Fish feed with low content of maillard products

Abstract

The present invention relates to a method for producing a fish feed by an extrusion process comprising the steps of preconditioning the feed composition to reach a pasting temperature, adjusting the pH to 5.0-6.5 by adding at least one acid, incorporating at least one AGE-inhibiting antioxidant, extruding, drying, and coating the feed; a fish feed produced according to the method; and use of the fish feed.

Description

Fish feed with low content of Maillard ProductsField of the inventionThe present invention relates to aquaculture, such as salmon farming and sea bass farming. More specifically, the invention relates to fish feed production in industrial scale, and health of fish in aquaculture.Background of the invention

[0001] In many parts of the world, aquaculture is a large industry, often vital for providing food for an ever-increasing population. Fish, crustaceans, mollusks, and marine plants are examples on what is produced by aquaculture. Currently, about 50% of all seafood is produced by aquaculture. Asian countries dominate the list of the largest producers. However, aquaculture of salmon and other salmonids, sea bass and other species are large industries in many countries outside Asia. For example, in Norway, about 1 .3 million of metric tons of salmon was produced by aquaculture in 2023, requiring about 1 .56 million of metric tons of fish feed for salmon in Norway alone.

[0002] The fish feed must be rich in nutrition, including nutrients, antioxidants and other contents required for the fish. Additionally, the fish feed must be feasible for industrial mass production, transport, handling, and storage if the resulting production cost shall be favorable, and aquaculture of for example salmon shall be economical viable.

[0003] Since about 1980 fish feed for salmonids have been produced by extrusion. However, when fish feed production by extrusion became industry standard in about 1980, it was discovered that the antioxidant vitamin C, added as ascorbic acid in the feed for extrusion, was lost in the finished feed pellet product. The problem was solved by replacing ascorbic acid with a stable version of vitamin C, namely C- monophosphate. This resulted in a final feed pellet product with healthy composition, including rich contents of vitamin C, meeting all requirements with respect to nutrition contents, in addition to feasibility for mass production and stability for handling and storage. Since all requirements for the fish feed have been met since about 1980, a prejudice exists in the fish farming aquaculture industry that the fish feed quality ismaintained and there is no reason to believe that the fish feed can have a significant role with respect to fish health.

[0004] On the websites and in other publications from the fish farming industry, or in publications from governments and other regulatory bodies, there seems to be no reason to believe for the industrial scale and fish feed producers or industrial scale fish farmers that the feed has a significant role with respect to fish health, since all regulatory requirements are met. Diseases and health problems are rather related to viruses, bacteria, parasites like salmon lice, too closely packed volumes of biomass, and stress reactions from the fish due to handling and weather issues. Instead, the health situation apparently is improving, since disease outbreaks have been reduced by 30%, from 1990 to 2007, despite the data from 1990 include fewer known diseases, cf. the Norwegian Seafood Federation. By the industry and government, fish health issues are apparently linked to environmental factors only.

[0005] Despite massive efforts to improve fish health, the mortality of salmon farming is still far higher than what should be acceptable and is apparently increasing. In 2023, 65 million salmon died in Norwegian fish farms, and increase from 58 million salmon in 2022. The mortality rate is above 15%, despite billions of Norwegian kroner (NOK) spent for achieving a reduced mortality and improved fish health. Figures for diseases and health problems are probably quite similar.

[0006] Patent publication EP 2445358 B1 relates to pigments in fish, and a related fish feed with pigment retention effect, as related to high carotenoid contents in the form of 10 to 40 mg of astaxanthin per kg fish feed. The purpose is to receive a positive quality evaluation and a positive cost factor, resulting in a commercial advantage, by red colored fish flesh and good skin color and shine. Fish health, such as healthy bone structure or tendency of inflammation are not linked specifically to the fish feed.Brief summary of the invention

[0007] The method of the invention relates to a method for producing an extruded fish feed.

[0008] More specifically, the invention provides a method for producing a fish feed, wherein the method comprises steps wherein process aids are added to the fish feedcomposition in the reactive phase to inhibit or reduce the formation of advanced glycation end-products (AGE). Thus, the invention further relates to an extruded fish feed with a reduced concentration of AGEs.

[0009] In one aspect, the present invention relates to a method for producing a fish feed, where the fish feed is produced by an extrusion process of a feed composition, comprising the steps of preconditioning the feed composition, wherein the preconditioned feed composition reaches a pasting temperature, adjusting the pH comprising adding at least one acid to the feed composition in an amount to obtain a pH in the range pf 5.0 to 6.5, adding at least one AGE-inhibiting antioxidant in a quantity equal to or greater than an AGE-formation potential, extruding the preconditioned feed composition, drying and coating the extruded fish feed, wherein adjusting the pH and adding the at least one AGE-inhibiting antioxidant are performed before or as part of the preconditioning, during the reactive phase of the extrusion process which is defined by the AGE-formation potential.

[0010] In a further aspect, the invention relates to a fish feed produced according to the method of the first aspect.

[0011] In another aspect, the invention relates to use of the fish feed for preventing bone deformities and improving fish health.

[0012] In aspects, the invention also relates to a method for producing feed and food by an extrusion process.

[0013] The invention further relates to a method for producing feed or food, to a method for producing fish feed, to the feed or food produced according to the methods, to the use of the methods and products, and the methods, products and use as described in the claims.Detailed description of the invention

[0014] The method of the invention relates to a method for producing an extruded fish feed. Extrusion is an advantageous method for producing fish feed due to the ability to incorporate all required nutrients in a feed which is easily digestible by the fish and sufficiently stable from production, transport, and storage until the feed is fed to the fish. Since the beginning of the 1980s, fish feed for example for salmon and trout has been produced by extrusion. Extrusion takes place by forcing heated and moist feed through one or more holes under high heat and high pressure. When the feed comes out of the extruder, the water will evaporate and cause the feed to expand, with a spongy hard pellet as a result.

[0015] In the step prior to extrusion, is a step of preconditioning wherein the feed is heated by adding steam to a conditioner. The temperature and the residence time of the feed in the preconditioning step depends on factors such as feed composition, and amount of feed in the preconditioner. The temperature thus rises to 85 to 90°C or even to 95°C during the 30 seconds to 180 seconds to 300 seconds the feed stays there. Water is also added so that the feed contains 25 to 30% water when it reaches the extruder.

[0016] In the extruder, the feed is exposed to frictional heat and pressure, and the starch and protein in the feed will paste and the moisture determines the balance between collapse and the rigid structure of the pellets. The temperature at the exit of the extruder will usually be between 95 and 130 °C, such as between 95-100 °C, alternatively between 110 and 130 °C. The residence time in the extruder is usually 15 to 20 seconds. In some cases, the residence time in the extruder is longer, typically in the timespan in the range of 15 to 90 seconds, such as 15 to 40 seconds, such as 30 to 60 seconds, such as 40 to 90 seconds. The temperature and residence time of the feed depends on the properties of the raw materials used in the feed and is adjusted to achieve the optimal pasting temperature for the feed. The extrudates are then dried and lipids are added during a vacuum coating. The physical quality and expansion of the final product are controlled by various factors, such as water and steam injection, viscous heat dissipation, and extruder screw and die configuration. The processing and quality of the final product is also influenced by the physicochemical and rheological properties of the feed ingredients. The starting material i.e. feed composition which is processed in the extrusion process typicallycomprises the nutrient sources such as proteins, carbohydrates, fats, and micronutrients which are mixed into a dry feed mixture.

[0017] Despite the many advantages of extrusion as described above, a challenge associated with the extrusion process is the production of undesirable advanced glycation end-products (AGEs). AGEs comprise a heterogeneous, chemically diverse group of compounds. Two types of reactions lead to formation of AGEs, namely the non-enzymatic glycation reaction (Maillard reaction) and the enzymatic glycosylation reaction. The biochemical, non-enzymatic glycation can be subdivided into three major stages: early, middle, and late. In the initial reaction, the carbonyl group (C=O) of a reducing sugar, such as glucose, reacts with the amino group (NH2) of the amino acid residue in a protein to form a Schiff base (C=N). This Schiff base is relatively unstable and eventually becomes an enol, causing Amadori rearrangement and finally leading to the formation of a stable Amadori compound (C-N). The Amadori compound then undergoes a repeated polycondensation reaction with an amino compound using ozone or furfural as an intermediate to produce a brown product, melanoidin, in late stages. Structures formed in the latter stage of the nonenzymatic glycation reaction between reducing sugars and proteins are collectively known as advanced glycation end products (AGEs), and are stable, irreversible products. The glycosylation reaction on the other hand, is not random, but rather mediated by enzymes in vivo. Maillard reactions are an integral and unavoidable part of feed manufacturing. Its adverse effects are experienced at least via reduced nutrient availability due to excess thermal processing and prolonged storage.

[0018] The Maillard reaction involves binding of amino groups to the carbonyl group of reducing sugars, such as those found in glucose and lactose. Browning is often confused with the intensity of the Maillard reaction. However, coloration and amino acid loss are two independent phenomena of the Maillard reaction process. The Maillard reaction may take place at room temperature, but its intensity increases as time, temperature, moisture (maximum at 40 to 70 percent), and alkalinity (linear between pH 3 and 8) increase. The conditions under which the glycation reaction or Maillard reaction is most likely to occur and proceed at a significant rate, is referred to as the reactive phase herein, and depends on parameters including time, temperature, moisture, alkalinity, and pressure.

[0019] In the extrusion process, the way fish feed is made today, proteins from sources including fishmeal and carbohydrates from various sources are mixed, and water and high temperature are added to get the pasting of the starches started. This is done so that the pellets get the physical strength they need and make room to hold fat. Throughout this process, the potential for a Maillard reaction is present. In other words, this is where potential reactions, oxidations, reductions, and changes can occur quickly in the feed mass, such as starting the Maillard reaction. Hence, when the feed composition is no longer in a dry state, the reactive phase is initiated.

[0020] AGE accumulates in connective tissue and in the skin during normal physiological metabolism with aging and with exogenously supplied AGE through food, mainly because connective tissue and skin cells have a long lifespan. Several AGEs have been identified as toxic, acting through their binding to AGE receptors (RAGE) which triggers the production of intracellular reactive oxygen species (ROS). Subsequently, ROS triggers various intracellular signaling pathways which in turn upregulate inflammatory cytokines, growth factors, and adhesion molecules which are involved in inflammatory diseases, cell apoptosis, and cell dysfunction. The interest in AGEs has grown in recent years due to increasing evidence suggesting their accumulation is associated with the involvement in pathophysiological processes including inflammatory diseases, diabetes, and cardiovascular diseases in humans.

[0021] As earlier disclosed herein, the challenges related to fish health the fish farming industry are facing, are largely attributed to environmental factors according to industry actors and others. However, given the increasing association of AGEs and human diseases, the large intake of harmful substances such as AGEs through feed is likely a contributing factor to health issues seen in the fish farming industry. Interestingly, ROS and oxidative stress are associated with various fish diseases, yet the connection between high AGE content in extruded fish feed and the challenges related to fish health faced by the fish farming industry remains underexplored.

[0022] Increasing evidence is suggesting that AGE introduced by food consumption accumulates in long-lived connective tissue and in the skin under normal physiological metabolism in humans. The effect of AGE on the dermis of the skin is that AGE and collagen are cross-linked, which causes the protein to brown and the fibers to deform. Elastin fibers become thinner, less rigid, and lose their biologicalproperties. Cross-linking of vimentin leads to loss of fibroblast contractility and an inability to maintain the basic cell shape.

[0023] In fish which only have a short lifespan of 1-3 years and are growing throughout their entire life cycle, accumulation of large amounts of AGEs through the extruded feed diet may negatively affect the fish health. Possibly, AGE accumulation subjects the fish to an artificial ageing, which leads to easily damaged skin, which in turn has difficulties in healing and thus easily gets bacterial infections. This, i.e. these health-related challenges, is a major problem and causes high mortality in salmon farming today.

[0024] The above-mentioned problem of connective tissue being weakened by AGE means that the issue is also a relevant topic regarding the bone structure of fish. Bone deformities is a common problem seen in fish farming today. Bone is built by the fact that where there is osteoblast activity, a collagen structure is first created which in turn is mineralized by hydroxyapatite (calcium phosphate) so that the structure becomes rigid, and which makes bone become "bone". Calcium makes up ten parts and phosphate six parts of hydroxyapatite (calcium phosphate), which is the building block for bone mineralization in salmon. Despite the fact that there is free access to calcium from the water that surrounds the fish, too low values are measured in the bones of farmed salmon. Calcium regulation is controlled by a small organ, the stannius body, whose task is to slow down absorption to avoid hypercalcemia. But the stannius body can be disturbed by what the fish eats and by the PD vaccine. The stannius body has calcium sensitive receptors and secretes the hormone stanniocalcin (STC1 ), which downregulates calcium uptake.

[0025] The conditions used in conventional extrusion processes to make fish feed, has a high potential for the glycation, or Maillard reaction, to occur. Temperature and humidity are a catalyst in such a process, so the reason for looking at the extrusion process is that in addition to the properties of the feed raw materials, it is through the steps of preconditioning, extrusion and drying that the feed is exposed to high humidity, temperature, and high pressure. And this is where potential reactions, oxidations, reductions, and changes can occur quickly in the feed mass, such as starting the Maillard reaction. This leads to the formation of components in the feed, i.e. AGEs, which in the metabolism can lead to disruption of calcium sensitive receptors (CaSR) and can affect the regulation of calcium uptake. The effect ofcollagen being cross-linked by AGE, which in turn causes the collagen fibers to weaken, can be one of the reasons why bones become weak and deformed. This has so far not been researched when it comes to fish but is a timely issue. Another scenario is to look at how "mimics" of calcium are formed, and how the feed is made in the extrusion process. So, in order to find triggers, agonists or calcimimetics that are formed, one should eliminate components and raw materials to see if these will produce changes in the excretion of stanniocalcin. And to eliminate process-formed components, i.e. AGEs, through use of process aids, to avoid formation of these AGEs.

[0026] Hence, AGEs formed in the conventional extrusion process for making fish feed is likely contributing to causing inflammatory conditions and bleeding in the intestine of the fish, as well as bone deformities and accelerating aging in the fish.

[0027] Despite large efforts in finding ways to inhibit AGE-content in foods consumed by humans and other mammals for health benefits, limited efforts have been made to do the same in the fish farming industry and there is no solution to this problem as of today.

[0028] The above-mentioned problems are solved by using process aids in the feed process during the extrusion of fish feed pellets, to inhibit AGE formation. Hence, the invention relates to the use of processing aids in specific control process steps in a method for producing an extruded fish feed. The processing aids are added in a specific timeframe so that they are present and perform their functions during the reactive phase of the extrusion process. The feed produced by the method containing reduced concentrations of AGEs is likely to positively affect fish health and fish welfare by inhibiting the development of bone deformities, inflammatory conditions, and bleeding in the intestines of farmed fish.

[0029] Furthermore, it is not only fish feed which is produced by an extrusion process. All feed and food products produced by an extrusion process, where the process comprises the above-mentioned conditions and ingredients for possible Maillard reactions, comprise the formation of advanced glycated end products (AGE).

[0030] Extrusion is the gold standard for fish feed production in the fish farming industry, and the extrusion process conditions leads to AGE formation which is suspected to negatively affect the fish health. Hence, there is a need to providealternative solutions for methods of producing a fish feed which meets the nutritional requirements, has the same stability during the product’s life cycle as today’s standard extruded fish feed, which inhibits or reduces the formation of harmful AGEs in the fish feed.

[0031] The present invention addresses the need for obtaining a method to produce a fish feed with reduced generation of AGEs, wherein the method comprises steps for inhibiting or reducing the formation of AGEs. The present invention further relates to a fish feed produced by the process with reduced AGE concentration which positively impacts the health and welfare of fish.

[0032] In a first aspect, the invention relates to a method for producing fish feed. Where the method for producing fish feed is aimed to prevent Maillard type reactions during the extrusion process. In other words, where the method for producing fish feed is aimed to prevent fish bone deformities. Where the fish feed is produced by an extrusion process of a mixture of feed ingredients, where the extrusion process comprises the steps of preconditioning, extrusion, drying, and coating, and where the extrusion process reaches a pasting temperature, and the extrusion process forces expanding of the feed, and where the method further comprises adding an antioxidant to the extrusion process before the preconditioning step, and adding an acid to the extrusion process adjusting the pH of the mixture before the preconditioning step. Hence, the method comprises use of process aids that can dampen the reaction processes.

[0033] Factors which promote the formation of AGEs are availability of reactants i.e. reducing sugars and amine groups in the fish feed mixture, temperature, time, pH, high humidity, and high pressure. In the feed composition, the concentration and types of sugars and proteins present in the feed mixture can impact the extent of glycation reactions and AGE formation. Reducing sugars include glucose, fructose, galactose, lactose (composed of glucose and galactose), and maltose (composed of two glucose molecules). Process-related parameters such as higher temperatures and longer processing times can accelerate glycation reactions, thus promoting AGE formation. Glycation reactions are pH-dependent, with higher rates occurring under alkaline conditions. Moisture content plays a crucial role in facilitating glycation reactions, as water molecules are essential for the formation of AGEs. Thus, higherio moisture levels can promote glycation. Hence, under certain conditions during the conventional extrusion process, the combination of feed composition properties and process-parameters define the AGE formation potential. The timespan within the extrusion process where the AGE formation is more likely to occur is defined herein as the reactive phase.

[0034] The invention relates to a method for producing fish feed, where the fish feed is almost free of Maillard type rection products. In other words, the method is aimed to prevent chemical reactions between amino acids and reducing sugars during the extrusion process. In food science, these reactions are named nonenzymatic browning reactions or Maillard reactions. This process is also known as protein glycation or glycoxidation. A professional in the art knows various strategies for controlling Maillard reactions. Maillard reactions contribute to desired changes such as generation of flavors, whereas in other cases undesired quality changes are obtained, especially if the Maillard reactions are too pronounced, producing bitter and burnt flavors, and unhealthy products. Thus, being able to control Maillard reactions during food production and storage is important from a feed quality perspective. The rate, extent, and course of Maillard reactions are influenced by several factors including, but not limited to, type of reactants, temperature / time combinations, pH, and water activity. The inventor of the present invention has planned experiments to test that adding antioxidants and acids to the extrusion process prevents bone deformities in fish and that this circumstance is due to the suppression of the Maillard reaction, Maillard type reaction products.

[0035] Technically, there will be no change to the feed. Use of the process aids will not impair the technical quality of the extruded fish feed. This will give significantly greater survival of fish in a life course and significantly better fish health / fish welfare and fewer quality defects on fish for slaughter. Operationally, there is a clearer difference between diseases caused by nutritional deficiencies and diseases caused by agents from the environment.

[0036] The method according to the invention is for producing an extruded fish feed. Prior to commencement of the extrusion process, raw materials comprising nutrient sources including fat sources, protein sources and carbohydrate sources such as sugars and starch, to obtain a dry feed composition. When existing in a dry stage, the components of the composition are not reactive. The pH of the dry feed compositionis typically slightly acidic to neutral, reflecting the natural pH of the ingredients comprised in the dry feed composition. The pH of such feed compositions is typically in the range of 6 to 7, such as typically 6.5 to 7. The specific pH of the dry feed composition varies depending on the exact formulation of the feed composition and the presence of any additives or preservatives used in the dry feed composition.

[0037] The present invention relates to providing a method for producing an extruded fish feed, wherein the method comprises specific control process steps wherein specific agents as processing aids are added to prevent or reduce the occurrence of the glycation reaction. The applicant has identified that use of specific agents in a defined timespan can be included as additional steps within the conventional extrusion process, wherein inclusion of the additional steps of the method according to the invention results in inhibition or strong reduction of AGE formation. These additional steps comprising adding said agents must be conducted during the reactive phase. Hence, the invention is based on an established method of producing nutritious and stable fish feed and comprises steps for improving the quality of the fish feed product which subsequently has beneficial effects on fish health.

[0038] In aspects, the process aids used in the method comprise at least one acid and at least one antioxidant. The method suggests that ascorbic acid, sulfite, and ferulic acid in the feed production process will be an inhibitor for the exogenous AGEs which are formed in the feed production process, and which contribute to the development of the aforementioned health challenges through the consumption and metabolism of feed in fish. The method requires that the pH in the feed mixture is on the acidic side, and addition of processing aids when feed ingredients are mixed. Examples of processing aids include crystalline ascorbic acid, sulfites, ferulic acid, y- oryzanol, lactic acid, propionic acid, phosphoric acid, acetic acid and citric acid. At least one of these is preferred to be present.

[0039] The method comprises a step of preconditioning the feed composition, comprising introducing heat and moisture in the form of water and steam to the dry feed composition. Herein, the temperature rises to 55 to 100°C, such as 65 to 85°C, such as 85 to 100°C, preferably 85 to 90°C. The preconditioning typically lasts for 30 to 180 seconds, such as 30 seconds, 60 seconds, 90 seconds, 120 seconds, or 180 seconds. In aspects, the preconditioning lasts for 30 to 300 seconds. The duration ofthe preconditioning step depends on factors including the feed composition and on the amount of feed composition in the preconditioner. In one embodiment, during the preconditioning step the temperature rises from 55 to 95°C, over a period of 30 to 180 seconds or to 300 seconds. During this time, the moisture is added so that the feed composition contains 25 to 30% water. In one embodiment, during the preconditioning step the feed composition reaches a pasting temperature in the range of 60 to 95°C, preferably at 85-95°C. The feed composition undergoes pasting wherein the starch of the feed composition contributes to gelation of feed ingredients, and the feed composition becomes viscous. The temperature at which a feed composition starts to paste, largely depends on the feed composition.

[0040] The method further comprises adjusting the pH comprising adding at least one acid in an amount to obtain a pH in the range of 5.0 to 6.5 of the feed composition. By an amount to obtain, it is meant an amount sufficient to obtain a pH in the range of 5.0 to 6.5 of the feed composition. Preferably the at least one acid is a weak acid. A weak acid herein means an acid with a pH in the range of 4.0 to 6.0 whose molecules do not fully dissociate when in contact with water. The adjustment of the fish feed composition to acidic pH of 5.0 to 6.5 contributes to reduce the availability of amino acids for reducible sugars and hence reduce the occurrence or reaction rate of the glycation reaction.

[0041] The at least one acid may be one or more of a group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, citric acid, lactic acid, propionic acid, phosphoric acid, and acetic acid. Preferably, the at least one acid is selected from the group comprising citric acid, lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid. In the preferred embodiment, the at least one acid is citric acid, intended to lower the pH in the mixture before the antioxidant is able to react in the feed mixture.

[0042] In all embodiments, the pH adjustment is carried out so that the at least one acid is present in the feed composition in the reactive phase of the extrusion process, i.e. in the extrusion step. In one embodiment, the pH adjustment is carried out during the preconditioning of the feed composition, preferably before the feed composition reaches its pasting temperature. Preferably, the pH adjustment step is carried out when the temperature of the preconditioning step is lower than 85°C, such as 65-84°C. In this embodiment, the water content of the feed mixture is in the range of 10- 25%.

[0043] Preferably, the pH adjustment step is carried out before the preconditioning of the feed composition, such as when the raw materials are mixed to obtain a dry feed composition before the extrusion process step starts. Herein the at least one acid is added in the feed composition comprising raw materials. Herein the temperature is in a range of 10-37°C, and humidity is in the range of 4-14%. In all embodiments, the total amount of acid added to the mixture comprises 0.5-3 wt%.

[0044] In yet another embodiment, the at least one acid is added in the liquid in which it is added to the preconditioner to lower the pH before reactions, i.e. before the reactive phase and pasting takes place.

[0045] In one embodiment, the at least one acid is selected from the group comprising citric acid, lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid. Preferably, the at least one acid comprises citric acid. In one embodiment, the at least one acid comprises citric acid and at least one of lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid. The amount of acid added to the feed composition is dependent on the AGE formation potential of the fish feed composition, defined by the contents of reactants i.e. reducing sugars and amine groups from lysine and arginine end-containing proteins and process parameters such as time, temperature, pH, moisture content, and pressure.

[0046] In the preferred embodiment, the acid is citric acid and is added in an amount of around 1000 ppm. In another embodiment, the acid is one or more of a group comprising ascorbic acid, ferulic acid, sulfite, and citric acid, in an amount of 200 to 1200 ppm. In aspects, the at least one acid may comprise citric acid and at least one of lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid in an amount of 100-1100 ppm, wherein the total amount of acid added is in the range of 1000-1200 ppm. In other aspects, the at least one acid may comprise citric acid and at least one of lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid, wherein the total amount of acid added is in the range of 1000-10000 ppm.

[0047] Generally, the amount of acid depends on the fish feed formulation and the extrusion process parameters, where parameters increasing the intensity of the Maillard reaction, as time, temperature, moisture, and alkalinity, increase theessential amount of the acid. The 1000 ppm citric acid of the preferred embodiment refer to standard extrusion process parameters as for example 120 to 160 °C of the extrusion step, and the preconditioning parameters between 55 to 90 °C of the preconditioning step. The amount of acid added must be sufficient to adjust the pH of the feed composition to the preferred range of 5.0 to 6.5. Hence, in some embodiments, the at least one acid is selected from the group comprising lactic acid, propionic acid, ferulic acid, and citric acid in a total amount of 10000 ppm.

[0048] Adding acid to the fish feed formulation is aimed to adjust the pH of the formulation between 5.0 to 6.5. A professional in the art will understand that different acids have different potential to decrease the pH and the intensity of the Maillard reaction, the professional will also understand synergetic effects and adjust the amount of the different acids to the process parameters to adjust the pH.

[0049] The method further comprises adding at least one antioxidant for inhibiting AGE formation, i.e. AGE-inhibiting antioxidant, in a quantity equal to or greater than the AGE-formation potential of the feed composition. The AGE-formation potential of the feed composition is defined by the contents of reactants i.e. reducing sugars and amine groups from lysine and arginine end-containing proteins, and process parameters including temperature, time, pH, water activity, and pressure as earlier disclosed herein. The primary function of the at least one antioxidant as used in the method disclosed herein is to specifically inhibit the initiation of the glycation reaction thus preventing Shiff base formation. In other words, the at least one antioxidant added to the feed composition blocks the reducible aldehyde group of the reducible sugars from reacting with free amino groups i.e. lysine and arginine side chains of proteins in the fish feed mixture, thereby preventing the formation of Schiff bases.

[0050] In aspects, the AGE-inhibiting antioxidant is one or more of a group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, and citric acid. Preferably, the antioxidant is ascorbic acid. In aspects, the antioxidant comprises at least one antioxidant, comprising ascorbic acid and sulfite.

[0051] As earlier disclosed herein, before the stable form of vitamin C (C-phosphate or stay-C) was used in feed production, only crystalline ascorbic acid was used as vitamin C in feed. Crystalline ascorbic acid was removed in the feed mixtures when the feed industry switched from wet feed and pellets to extruded feed because alarge part of the ascorbic acid disappeared in the extrusion process. It has never been explained other than that it is oxidized and disappears in the process, but it was suspected that it then acted as a reducing agent against the other feed components, and this should be addressed. When someone in the food industry is to use Vit C as an antioxidant to prevent the formation of / endings of aroma, taste, appearance, browning reactions, and nutritional value, then crystalline ascorbic acid is used. It is slightly reactive and will protect against the reactions that heat and processing contribute to. By using a stable form of Vit C, such as ascorbic acid phosphate, the vitamin will be stable throughout the processing process and thus cannot protect against reactions between the ingredients. Hence, crystalline ascorbic acid, i.e. ascorbic acid, may be used as an antioxidant to prevent AGE formation in the method for producing a fish feed as described herein.

[0052] Sulfites may also act as an antioxidant in that it reacts easily with water and forms sulfur dioxide. This is known from the wine industry where sulfites prevent fermentation and some chemical reactions in wine, sulfites are also used as an antioxidant on fruit where sulfites prevent fermentation and deterioration of pigments and the formation of browning substances which in turn can form melanin (for example as when drying apricots). Sulfites has also been tested as an inhibitor of lipase in offal from the salmon industry, to reduce the amount of free fatty acids in salmon oil. Sulfites will also inhibit polyphenol oxidase from reacting with phenols which in turn act as important antioxidants in several processes. In the present invention, sulfites may be used as an antioxidant to prevent AGE formation in the method for producing a fish feed as described herein. Sulfites may inactivate thiamine (vitamin B1 ) by reacting with thiol groups of thiamine resulting in thiaminesulfonate compounds. Hence, in aspects wherein the at least one antioxidant of the method comprises sulfites, the thiamine dosage should be adjusted to compensate for thiamine inactivation.

[0053] The method comprises the use of process aids that can dampen the glycation reaction processes and subsequent AGE formation in the extruded fish feed. The method according to the present invention proposes the use of crystalline ascorbic acid as an antioxidant, i.e. a reducing agent that inhibits glycation i.e. the Maillard reaction in the feed mass. The method suggests the use of sulfites and, or ferulic acid which additionally inhibits glycosylation (the above-mentionedglycosylation with enzymes). Glycosylation which breaks down pigment’s forms tanning substances which in turn can react and form melanin. The method requires that the pH in the feed mixture or composition is on the acidic side. Addition of processing aids must take place when feed ingredients are mixed and must be present and apply in the process steps preconditioning, extrusion, drying, coating, and storage.

[0054] In all embodiments, the addition of the at least one antioxidant is carried out so that the at least one antioxidant is present in the feed composition in the reactive phase of the extrusion process to inhibit AGE formation.

[0055] In a preferred embodiment, the antioxidant and the acid are added to the fish feed composition in the extrusion process before the preconditioning step, preferably in the mixing step wherein the temperature is in a range of 10-37°C, and humidity is in the range of 4-14%. Hence, the at least one AGE-inhibiting antioxidant is present in the feed composition and begin to work when water activity increases due to the addition of water and / or steam during the preconditioning. In all embodiments, the total amount of AGE-inhibiting antioxidant added to the mixture comprises 0.5-4 wt%. In another embodiment, the at least one antioxidant and the at least one acid are added in premixes of micronutrients. In yet another embodiment, the at least one antioxidant and the at least one acid are added in a liquid phase in which it is added to the preconditioner with water before pasting of starch is starting.

[0056] In a preferred embodiment, addition of the at least one antioxidant occurs where they are dispersed in the fish feed mixture, in the mixer before preconditioning, and a reaction process starts at the beginning of water activity when water / steam is added in preconditioner, and where the pH is adjusted with citric acid, in an amount of 200 to 1200 ppm. In another embodiment, the antioxidant as for example ascorbic acid, ferulic acid, and / or sulfite is added together with the protein sources and micronized before mixing with other ingredients, and a reaction process starts at the beginning of water activity when water / steam is added in preconditioner, and where the pH is adjusted with citric acid, in an amount of 200 to 1200 ppm.

[0057] In aspects, the amount of the at least one antioxidant added to the fish feed composition is in the range of 20 to 500 ppm. Under certain circumstances ascorbic acid may undergo ascorbic acid oxidation leading to the formation ofdehydroascorbic acid which upon cleavage and further processing of intermediates may lead to formation of AGE products CML and / or CLE (Li et al, 2023). However, in small amounts, the antioxidant function of ascorbic acid will prevent the formation of Schiff bases. Moreover, synergistic effects of the different antioxidants added may protect ascorbic acid from undesirable oxidation. Hence, in some aspects the at least one antioxidant comprises ascorbic acid in an amount of 20 to 500 ppm and further comprises at least one antioxidant selected from the group comprising ferulic acid, y- oryzanol, sulfite, and citric acid. Preferably, the at least one further antioxidant is sulfite in an amount of 100 to 2000 ppm, such as in an amount of 200 to 1200 ppm. Preferably, the amount of sulfite is higher than the amount of ascorbic acid.

[0058] In yet another embodiment, the group of antioxidants are added in such a way that the antioxidants are present in the mixture and begin to work when water activity increases due to the addition of water / steam in the preconditioner. The antioxidant is one or more of a group comprising ascorbic acid, sulfite, y-oryzanol, ferulic acid and citric acid. In yet another embodiment the antioxidant comprises at least ascorbic acid and citric acid.

[0059] In one embodiment, the at least one AGE-inhibiting antioxidant is added to the feed composition during the preconditioning, preferably before the feed composition reaches its pasting temperature i.e. when the temperature of the preconditioning is lower than 85°C, such as 65-84°C. Herein, the water content of the feed mixture is typically in the range of 10-25%.

[0060] Preferably, the at least one AGE-inhibiting antioxidant is added after adjustment of the pH, i.e. the at least one AGE-inhibiting antioxidant is added when the pH of the feed mixture is in the range of 5.0 to 6.5. In all embodiments, the at least one AGE-inhibiting antioxidant is added to the feed mixture at a timepoint so that the antioxidant activity is carried out in the reactive phase of the extrusion process, as earlier disclosed herein.

[0061] In aspects, the at least one AGE-inhibiting antioxidant is one or more of a group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, and citric acid, preferably ascorbic acid, and sulfite. In some embodiments, the at least one AGE- inhibiting antioxidant comprises ascorbic acid and one or more antioxidants selected from the group comprising ferulic acid, y-oryzanol, sulfite, and citric acid, preferablysulfite. The amount of AGE-inhibiting antioxidant added herein is dependent on the AGE formation potential of the fish feed composition, defined by the contents of reactants i.e. reducing sugars and amine groups from lysine and arginine endcontaining proteins and process parameters such as time, temperature, pH, moisture content, and pressure.

[0062] The type and amounts of antioxidants used in the method may vary. In a preferred embodiment, the antioxidant is ascorbic acid in an amount of 500 ppm. In another embodiment, the antioxidant is one or more of a group comprising ascorbic acid, ferulic acid, sulfite, y-oryzanol, and citric acid, in an amount of 200 to 800 ppm. In aspects, ascorbic acid is preferably in an amount of 20-500 ppm. Preferably, the amount of sulfite should be higher than the amount of ascorbic acid. The at least one AGE-inhibiting antioxidant may comprise ascorbic acid and sulfite in a 1 :2 ratio, or a 1 :5 ratio, or a 1 :10 ratio, alternatively a 1 :25 ratio of ascorbic acid to sulfite.

[0063] Generally, the amount of antioxidant depends on the fish feed formulation, the extrusion and process parameters, where parameters increasing the intensity of the Maillard reaction, as time, temperature, moisture, and alkalinity, increase the essential amount of the antioxidant. In all aspects, the at least one AGE-inhibiting antioxidant is added in a quantity equal to or greater than an AGE-formation potential, wherein the AGE-formation potential is defined by the contents of reducing sugars plus amino acids of the feed composition and extrusion process parameters comprising temperature, pressure, and moisture content. The 500 ppm ascorbic acid of the preferred embodiment refers to standard extrusion process parameters as for example 120 to 160°C, and the preconditioning parameters between 55 to 90°C. A professional in the art will understand that different antioxidants have different potential to decrease the intensity of the Maillard reaction, the professional will also understand synergetic effects and adjust the amount of antioxidants to the process parameters to be adapted to a product that in the end gives a low content of Maillard product.

[0064] In one embodiment, the component added to adjust the pH and the AGE- inhibiting antioxidant is the same component and has dual functions. For example, in aspects citric acid is added to adjust the pH wherein it dissociates and lowers the pH to the range of 5.0-6.5 and if citric acid is in excess after pH adjustment theremaining citric acid may function as an antioxidant to inhibit or reduce AGE formation as earlier described about antioxidants herein.

[0065] Hence, in one embodiment the pH adjustment to and the AGE-inhibition are carried out at the same time, and the component used to adjust the pH may also be used as an AGE-inhibiting antioxidant. For example, citric acid may be used to adjust the pH to 5.0-6.5 and as an AGE-inhibiting antioxidant. Herein, the amount of citric acid added to the fish feed composition must be sufficient to adjust the pH and function as an AGE-inhibiting antioxidant. Hence, in aspects the amount of citric acid is added to the feed composition is more than 0.5 wt%, such as 0.5-3 wt%, such as 1-2 wt%, 1 ,5-3wt%, or 2-3 wt%, calculated on the whole extrusion mass..

[0066] In preferred embodiments, the pH adjustment is carried out before the addition of the at least one AGE-inhibiting antioxidant. In such embodiments, the at least one acid is added in an amount of 0.5-4wt%, such as 0.5-1 .5 wt%, or 2-4 wt%, preferably 0.8-1 wt%, calculated on the whole extrusion mass, to adjust the pH to 5.0- 6.5. In such embodiments, the at least one acid is selected from the group comprising citric acid, lactic acid, propionic acid, ferulic acid, phosphoric acid, and acetic acid.

[0067] In such embodiments, the at least one AGE-inhibiting antioxidant is added in an amount of 0.5-4 wt%, such as 0.5-1 ,5wt%, or 2-4 wt%, preferably 0.8-1 wt%, calculated on the whole extrusion mass. In such embodiments, the at least one AGE- inhibiting antioxidant is selected from the group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, and citric acid.

[0068] Following extrusion, the extruded fish feed is subsequently dried. During the drying, in embodiments wherein sulfite the at least one AGE-inhibiting antioxidant comprises sulfite, the sulfur dioxide will evaporate during the drying so that only trace amounts of sulfite will be present in the final fish feed. The fish feed is further vacuum coated comprising coating the dried, extruded fish feed with lipids.

[0069] The physical quality and expansion of the final product are controlled by various factors, such as water and steam injection, viscous heat dissipation, and extruder screw and die configuration. The processing and quality of the final product is also influenced by the physicochemical and rheological properties of the feed ingredients.

[0070] The present invention can be used in / for all kinds of fish feed, but for sake of clarity the description refers to typical fish feed formulations containing the following category of ingredients: protein sources (for example fishmeal, feather meal, krill meal, insect meal, soy bean meal, legumes, maize gluten, wheat gluten other marine byproduct meals); starch sources (for example wheat flour, tapioca and rice flour); water; fats and oils (for example fish oil, rapeseed oil); fiber (as part of some ingredients); and minor ingredients like vitamins, minerals, binders, expanders, and so on. Hence, the method relates to production of fish feed comprising a typical fish feed formulation comprising the following categories of ingredients: protein sources selected from the group comprising fishmeal, feather meal, krill meal, insect meal, soybean meal, legumes, maize gluten, wheat gluten, and other marine byproduct meals, starch sources selected from the group comprising wheat flour, tapioca and rice flour, water, fats, lipids, and oils selected from fish oil and rapeseed oil, fiber (as part of some ingredients), vitamins, minerals, binders, and expanders. Each group of ingredients may influence the final product and processing conditions. As ingredient formulation is changed, one may be aware that processing conditions in the extruder may also have to change.

[0071] The feed composition used in the method may comprise at least one protein source, wherein fish meal comprises up to 30% of the protein source, and the remaining protein is derived from at least one protein source selected from the group comprising insect meal, soybean meal, legumes, maize gluten, wheat gluten, and other marine byproduct meals. The ratio of protein and fat content of the fish feed composition depends on factors such as age and size of the fish which will consume the feed. Other factors include the considerations of the feed manufacturer, properties of the feed ingredients, and availability of ingredients. In aspects, when the recipient fish is a fatty fish such as salmon and is in early life stage or is small in size, the content is typically 40 to 50% protein and 20 to 30% fat, preferably 45% protein and 25% fat. In aspects when the recipient fish is older or larger in size, the fish feed comprises 35 to 45% protein and 30 to 40% fat, preferably 40% protein and 38% fat. Hence, the feed composition may comprise a protein to fat content ratio in the range of 9:5 to 40:38. In lean fish such as white fish, the protein and fat content will be different and must be adapted according to the needs for specific fish species. Importantly, commercially manufactured fishmeal which is a source of fats for thefeed composition herein have variable fat contents due to seasonal variations and the species the fishmeal is made from.

[0072] Table 1 and 2 show typical compositions of a salmon feed that have the potential for a Maillard reaction i.e. glycation in or during the extrusion process, where both reducing sugars and the formation of reducing sugars occur because of heat, pressure, and shearing forces, and which can glycate with amino acids and form large amounts of AGEs in the feed, and where in addition a pH on the alkaline side will increase these reactions. And when fish eats this feed over time, health problems such as poor bone formation, bleeding in the intestine, inflammation, and poor healing of wounds in skin and skin damage is susceptible to occur.

[0073] Table 1 : Typical composition of the main components from the raw materials in the fish feed composition used in the method as described herein.

[0074] Table 2: Typical recipe of a growth feed for Salmon.

[0075] The same recipes or compositions as disclosed in Table 1 and 2, with the pH adjusted to the acidic side, according to the present invention, have the potential to inhibit the Maillard reactions during the reactive phase until a finished processed feed is made and to avoid the formation of AGEs (Maillard products). Hence, the compositions may be used in the method to produce fish feed comprising reduced concentrations of AGEs.

[0076] In a second aspect, the invention relates to a fish feed produced via the above-mentioned method. Wherein the fish feed is almost free of Maillard-type- rection-products. In other words, where the fish feed comprises low concentrations of AGEs and thus is thought to avoid the forementioned nutritional-related growth disorders of the fish. Hence, the fish feeds produced according to the method of the first aspect is aimed to positively affect the overall fish health and welfare, to prevent fish bone deformities, inflammation, and poor healing of wounds in skin. Wherein the fish feed is produced by adding at least one antioxidant and / or at least one acid to the fish feed formulation during the extrusion production process.

[0077] The fish feed produced by the method according to the first aspect, contains reduced concentrations of AGEs. The above-mentioned problems relating to fish health and welfare are largely solved by preventing the formation of AGEs in the extrusion process, as described in the present invention. Thousands of Maillard-type- reaction-products are known, and it is therefore not possible at this present time to specify the one or more AGE products responsible for the described bone deformities. As mentioned above, the inventor of the present invention has found a specific time-window wherein adding process agents comprising antioxidants and acids to the extrusion process prevents or reduces the AGE formation. The applicant has plans for testing the effect of the feed produced according to the method according to the first aspect through experiments in fish farming facilities. It is expected that a reduction in bone deformities and a generally improved fish health will be achieved and that this circumstance is due to the reduced concentration of Maillard-type-reaction-products i.e. AGEs due to suppression of the Maillard-reaction in the extrusion process. Therefore, the present invention is aimed to prevent theunderlying chemical mechanisms, and hence formation of Maillard-type-reaction- products in the fish feed.

[0078] In a third aspect, the invention relates to the use of a fish feed, where the fish feed is produced according to the above-mentioned method, according to the present invention. Where the fish feed is used to feed the fish and to positively affect the overall fish health and welfare, to prevent fish bone deformities, inflammation, and poor healing of wounds in skin. Where the fish feed is used solely or partially.

[0079] The fish feeds produced according to the method of the first aspect are intended for use for feeding teleosts, bony fish that are easily adapted to dry feed. In one embodiment, the fish feed produced according to the method of the first aspect is for feeding teleost fish, including eels, catfish, tarpon, tuna, flounder, trout, herring, yellowtail, salmon, seabream, sea bass, wrasses / cleaner fish, codfish, halibut, turbot, and salmonids. In one embodiment, the produced fish feed according to the method of the first aspect is for feeding salmonids, including but not limited to trout, salmon, char, and whitefishes. In one embodiment the fish feed produced according to the method of the first aspect is for feeding at least one of cold-water fish such as salmon, bream, brass, and warm -water fish such as tilapia, carp, and catfish.

[0080] In a fourth aspect, the invention relates to a method for producing feed or food, the product, and the use of it. Where the fourth aspect of the invention is related to all kinds of feed and food, aimed to prevent Maillard reaction, and Maillard products. Hence, in aspects, the invention relates to a method for producing feed and food by an extrusion process. Hence, the invention relates to a method for producing any extrudate. All feed and food products, i.e. extrudates, produced by an extrusion process, wherein the process comprises the above-mentioned conditions and ingredients for possible Maillard reactions, i.e. reducible sugars, and amino groups, comprise the formation of AGEs. The method according to the invention is aimed to prevent the Maillard reaction, the chemical reactions between amino acids and reducing sugars during the extrusion process. Non-limiting examples include extruded pelleted feed for pets, such as dog feed and cat feed.

[0081] It is to be understood that every embodiment of the disclosure can optionally be combined with any one or more of the other embodiments described herein.Elements disclosed for one aspect also apply for other aspects, hence the details provided for the composition also apply for the method or use when relevant.

Claims

Claims1 . A method for producing a fish feed, the fish feed is produced by an extrusion process of a feed composition, comprising the steps of preconditioning the feed composition, wherein the preconditioned feed composition reaches a pasting temperature, adjusting the pH comprising adding at least one acid to the feed composition in an amount to obtain a pH in the range of 5.0 to 6.5, adding at least one AGE-inhibiting antioxidant in a quantity equal to or greater than an AGE-formation potential, extruding the preconditioned feed composition, drying and coating the fish feed, wherein adjusting the pH and adding the at least one AGE-inhibiting antioxidant are performed before or as part of the preconditioning, during the reactive phase of the extrusion process which is defined by the AGE-formation potential.

2. The method according to claim 1 , wherein the AGE-formation potential is defined by the contents of reducing sugars plus amino acids of the feed composition and extrusion process parameters comprising temperature, pressure, and moisture content.

3. The method according to claim 1 , wherein the pH adjustment is performed before or simultaneously as the addition of at least one AGE-inhibiting antioxidant.

4. The method according to any of the preceding claims, wherein the at least one acid is selected from the group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, citric acid, lactic acid, propionic acid, phosphoric acid, and acetic acid.

5. The method according to any of the preceding claims, wherein the at least one acid is citric acid.

6. The method according to any of the preceding claims, wherein the at least one AGE-inhibiting antioxidant is selected from the group comprising ascorbic acid, ferulic acid, y-oryzanol, sulfite, and citric acid.

7. The method according to any of the preceding claims, wherein the at least one AGE-inhibiting antioxidant is sulfite.

8. The method according to any of the preceding claims, wherein the feed composition is preconditioned for 30-300 seconds, and the pasting temperature is in the range of 60-95°C.

9. The method according to any of the preceding claims, wherein the preconditioned feed composition is extruded for 10-60 seconds, and the temperature at the exit of the extruder is in the range of 100-140°C.

10. The method according to any of the preceding claims, wherein the quantity of the at least one acid added to the feed composition is in the range of 0.5-3 wt%, calculated on the whole extrusion mass.11 . The method according to any of the preceding claims, wherein the quantity of the at least one AGE-inhibiting antioxidant added to the feed composition is in the range of 0.5-4 wt%, calculated on the whole extrusion mass.

12. The method according to any of the preceding claims, wherein the feed composition comprises a total carbohydrate content in the range of 15-35%, and a protein to fat content ratio in the range of 9:5 to 40:38.

13. A fish feed produced according to the method of any of claims 1-12, wherein the pH of the fish feed is in the range of 6-7.

14. A fish feed according to claim 13 for feeding fish selected from the group comprising eels, catfish, tarpon, tuna, halibut, flounder, trout, cod, herring, salmon, salmonids, bream, char, whitefishes, brass, tilapia, yellowtail, and carp.

15. Use of the fish feed according to any of claims 13-14 for preventing bone deformities and improving fish health.

Images