GB2617574A - Supplemented krill meal and uses thereof - Google Patents

Abstract

A process for preparing supplemented krill meal comprises the steps of (a) providing krill A, (b) subjecting the krill to a heat treatment B, D, (c) separating E the heat-treated krill into press cake O and press water F, (d) separating G stick water from the press water, (e) mixing the stick water and the press cake, and (f) drying M the mixture to obtain a supplemented krill meal N. Step (f) preferably comprises drying the mixture using a vacuum dryer below 95°C. A process for preparing aquaculture feed is also claimed, comprising steps (a) to (f) and the further step of (g) formulating the supplemented krill meal into aquaculture feed. An aquaculture feed formed by the process, a method of aquaculture comprising making the aquatic feed as in steps (a) to (g) and feeding it to aquatic organisms such as fish, and krill meal augmented by stick water wherein the meal has a free amino acid concentration of at least 1g/100g of krill meal, are also claimed. The meal is preferably used for feeding salmon or shrimp, and steps (a) to (f) may be carried out on a ship, with step (g) being carried out on shore.

Description

SUPPLEMENTED KRILL MEAL AND USES THEREOF Field of the invention The present invention relates to processes for preparing aquaculture feed that involve supplementing krill meal with stick water, processes for preparing supplemented krill meal and krill meal compositions that have been supplemented with stick water. By utilizing not only the conventional krill meal, but also the resources available in stick water, the present invention provides a more sustainable ingredient to feed for the fish farming industry. Further, in addition to being more sustainable, the supplemented krill meal produced by the process of the invention has been shown to increase feed intake and improve growth in fish feeding trials.

Background of the invention

Global human consumption of salmonids has increased substantially in the last decades, which has been made possible by extensive fish farming, where Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the dominant species.

Originally, salmonid feeds were mainly based on marine resources and therefore naturally rich in omega-3 fatty acids and other marine nutrients. However, through the increase in global fish farming (including non-salmonid species) and increased direct human consumption of marine oils, the marine resources from capture fisheries can no longer meet the demand of the fish farming industry. In order to understand how aquaculture contributes to global seafood, efficiency assessments of marine ingredient are commonly applied. The main principle of these assessments is to ensure that aquaculture does not negatively impact wild fish stocks. Feed conversion ratio (FCR), fish-in-fish out (FIFO), forage-fish dependency ratio (FFDR) are methods that are used to analyze effectiveness of a feed. Previous assessments have demonstrated that most aquaculture species groups are net producers of fish, but salmon and trout aquaculture are net neutral, producing as much fish biomass as is consumed. Overall, global fed-aquaculture currently produces three to four times as much fish as it consumes (Aquaculture Vol 528, 15 Nov 2020, 735474). To ensure a positive contribution and net production of fish, both FIFO and FFDR are parameters that limits the use of marine ingredients from caught fish in the feed. In addition, life-cycle analysis (LCA) is used to evaluate the environmental impact of feed and feed ingredients.

As the importance of salmonids in the aquaculture industry increases, salmonid farmers are compelled to deliver more sustainable, healthy, and high-quality products for human consumption. This means replacing current marine raw ingredients with new, sustainable products rich in alternative protein sources that ensure fish zootechnical performance. Significant resources have been invested to find substitutes for the marine resources, and during the last decade the composition of salmonid feeds has shifted from being largely of marine origin to being largely of agricultural origin. Coinciding with the shift in composition of salmonid feed, a decrease in omega-3 content of farmed salmon has been observed. Without being bound by theory, it is believed that the observed decrease in omega-3 is due to the increased amount of vegetable oils less rich in omega-3 in the feed.

In addition to the omega-3 issue, the fish farming industry have been facing several other challenges caused by the increased amount of vegetable products in fish feed. Vegetable products, such as soybean meal has an organoleptic profile that is different from fish meal. Reduced palatability, as well as anti-nutritional factors causing low digestibility, can lead to impaired growth performance of the fish.

Although plant-based ingredients are available, they fail to meet nutritional requirements of fish, and include anti-nutritional factors affecting the palatability of aquafeeds and triggering unwanted immune responses.

Thus, there is a need for both alternative as well as effective and viable ingredients to replace the marine resources from capture fisheries.

In the quest of finding such alternatives, the use of marine algae and krill have attracted considerable interest in recent years. Krill is a shrimp-like swarming pelagic crustacea which is known to represents a viable alternative to fish meal due to its high levels of marine protein, phospholipid-bound omega-3 fatty acids and feeding stimulants. Krill is a proven solution to address the challenges faced by the aquaculture industry. It supports a sustained, healthy and fast fish growth, in addition it shows a nutritional profile and taste that promotes feed intake, improves feed conversion ratio, and enhances fillet quality. In addition, krill is also known to strengthen the fish immune system, thereby playing a significant role in reducing disease and mortality.

Even though krill meal has been identified as a promising candidate to be included in fish feed, there is still a need in the art for a more sustainable fish feed with improved properties.

Accordingly, there is a need in the art for optimization in harvesting and processing technology in order to produce sustainable ingredients in fish and shrimp feed.

Summary of the invention

In a first aspect the present invention provides process for preparing aquaculture feed, wherein the process comprises the following steps (a) providing krill (Figure 1, A); (b) subjecting the krill to a heat treatment (Figure 1, B and Figure 1, D), (c) separating the heat-treated krill (Figure 1, E) into a press cake (Figure 1, 0) and press water (Figure 1, F); (d) separating (Figure 1, G) stick water from the press water (Figure 1, F); (e) mixing the stick water and the press cake (Figure 1, 0); (f) drying (Figure 1, NI) the mixture to obtain a supplemented krill meal (Figure 1, N); and (g) formulating the supplemented krill meal into aquaculture feed.

The examples demonstrate that the aquaculture feed produced by the process of the invention significantly increased the total feed intake in fish in feeding trials, increased the biomass of fish, and lead to reduced mortality, compared to comparative aquaculture feeds comprising krill meal which has not been supplemented with stick water. Without wishing to be bound by any particular theory, it is hypothesized that the aquaculture feed has improved digestibility which makes it easier for aquatic organisms to utilize the ingredients in the aquaculture feed.

In a second aspect, the invention provides an aquaculture feed prepared by the processes of the invention.

In a third aspect the invention provides krill meal supplemented with stick water, wherein the krill meal has a free amino acid concentration of at least lg per 100g of krill meal, optionally between lg-3g per 100g of krill meal.

In a fourth aspect the invention provides processes for preparing supplemented krill meal, wherein the process comprises the following steps: (a) providing krill (Figure 1, A); (b) subjecting the krill to a heat treatment (Figure 1, B and Figure 1, D), (c) separating the heat-treated krill (Figure 1, E) into a press cake (Figure 1, 0) and press water (Figure 1, F); (d) separating (Figure 1, G) stick water from the press water (Figure 1, F); (e) mixing the stick water and the press cake (Figure 1, 0); (0 and drying (Figure 1, M) the mixture using a vacuum drier at a temperature below 95°C to obtain a krill meal (Figure 1, N).

Brief description of drawings

Figure 1 illustrates production line for processing krill (A) into krill meal (Q), Oil (I), Sludge (H), Stick water (7) and the supplemented krill meal (N).

Figure 2 illustrates seasonal variation in total protein content of krill meal supplemented with stick water at different relative proportions of krill meal and stick water.

iM Krill meal A Krill meal + 6% by dry weight stick water Krill meal + 12% by dry weight stick water 1/4 Krill meal + I S% by dry weight stick water A timeline is presented along the X-axis. Each month is represented by two measuring points, one representing the first half of the month and the second representing the second half of the month. The first two measuring points along the X-axis represent first and second half of January while the last two measuring points represent first and second half of August. Percentage total protein at a moisture level of about 6 % by weight is denoted on the Y-axis.

Figure 3 illustrate the set-up in the feeding trial as demonstrated in Example 2 Figure 4 illustrates reduced mortality in feeding trial with inclusion of SW corresponding to 5-7 %.

Figure 5 illustrate higher feed intake and growth (biomass) with QSW in comparison to QA and control.

Figure 6 illustrates the higher percentage of water soluble proteins in the 2 batches of QSW meals versus QA meal.

Figure 7: Higher percentage of appetite stimulating free amino acids in QSW meals versus QA meal.

Figure 8: Higher percentage of appetite stimulating free amino acids in feeds with QSW in comparison to QA and control feeds.

Figure 9: A process for preparing supplemented krill meal that involves recycling the excess heat from the drying to heat the stick water.

Definitions The term "indirect heating" refers to a process where a material is not allowed to come in direct contact with a heating agent, such as steam, to change the final temperature of the material.

The term "direct heating" refers to a process where a material comes into direct contact with a heating agent, such as steam, to change the final temperature of the material.

The term "feed intake" as used herein is measured as percentage of body weight per day.

The term "feed conversion ratio (FCR)" as used herein refers to mass of the input (mass of fish feed) divided by the output (bodyweight of the fish).

The term "specific growth rate (SGR)" is calculated according to the following formula: (In (final weight in grams) -In (initial weight in grams) x100) / t (in days).

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of medicine, pharmacology, pharmaceutical chemistry, biology, biochemistry and physiology.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail.

Headings have been used for organizational purposes and should not be construed as limiting the subject-matter herein.

Detailed description of the invention Providing the krill Krill lives in all the major oceans world-wide. For example, it can be found in the Pacific Ocean (Ettphansia pactfica), in the Northern Atlantic (Meganycilphanes norvegica) and in the Southern Ocean off the coast of Antarctica (Euphausia superba). As a key species in the ocean, it is a food source for many animals such as fish, birds, sharks and whales. The total biomass of Antarctic krill (E. superba) is estimated to be in the range of 300-500 million metric tons. Preferably, the krill utilized in the invention is Eztphattsia superba.

Antarctic krill feeds on phytoplankton during the short Antarctic summer. During winter, however, its food supply is limited to ice algae, bacteria, marine detritus as well as depleting body protein for energy (Virtue et al., Mar. Biol. 126, 521-527). For this reason, the nutritional values of krill vary during the season and to some extent annually (Phleger et al., Comp. Biochem. Physiol. 131B (2002) 733). In order to accommodate variations in food supply, krill has developed an efficient enzymatic digestive apparatus resulting in a rapid breakdown of the proteins into amino acids (Ellingsen et al., Biochem. J. (1987) 246, 295-305). This auto proteolysis is highly efficient also postmortem, making it a challenge to catch and store the krill in a way that preserves the nutritional quality of the krill.

The krill in step (a) can be provided by catching wild krill from a sea or an ocean. A ship can tow a trawl that is configured to catch the krill. The krill is then transferred from the trawl to the ship and processed.

Overall, harvesting krill in a commercially viable and environmentally sound way is challenging. Traditional trawling methods where the catch is hauled up on deck and emptied into holding tanks before processing is unsuitable.

Krill have a fragile outer shell which when netted is easily crushed. Once the shell is crushed, the enzymes trapped in its digestive system putrefy the flesh quickly.

Therefore, it is preferably to pump the krill out of the bottom of the net while in the water and straight into the vessel to ensure that the krill remain intact. The pump system can comprise a tube that extends below the water the trawl and a pumping action is provided by injecting air into the tube below the waterline so that the krill is continuously drawn or pumped from the trawl, through the tube and on board the ship. Preferred trawling systems with pumps are described in PCT Applications WO 07/108702 and WO 05/004593, which are incorporated herein by reference.

The krill that can be used in step (a) include Antarctic krill (Euphausia superba), Pacific krill (Euphausia pacifica) and Northern krill (Illeganyctiphanes uorvegica).

Preferably, the krill that can be used in step (a) is Antarctic krill (Euphausia superba). The krill in step (a) can be fresh or frozen, but are preferably fresh.

To prevent the degradation of krill and to preserve the nutritional quality, krill may be processed into a krill meal as an alternative to reduce enzymatic activity. This process is preferably done on the fishing vessels, immediately after catch, and is generally a highly energy-demanding process. In order to meet the requirements from the aquaculture industry and be a competitive alternative to agricultural ingredients there are significant advantages in reducing the energy consumption in the off-shore processing of krill into krill meal. This will also reduce the CO2 footprint of the final products and make krill a more attractive and sustainable ingredient.

Therefore, steps (a) to (f) can be conducted immediately after the krill has been caught. Steps (a) to (f) can be conducted on a vessel while step (g) is conducted on shore. The time between steps (0 and (g) can be at least 1 week or at least 1 month. Advantageously, the supplemented krill meal may be stored on the vessel for a period of time before it is then formulated into aquaculture feed, for example the time between steps (0 and (g) can be up to 36 months.

The reduced energy consumption and increased yield of the final product have been demonstrated to result in a reduction of CO2 emissions per unit meal produced.

Subjecting the krill to a heat treatment The krill can be heat-treated in step (b) in a number of ways. For example, the heat treatment in step (b) can comprise increasing the temperature to about 80 °C to 100 °C. Alternatively, the heat treatment in step (b) comprises a first heat treatment (Figure 1, B) by increasing the temperature to about 40 °C to 60 °C; and then subjecting the krill to a second heat treatment (Figure 1, D) by increasing the temperature to about 80 °C to 99 °C. Increasing the temperature to a specific temperature involves increasing the temperature so that the krill reaches the desired temperature before the krill is further processed. Optionally the krill may be held at the desired temperature for at least 1 minute, for example for 1-10 minutes. For example, the heat treatment in step (b) may comprise a first heat treatment by increasing the temperature to about 40 °C to 60 °C and holding the krill at that temperature for at least 1 minute, or 1-10 minutes, or 3-5 minutes; and then subjecting the krill to a second heat treatment by increasing the temperature to about 80 °C to 99 °C and holding the krill at that temperature for at least 1 minute, or 1-10 minutes, or 1-2 minutes.

It is known in the art that krill meal production can involve a two-step separation process. In W02009/027692 the first step involves removing proteins and phospholipids from the krill, precipitated as a coagulum in a process in which substantial volumes of liquid water are added. In the second stage the krill without the proteins and phospholipids are cooked. Following this, residual fat and astaxanthin are removed from the krill using mechanical separation methods.

In contrast, the examples demonstrate that supplementing krill meal with stick water that has been produced by a process that does not remove fats and proteins from the heat-treated krill has a higher amount of free amino acids which are known to be appetite stimulating. Without wishing to be bound by any particular theory, by ensuring that no fats and proteins are removed from the heat-treated krill this results in stick water that has a particularly advantageous composition for supplementing krill meal for use in aquaculture.

Therefore, preferably no filtration steps are performed between steps (b) and (c).

Preferably no steps are performed between steps (b) and (c) that would remove fats and proteins from the heat-treated krill. The heat-treated krill from step (b) may be directly introduced into the separating apparatus used in step (c).

Furthermore, processes known in the art, such as those described in W02009/027692, require a large amount of water such as fresh water. As discussed above, it is preferable for krill to be processed on a vessel directly after they are caught. It is therefore difficult to store the required fresh water for these processes on the vessel and it is energy intensive to produce fresh water from sea water.

The claimed processes preferably do not require the addition of large amounts of fresh water. For example, preferably during steps (a)-(c) fresh liquid water, if added to the krill, is added in amounts of no more than 2 parts water to 1 part krill by mass, or no more than 1 part water to 1 part krill by mass, or no more than 0.5 parts water to 1 part krill by mass. Steps (a)-(c) may be performed without the addition of fresh liquid water to the krill. The term "fresh water" may be understood as excluding sea water, for example water containing low concentrations of dissolved salts, and may encompass water recovered from an evaporation process.

Separating the heat-treated krill into a press cake and press water Separating the heat-treated krill into a press cake and press water can be performed with a centrifuge such as for example 2-or 3-phase decanter centrifuges (e.g. a tricanter), or with a press such as a screw press. Centrifuging or pressing the krill produces a liquid fraction known as press water and a solid fraction known as press cake.

Se)aratint slick water torn the ress water The step of separating stick water from the press water (step (d)) can comprise separating the press water (Figure 1, F) into stick water (Figure 1, 7), an oil fraction (Figure 1, I) and a sludge fraction (Figure 1, H). The separation can be achieved by centrifuging the press water to stick water, an oil fraction and a sludge fraction, for example using a 2-or 3-phase decanter centrifuge.

Stick water is the water-soluble fraction of krill generated as an intermediate product during krill meal production (Figure 1, J). It represents from about 20 to 50% of the wet weight of the krill raw material and is typically discharged to sea. The stick water produced can contains from 57 g to 87 g water soluble proteins per 100 g protein containing material on a dry weight basis. Accordingly, the use of the stick water in the invention provides a supplemented krill meal which is more sustainable, since it utilises a krill fraction which might otherwise be discarded.

The examples demonstrate that stick water produced during the claimed processes has a high level of the level of water-soluble proteins and the amount of trimethylamine (TMA) + Trimethylamine N-oxide (TMAO). Further, with reference to table 3 provided herein the molecular weight distribution of water-soluble peptides are also different in stick water and krill meal. Based on the composition of stick water, adding stick water condensate back into the krill meal increases the content of ingredients such as free amino acids, peptides, small proteins, minerals, water soluble vitamins and low molecular weight (below 4000 Da) components such as taurine, proline, carnosine, (see Tables 2-5).

Therefore, the amount of trimethylamine and the amount of trimethylaminooxide in the stick water can be in the range less than 2500 mg N per 100 g, such as 1100 mg N to 2500 mg N per 100 g the stick water on a dry weight basis. The amount of trimethylamine and the amount of trimethylaminooxide in the supplemented krill meal can be in the range of less than 250 mg N per 100 g, such as 100 mg N to 250 mg N per 100 g supplemented krill meal on a dry weight basis.

In some embodiments, 0.1 % to 0.5 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 10kDa to 15kDa. In some embodiments, 0.2 % to 1 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 8kDa to 10kDa. In some embodiments, 1 % to 2.4 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 6kDa to 8kDa. In some embodiments, 70 % to 80 % by dry weight of the water-soluble proteins in the stick water have a molecular mass 200 Da.

Prior to step (e) the method can comprise the step of reducing the amount of water (Figure 1, IC) in the stick water (Figure 1, J) thereby obtaining a stick water concentrate (Figure 1, L). The stick water concentrate produced can have a moisture content of 40-80 4/0, or 50-70 % or 55-65 %. Step (e) can comprise mixing the stick water concentrate and the press cake.

Being sustainable is all about reducing energy consumption and reducing waste.

One of the most effective ways of reducing waste is by utilizing as much of the wild catch as possible. In a first step towards more sustainable harvesting of krill, the inventors of the present invention have discovered improved means for stick water recovery. One suggested solution is to include more energy effective and gentle ways of reducing moisture content of stick water (Figure 1, J) and production of the final product (Figure 1, N). The technological solution includes using a dryer (Figure 1, M) under vacuum, such as a disc dryer, for reducing the moisture content in the new compositions. In addition, the inventors have proposed to reuse the water evaporated from stick water. The water evaporated off the krill is recovered through a condenser (Figure 1, R). This water is then used as water source in a boiler (Figure 1, S) and to be made pressurized steam of, which is injected into the heat treatment #2 (Figure 1, D). The need for desalination of sea water to produce water for the direct steam heater is then reduced, as the water recovered in the stick water evaporator is used instead.

The inventors have also realised that the efficiency of the process can be further improved by using the heat produced by the drying step (step (f)) to concentrate the stick water in step (d). In this way, the CO2 emissions of the process may be further reduced.

For example, reducing the amount of water in the stick water can comprise using the excess heat from the drying of step (f) to heat the stick water and reduce the amount of water in the stick water, as shown in Figure 9.

The different solutions have provided cost effective access to ingredients which in the past are discharged to sea.

By including stick water in the krill meal, the inventors were also able to significantly increase the yield. In sum the reduced energy consumption and increased yield of the final product have been demonstrated to result in a reduction of CO2 emissions per unit meal produced.

Mixing the stick wafer and the press cake The amount of stick water in the supplemented krill meal can vary. For example, the relative proportions of the stick water and the press cake mixed in step (e) can be in the range of 1 part by dry weight of the stick water and 25 parts by dry weight of the press cake to 1 part by dry weight of the stick water and 3 parts by dry weight of the press cake. The supplemented krill meal produced in step (f) can comprise stick water at a concentration of 3-25 % w/w, or 3.5-10 % w/w, or 4-7 % w/w of the total dry matter.

Drying the mixture to obtain a supplemented krill meal The drying step is discussed in detail below in relation to the process for preparing supplemented krill meal. It will be understood that the discussion of the drying step in relation to processes for preparing supplemented krill meal applies equally to the process for preparing aquaculture feed as disclosed herein.

The examples demonstrate that the supplemented krill meal produced by the processes of the invention has a higher amount of free amino acids that are known to be appetite stimulating. The supplemented krill meal therefore can have a free amino acid concentration of at least 1g per 100g of supplemented krill meal. In preferred embodiments, the supplemented krill meal has a free amino acid concentration of between lg-3g per 100g of supplemented krill meal. The free amino acids can comprise one or more amino acids selected from the group consisting of alanine, proline, arginine, leucine, glutamine and glycine. Preferably, the free amino acids comprise alanine, proline, arginine, leucine, glutamine and glycine. The combined concentration of alanine, proline, arginine, leucine, glutamine and glycine in the supplemented krill meal can be at least 2.5, or 3.0-15, or 3.2-10 (3'w/w of the total protein in the supplemented krill meal.

Formulating the supplemented krill meal into aquaculture feed Results that have been reported so far indicate that inclusion of krill meal into fish feed improves both growth and yield. Further, krill meal has also been shown to, increase i) growth performance, ii) feed utilization efficiency, iii) diet digestibility and improve i) palatability, ii) intestinal development, iii) innate immunity and iv) disease resistance of olive flounder (Aquaculture Research, February 2020, DOI: 10.1111/are.14573). Some reports also demonstrate improved fillet quality, and in particular decreased gaping score and increased flesh and fillet firmness, when fish are fed a diet supplemented with krill meal (W02015185993). Further, there are also publicly available data demonstrating that krill meal has a positive effect on heart health of fish and also that there is a positive effect on preventing viral diseases in fish (W02017029558). Thus, all data published so far points at conventional krill meal as a promising substitute for fish meal in feed for fish.

By supplementing the krill meal with stick water, the inventors were also able to significantly increase the yield from the krill that is processed.

The supplemented krill meal can be formulated into aquaculture feed by mixing it with additional components, such as one or more of marine protein (e.g. fish meal), vegetable protein (e.g. soya protein, sunflower extract, wheat gluten, pea protein, Guar meal), fish oil, vegetable oil (e.g. rapeseed oil, soybean oil, camelina oil), starch (e.g. wheat), vitamins, minerals, antioxidants, fats and sealers. The formulation of the aquaculture feed will depend on the species of aquatic organism that the aquaculture feed is intended to be used for and suitable formulations are known in the art.

The aquaculture feed can comprise different amounts of supplemented krill meal. For example, the aquaculture feed can comprise supplemented krill meal at a concentration of at least 2 % w/w, at least 4 % w/w, at least 5 % w/w, at least 6 % w/w, at least 10 % w/w, at least 20 % w/w, at least 30 % w/w or at least 40 % w/w.

In preferred embodiments, the aquaculture feed comprises supplemented krill meal at a concentration of at least 2 % w/w. The aquaculture feed can comprise supplemented krill meal at a concentration of 3-50 % w/w, or 4-30 9/Ow/w, or 5-15 %w/w. The combined concentration of alanine, proline, arginine, leucine, glutamine and glycine in the aquaculture feed may be at least 0.60 %w/w of the total protein in the aquaculture feed.

Formulating the aquaculture feed can comprise substituting some or all of the fish meal in a typical aquaculture feed for the supplemented krill meal. For example, the aquaculture feed may contain no fish meal. Alternatively, the aquaculture feed can comprise both fish meal and supplemented krill meal. The relative proportions of fish meal and supplemented krill meal in the aquaculture feed can be in the range of parts supplemented krill meal to 1 parts fish meal to 1 part supplemented krill meal to 5 parts fish meal.

The invention also includes processes according to the first aspect that do not involve step (g), for example the process may omit the step of formulating the supplemented krill meal into aquaculture feed. In this way, the invention provides a process for preparing a supplemented krill meal.

Methods of aquaculture The invention also provided methods of aquaculture, that comprise preparing aquaculture feed by the processes of invention, and feeding aquatic organisms with the aquaculture feed. An aquatic organism can be any marine fish, for example an organism such as salmon from the family Salmonidae, halibut, seabream, seabass, flounder, tilapia. Preferably, the aquatic organism is from the family Salmonidae, such as salmon. Types of salmon include Sahno salar (Atlantic salmon), Oncorhynchus Ishawyischa (Chinook salmon), Oncorhynchus keta (Chum salmon), Oncorhynchus kisulch (Coho salmon), Oncorhynchus mason (Masu salmon), Oncorhynchus gorbuscha (Pink salmon) and Oncorhynchus nerka (Sockeye salmon). Preferably, the salmon is Salmo salar (Atlantic salmon).

The aquatic organism may be a shrimp. Types of shrimp include Lilopenaeus rannamei (Whiteleg shrimp), Penaens monodon (Giant tiger prawn), Aceles japonicus (Akiami paste shrimp), Trachysalambria curvirostris (Southern rough shrimp), Fenneropenaeus chinensis (Fleshy prawn), Fenneropenaeus merguiensis (Banana prawn) and Pandalus borealis (Northern prawn). Preferably, the shrimp is Litopenaeus Valli7C1MCi (Whiteleg shrimp).

The aquaculture feed and supplemented krill meal produced by the processes of the invention are particularly useful for feeding aquatic organisms that are undergoing periods of stress. During these stressful periods the aquatic organisms lose their appetite. The examples demonstrate that supplemented krill meal increased feed consumption, which is thought to be due to the presence of appetite stimulating amino acids in the stick water. An example of a stressful period for anadromous fish such as salmon is when they are transferred from fresh water to saltwater, a physiological process termed smoltification. Hence, palatability plays an important role in aquaculture feeds for salmon during their transfer to sea water. Therefore, the aquaculture feed is preferably fed to aquatic organisms that are being transferred or have been transferred from fresh water to saltwater, such as during the smoltification process. For example, the methods of aquaculture can include the step of transferring an aquatic organism from fresh water to saltwater and feeding the aquatic organism with aquaculture feed prepared by the processes of invention.

Process for preparing supplemented krill meal The invention provides processes for preparing supplemented krill meal. These processes comprise the following steps: (a) providing krill (Figure 1, A); (b) subjecting the krill to a heat treatment (Figure 1, B and Figure 1, D), (c) separating the heat-treated krill (Figure 1, E) into a press cake (Figure 1, 0) and press water (Figure 1, F); (d) separating (Figure 1, G) stick water from the press water (Figure I, F); (e) mixing the stick water and the press cake (Figure 1, 0); (f) and drying (Figure 1, NI) the mixture using a vacuum drier at a temperature below 95°C to obtain a krill meal (Figure 1, N).

Surprisingly, it is also demonstrated that the upgrades to the drying process with more gentle drying have a positive effect on meal composition with reduced risk of aggregation of proteins and browning.

Thermal processing of krill meal at 100°C for 5-15 min was thought to be critical to ensure food safety. It was believed that this high temperature was required to reduce the viral load in wild krill, and thus achieving a temperature of 100 °C during processing can be a regulatory requirement. However, the inventors have theorised that this thermal processing alters the proteins in the krill meal and can even result in the proteins being inaccessible for digestion due to heat-induced non-enzymatic browning, so called Maillard reactions, oxidation and exposure of hydrophobic surfaces. Maillard reactions are a complex group of chemical reactions that are induced by heat and occur mainly between amino acids and sugars. Here, heat trigger a great number of reactions that can lead to the formation of flavor compounds and the characteristic brown color, but also toxic compounds such as acrylamide. The main amino acid reacting in Maillard reactions is the essential amino acid lysine, meaning that Maillard reactions leads to a reduction in essential amino acids. Excessive heat also drives the formation radicals, i.e., formation of reactive oxidative species (ROS). Radicals can alter the amino acid side chain groups by generating e.g., protein carbonyls, disulfides, and dityrosines. These changed amino acids can form a network of stable covalent bonds between proteins that have been heat denatured, hereby reducing the number of these amino acids and forming stabilized aggregates shielding other parts of the proteins which becomes inaccessible for digestive enzymes. Aggregate formation can also be driven by heat-denaturation of proteins. In a hydrophilic environment this drives formation of aggregates of proteins, where the hydrophobic moieties within the protein structures strive to associated, held together by van der Waals interactions.

The inventors have surprisingly found that lower temperatures are sufficient to endure the safety of the krill meal. Based on this realization they have designed an improved process for preparing supplemented krill meal. This new process uses a vacuum drier at a temperature below 95°C. A vacuum dryer has several advantages, for example that it allows for drying at lower temperatures. As described above, subjecting krill meal to high temperatures have several drawbacks. A result of the new drying process is krill meal with improved digestibility because of reduced denaturation and aggregation of proteins, as well as improved visual properties due to less browning.

Accordingly, the invention provides a process for making supplemented krill meal compositions wherein the step of drying is performed using a vacuum drier at a temperature below 95°C. This step is preferably also implemented as part of the processes for preparing aquaculture feed of the first aspect. For example, the drying in step (0 can comprise a vacuum drier at a temperature below 95°C.

Preferably, the temperature is in the range from 80°C to 90°C, or 80 to 85 °C, such as at about 85 or 82°C. Vacuum drying occurs below atmospheric pressure, for example at < 0.9 bar, <0.75 bar, or <0.6 bar.

The moisture content of the supplemented krill meal may be in the range of 0-25 wt%, 1-15 wt%, or 3-10 wt%.

In a further step towards being an attractive alternative ingredient in aquaculture, the inventors of the present invention decided to test the new composition comprising krill meal with added stick water in a feeding trial, as shown in the examples. The hypothesis was that improved digestibility because of reduced denaturation and aggregation of proteins would make it easier for the test subjects to utilize the ingredients.

As will be shown in the subsequent examples, the krill meal supplemented with stick water had an effect on feed intake, specific growth rate, feed conversion ratio and thermal growth coefficient. 9 different diets were produced and formulated according to the recipes presented in table 4 in example 2. QA refers to krill meal (analysis provided in Table 1). QSW refers to supplemented krill meal with dry matter originating from stick water corresponding to either 6.5% or 4.9% w/w of the total dry matter.

After analysing the QA and QSW, the inventors found a general higher percentage of water-soluble proteins, in particular higher levels of the free amino acids alanine, proline, arginine, leucine, glutamine and glycine in QSW (Figure 6). These amino acids belong to a group of amino acids that are known to be feed stimulating amino acids. Besides, the inventors found a dose response effect of QSW inclusion on the percentage of appetite stimulating free amino acids. There was higher percentage of these free amino acids with higher inclusion of QSW (Figures 7 and 8). Based on the analysis, the inventors suggest that higher feed intake with QSW was due to the higher percentage of water-soluble proteins and appetite stimulating free amino acids. Interestingly, the results obtained from the feeds used during the feeding trial support this.

In view of the above it is clear that a composition comprising krill meal supplemented with stick water according to the claimed invention provides increased feed intake. By increasing the feed intake, the fish will be provided with more krill derived products, including ingredients derived from stick water and krill meal. Thus, all the positive effects of krill meal on fish, such as: improved growth performance (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); increased feed utilization efficiency (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); improved diet digestibility (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); improved intestinal development (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); improved innate immunity (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); increased disease resistance (Aquaculture Research, February 2020, DOI: 10.1111/are.14573); -improved fillet quality (W02015185993); positive effect on heart health (W02017029558); and positive effect on preventing viral diseases (W02017029558); will be enhanced.

In another aspect, the invention therefore provides krill meal supplemented with stick water, wherein the krill meal has a free amino acid concentration of at least lg per 100g of krill meal, e.g. a free amino acid concentration of between 1g-3g per 100g of krill meal. The free amino acids preferably comprise one or more amino acids selected from the group consisting of alanine, proline, arginine, leucine, glutamine and glycine; optionally wherein the free amino acids comprise alanine, proline, arginine, leucine, glutamine and glycine. Accordingly, the invention also provides krill meal supplemented with stick water, wherein the combined concentration of alanine, proline, arginine, leucine, glutamine and glycine in the supplemented krill meal is at least 2.5, or 3.0-15, or 3.2-10 %w/w of the total protein in the supplemented krill meal.

The level of water-soluble proteins in the krill meal can be greater than 15% of total protein, optionally wherein the level of water-soluble proteins in the krill meal is 17-25%.

A comparison between the content of krill meal (analytic data presented in table 1) and the content of stick water (analytic data presented in table 2), when the level of moisture is accounted for, shows that the amount of certain ingredients is very different between the two products. In particular, the amounts of water-soluble proteins and trimethylamine (TMA) + Trimethylamine N-oxide (TMAO) are much higher in stick water compared to krill meal and the molecular weight distribution of the water-soluble peptides are also different in the two products.

Based on the latter analysis it is plausible to conclude that increasing amount of water-soluble proteins has a positive effect on feed intake which demonstrates the advantage of the composition according to the claimed invention. Further, it is also reasonable to believe that high levels of TMA and TMAO have a positive effect on feed intake, further substantiating the use of the compositions as claimed herein.

Furthermore, stick water contains significant higher levels of short water-soluble peptides having a molecular mass equal to or below 200 Da (table 3) as compared to krill meal, and these short peptides is also expected to contribute to the positive effect on feed intake. It is also to be mentioned that intake of the feed has been demonstrated to be positively correlated with thermal growth coefficient (figure 2).

Previous studies support that some free amino acids are linked to higher appetite in salmons (Kousoulaki et al, Aquaculture Nutrition, 2013 19; 47-61).

In example 2, the amino acids belonging to the group of alanine, proline, arginine, leucine, glutamine and glycine were found at higher levels as free amino acids in the QSW in comparison to QA, see Ex 2. The results of example 2 support that adding krill meal supplemented with the water-soluble krill protein containing material recovered from stick water to fish feed increase feed intake and improve growth in aquaculture.

In specific embodiments, the water-soluble krill protein containing material comprises free amino acids selected from the group of alanine, proline, arginine, leucine, glutamine and glycine, The effect of water-soluble amino acids from krill has also been confirmed by another study where the different protein fractions of conventional krill meal was prepared in feed to shrimps. In this study (unpublished results) the proteins of ordinary krill meal (without inclusion of stick water) were separated into a fraction of water-soluble krill proteins (krill protein hydrolysate) and a fraction of water-insoluble proteins and compared to the krill meal starting material. The conventional krill meal, the water-soluble protein fraction from krill (krill hydrolysate) and the remaining fraction of insoluble proteins from krill was added to feed for shrimps and tested against a negative control feed with no inclusion of krill derived material. The result demonstrated that adding 0.25% w/w of the water-soluble protein fraction had a significantly improved effect on shrimp growth, the same as seen with 3 % w/w of conventional krill meal, while the insoluble krill proteins had the same growth as the negative control. Since the effects is only demonstrated for the water-soluble protein product, the effect is likely to be associated with an enhancement of shrimp feed intake due to the water-soluble protein fraction functioning as an attractant and palatability enhancement, rather than a nutrient enrichment of the diets. The findings support that addition of krill meal supplemented with stick water, e.g. additional water-soluble krill proteins, will result in increased feed intake and improved growth of shrimps.

In conclusion, the inventors have demonstrated that inclusion of ingredients from stick water has an overall positive effect on the feed conversion ratio as it increased growth in the feeding trial and reduced waste during production. Using life-cycle analysis (LCA), it was also found that krill meal with inclusion of stick water has a significantly lower CO2 footprint compared to conventional krill meal produced without inclusion of stick water.

Having generally described this invention, a further understanding can be obtained by reference to the examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

Examples

Example 1: Production of krill meal (QA), stick water (SW) and combined product (QA+SW) A mixture of krill in water (figure 1, A) was subjected to indirect heating, using a scraped surface heater (figure 1, B), thereby increasing the temperature of the mixture to about 40 °C. Materials accumulating on the surface of the scraped surface heater were continuously removed from the surface to prevent burn-on or material build-up (Figure 1, C). The use of scraped surface heater ensure that the heating surface is always clean to achieve an optimal heat transfer.

The mixture of krill and water was then subjected to direct heating (Figure I, D) by injecting steam directly into the mixture thereby increasing the temperature of the mixture to about 92 °C.

Thereafter the mixture was separated into a press cake (Figure 1, 0) and a press water (Figure 1, F) using a decanter. Analysis of the press cake (Figure 1, 0) indicating a moisture content of from about 58 to about 62 % by weight.

The temperature of the press water (Figure 1, F) was adjusted to about 95 °C before the press water (Figure 1, F) was subjected to high-speed centrifugation (Figure 1, G) to separate the press water (Figure 1, F) into a sludge fraction (Figure 1, 1-1), an oil fraction (Figure 1, 1) and stick water (Figure 1, J).

The stick water (Figure 1, J) was then subjected to treatment on an evaporator (Figure 1, K) to reduce the water content and thereby obtain a stick water concentrate (Figure 1, L). The evaporator utilises excess heat from the disc dryer (Figure 1, M), improving the efficiency of the process. Analysis of the stick water concentrate (Figure 1, L) indicating a moisture content of about 62 % by weight. In an optional embodiment, the water evaporated from stick water can be re-used to heat the mixture of krill and water (Figure 1, D). The water evaporated off the krill is recovered through a condenser (Figure 1, R). This water is then used as water source in a re-boiler (Figure 1, S) and made pressurized steam of, which is injected into the heat treatment #2 (Figure 1, D).

Some of the press cake (Figure 1, 0) was subjected to drying on a Disc dryer (Figure 1, P) to form a krill meal (Figure 1, Q) having a moisture content of about 6 °,O by weight. The remaining part of the press cake (Figure 1, 0), having a moisture content of about 62 % by weight, was mixed with the stick water concentrate (Figure 1, L), also having a moisture content of about 62 % by weight, at different ratios in the range 1 part by dry weight of the stick water concentrate (Figure 1, L) and 25 parts by dry weight of the press cake (Figure 1, 0) to 1 part by dry weight of the stick water concentrate (Figure 1, L) and 3 parts by dry weight of the press cake (Figure 1, 0). The combined product was then subjected to drying on a Disc dryer under vacuum (Figure 1, NI) to form a combined product (Figure 1, N) having a moisture content of about 6 14) by weight. A vacuum of -0,48 bar allows for drying at lower temperature from about 81°C to about 95 °C instead of drying at 100 °C. This process reduces the risk of protein denaturation and browning and ensures improved digestibility and visual properties of the final product.

The content of krill meal (Figure I, Q) was analyzed, and the provided data are presented in table 1 below. The stick water (Figure 1, J) was analyzed, and the data obtained are presented in table 2 below. Further the molecular weight distribution of the water-soluble proteins and peptides in krill meal and stick water is presented in table 3.

Table I: Analysis of krill meal (QA) A25 Crude protein combustion °A 61,2 Moisture % 6,1 Ash % 10,6 Salt % 2,9 Watersoluble crude protein g/100g 7,3 Cadaverine mg/kg <10 Histamine mg/kg <10 Phosphorus % 1,4 Fluorine (acid soluble) g/kg 1,5 Free Astaxanhin mg/kg <2 Astaxanthin esters mg/kg 122 Total Volatile Nitrogen mg N/mog 15 Trimethylamin-N mg N/100g <1 Trimethylaminooxide-N mg N/mog 150 Triacylglyserol g/100 extracted fat 37 Diacylglycerol g/100 extracted fat 2 Monoacylglycerol g/100 extracted fat <1 Free fatty acids g/100 extracted fat 4,5 Cholesterol g/100 extracted fat 1,5 Cholesterol esters g/100 extracted fat 0,5 Phosphatidyletanolamin g/100 extracted fat 5 Phosphatidylinositol g/100 extracted fat <1 Phosphatidylserin g/100 extracted fat <1 Phosphatidylcholin g/100 extracted fat 29 Lyso-phosphatidylcholin g/100 extracted fat 2,9 Total polar lipids g/100 extracted fat 37,3 Total neutral lipids g/100 extracted fat 45,8 Total sum lipids g/100 extracted fat 83,2 14:00 g/100 extracted fat 8,8 16:00 g/100 extracted fat 17,7 18:00 g/100 extracted fat 1,3 20:00 g/100 extracted fat 0,1 22:00 g/100 extracted fat 0,1 16:1 n-7 g/100 extracted fat 4,6 18:1 (n-9)+(n-7)+(n-S) g/100 extracted fat 14,9 20:1 (n-9)÷(n-7) g/100 extracted fat 0,9 22:1 (n-11)+(n-9)÷(n-7) g/100 extracted fat 0,5 24:1 n-9 g/100 extracted fat 0,1 16:2 3 n-4 g/100 extracted fat 0,7 16:3 n-4 g/100 extracted fat 0,4 18:2 n-6 g/100 extracted fat 1,5 18:3 n-6 g/100 extracted fat 0,2 20:2 n-6 g/100 extracted fat <0,1 20:3 n-6 g/100 extracted fat 0,1 20:4 n-6 g/100 extracted fat 0,4 22:4 n-6 g/100 extracted fat <0,1 18:3 n-3 g/100 extracted fat 0,9 18:4 n-3 g/100 extracted fat 2,9 20:3 n-3 g/100 extracted fat <0,1 20:4 n-3 g/100 extracted fat 0,4 20:5 n-3 (EPA) g/100 extracted fat 12,6 21:5 n-3 g/100 extracted fat 0,4 22:5 n-3 g/100 extracted fat 0,4 22:6 n-3 (DHA) g/100 extracted fat 7,7 Fat Bligh & Dyer % 21 Acid value in BI&D ex. mg KOH/g extracted fat 11,5 Peroxide value in BI&D ex. meq peroxide/kg oil 9,6 Sum saturated fatty acids g/100 extracted fat 28 Sum monoenoic fatty acids g/100 extracted fat 21 Sum PUFA (n-6) fatty acids g/100 extracted fat 2,2 Sum PUFA (n-3) fatty acids g/100 extracted fat 25,3 Sum total-PUFA fatty acids g/100 extracted fat 28,6 omega-6/omega-3 ratio g/100 extracted fat 0,09 Sum EPA + DHA g/100 extracted fat 20,3 Sum identified fatty acids g/100 extracted fat 77,6 Sum unidentified fatty acids g/100 extracted fat 8,2 A42 Aspartic acid g/100g sample 6,6 A42 Glutamic acid g/100g sample 8,4 A42 Hydroksyproline g/100g sample <0,10 A42 Serine g/100g sample 2,5 A42 Glycine g/100g sample 3 A42 Histidine g/100g sample 1,4 A42 Arginine g/100g sample 4,1 A42 Threonine g/100g sample 2,9 A42 Alanine g/100g sample 3,3 A42 Proline g/100g sample 2,1 A42 Tyrosine g/100g sample 2,3 A42 Valine g/100g sample 3,2 A42 Methionine g/100g sample 1,9 A42 Isoleucine g/100g sample 3,2 A42 Leucine g/100g sample 4,9 A42 Phenylalanine g/100g sample 2,7 A42 Lysine g/100g sample 5,3 A99 Cysteine/Cystine g/100g sample 0,65 A100 Trypthophane g/100g sample 0,74 *A25: 15016634-1-Dumas analysis of N/100g through combustion Total amino acids in meal are measured by performing hydrolysis of protein first and quantifying whole amino acids (A42 method).

Table 2 Analysis of Stick Water (SW) Crude protein combustion % 16,3 Moisture % 76,4 Ash % 7,8 Salt % 7 Watersoluble crude protein g/100g 17 Cadaverine mg/kg <20 Histamine mg/kg <20 Phosphorus % 0,24 Fluorine (acid soluble) g/kg 0,015 Free Astaxanhin mg/kg <2 Astaxanthin esters mg/kg <5 Total Volatile Nitrogen mg N/100g 10 Trimethylamin-N mg N/100g 5 Trimethylaminooxide-N mg N/100g 431 Fat Bligh & Dyer % 0,5 Acid value in BI&D ex. mg KOH/g extracted fat 20,7 Peroxide value in BI&D ex. meq peroxide/kg oil <2,0 A42 Aspartic acid g/100g sample 0,61 A42 Glutamic acid g/100g sample 1,1 A42 Hydroksyproline g/100g sample <0,10 A42 Serine g/100g sample 0,23 A42 Glycine g/100g sample 1,3 A42 Histidine g/100g sample 0,1 A42 Arginine g/100g sample 1,3 A42 Threonine g/100g sample 0,19 A42 Alanine g/100g sample 0,47 A42 Proline g/100g sample 0,7 A42 Tyrosine g/100g sample 0,21 A42 Valine g/100g sample 0,24 A42 Methionine g/100g sample 0,14 A42 Isoleucine g/100g sample 0,17 A42 Leucine g/100g sample 0,34 A42 Phenylalanine g/100g sample 0,18 A42 Lysine g/100g sample 0,56 A99 Cysteine/Cystine g/100g sample 0,21 A100 Trypthophane g/100g sample <0,10 Table 3: Analysis of Stick Water (SW) Krill Meal Stick Water Mw-peptide >20000 % of water sol. Peptides 0,1 <0,1 Mw-peptide 20000-15000 % of water sol. Peptides 0,2 0,1 Mw-peptide 15000-10000 % of water sol. Peptides 1,1 0,3 Mw-peptide 10000-8000 % of water sol. Peptides 1,6 0,6 Mw-peptide 8000-6000 % of water sol. Peptides 4 1,7 Mw-peptide 6000-4000 % of water sol. Peptides 7,3 4,5 Mw-peptide 4000-2000 % of water sol. Peptides 8,5 7,9 Mw-peptide 2000-1000 % of water sol. Peptides 4,1 4,2 Mw-peptide 1000-500 % of water sol. Peptides 2,6 2,6 Mw-peptide 500-200 % of water sol. Peptides 4,4 3,2 Mw-peptide 200- % of water sol. Peptides 66 74,9 Table 4: Average based on analysis of 5 different batches of krill meal with inclusion of ingredients from stick water (QSW) compared to conventional krill meal (krill meal without stick water ingredients = QA) Parameter/Method Units QSW QA A25 Crude protein % 57,9 56,2 combustion A20 Water-soluble crude g/100g sample 12,7 8,5 protein Water-soluble crude protein % total protein 22 15 Mw-peptide > 20000 % of water sol. Peptides 0,3 0,9 Mw-peptide 20000-15000 % of water sol. Peptides 0,4 0,7 Mw-peptide 15000-10000 % of water sol. Peptides 2,0 2,7 Mw-peptide 10000-8000 % of water sol. Peptides 2,5 3,1 Mw-peptide 8000-6000 % of water sol. Peptides 4,3 5,3 Mw-peptide 6000-4000 % of water sol. Peptides 6,6 7,6 Mw-peptide 4000-2000 % of water sol. Peptides 10,5 10,4 Mw-peptide 2000-1000 % of water sol. Peptides 7,5 6,6 Mw-peptide 1000-500 % of water sol. Peptides 5,4 4,7 Mw-peptide 500-200 % of water sol. Peptides 8,0 7,6 Mw-peptide 200- % of water sol. Peptides 52,4 50,5 Mw-peptide > 20000-4000 Sum in the defined range of % of water sol. Peptides 16,3 20 Mw-peptide 200-4000 Sum in the defined range of % of water sol. Peptides 84 80 *A25: 15016634-1-Dumas analysis of N/100g through combustion.

A20: Water extraction followed by Dumas analysis.

Table 5: Comparison of the composition of krill meals supplemented with stick water with krill meal that is not supplemented with stick water Parameter/Method* Units QSW 6.5 QSW 4.9 QA no stick water (control) A25 Crude protein g/100g sample 58.7 57.8 56.2 combustion A42 Alanine g/100 g amino acid 5.9 6.0 5.4 A42 Arginine g/100 g amino acid 6.4 6.8 6.4 A42 Aspartic acid g/100 g amino acid 11.0 11.1 10.8 A42 Glutamic acid g/100 g amino acid 13.8 13.6 13.8 A42 Glycine g/100 g amino acid 5.1 5.3 5.0 A42 Histidine g/100 g amino acid 2.4 2.3 2.2 A42 Hydroksyproline g/100 g amino acid 0.2 0.2 0.2 A42 Isoleucine g/100 g amino acid 5.5 5.5 5.6 A42 Leucine g/100 g amino acid 8.3 8.6 8.6 A42 Lysine g/100 g amino acid 7.9 8.0 7.8 A42 Methionine g/100 g amino acid 3.3 3.3 3.2 A42 Phenylalanine g/100 g amino acid 5.0 5.1 5.2 A42 Proline g/100 g amino acid 4.2 4.5 4.4 A42 Serine g/100 g amino acid 4.4 4.5 4.4 A42 Threonine g/100 g amino acid 4.8 4.7 4.6 A42 Tyrosine g/100 g amino acid 4.0 4.1 4.4 A42 Valine g/100 g amino acid 5.5 5.5 5.4 A99 Cystein/cystine g/100 g amino acid 1.2 NA 1.4 A100 Tryptophan g/100 g amino acid 1.2 NA 1.5 A105 Creatinine (free) % of protein 0.02 0.02 0.02 A105 Aspartic acid (free) % of protein 0.05 0.03 0.02 A105 Glutamic acid (free) % of protein 0.15 0.07 0.07 A105 Hydroxyproline (free) % of protein 0.02 0.02 0.02 A105 Serine (free) % of protein 0.09 0.07 0.04 A105 Asparagine (free) % of protein 0.03 0.02 0.02 A105 Glycine (free) % of protein 0.92 0.80 0.48 A105 Glutamine (free) % of protein 0.05 0.02 0.02 A105 3-amino-propanic acid (free) % of protein 0.87 0.76 0.44 A105 Taurine (free) % of protein 1.31 0.93 0.69 A105 Histidine (free) % of protein 0.02 0.02 0.02 A105 4-amino-butanoic acid (free) % of protein 0.02 0.02 0.02 A105 Citrulline (free) % of protein 0.02 0.02 0.04 A105 Threonine (free) % of protein 0.07 0.05 0.02 A105 Alanine (free) % of protein 0.34 0.31 0.18 A105 Carnosine (free) % of protein 0.02 0.02 0.02 A105 Arginine (free) % of protein 1.35 1.28 0.69 A105 Proline (free) % of protein 1.31 1.18 0.78 A105 Anserine (free) % of protein 0.03 0 0 A105 Tyrosine (free) % of protein 0.12 0.10 0.07 A105 Valine (free) % of protein 0.10 0.10 0.05 A105 Methionine (free) % of protein 0.07 0.02 0.05 A105 Cystine (free) % of protein 0.02 0.02 0.02 A105 Isoleucine (free) % of protein 0.89 0.47 0.05 A105 Leucine (free) % of protein 0.17 0.12 0.09 A105 Phenylalanine (free) % of protein 0.10 0.07 0.05 A105 Trypthophan (free) % of protein 0.03 0.02 0.02 A105 Ornithine (free) % of protein 0.02 0.02 0.02 A105 Lysine (free) % of protein 0.22 0.17 0.11 *Total amino acids in meal is measured by performing hydrolysis of protein first and quantifying whole amino acids (A42, A99 and A100 method). To determine free amino acids (A105 method), protein hydrolysis step is omitted allowing to measure only free amino acids present in the meal.

Example 2: Feeding trial The main objective of this feeding trial was to compare the effects of stick water inclusion in krill meal on the feed intake and growth performance in Atlantic salmon. Figure 3 illustrates the set up.

A growth trial with salmon was carried out for a total of 9 months during the months July -May. After 9 months of feeding trial, following parameters were calculated: feed intake (in grams), FCR, and growth (weight in grams). The results showed a positive effect of QSW in the feed intake, and growth (biomass in grams).

Experimental diets Nine isoprotein and isolipid diets were formulated and produced. The recipes of the feeds are provided in Table 6.

Table 6: Diet recipes, total feed Ingredients Diet # 1 2 3 4 5 6 7 8 9 Raw Material Name % 9-6 9,6 991 991 % 991 991 % (Allix) Fish meal (LT, NSM) 30 22,5 22,5 15 7,5 7,5 7,5 0 0 QA 7,5 7,5 7,5 QSW 7,5,- 7,5 7,5 Soya SPC >62%, non 20,8 22,3 22,8 27,6 28,9 29,6 21,6 21,3 21,5

GMO

Sunflower Extracted, low fiber 2,0 2,0 2,0 2,0 2,0 2,0 2,0 2,0 2,0 Wheat Gluten 80 6,3 5,9 5,4 8,7 8,4 7,8 10,3 11,2 11,0 Pea Protein 65% 5,0 5,0 5,0 5,0 5,0 5,0 Guar meal 65% 10,0 10,0 10,0 Wheat Milling quality 11,2 11,2 11,2 11,2 11,2 11,2 11,2 11,2 11,2 Fish Oil 13,5 12,5 12,6 9,0 7,9 8,0 9,4 9,3 9,4 Rapeseed Oil 8,3 8,2 8,3 17,5 17,4 17,5 22,0 21,7 21,9 Premixes (vitamins, 1,16 1,3 1,3 1,52 1,66 1,66 2,76 3,06 3,04 minerals, antioxidants) Premixes (fats, sealers) 1,68 1,51 1,53 1,78 1,62 1,64 1,54 0,80 0,89 Diets 1, 4 and 7 are fish feed containing 30, 15 and 7.5% by weight fish meal.

Diets 2, 5 and 8 were similar to fish meal diets (1, 4 and 7), except 7.5 % percentage points of the fish meal was replaced by krill meal (QA). Diets 3 and 6 were similar to fish meal diets (1, 4 and 7), except 7.5 percentage points of the fish meal was replaced by QA+ 6.5% SW; and diet 9 was similar to fish meal diets (1, 4 and 7), except 7.5 percentage points of the fish meal was replaced by QA+ 4.9% SW.

Diet 3, 6 and 9 are herein referred to as QSW diets, and these were made by adding QSW compositions (Figure 1, N) in accordance with the invention.

Diets were analyzed for moisture (drying at 103°C to stable weight; ISO 6496:1999), crude fat (Soxhlet, with acid hydrolysis; EC 152/2009), crude protein (Nx6.25, Kjeltech Auto System; Tecator, flegands, Sweden), ash (combustion at 550°C, ISO 5984: 2002), starch, astaxanthin, and gross energy (bomb 20 calorimetry; ISO 9831:1998). Additionally, the composition of amino acids (ISO 13903:2005, EU 152/2009; tryptophan EU 152/2009) in the feeds and fatty acid composition of the feeds were analyzed.

Feeding trial The experiment was performed according to the guidelines and protocols approved by the European Union (EU Council 86/609; D.L. 27.01.1992, no. 116) and by the National Guidelines for Animal Care and Welfare published by the Norwegian Ministry of Education and Research.

A growth trial with Atlantic salmon was carried out at LetSea trial station at Donna (Norway), for a total of 9 months during the period July 2020 -May 2021. Fish arrived and were acclimated in the net pens (temperature, oxygen and salinity) for a period of 2 weeks prior to distribution.

Feeding, trial, start sampling The fish was bulk weighted at start, and 20 fish were individually measured for length and weight. The fish were fed ad libitum.

Feed intake Daily feeding and feed losses were registered to estimate feed conversion ratio (FCR).

Feeding trial, mid -1, mid-2 and final sampling The fish were bulk weighted after two months of feeding trial (mid 1), four months of feeding trial (mid 2) and nine months of feeding trial (final sampling). In addition, 12 fish/tank were sampled and individually measured for fork length, weight, gutted weight to measure Condition factor (CF) and Dressout (,)%0 (D%).

After mid-2 and final sampling, 10 fish/tank were filleted immediately after slaughtering in the pre-rigor state. The fillets were packed individually in sealed plastic bags and stored on ice for one week before analyzing. Fillet quality analyses included suitability for processing, analyzed as degree of fillet gaping (score 0-5) after simulating commercial processing conditions (FFIF Industry standard), fillet firmness (instrumentally using a TA-XT2, Stable Micro Systems Ltd., Surrey, England and flat-ended 189 12.5mm 20 probe at 1mm s-1 compression speed), fillet color (visual assessment, SalmoFan color score, DSM (Heerlen, Netherlands) under standardized light conditions in a Salmon Color Box, and number of melanized muscle segments (myomers). Firmness and color were analyzed above the lateral line just below the cranial part of the dorsal fin and between the caudal part of the dorsal fin and the gut (i.e. 25 Norwegian Quality Cut, NQC). The NQC was used for analyzing content of protein, fat and astaxanthin, fatty acid composition and collagen characteristics.

Results After 9 months of feeding trial, following parameters were calculated: mortality, feed intake (in grams), FCR, and growth (weight in grams). The mortality was lower in the group having been feed QSW compared to QA and control, see Figure 4. The results showed a positive effect of QSW in the total feed intake, and growth (biomass in grams) compared to QA and Control as shown in Figure 5.

After analysing the QA and QSW, the inventors found a higher percentage of water soluble proteins (Figure 6) and feed stimulating free amino acids (Figure 7) in QSW. The results illustrated in the Figure 6-8 is based on data found in Table 5. The free amino acids linked to appetite that are summarized in the figures are alanine, proline, arginine, leucine, glutamine and glycine. The difference between the amount of the appetite stimulating free amino acids in 2 batches of QSW and QA is illustrated in Figure 7. The inventors also found a dose response effect of QSW inclusion on the percentage of the free appetite stimulating amino acids. There was higher percentage of these free amino acids with higher inclusion of QSW (Figure 7). Based on these results, the inventors suggested that higher feed intake with QSW was due to the higher percentage of water soluble proteins and appetite stimulating free amino acids. Interestingly, the similar results were found in the feeds used during the feeding trial (see Figure 8).

Figure 7 shows the higher percentage of appetite stimulating free amino acids (alanine, proline, arginine, leucine, glutamine and glycine) in QSW meals versus QA meal.

Figure 8 show higher percentage of appetite stimulating free amino acids (alanine, proline, arginine, leucine, glutamine and glycine) in feeds with QSW in comparison to QA and control feeds.

Conclusion

The results clearly indicate that increased feed intake and improved growth may be achieved by increasing the level of water-soluble proteins and appetite stimulating free amino acids in fish feed. Those water-soluble proteins may be sustainably obtained by utilizing ingredients present in stick water. This could be beneficial to the fish farmers, especially during the stressful periods. The stressful periods such as transfer to sea, treatment periods against salmon louse or other diseases, handling etc., could have a negative impact on the appetite of fish. The reduced appetite could negatively affect the growth and welfare of fish. Hence, by including stick water in the feeds, the appetite could be stimulated during these stressful periods, and that could eventually lead to better growth and welfare of the fish. In addition, the quality of fillet, in terms of pigmentation and firmness, was equally good in QA and QSW (with both the inclusion levels). Besides, the FCR was numerically better in QA and QSW in comparison to the control group. Based on these results, the inventors have concluded that QSW is relatively more sustainable raw material with the same benefits as demonstrated for QA.

Example 3: CO2 Emissions Fuel consumption and krill meal production data from excursions to catch krill and process into meal on-board a vessel was used to calculate the CO2 emissions per units of meal produced. A comparison was performed between excursions where stick water was not used to supplement the meal, with an excursion where stick water was used to supplement the meal in accordance with the invention using the excess heat from the disc dryer to heat the stick water. It was found that the process of supplementing with stick water reduced CO2 emissions, as noted in Table 7.

Table 7: Comparison of meal production and fuel consumption from three consecutive excursions. Stick water inclusion was implemented in Excursion 3 Excursion 1 Excursion 2 Excursion 3 Meal produced MT 2237.85 2036.525 3148.5 Fuel Consumed MT 905.1 840.17 1006.57 Fuel / Meal 0.404 0.413 0.32 CO2e/Kg Meal* 1.313 1.34225 1.04 *Based on calculation of emissions from the vessel. Several parameters affect the CO2 equivalents per kg meal but part of the reduction is attributed to the inclusion of stick water to the meal.

Claims (37)

1. CLAIMSA process for preparing aquaculture feed, wherein the process comprises the following steps: (a) providing krill (Figure 1, A); (b) subjecting the krill to a heat treatment (Figure 1, B and Figure 1, D), (c) separating the heat-treated krill (Figure 1, E) into a press cake (Figure 1, 0) and press water (Figure 1, F); (d) separating (Figure 1, G) stick water from the press water (Figure 1, F); (e) mixing the stick water and the press cake (Figure 1, 0); (f) drying (Figure 1, M) the mixture to obtain a supplemented krill meal (Figure 1, N); and 2. 3. 4. 6.(g) formulating the supplemented krill meal into aquaculture feed.
2. The process according to claim 1, wherein the aquaculture feed comprises the supplemented krill meal at a concentration of at least 2 °;Ow/w, or 3-50 %w/w, or 4-30 %w/w, or 5-15 ?4)w/w.
3. The process according to any one of the preceding claims, wherein the relative proportions of the stick water and the press cake mixed in step (e) are in the range of 1 part by dry weight of the stick water and 25 parts by dry weight of the press cake to 1 part by dry weight of the stick water and 3 parts by dry weight of the press cake.
4. The process according to any one of the preceding claims, wherein the supplemented krill meal produced in step (f) comprises stick water at a concentration of 3-25 %w/w, or 3.5-10 9,0w/w, or 4-7 9/0w/w of the total dry matter.
5. The process according to any one of the preceding claims, wherein the stick water contains from 57 g to 87 g water soluble proteins per 100 g protein containing material on a dry weight basis.
6. The process according to any one of the preceding claims, wherein the amount of trimethylamine and the amount of trimethylaminooxide in the stick water is in the range 1100 mg N to 2500 mg N per 100 g the stick water on a dry weight basis.
7. The process according to any one of the preceding claims, wherein: a. 0.1 94 to 0.5 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 10kDa to 15kDa; b. 0.2 % to 1 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 8kDa to 10kDa; c. 1 % to 2.4 % by dry weight of the water-soluble proteins in the stick water have a molecular mass in the range 6kDa to 8kDa; and/or d. 70 % to 80 % by dry weight of the water-soluble proteins in the stick water have a molecular mass < 200 Da.
8. 8. The process according to any one of the preceding claims, wherein the amount of trimethylamine and the amount of trimethylaminooxide in the supplemented krill meal is in the range 100 mg N to 250 mg N per 100 g supplemented krill meal on a dry weight basis.
9. 9. The process according to any one of the preceding claims, wherein the supplemented krill meal has a free amino acid concentration of at least lg per 100g of supplemented krill meal, optionally between lg-3g per 100g of supplemented krill meal.
10. 10. The process according to claim 9, wherein the free amino acids comprise one or more amino acids selected from the group consisting of alanine, proline, arginine, leucine, glutamine and glycine; optionally wherein the free amino acids comprise alanine, proline, arginine, leucine, glutamine and glycine.
11. 11. The process according to any one of the preceding claims, wherein the combined concentration of alanine, proline, arginine, leucine, glutamine and glycine in the supplemented krill meal is at least 0.60 94w/w of the total protein in the supplemented krill meal.
12. 12. The process according to any one of the preceding claims, wherein the heat treatment in step (b) comprises increasing the temperature to about 80 °C to 100 °C, and optionally holding the krill at that temperature for at least 1 minute or for 1-10 minutes.
13. 13. The process according to any one of the preceding claims, wherein the heat treatment in step (b) comprises a first heat treatment (Figure 1, B) by increasing the temperature to about 40 °C to 60 °C; and then subjecting the krill to a second heat treatment (Figure 1, D) by increasing the temperature to about 80 °C to 99 °C.
14. 14. The process according to any one of the preceding claims, wherein prior to step (e) the method comprises the step of reducing the amount of water (Figure 1, K) in the stick water (Figure 1, J) thereby obtaining a stick water concentrate (Figure 1, L), and step (e) comprises mixing the stick water concentrate and the press cake.
15. 15. The process according to claim 14, wherein the stick water concentrate has a moisture content of 40-80 %, or 50-70 % or 55-65 %.
16. 16. The process according to any one of claims 14 and 15, wherein reducing the amount of water in the stick water comprises using the excess heat from the drying of step (0 to heat the stick water.
17. 17. The process according to any one of claims 14-16, wherein water evaporated from the stick water is recovered, converted to steam, and the steam used during the heat treatment of step (b).
18. 18. The process according to any one of the preceding claims, wherein step (d) comprises separating the press water (Figure 1, F) into stick water (Figure 1, J), an oil fraction (Figure 1, I) and a sludge fraction (Figure 1, H).
19. 19. The process according to any one of the preceding claims, wherein the drying in step (f) comprises vacuum drying, optionally at a pressure of about <0.9 bar, <0.75 bar, or <0,6 bar.
20. 20. The process according to claim 19, wherein the vacuum drying is at a temperature below 95 °C, or 80-90 °C, or 80-85 °C.
21. 21. The process according to any one of the preceding claims, wherein the relative proportions of fish meal and supplemented krill meal in the aquaculture feed are in the range of 5 parts supplemented krill meal to 1 parts fish meal to 1 part supplemented krill meal to 5 parts fish meal.
22. 22. The process according to any one of claims 1-20, wherein the aquaculture feed does not contain fish meal.
23. 23. The process according to any one of the preceding claims, wherein steps (a) to (f) are conducted immediately after the krill has been caught.
24. 24. The process according to any one of the preceding claims, wherein steps (a) to (f) are conducted on a vessel and step (g) is conducted on shore.
25. 25. The process according to any one of the preceding claims, wherein the time between steps (f) and (g) is at least 1 week or at least 1 month, optionally wherein the time between steps (f) and (g) is up to 36 months.
26. 26. An aquaculture feed prepared by the method of any one of the preceding claims.
27. 27. A method of aquaculture, the method comprising preparing aquaculture feed by the process of any one of claims 1-25, and feeding aquatic organisms with the aquaculture feed.
28. 28. The method according to claim 27, wherein the aquatic organism is from the family Salmonidae, such as salmon; optionally wherein the aquatic organism is Salmo salar (Atlantic salmon).
29. 29. The method according to claim 27, wherein the aquatic organism is shrimp.
30. 30 Krill meal supplemented with stick water, wherein the krill meal has a free amino acid concentration of at least lg per 100g of krill meal, optionally between lg-3g per 100g of krill meal.
31. 3 I. The krill meal according to claim 30, wherein the free amino acids comprise one or more amino acids selected from the group consisting of alanine, proline, arginine, leucine, glutamine and glycine; optionally wherein the free amino acids comprise alanine, proline, arginine, leucine, glutamine and glycine.
32. 32. The krill meal according to any one of claims 30 or 31, wherein the level of water-soluble proteins in the krill meal is greater than 15% of total protein, optionally wherein the level of water-soluble proteins in the krill meal is 17-25%.
33. 33. The krill meal according to any one of claims 30-32, wherein the combined concentration of alanine, proline, arginine, leucine, glutamine and glycine in the meal is at least 2.5, or 3.0-15, or 3.2-10 %w/w of the total protein in the meal.
34. 34. A process for preparing supplemented krill meal, comprising following the process of any one of claims 1-23, but omitting step (g).
35. 35. A process for preparing supplemented krill meal, wherein the process comprises the following steps: (a) providing krill (Figure 1, A); (b) subjecting the krill to a heat treatment (Figure 1, B and Figure 1, D), (c) separating the heat-treated krill (Figure 1, E) into a press cake (Figure 1, 0) and press water (Figure 1, F); (d) separating (Figure 1, G) stick water from the press water (Figure 1, F) (e) mixing the stick water and the press cake (Figure 1, 0), (f) and drying (Figure 1, M) the mixture using a vacuum drier at a temperature below 95°C to obtain a supplemented krill meal (Figure 1, N).
36. 36. The process according to claim 35, wherein the drying in step (f) comprises vacuum drying, optionally at a pressure of about <0.9 bar, <0.75 bar, or <0.6 bar.
37. 37. The process according to claim 36, wherein the vacuum drying is at a temperature below 95 °C, or 80-90 °C, or 80-85 °C.