules that may be responsible for the slight odor of the collagen hydrolysate solution, thus further improving the quality of the final product. When using dialysis, the effect of repeatedly adding water also has the effect of washing the collagen solution, further reducing undesirable components. This measure also improves the removal of salts from the collagen solution. Dialysis may be performed with or without subsequent filtration of the retentate (collagen solution) on activated carbon. Alternatively, the collagen solution may first be filtered with activated carbon and then subjected to dialysis.

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

[0052] It has been observed by the inventors that nanofiltration and dialysis have a good effect on collagen deodorization. Therefore, by using these additional steps, a better collagen solution / powder can be produced.

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

[0054] In the sense of the present invention, the expression "protein rich" refers to proteins derived from meat in the context of the present invention. Moreover, 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 carried out using routine methods and equipment. Preferably, the separation is carried out by a three-phase decanter centrifugation, as described above for the method of producing collagen hydrolysate. In particular, the parameters of operating the three-phase decanter are adapted as necessary for the protein-rich fraction, which are described in the manufacturer's operating instructions for the respective equipment 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 results in 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 collagen hydrolysate. However, the proteins of the protein-rich fraction are more accessible to hydrolysis. Thus, the incubation time of the 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 raw protein hydrolysate is subjected to a separation step (e) in which the raw protein hydrolysate is separated into (i) a (second) lipid fraction, (ii) an aqueous protein hydrolysate fraction, and (iii) a (second) solid fraction. As indicated above, any suitable means for achieving this separation may be used. Preferably, the separation is carried out by a further three-phase decanter centrifugation as described above, adapting the decanter parameters to the softer type of material fed.

[0058] (f) The aqueous protein hydrolysate is then collected (step (f)) for storage or further processing as described below. One (optional) further step to improve the quality of the protein hydrolysate is to carry out a demineralization (demineralization) step (g). As described above for collagen hydrolysates, demineralization may be carried out by nanofiltration and / or dialysis using membranes 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. type of membrane) applied for nanofiltration of collagen hydrolysates apply fully to the demineralization of protein hydrolysates.

[0059] The present invention further relates to a method as described above, further comprising a step of concentrating and sterilizing the aqueous protein hydrolysate or the demineralized aqueous protein hydrolysate, preferably by evaporation, for example by direct steam injection as described above.Furthermore, the method for producing a protein hydrolysate may further comprise a step of drying the aqueous protein hydrolysate, the demineralized aqueous protein hydrolysate or their respective concentrates to produce a protein hydrolysate powder.In one embodiment, the drying is carried out by spray drying, freeze drying, lyophilization or using a nozzle atomizer as described above.Most preferably, the drying is carried out by continuous horizontal spray drying, as described in the method for producing a collagen hydrolysate. The present invention further relates to a method for producing a protein hydrolysate as described above, further comprising the steps of: (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 (most preferably between 150 and 300 Da) and (c") adding the retentate of step (c') to the protein-rich solid fraction prior to step (d). These further steps improve the yield of the protein hydrolysate.

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

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

[0062] The above-mentioned method produces a collagen (hydrolysate) solution and / or a collagen (hydrolysate) powder. The collagen hydrolysate produced by the above-mentioned method is found to be of very high quality. Thus, the present invention relates to a purified collagen (hydrolysate) solution or collagen (hydrolysate) powder produced by the method of the present invention. In one embodiment, the collagen (hydrolysate) solution and / or collagen (hydrolysate) powder comprises at least one matrikine. The at least one matrikine of the collagen (hydrolysate) solution and / or powder may be any one or more of arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lamstatin, hexastatin 1, hexastatin 2, NC1 domain of collagen IV or XIX, endotropin, ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin 7, neostatin 14, palmitoyl pentapeptide-4, 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 invention relates to a collagen (hydrolysate) solution or powder produced by the method of the 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 (hydrolysate) solution or powder of the invention is characterized in that it has a residual salt content of 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 comprises at least 3% (by weight) omega-3 lipids and at least 1% (by weight) phospholipids, preferably the hydrolysate or powder comprises at least 2% (by weight) monosaturated lipids, at least 3% (by weight) polysaturated lipids and at least 2.5% (by weight) omega-3 lipids. In one embodiment, the purified protein hydrolysate or powder is characterized by a residual salt content of less than 15% by dry weight, preferably less than 10%, 7%, 6%, 5% or 4%, a lipid content of less than 20% by dry weight, preferably less than 15%, 12% or 10%, and a protein content of more than 60% by dry weight, preferably more than 70%, 75% or 80%. In particular, the protein hydrolysate end product contains about 17-18% lipids in the hydrolysate in the powder, along with about 3.7% phospholipids and about 2% omega-3 lipids.

[0065] A typical protein powder end product produced by the method of the present invention has between 70-80% protein, 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% chloride) with a residual moisture content of about 2-4%. The product may be referred to as Fish Protein Hydrolysate Powder or FPHP. By way of example, an FPHP end product has a total lipid content of about 73-76% total protein, 13-16% total lipid (2.5%-3.5% phospholipids), and a total mineral content of 7-9% (about 2.5%-3.5% chloride). All percentage values ​​in this paragraph are by weight of the end product.

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

[0067] The collagen and protein hydrolysates produced by the methods of the present invention exhibit a specific and distinctive peptide size distribution, as described below.

[0068] In particular, the collagen hydrolysate of the present invention contains about 12-18% peptides with a size of 4-10 kDa, about 60-70% peptides with a size of 1-4 kDa, about 12-18% peptides with a size of 0.5-1 kDa, and about 3-7% peptides with a size of less than 0.5 kDa. The collagen hydrolysate of the present invention contains less than 1% and preferably essentially no peptides larger than 35 kDa, and less than 5%, preferably less than 2%, and more preferably essentially no peptides larger than 10 kDa. The relatively high content of small size peptides has the advantage of reproducibility, enhanced biological properties and higher digestibility.

[0069] The heavy metal and PCB / dioxin contents are below the threshold values ​​defined in the European Union and therefore the collagen hydrolysate is suitable for human and animal nutrition and / or cosmetology.

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

[0071] The fatty acid profile of the protein hydrolysates 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 content are below the thresholds defined in the European Union, therefore the protein hydrolysate is suitable for human and animal nutrition.

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

[0075] Usefulness and industrial applicability

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

[0077] Collagen injections can improve the contour of the skin. Fillers containing collagen can be used cosmetically to remove lines and wrinkles from the face. Collagen can also improve scarring and aid 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 fluids such as urine or sweat, granulated wounds, necrotic or decaying wounds, partial and full thickness wounds, second degree burns, and areas of skin transplantation and skin grafting. Collagen-based membranes have also been used in periodontal and transplantation therapy. In oral surgery, collagen barriers prevent rapidly proliferating cells from migrating into dental wounds, thereby preserving space where dental cells have a chance to regenerate. Donor-derived collagen tissue grafts have been used in peripheral nerve regeneration, artificial blood vessels, and arterial reconstruction. Collagen supplements or formulations can be used to treat osteoarthritis. Collagen can reduce pain symptoms and improve joint function in osteoarthritis patients. For cosmetic uses, collagen can be used for skin revitalization.

[0078] Due to the fact that collagen hydrolysate mainly contains smaller peptides, it can be consumed in the form of food supplements or functional foods or beverages to support joint and bone health and promote skin health.Hydrolyzed collagen, such as that resulting from the method of the present invention, has a much smaller molecular weight compared to natural collagen or gelatin.More than 90% of hydrolyzed collagen is digested within 1 hour and is available as small molecule peptides in the bloodstream.Peptides are transported from blood to target tissues (e.g. skin, bone and cartilage), where they act as building blocks for local cells and help boost the production of new collagen fibers.

[0079] definition

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

[0081] "Solids" are components that undergo sedimentation in their respective solutions and / or can be removed from solution by filtration through a sieve with a mesh size of 0.5 mm or by centrifugation at g-forces of up to 20 x g.

[0082] The term "ultrafiltration" is used to refer to filtration through a semi-permeable membrane in which high molecular weight suspended solids and solutes are retained in a 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 similarly to microfiltration, except that nanofiltration retains molecules with molecular weights between 100 Da and 1000 Da and requires higher pressures inside the membrane.

[0084] The term "pore size" in relation to ultrafiltration and nanofiltration describes the molecular weight cut-off of the respective membrane or filter, e.g., a pore size of 300 Da will retain peptides and other solutes with a MW of >300 Da, while allowing peptides and amino acids and other solutes with a MW of <300 Da to pass through the membrane.

[0085] "Filtration membrane" refers to sheets of membrane as well as other configurations of materials (filter / membrane stacks, ceramic cylinders, etc.) that perform the function of filtration.

[0086] A "permeate" is the solution that exits or passes through a given filter or membrane in a filtration step. Correspondingly, a "retentate" is the fraction that does not pass through a given filtration membrane at the end of the filtration; the retentate may be a solution or a solid fraction, and if 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 originated.

[0087] The terms "demineralizing" as well as "desalting" refer to the removal of salts (such as, 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 molecule components by diffusion through a semipermeable membrane by adding water and pushing 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. Solutions processed by dialysis are also diluted, while solutions subjected to nanofiltration are more concentrated.

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

[0090] The term "deodorizing" in the context of the present invention denotes a process that removes odor-causing components from a composition.

[0091] The term "concentrate" in the context of the present invention denotes an increase in the dry matter of a solution by 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 such an 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 is a solution that contains no organic solvent or less than 10% or less than 5% organic solvent, ie, a solution that is essentially based on water.

[0094] Extracellular proteins and glycosaminoglycans (GAGs) can undergo limited enzymatic cleavage, usually resulting in the release of fragments that exert 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 matrikines. These fragments control many physiopathological processes, including angiogenesis, cancer, fibrosis, inflammation, neurodegenerative diseases, and wound healing. Exemplary matrikins include arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lamstatin, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endotropin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin 7, neostatin 14, palmitoyl pentapeptide-4, 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 certain 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 a cannery), protein dough, charge water and cartilage and skin residues (from the oil recovery process from tuna heads).

[0098] raw materials The study was carried out using whole tuna heads from Seychelles and the Indian Ocean. The heads were packaged in cardboard pallets and frozen in cold storage before processing. The tuna heads from Seychelles were derived from tuna that was not stored in salt water and frozen, resulting in significant differences in composition and therefore salt content.

[0099] The heads were then processed on an AM2C Barracuda Beluga 1830 separator to produce a protein dough and cartilage residue.

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

[0101] Raw material characteristics: Upon receipt, some heads were not frozen, the majority were albacore. Random temperature controls produced values ​​between -15°C and 2.7°C. Yellowfin heads were frozen and glued together, and mallets and crowbars had to be used to perform crush testing and quality control sampling. Albacore heads were mostly frozen at the outer edge of the pallets, with internal temperatures ranging from -3.5°C to 2.7°C. One pallet had blood running out. According to the supplier, all heads had been placed in a freezer. Albacore heads were smaller, settling occurred, and the cold did not appear to have diffused sufficiently to ensure rapid and complete freezing. Upon use of the heads, some heads showed browning and even clear signs of spoilage. Blood had run to the bottom of the container.

[0102] [Table 1] Table 1: Sensory properties of ingredients

[0103] Histamine standards according to Regulation (EC) No 2073 / 2005 and its applicable amendments:For fishery products: the average content of nine samples of fish shall not exceed 100 mg / kg; two samples may contain more than 100 mg but not more than 200 mg / kg; none of the samples shall exceed 200 mg / kg.For enzymatically or brine matured products: the upper limit shall be multiplied by 2.TCB-N=total volatile basic nitrogen.

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

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

[0106] process A simplified material balance for the main steps of the process to produce protein hydrolysate (FIG. 1A) and collagen hydrolysate (FIG. 1B) is shown in FIG. 1. The expected products are: Collagen Demineralized Powder Demineralized Protein Hydrolysate (Dough) Mineral Residue It is.

[0107] The balances depicted in FIG. 1 are given per tonne of raw material (FIG. 1A is the balance for the protein hydrolysate process, FIG. 1B is the balance for the collagen hydrolysate process).

[0108] Equipment used Shredder: The raw material was processed into marine by-product pieces using a Pallman 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) x 750 mm (width). Separator: AM2C supplied with a Barracuda Beluga 1830 separator with a 3mm sieve was used as the separator to produce a protein rich fraction and a fraction containing skin and cartilage.

[0109] Grinder (optional): A BIRO crusher AFM type G52 was used with 12-14 mm grids on the cartilage and skin from the separator to ensure that the cartilage was finely ground to facilitate hydrolysis.

[0110] 5m with variable speed drive and half shell heating system 3 Enzymatic hydrolysis was achieved using a stirred reactor. The device contains a manual bottom valve coupled with a pneumatic discharge valve. At the outlet of the reactor, a sieve screw is used to collect the cartilage residue. A minimum volume of 700 liters is required to start stirring. Promod 950L was used as the enzyme.

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

[0112] One skid ultrafiltration (UF) / nanofiltration (NF): The UF / nano skid may be equipped with a variety of membranes of different porosity. In this example, two different membranes: (a) 4.2 m 2 and porosity of 10,000 Daltons, and (b) 5.4 m 2 A nanofiltration GE Waters and Process Technologies membrane of type DK3840C-50D with a surface area of ​​100 nm and a porosity of 150–300 Daltons was used.

[0113] Or 4.3m 2 Ultrafiltration membranes of the HFK-328 type Food & Dairy UF element with a surface area and porosity of 5,000 Daltons may be used. The filtration unit is equipped with a 100 liter feed tank, limiting the volume to be processed, but can be equipped with an external tank to increase the volume that can be processed. The filtration unit makes it possible to produce and purify collagen hydrolysate (1 kDa), demineralize the collagen with a nano membrane (150-300 Da) and perform a preconcentration and demineralization of the hydrolysate (with a nano 150-300 Da membrane).

[0114] In this example, a filter press with activated carbon was used. A filter press was used to remove certain coloring or odor particles. 3% of each dry matter of coal and Smellrite (deodorant) were mixed with the hydrolysate. Filtration was then performed on a filter plate containing adjuvanted Pall K200, and the coal and Smellrite were retained in the press cake.

[0115] As an evaporator, a single-effect evaporator with thermal compression was used. This plate evaporator has a capacity of about 1 T / h and increases the solution concentration to between 35-40% of the dry weight content. A minimum volume of 300 liters is required.

[0116] Drying: For the final drying step, a nozzle sprayer with a 200 bar HP pump and equipped with a 800μ prefilter was used, the nozzle sprayer was adapted for drying soluble protein and fish protein products. Other sprayers may be used, allowing the injection of anti-caking agents in the pipes of the pneumatic transport, which facilitates the extraction of the moving powder. The nozzle selection takes into account the viscosity and dry matter content of the product to obtain sufficient spray pressure in the chamber.

[0117] Packaging: The final product was packaged using a vacuum packaging machine (Bernhardt) to create small vacuum bags that protect the product against oxidation.

[0118] Methodology Timeline Tests were carried out on two similar batches of 3-4 tonnes of raw material (heads). Each test took a total of two days and the second batch was allowed to be optimised compared to the first batch.

[0119] Day 1 Raw materials management

[0120] Day 2: First batch Yellowfin tuna head mash / Pallman (approx. 1.5T) Separation of protein paste / cartilage from albacore tuna head (1.8T) and then mashing of yellowfin tuna head Hydrolysis of albacore tuna head protein paste (1.9T) followed by decantation Nanofiltration / hydrolysate sample protein paste after salt addition Atomisation of protein paste hydrolysates (batch 1)

[0121] Day 3: 1st and 2nd batches Cartilage Hydrolysis (Batch 1) Ultrafiltration of Cartilage Hydrolysate (Hydrolysate 1) Deodorization and decolorization of collagen hydrolysate Yellowfin tuna head mash / Pallman (approx. 0.5T) Protein paste / cartilage separation for yellowfin tuna head and albacore tuna head mash (3.4T) After hydrolysis of yellowfin tuna head dough (1.4T), decantation

[0122] Day 4: Second batch Concentration and micronization of hydrolysate protein paste in two batches (Batch 2 and 3) using different antioxidants Collagen hydrolysate sample (batch 1) nanofiltration followed by salt addition 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 samples

[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-mashed yellowfin tuna heads: from 638 kg of heads (+150 L water) 675 kg of protein paste (approximately 82% of the raw material) is obtained. Albacore tuna head isolation: Approximately 3300 kg of protein paste was obtained from 3277 kg of head (+250 L water). For batch 2, the weight of the head used was approximately 7% skin / cartilage (283 kg) was obtained.

[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 an improved separation obtained by the different settings (increased pressure and temperature) allowing a better separation. The skin / cartilage fraction of batch 2 is less "lean" and contains less meat compared to batch 1.

[0131] Across the two batches, this equates to an average of 13% skin / cartilage by head weight.

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

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

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

[0135] Skin / cartilage fraction composition:

[0136] [Table 3] Table 3: Composition of skin / cartilage fraction

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

[0138] Cartilage and skin mash To optimize the hydrolysis of the collagen contained in the cartilage, a fine grinding of the cartilage is important (the finer the grinding, the better the performance). 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] Table 4: Protein hydrolysis conditions and yields (RM = raw material, DM = dry matter).

[0141] The composition of the extracted fatty phase (batch 2) was: dry matter: 98.5%, minerals: 0%, protein: 0.7%. The process therefore produces an oil of relatively high purity.

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

[0143] [Table 5] Table 5: Collagen hydrolysis conditions and yields (RM = raw material, DM = dry matter).

[0144] Hydrolysis was carried out without any particular problems and discharge of the reactor for both batches was achieved in a fairly well controlled manner, with only a small amount of water "pushed out".

[0145] The collagen hydrolysate obtained was fairly clear. It was possible to obtain a fairly white fraction (tailings) 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 nutritional market.

[0146] filtration Hydrolysates from protein pastes, nanofiltration (desalting) of protein hydrolysates Before filtration, the protein hydrolysates had a mineral content of about 15% in dry matter. For nanofiltration of the protein hydrolysates, organic nanofiltration membranes (150-300 Da) were used.

[0147] The initial volume of hydrolysate was 73 liters; water was added during the process to maintain the flow rate and improve desalting. The total volume of hydrolysate processed was 110 liters. The fraction resulting from the protein hydrolysate nanofiltration is treated as follows: permeate in a plastic container (a fraction that contains high concentrations of salt and very little protein and is subsequently discarded), holding thing was recycled into the pilot feed tank (noble protein fraction). After the retentate concentration step, distilled water was added to further extract 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 performed at the outlet. The resistance to dry matter (R / ES) ratio is a good indicator of the soluble mineral content. During recycling, the process was continued for 12 hours and the membrane performance was monitored over time.

[0149] During the nanofiltration, samples were taken at regular intervals. Some of these samples were analyzed (dry matter, protein, minerals) and the conductivity / dry matter ratio was thereby monitored. In the retentate, the ratio was reduced 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 The permeate flow rate was 15 l / h / m after 15-18 hours. 2 From 5 to 6 l / h / m 2 and variety, which is excellent and very classical for this type of membrane.

[0151] The following characteristics of the protein hydrolysate fractions produced from NF were obtained:

[0152] [Table 6]

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

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

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

[0156] 2) Nanofiltration NF 300Da on the permeate from the UF. This step allows the demineralization of the collagen hydrolysate: valuable fraction = retentate; permeate (= "brine"). The membranes used were thin-layer nanofiltration membranes 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 in continuous UF without recycling or dialysis (no recirculation or water addition). Ultrafiltration was started at 80 liters volume (tank volume). This volume was reduced by a volume corresponding to the permeate volume. The volume was then completed to continue ultrafiltration.

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

[0159] Lot 1: Processing hydrolysate 366 liters Lot 2: Processing hydrolysate 280 liters

[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 At the end, it is 30 l / h / m 2 It was.

[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 the different analyses weighted by the volume obtained during the analysis. The composition of the retentate was reproducible. More than half of the protein is in the permeate (collagen hydrolysate).

[0164] A total of 646 liters of cartilage hydrolysate was processed by UF in 2 h41. The flow rate decreased over time: between 216 and 266 L of permeate / h at the start of UF compared to 160 L / h at the end of UF (1 h later); i.e., 49 L / h / m at the start. 2 , at the end, 30L / h / m 2 The flow rate was .

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

[0166] Ultrafiltration (Batch 2) Due to the amount of water added for hydrolysis to ensure good mixing, the dry matter content of the hydrolysate of batch 2 was rather 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 membranes were the same as for batch 1. The permeate contained about 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 cartilage hydrolysate batch: the flow rate was about 23 L / h / m at the start. 2 and 7 L / h / m 2 This difference is due to the difference in hydrolysate concentration and viscosity, since 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 input was estimated to be identical to the first batch, while its dry matter was higher because the hydrolysate had been pre-concentrated.

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

[0172] Nanofiltration of Collagen Hydrolysate (Batch 1) After the above mentioned ultrafiltration, the permeate obtained from the ultrafiltration is subjected to nanofiltration in order to remove the minerals present.

[0173] At the end of the nanofiltration, the retentate is recycled to the feed tank, allowing several passes of the liquid through the membrane, thereby concentrating the retentate while continuously removing the permeate (salts). After concentration, water is added and dialysis is performed to further improve the removal of soluble minerals. This process is carried out in a closed circuit for 12 hours, and the membrane performance is monitored over time.

[0174] This test was carried out on a sample of the first batch of collagen hydrolysate after adding salt in order to obtain a mineral content equivalent to that of collagen obtained from heads stored in salt water.

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

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

[0177] The mineral content in the retentate (collagen hydrolysate) decreased rapidly: by a factor of 2 in 15 min. After 3 h, a dry mineral content of about 3% was reached.

[0178] The initial flow rate is 78 l / h / m 2 After triple concentration (FVC3), this was 42 l / h / m 2 After adding water, the flow rate increases to 74 l / h / m 2 After 12 hours in a closed loop, the flow rate 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 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 probably overestimated as well. The sum of the protein and ash amounts in the retentate and permeate is greater 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 was in the retentate. Nanofiltration therefore enabled an effective separation of minerals from the collagen hydrolysate and the demineralization was very effective. A mineral content of 3.8% was achieved in the collagen hydrolysate, which corresponds to market standards. It was also observed that nanofiltration and dialysis had a positive effect on collagen deodorization.

[0183] Deodorization and decolorization on plate filter press After nanofiltration was completed, a portion 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 material 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 first test.

[0184] Concentration and drying Protein Hydrolysates The various drying operations were carried out on a spray-drying tower. The spray-drying equipment consists of a tower with a conical base, a nozzle for spraying and a high-pressure pump. The spray-drying equipment was constructed by TGE and has a capacity of 250 l / h evaporation capacity.

[0185] The first batch of protein hydrolysate was directly dried (i.e., no further concentration was performed for the separation step). Due to the low dry matter content (15%), it was not possible to obtain sufficient pressure from the HP pump (high pressure) due to the nozzle arrangement and low viscosity. Due to the high fat content of the first batch of protein hydrolysate (24% lipid content), the spray in the drying chamber was poor and it became sticky in the tower. This problem could be solved by using an anti-caking agent.

[0186] The second batch of protein hydrolysate was previously concentrated by evaporation and had a content of 33% of dry matter. A longer hydrolysis, driven by a decanter centrifuge at high temperature, made it possible to extract part of the oil in the form of fluid oil. Finally, the initial content of the yellowfin tuna protein paste (used in this test batch) was lower than that of the albacore tuna. Thus, the drying process was better than the first batch and very little sticking was observed on the tower.

[0187] The addition of anti-caking agents improves the flowability in pneumatic conveyors and various rotary valves, leading to improved drying processes for protein hydrolysates.

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

[0189] Composition of the obtained hydrolysate

[0190] Characteristics of Protein Hydrolysate (Powder)

[0191] Nutritional composition [Table 10] * Eurofins analysis (Eurofins NDSC Food Testing Germany GmbH)

[0192] The only difference between batches 2 and 3 is the type of antioxidant and the drying level (dry matter content). Hydrolysates 2 and 3 contain 72.6% protein and 12.3% fat on dry matter. The mineral content (12.8%) is higher than in the batch 1 hydrolysate (5%), for no other reason than that protein paste from albacore tuna heads was used in the first batch and yellowfin tuna in 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 brine frozen, so the salt content was lower, but the final mineral content was considered to be relatively high. Therefore, a nanofiltration step is recommended to reduce the overall mineral content of the protein hydrolysate.

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

[0194] [Table 11]

[0195] Assays for biogenic amines, PCBs and heavy metals were initiated on the hydrolysates of batch 2.

[0196] [Table 12]

[0197] Heavy metal levels were in line with regulations, with only the mercury content close to the limit.

[0198] [Table 13]

[0199] PCB and dioxin levels comply with European regulatory thresholds (2017 / 644).

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

[0201] [Table 14]

[0202] The biogenic amine levels were quite high, which was due to the raw materials not being very fresh.

[0203] [Table 15]

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

[0205] Fatty Acid Profile [Table 16]

[0206] Thirty-one percent of the fatty acids (11.2%) are omega-3. There are 4.1 grams of polyunsaturated fatty acids per 100 g of protein hydrolysate. A detailed fatty acid profile is provided in the Appendix (Batch 2).

[0207] Characteristics of collagen hydrolysate (powder) Nutritional composition

[0208] [Table 17]

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

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

[0211] Amino acid profile [Table 18]

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

[0213] quality [Table 19] [Table 20]

[0214] Molecular Weight Profile The protein size of the samples was estimated by HPLC analysis. The samples were diluted in an appropriate buffer and analyzed through a filtration gel column that allows the molecules to be sorted by size: during the analysis, the largest molecules come out of the column first and the smallest ones last. The protein compounds are detected using a detector set at 214 nm. By using standard proteins it is possible to calibrate the column. The table below shows the correspondence between the retention time in the column and the size of the proteins / peptides of the collagen and protein hydrolysates produced.

[0215] [Table 21]

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

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

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

[0219] Matrikine Without being bound by theory, MMPs (matrix metalloproteinases) are very important for generating matrikines from ECM (extracellular matrix) collagen components. Therefore, it is expected that the collagen products of the present invention contain these valuable components. Exemplary matrikines are arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lamstatin, hexastatin 1, hexastatin 2, NC1 domain of collagen IV or XIX, endotropin, ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin 7, neostatin 14, palmitoyl pentapeptide-4, collagen hexapeptide GFOGER, and CLAC.

[0220] Schedule

[0221] [Table 22]

[0222] [Table 23]

[0223] [Table 24]

Claims

1. 1. A method for extracting collagen from marine animal by-products comprising: a) providing marine animal by-product pieces, said by-product pieces comprising at least two components selected from meat, skin, fish bones, fins and cartilage of said marine animal; b) separating the animal by-product pieces into at least two fractions including a protein rich fraction and a fraction containing skin and cartilage, the skin and cartilage fraction being essentially meat free; c) optionally grinding said fraction containing skin and cartilage to produce a particle size preferably between 0.5 mm and 1 cm, more preferably between 1 mm and 4 mm; d) enzymatically hydrolyzing said fraction containing skin and cartilage to produce a collagen raw hydrolysate, said hydrolysis being preferably carried out at a pH between 5 and 9; e) separating the raw collagen hydrolysate into a lipid fraction, a solid-free aqueous collagen hydrolysate fraction and a solid-containing fraction, preferably 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 between 2000 and 100,000 Da, preferably between 4000 and 50,000 Da, more preferably about 10,000 Da to produce a first permeate and a retentate containing collagen hydrolysate; 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 150-300 Da, to produce a purified collagen solution; A method comprising:

2. 2. The method of claim 1, further comprising the step of deodorizing the purified collagen solution of step g), preferably wherein said deodorization is performed using activated carbon and / or dialysis.

3. 3. The method according to claim 1 or 2, further comprising concentrating and sterilizing the purified collagen solution or the deodorized purified collagen solution, preferably wherein the concentration and sterilization is carried out by evaporation, such as by direct steam injection.

4. The method according to any one of claims 1 to 3, further comprising the step of drying the purified collagen solution or the purified deodorized collagen solution or the concentrate thereof to produce a collagen powder, preferably wherein the drying is carried out by spray drying, freeze drying, lyophilization, nozzle atomizer, most preferably wherein the drying is carried out by continuous horizontal spray drying.

5. 1. A method for producing a protein hydrolysate from a marine animal by-product, comprising: a) providing marine animal by-product pieces, said by-product pieces comprising at least two components selected from meat, skin, fish bones, fins and cartilage of said 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 said 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 produce a protein hydrolysate solution; e) separating said 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; A method comprising:

6. 6. The method according to claim 5, further comprising the step of: g) demineralizing the aqueous protein hydrolysate of step f) by filtration and / or dialysis using a membrane with a pore size of less than 1000 Da, preferably between 100 and 500 Da.

7. 7. The method according to claim 5 or 6, further comprising the steps of concentrating and sterilizing the aqueous protein hydrolysate or the demineralized aqueous protein hydrolysate, preferably wherein the concentration and sterilization is carried out by evaporation, such as by direct steam injection.

8. 8. The method according to any one of claims 5 to 7, further comprising the step of drying said aqueous protein hydrolysate, said demineralized aqueous protein hydrolysate, or said respective concentrates thereof to produce a protein powder, preferably said drying being carried out by spray drying, freeze drying, lyophilization, nozzle atomizer, most preferably said drying being carried out by continuous horizontal spray drying.

9. c') collecting the aqueous fraction of step c) and subjecting this fraction to filtration and / or dialysis using a membrane with a pore size 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 of any one of claims 5 to 8, further comprising:

10. 1. An integrated process for upcycling marine animal by-products, comprising: a) providing marine animal by-product pieces, said by-product pieces comprising at least two components selected from meat, skin, fish bones, fins and cartilage of said 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, the skin and cartilage fraction being essentially meat free; c1) subjecting said protein-rich fraction to step c) and subsequent steps according to any one of claims 5 to 9; c2) subjecting said fraction containing skin and cartilage to step c) and subsequent steps according to any one of claims 1 to 4; The process includes:

11. 11. The method according to any one of claims 1 to 10, wherein the marine animal by-products are fish by-products such as heads, organs, fish bones, scraps and / or skins, preferably the marine by-products are tuna by-products.

12. 12. A purified collagen solution or powder produced by the method of any one of claims 1 to 4, 10 or 11, said collagen solution or powder comprising at least one matrikine.

13. 13. The collagen solution or powder of claim 12, wherein the at least one matrikine is selected from the group consisting of arresten, canstatin, tumstatin, tetrastatin, pentastatin 1, pentastatin 2, pentastatin 3, lamstatin, hexastatin 1, hexastatin 2, the NC1 domain of collagen IV or XIX, endotropin, the ectodomain of collagen XIII, XVII, XXIII or XXV, restin 1, restin 2, restin 3, restin 4, endostatin, neostatin 7, neostatin 14, palmitoyl pentapeptide-4, collagen hexapeptide GFOGER, and CLAC.

14. 14. A collagen solution or powder according to claim 12 or 13, wherein at least 55% of the peptides of the hydrolysate or powder are <2kDa, preferably at least about 60% of the peptides are <2kDa, more preferably at least about 65% of the peptides are <2kDa.

15. 12. A protein hydrolysate or protein powder produced by the method according to any one of claims 5 to 11, wherein the hydrolysate or powder comprises at least 3% (by weight) omega-3 lipids and at least 1% (by weight) phospholipids, preferably wherein the hydrolysate or powder comprises at least 2% (by weight) monosaturated lipids, at least 3% (by weight) polysaturated lipids and at least 2.5% (by weight) omega-3 lipids.

Images