Feed compositions comprising microbial biomass for fish, crustaceans and / or cephalopods

Abstract

The present invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a bacterial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25 %w / w protein with respect to the dry mass and at least 5 % w / w nucleic acid with respect to the dry mass. The feed of the present invention is particular useful in promoting the growth of the marine organisms and in promoting their resistance against infectious diseases. Accordingly, the feed of the present invention is particularly applicable in aquaculture.

Description

[0001] Feed compositions comprising microbial biomass for fish, crustaceans and / or cephalopods

[0002] Field of the invention

[0003] The present invention relates to a feed composition for fish, crustaceans and / or cephalopods, comprising a bacterial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25 % w / w protein with respect to the dry mass, at least 5 % w / w nucleic acid with respect to the dry mass and, optionally, at least 3 % w / w peptidoglycans with respect to the dry mass. The feed of the present invention is particularly useful in promoting the growth of marine organisms and in promoting their resistance against infectious diseases. Accordingly, the feed of the present invention is particularly suitable for application in aquaculture.

[0004] Background of the invention

[0005] Aquaculture is a rapidly growing industry, providing a significant portion of the world's food supply. However, microbial and / or viral infections pose a major threat to the health and productivity of aquaculture species, leading to significant economic losses. Diseases can spread rapidly within aquaculture facilities, causing high mortality rates and compromising the overall yield. Reducing the risk of infections is crucial for ensuring the sustainability and profitability of aquaculture operations.

[0006] In the field of shrimp farming, diseases such as White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), Yellow Head Virus (YHV) and Enterocytozoon hepatopenaei disease (EHP) have caused devastating losses worldwide. These infections can spread quickly through water and can be challenging to control, once an outbreak occurs.

[0007] Enterocytozoon hepatopenaei disease, also referred to as EHP, for instance, is a microsporidian parasite that affects hepatopancreas tubule cells in Penaeid shrimp species. This disease has become endemic in major shrimp farming countries, significantly impacting economic sustainability, production, and profitability. Although EHP does not cause significant mortality, it impacts shrimp production and yield, and has great economic implications. EHP infection spreads easily through horizontal transmission and implications of the disease include: (i) worse immune parameters, compromising the overall health of shrimp; (ii) higher feed conversion ratio; (iii) reduced growth rates; (iv) uneven size distribution; and (v) lower nutritional value of EHP-infected shrimp.

[0008] In the field of finfish aquaculture, viral diseases like Viral Hemorrhagic Septicemia (VHS), Infectious Hematopoietic Necrosis (IHN), and Infectious Pancreatic Necrosis (IPN) can severely impact salmon, trout, and other commercially important species. These viral infections can lead to high mortality rates, reduced growth rates, and compromised product quality.

[0009] While cephalopod aquaculture, such as that for Octopus vulgaris and Sepia officinalis, is still in its infancy compared to finfish culture, ongoing research and advancements offer exciting prospects for sustainable production in the future. Cephalopods have a short life span but rapid growth rates and high food conversion rates, making them in theory valuable for aquaculture. But understanding cephalopod physiology and nutrition remains a significant challenge for large-scale culture. MX338866B describes the challenges of using standard crabmeat and / or other crustaceans, and provides a feed composition comprising muscle flours, a mixture of vitamins and a binder. Developing sustainable artificial diets is crucial to support growth, control reproduction and avoid infections. Gestal C. et al (Handbook of Pathogens and Diseases in Cephalopods, Springer Open, 2019) gives an overview on what is known regarding diseases and its management. An example is infections caused by Labyrinthulomycetes. They can affect captive cephalopod populations and persist for several years. Factors such as stress play a big role in its spread and persistence.

[0010] Developing effective strategies to reduce the risk of infections in aquaculture is crucial for ensuring the long-term sustainability and profitability of the industry. This includes implementing biosecurity measures, developing disease-resistant strains, and exploring the potential of feed additives and immunostimulants to enhance the immune response of aquaculture species. Researchers and companies are continuously exploring new compositions, formulations, and ingredients to enhance the immunostimulant properties of aqua feed, potentially leading to more effective and efficient products. However, reducing the risk of infections in aquaculture through feed composition presents several technical characteristics and challenges: Identifying and incorporating antiviral compounds or immunostimulants into aqua feed formulations is a promising approach. However, challenges include ensuring the stability and bioavailability of these compounds, as well as potential negative impacts on the growth and health of the aquaculture species. Also their efficacy can vary depending on the species, life stage, and environmental conditions, making it challenging to develop a one-size-fits-all solution.

[0011] Incorporating prebiotics and probiotics into aqua feed can modulate the gut microbiome, potentially enhancing the immune response and reducing the risk of viral infections. However, maintaining the viability and efficacy of these compounds during feed processing and storage can be challenging.

[0012] For any of these solutions, balancing nutrient levels together with the additives while maintaining palatability and cost-effectiveness can be a challenge.

[0013] Of the above-mentioned solutions, there is an increasing body of evidence showing the benefits of nucleotides in aquaculture feed. They are multifunctional in nature, being the building blocks of tissue RNA and DNA, functioning as enzyme cofactors and participating in intra and extra cellular signaling. A growing body of evidence suggests that when externally added to the diet they can modify immune responsiveness in fish and crustaceans. Burrells C. et al (Dietary nucleotides: a novel supplement in fish feeds: 1. Effects on resistance to disease in salmonids, Aquaculture 199, pages 159-169) describes the effect of including exogenous nucleotides in aquaculture diets in increasing resistance to challenge infections with bacterial and viral diseases in salmonids. Ancieta-Probstl D. K. et al (Enhancing growth performance of shrimp with nucleotide-supplemented diets, Aqua Culture Asia Pacific Magazine, Issue Jul / Aug 26-28) report that supplementing the feed of shrimp with nucleotides increases the total haemocyte count, cells that are important in the response against pathogens.

[0014] Besides the immunostimulatory effects, nucleotides also have an effect in helping the development of the Gl tract. US6987095B1 discloses a method to improve the development of the intestinal tract in fish comprising the administration of a diet containing augmented nucleotide levels.

[0015] Other immunostimulants are Peptidoglycans, which are components of the bacterial cell wall that can be used to immunopotentiate crustaceans and fishes and to prevent infectious diseases thereof. EP1082908A1 discloses a feedstuff additive with low molecular weight lipopolysaccharide that is an effective component in activating immunity or preventing infection in crustaceans or fishes. EP2293687B1 discloses a feed composition to which both nucleotides and peptidoglycans are added. This feed composition can be used to prevent or reduce infectious diseases as well as symptoms related to an infectious disease in an animal

[0016] The success of a feed diet in reducing the risk of infections is dependent on the consumption of such a diet. Certain ingredients affect the palatability of the feed and consequently its consumption, leading to a reduced growth of the animals. Solutions that increase the acceptance of a given feed include the addition of palatants that can play a crucial role in efficient nutrient utilization and in the minimization of feed loss. EP2895006A1 discloses a method to increase palatability of fish feed in which specific protein hydrolysates and nucleotides are added during the preparation of fish feed pellets. W02022043527 discloses a method of preparing a composition with high levels of nucleotides and amino acids derived from microbial biomass

[0017] The present invention provides a feed composition comprising a microbial biomass or an extract or hydrolysate thereof that can be used in the prevention of infections in fish, crustaceous and cephalopods and that at the same time promotes body growth and survival. The microbial biomass provides at the same time several of the abovementioned compounds in a single ingredient, with the advantage of simplifying the production of aquaculture feed.

[0018] Document US 2015 / 216915 discloses that the administration of purified Bacillus or Lactobacillus compositions to post-larvae shrimp increases survival, body weight gain, length, feed intake, immunity and survival in certain challenges when incorporated into feed at levels of 0.05% to 0.1 %.

[0019] Document CN 112 970 968 A discloses the incorporation of probiotics (i.e., living microorganism) such as a Gram-positive Bacillus stratosphericus, into fish feed. As a result, resistance and survival against infectious diseases such as the infectious disease caused by Pseudomonas mutans, as well as growth rate, are increased.

[0020] Document WO 2024 / 076289 provides the use of Brevundimonas spp. as a probiotic supplement in shrimp farming, and demonstrates that the incorporation of said bacterial species into crustacean feed improves resistance to diseases caused by different pathogens. Document Linan Vidriales et al. (Aquaculture, vol 531 , DOI: 10.1016 / J.AQUACULTURE.2020.735958) discusses administration of rice bran fermented with a consortium of Bacillus and Lysinibacillus species to shrimp culture, which has been shown to reduce the abundance and impact of potential pathogenic species as well as to provide beneficial effect on shrimp culture.

[0021] Summary of the invention

[0022] It was an objective technical problem of the present invention to provide a feed composition for marine animals, in particular fish, crustaceans and / or cephalopods, with advantageous properties in the context of reducing the risks or alleviating the consequences of infections.

[0023] The problem is solved by embodiments disclosed herein and as characterized by the hereto appended claims.

[0024] It has been shown, surprisingly, that the feed compositions supplemented with bacterial biomass leads to accelerated weight gain in certain marine animals, such as particular crustaceans. Furthermore, feeding the marine animals with the feed of the present invention prevents any negative effects associated with particular infections of the cultures, which lead e.g., to lower gain of weight over time. Accordingly, the feed composition of the present invention has been shown to be useful to treat the symptoms of infectious diseases in certain marine animals, including crustaceans.

[0025] More specifically, it has been demonstrated in Example 1 that feeding the shrimp with the feed composition of the present invention has led to improved survival rate and improved growth rate. It has been further demonstrated that the feed compositions of the present invention significantly protect the shrimp from EHP infection, as demonstrated in Example 2. Also, the shrimp fed on the feed composition of the invention have shown high survival rates.

[0026] The invention is summarized in the following embodiments.

[0027] In a first embodiment, the present invention relates to a feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25%w / w protein with respect to the dry mass and at least 5% w / w nucleic acid with respect to the dry mass. In this first embodiment, it is preferred that at least 3 % w / w peptidoglycans with respect to the dry mass are further present.

[0028] In a second embodiment, the present invention refers to the feed composition of the present invention for use as functional food in a therapeutic application.

[0029] In a third embodiment, the present invention relates to the feed composition of the present invention for use in treating or preventing an infectious disease in fish, crustacean and / or cephalopod.

[0030] In a fourth embodiment, the present invention relates to use of the feed composition of the present invention in the manufacture of functional food for treating or preventing an infectious disease in fish, crustacean and / or cephalopod.

[0031] In a sixth embodiment, the present invention relates to a method of treating an infectious disease in fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof with the feed composition of the present invention. It is to be understood that therapeutically effective amount of feed is provided.

[0032] In a seventh embodiment, the present invention relates to the feed composition of the present invention for use in a method of stimulating immune system of fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof with the feed composition of the present invention.

[0033] In an eighth embodiment, the present invention relates to a method of stimulating immune system of fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof with the feed composition of the present invention.

[0034] In a ninth embodiment, the present invention relates to use of the feed composition of the present invention for increasing the growth rate of fish, crustacean and / or cephalopod.

[0035] Brief description of figures The invention is further illustrated using the following drawings. These are not meant to be construed as limiting the scope of the invention in any way, which is defined by the hereto appended claims. Instead, the appended drawings serve merely illustrative purposes.

[0036] Fig. 1 presents images of the shrimp that belong to a replicate of each group at the end of the Grow Out trial. For each square (1 to 6) T represents the number of the replicate (T4 stands as replicate 4) and the number after represents the Diet Group to which shrimp belong to. So T4-1 shows shrimp from the 4threplicate fed with the Control Diet (Diet 1).

[0037] Fig. 2 shows Shrimp Survival Rate after EHP challenge.

[0038] Fig. 3 presents images of the shrimp from the different groups after EHP challenge.

[0039] Fig. 4 presents hepatopancreas of shrimp in each group on termination day.

[0040] Detailed description of the invention

[0041] As mentioned before, the present invention pertains to microbial biomass ingredients intended for aqua feed compositions. These ingredients enhance resistance and recovery against infections. Additionally, the invention covers feed compositions that incorporate these ingredients and outlines their preparation. More specifically, the invention highlights the utilization of bacterial biomass containing over 25% w / w protein and more than 5% nucleic acids as a constituent in feed compositions. This inclusion aims to bolster the resistance of aquatic organisms (hydrobionts) against bacterial, viral, fungal, and protozoal infections.

[0042] In one embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25%w / w protein with respect to the dry mass and at least 5% w / w nucleic acid with respect to the dry mass. Preferably, the biomass or an extract or hydrolysate thereof further comprises at least 3% peptidoglycans with respect to the dry mass.

[0043] Thus, accordingly, in particularly preferred embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25%w / w protein with respect to the dry mass, at least 5% w / w nucleic acid and at least 3% peptidoglycans with respect to the dry mass.

[0044] As understood herein, the biomass comprised in the feed composition of the invention comprises at least 5% w / w nucleic acid with respect to the dry mass. Thus, said biomass comprised in the compositions encompassed by the present invention may comprise 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 (or more) % w / w of nucleic acid with respect to dry mass. Preferably, said biomass comprises at least 7% w / w nucleic acid with respect to the dry mass. More preferably, said biomass comprises at least 9% w / w nucleic acid with respect to the dry mass. Even more preferably, said biomass comprises at least 11 % w / w nucleic acid with respect to the dry mass. Again more preferably, said biomass comprises at least 13% w / w nucleic acid with respect to the dry mass. Still more preferably, said biomass comprises at least 15% w / w nucleic acid with respect to the dry mass.

[0045] It follows that, in one embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25%w / w protein with respect to the dry mass and at least 15% w / w nucleic acid with respect to the dry mass. Preferably, the biomass or an extract or hydrolysate thereof further comprises at least 3% peptidoglycans with respect to the dry mass. Accordingly, in this embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 25%w / w protein with respect to the dry mass, at least 15% w / w nucleic acid and at least 3% peptidoglycans with respect to the dry mass.

[0046] As further understood herein, the biomass comprised in the feed composition of the invention comprises at least 25% w / w protein with respect to the dry mass. Thus, said biomass comprised in the compositions encompassed by the present invention may comprise 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 (or more) % w / w of protein with respect to dry mass. Preferably, said biomass comprises at least 30% w / w of protein with respect to dry mass. More preferably, said biomass comprises at least 35% w / w of protein with respect to dry mass. Even more preferably, said biomass comprises at least 40% w / w of protein with respect to dry mass. Again more preferably, said biomass comprises at least 45% w / w of protein with respect to dry mass. Still more preferably, said biomass comprises at least 50% w / w of protein with respect to dry mass. Even more preferably, said biomass comprises at least 55% w / w of protein with respect to dry mass. It follows that, in one embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 55%w / w protein with respect to the dry mass and at least 5% w / w nucleic acid with respect to the dry mass. Preferably, the biomass or an extract or hydrolysate thereof further comprises at least 3% peptidoglycans with respect to the dry mass.

[0047] Thus, in one embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 55%w / w protein with respect to the dry mass, at least 5% w / w nucleic acid and at least 3% peptidoglycans with respect to the dry mass.

[0048] In one specific embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 55%w / w protein with respect to the dry mass and at least 15% w / w nucleic acid with respect to the dry mass. Preferably, the biomass or an extract or hydrolysate thereof further comprises at least 3% peptidoglycans with respect to the dry mass.

[0049] It follows that in one embodiment, the invention relates to feed composition for fish, crustaceans and / or cephalopods, comprising a microbial biomass or an extract or hydrolysate thereof, wherein the biomass or an extract or hydrolysate thereof comprises at least 55%w / w protein with respect to the dry mass, at least 15% w / w nucleic acid and at least 3% peptidoglycans with respect to the dry mass.

[0050] As understood herein, the term feed can be understood broadly and refers to any composition that can be used by specified organisms as food. Accordingly, it is required that the feed is palatable to the particular organisms and that it provides sufficient caloric value so that the organism can survive upon feeding itself with said feed composition. Particular preferred ingredients in the feed of the present invention are provided herein. However, no particular limitation is imposed herein, except those on the microbial biomass content, as recited in the claims. Furthermore, the application of the feed composition of the invention is not limited in any way. Accordingly, while the feed is required to be suitable for fish, crustacean and cephalopod, it is not excluded that said feed is also fed to other organisms, insofar practical. It is preferred that the feed composition of the invention is suitable for young (or, in other words, not adult, or juvenile) fish, crustacean or cephalopod. Accordingly, the present invention further relates to use of the feed composition of the invention for feeding juvenile fish, and crustacean larvae.

[0051] As apparent from the foregoing, the feed composition of the present invention comprises a microbial biomass or an extract or hydrolysate thereof.

[0052] As it is to be understood herein, the biomass is preferably a composition comprising the microorganism cells as its major component.

[0053] The term “biomass” is understandable to the skilled person. Accordingly, the skilled person is capable of growing the biomass and performing all the standard operations related thereto, for example harvesting the biomass or storing the biomass. Further description and specific examples of such operations are disclosed herein and discussed in the following. The cells of the microorganism in the biomass may be capable of further reproduction and multiplication. Accordingly, the biomass may comprise living cells.

[0054] However, it is preferred that the cells present within the biomass are not capable of further reproduction and multiplication. Thus, preferably, the cells present in the biomass are dead. For example, at least 98% of all cells in the biomass are dead. More preferably, at least 99% of all cells in the biomass are dead. Even more preferably, at least 99.8% cells in the biomass are dead. Again more preferably, at least 99.9% cells in the biomass are dead. Still more preferably, substantially all cells in the biomass are dead.

[0055] Accordingly, it is preferred that the biomass in the composition of the present invention does not play any role as a probiotic. In other words, the composition of the present invention is not a probiotic.

[0056] As apparent to the skilled person, the so defined biomass may also be referred to as single cell protein, or SCP.

[0057] As mentioned previously, the feed composition of the present invention comprises microbial biomass, or an extract or hydrolysate thereof. Preferably, the microbial biomass or an extract or hydrolysate thereof is a microbial biomass, as defined herein. However, the present invention does not exclude the use of biomass which has been processed by breaking the cells contained therein, as disclosed herein. Accordingly, an extract of the microbial biomass, as defined herein, can be used in the feed composition of the present invention. Alternatively, a hydrolysate of the microbial biomass, as defined herein, can be used in the feed composition of the present invention.

[0058] The microbial biomass of the present invention is preferably a bacterial biomass. It is to be understood that bacterial biomass is a biomass comprising bacterial cells as the only type of cells.

[0059] The bacterial biomass may comprise gram positive bacteria. Alternatively or additionally, the bacterial biomass may comprise gram negative cells.

[0060] The biomass may comprise a bacterial strain selected from Bacillus spp., Priestia spp., Vibrio spp., and Geobacillus spp.

[0061] It is particularly preferred that the bacterial strain is Bacillus spp. or Priestia spp. strain. It is to be understood that several organisms are described as belonging to both these genera, or have been reclassified from one to another genera. For example, Bacillus flexus is presently referred to as Priestia flexa. Similarly, Bacillus megaterium, an organism very important from the biotechnological point of view due to its ability of producing vitamin B12, is more recently referred to as Priestia megaterium.

[0062] Thus, in one embodiment, the bacterial strain may be Bacillus spp. strain. In one embodiment, the bacterial strain may be Priestia spp. strain.

[0063] Accordingly, one exemplary and particularly preferred strain of bacterium is Bacillus flexus, which may also be referred to as Priestia flexa. Priestia flexa is an aerobic, Gram-variable, rod-shaped, endosporeforming, oxidase-positive bacterium. The endospores are ellipsoidal, located in central / paracentral, unswollen sporangia. In laboratory conditions, it produces opaque, creamish, raised-margin colonies at 30 ± 2°C when incubated for 24-72 hrs. on tryptic soy agar. This bacterial species may be isolated from feces (poultry) and soil.

[0064] However, other Bacillus spp can also be used in the feed composition of the present invention. Accordingly, other suitable Bacillus strains include Bacillus megaterium, Bacillus stearothermophilus, and Bacillus coagulans and Bacillus licheniformis, preferably Bacillus megaterium, Bacillus stearothermophilus, Bacillus licheniformis. Alternatively, suitable Bacillus strains may include Bacillus megaterium, Bacillus stearothermophilus, and Bacillus coagulans. When reference is made to a Bacillus strain, preferably Bacillus subtilis is excluded. In particular, it is preferred that the present invention does not encompass embodiments wherein the bacterial strain is Bacillus subtilis B1144.

[0065] Alternatively, the bacterial strain may be selected from Vibrio spp., such as Vibrio natriegens. Vibrio natriegens is a Gram-negative marine bacterium. It was first isolated from salt marsh mud. It is a saltloving organism (halophile) requiring about 2% NaCI for growth. It reacts well to the presence of sodium ions which appear to stimulate growth in Vibrio species, to stabilise the cell membrane, and to affect sodium-dependent transport and mobility. Under optimum conditions, and all nutrients provided, the doubling time of V. natriegens can be less than 10 minutes. V. natriegens is able to successfully live and rapidly divide in its coastal areas due its large range of metabolic fuel. Recent research has displayed that Vibrio natriegens has a flexible metabolism, which allows it to consume a large variety of carbon substrates, reduce nitrates, and even fix nitrogen from the atmosphere under nitrogen-limiting and anaerobic conditions. In the laboratory, the growth medium can be easily changed, thus affecting the growth rate of a culture. V. natriegens is commonly found in estuarine mud.

[0066] Alternatively, the bacterial strain may be selected from Geobacillus spp., such as Geobacillus LC300.

[0067] Preferably, the bacterial biomass in the present invention is not biomass comprising E. coll bacteria or Corynebacteria. Accordingly, preferably the biomass used in the feed composition of the present invention does not comprise E. coll bacteria or Corynebacteria.

[0068] The present invention may also refer to microbial biomass which is not bacterial biomass, provided that the requirements of the protein and nucleic acid content are fulfilled. For example, the microbial biomass may be fungal biomass, such as yeast biomass. It is however preferred that said yeast biomass is not biomass comprising Pichia species or Saccharomyces species.

[0069] As referred to herein, the %w / w values are preferably provided as values relating to the dry weight, i.e. weight of the biomass upon removal of water from the biomass. Such normalization of the content studies of the biomass is routine in the field and known to the skilled person. As provided herein, the recitation of dry mass refers to dry biomass, and provided %w / w values are not provided with respect to the whole feed composition, but with respect to the biomass only (or, as applicable, an extract or hydrolysate thereof).

[0070] Nucleic acids as understood herein include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), also understood as polydeoxyribonucleotides and / or polyribonucleotides, without particular limitation on size of the polymers. It is noted that nucleic acids are formed of nucleotides.

[0071] It is known by the skilled person that RNA in the cell is in part complexed with proteins (among others, RNA polymerase and sigma factor). It is therefore possible that not all RNA is readily available to the nucleases, hampering a full conversion from RNA into nucleotides and nucleosides. Adequate treatment with a protease prior to nuclease enzymatic treatment may degrade the RNA-protein complexes and may improve the nucleotide yield.

[0072] Nucleic acid content, as referred to herein, refers to % w / w content of nucleic acids in the cell when normalized with respect to dry mass. Nucleic acids as understood herein include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), also understood as polydeoxyribonucleotides and / or polyribonucleotides, without particular limitation of size of the polymers. It is noted that nucleic acids are formed of nucleotides. Accordingly, unless indicated to the contrary nucleic acid content refers to combined content of nucleic acids and nucleotides, as defined herein. Nucleotides as understood herein refer to deoxyribonucleotides and ribonucleotides that are monomers which upon polymerization form polydeoxyribonucleotides and / or polyribonucleotides, respectively. It is noted that the term also encompasses modified nucleotides, for example IMP which comprises inosine base. Preferably, when referring to a nucleotide content of a composition, a reference is made to nucleotides, nucleosides and nucleobases (a pyrimidine or a purine) content of the composition, even more preferably a reference is made to nucleotides and nucleosides content of the composition. Nucleosides are known to the skilled person as nucleotides lacking the phosphate moiety. Nucleobases are understood herein preferably as including pyrimidine bases and purine bases, more preferably nucleobases are adenine, cytosine, guanine, thymine and uracil.

[0073] The term “protein” or "proteins" as used herein covers proteins, peptides and polypeptides, wherein said proteins, peptides or polypeptides may or may not have been post-translationally modified. Post- translational modification may for example be phosphorylation, methylation, glycosylation. The term “protein” is preferably used in reference to a fraction or a composition comprising proteins, as defined herein. Upon enzymatic treatment with protease(s), that is enzymes capable of catalyzing the hydrolysis of peptide bonds, peptide bonds in proteins are hydrolyzed and larger polypeptides are turned into smaller polypeptides and / or amino acids. Accordingly, the term protein, as referred to herein, further encompasses peptides and amino acids. Thus, when referring to protein content of the compositions, protein content, as referred to herein, refers to %w / w content of proteins (including peptides and amino acids) in the cell when normalized with respect to dry mass.

[0074] Peptidoglycans are essential components of the bacterial cell wall, giving it structural strength and counteracting the osmotic pressure of the cytoplasm. Peptidoglycans are known to the skilled person. Structurally, they include linear chains of repeating disaccharide units made of N-acetylglucosamine (GIcNAc or NAG) and N-acetylmuramic acid (MurNAc or NAM), wherein each MurNAc is attached to a short amino acid chain of typically 4 to 5 amino acid residues. Optionally, said amino acid chains can be cross-linked, e.g. by activity of D,D-transpeptidase, resulting in linear polysaccharide chain that are crosslinked by short peptide chains to form a mesh like layer or three-dimensional structure.

[0075] Accordingly, as mentioned above, it is required that the biomass used in the feed composition of the present invention comprises at least 25%w / w protein with respect to the dry mass and at least 5% w / w nucleic acid with respect to the dry mass. It is preferred that the biomass further comprises at least 3% w / w peptidoglycans with respect to the dry biomass.

[0076] Preferably, the biomass comprises at least 25%w / w protein with respect to the dry mass of the biomass, more preferably, the biomass comprises at least 27%w / w protein with respect to the dry mass of the biomass, even more preferably, the biomass comprises at least 29%w / w protein with respect to the dry mass of the biomass, again more preferably, the biomass comprises at least 31 %w / w protein with respect to the dry mass of the biomass.

[0077] Preferably, the biomass comprises at least 5% w / w nucleic acid with respect to the dry mass of the biomass. More preferably, the biomass comprises at least 9% w / w nucleic acid with respect to the dry mass of the biomass. Even more preferably, the biomass comprises at least 11% w / w nucleic acid with respect to the dry mass of the biomass. Again more preferably, the biomass comprises at least 12% w / w nucleic acid with respect to the dry mass of the biomass. Preferably, the biomass comprises at least 3% w / w peptidoglycans with respect to the dry mass of the biomass. More preferably, the biomass comprises at least 4% w / w peptidoglycans with respect to the dry mass of the biomass. Even more preferably, the biomass comprises at least 5% w / w peptidoglycans with respect to the dry mass of the biomass.

[0078] It is noted that in case an extract or hydrolysate of the biomass is used, the values describing protein content and / or nucleic acid content in %w / w preferably refer to the biomass itself, and not the extract or hydrolysate thereof. Accordingly, the reference to extract or hydrolysate of the biomass merely indicates that the biomass may be further processed before it is used in the feed composition of the present invention.

[0079] The bacteria that can produce biomass characterized by said nucleic acid, protein content and peptidoglycan content are exemplified herein. Alternatively, the bacteria capable of producing biomass comprising at least 25%w / w protein with respect to the dry mass, at least 5% w / w nucleic acid with respect to the dry mass and at least 3% w / w peptidoglycans with respect to dry mass are characterized by a growth rate of at least 0.4 IT1, preferably by a growth rate of at least 0.85 IT1. Preferably, fast-growing bacterial strains, for which the growth rate in the process, also referred to as the growth rate, as defined herein, is >0.4 IT1, are used within the present invention. Preferably, the bacteria, or the bacterial strain, used in the biomass is characterized by a growth rate in process of at least 0.8 IT1, preferably 0.9 IT1, more preferably at least 1.1 IT1, even more preferably at least 1 .2 IT1, still more preferably at least 1 .4 h-1. Also encompassed by the present invention is an embodiment, wherein said bacteria / said bacterial strain are / is characterized by a growth rate in process of at least 2 IT1, at least 3 IT1, or of at least 4 IT1. The growth rate as understood herein, unless stated otherwise, is the growth rate in the process, as defined herein. As it is apparent to the skilled person, the growth rate in the process may depend on the growth medium, and be different for minimal medium and rich medium, as understood to the skilled person.

[0080] The growth rate in the process is defined herein as the growth rate achievable under the fermentation conditions. In other words, for a microorganism, such a bacterium to be suitable to produce biomass to be used in the feed composition of the present invention it must be possible to cultivate it with the growth rate as defined herein in the fermentation conditions, in particular on a specific growth medium. The growth rate in the process as defined herein refers to a growth rate of a particular microorganism, herein particular bacterium, unless indicated otherwise. In the case of co-cultivation of more than one microorganism, the growth rate may refer to a combined growth rate of all the microorganism in the culture, and describe the increase in combined biomass over time.

[0081] As apparent to the skilled person, the growth rate in the process is preferably determined by plotting the In (natural logarithm) of biomass amount as a function of time and using a linear regression to calculate the slope in the linear range that corresponds to the exponential growth phase.

[0082] The growth rate in the process may also be referred to as p [h1].

[0083] Suitable examples of such bacteria include Vibrio spp., Bacillus spp., Priestia spp. and Geobacillus spp. strains. Thus preferably, in the method of the present invention the bacteria that can produce biomass are selected from Vibrio spp, in particular Vibrio natriegens, Bacillus spp., in particular Bacillus stearothermophilus, Priestia spp., in particular Priestia Flexa and Geobacillus spp, in particular Geobacillus LC300. Preferably, the at least one microorganism strain is selected from Vibrio spp, in particular Vibrio natriegens. Further suitable examples of Geobacillus spp. may include Geobacillus uralicus, and Geobacilus stearothermophilus. Further suitable examples of Bacillus spp may include Bacillus licheniformis and Bacillus coagulans. Alternatively, further suitable examples of Bacillus spp may include Bacillus megaterium, Bacillus stearothermophilus Bacillus licheniformis and Bacillus coagulans. It is preferred that Bacillus spp is not Bacillus subtilis. Further suitable examples of Priestia spp may include Priestia megaterium.

[0084] The production of the biomass is described herein.

[0085] The microbial biomass is obtainable in a process of cultivation / fermentation of a microorganism, or more specifically, at least one microorganism strain (preferably a bacterial strain). As understood herein, at least one microorganism strain(s) is to be cultivated on a substrate. The skilled person would be capable ofselecting, adjusting and / or applying particular medium / mediato the at leastone microorganism strain(s) as used in the invention. The substrate used can be any commercial medium including carbon and nitrogen source as well as trace elements and / or vitamins. Preferably, the substrate can be one or several side-streams from the industry, either extracted by thermal and / or enzymatic methods or directly used for fermentation and containing either proteins, C5 sugars, C6 sugars or a combination of those. The one or several side-stream(s) can then be supplemented with other compounds to obtain a suitable growth medium (e.g. additional sugars as carbon source or nitrogen sources such as yeast extracts as well as vitamins and trace elements). Medium is preferably sterilized before inoculation of fermenters as known to the skilled person familiar with industrial fermentation process.

[0086] As known to the skilled person, the fermentation may be performed as a batch process, fed-batch process or (semi)continuous process.

[0087] The batch processes are characterized by lack of inflow of material into the fermentation vessel. In a batch process, all nutrients are provided at the beginning of the cultivation, without adding any more in the subsequent bioprocess. During the entire bioprocess, no additional nutrients are added with the exception of gases, acids and bases. In certain embodiments, an antifoaming agent and / or or pH correction agent may also be added. The bioprocess then lasts until one of the nutrients required for growth becomes limiting. This strategy is suitable for rapid experiments such as strain characterization or the optimization of nutrient medium. The disadvantage of this convenient method is that the biomass and product yields are limited. Since the carbon source and / or oxygen transfer are usually the limiting factor, the microorganisms are not in the exponential growth phase for a long time. After the end of a bioprocess run in batch mode, only the biomass or medium is harvested and appropriately processed to obtain the desired product. From the bioreactor point of view, the process is repeatedly interrupted by cleaning and, when needed, sterilization steps, and the biomass is only produced in stages.

[0088] In the fed batch process, substrate, nutrients and other substances may be added (preferably, in a form of a concentrated solution) into the fermentation vessel, to extend the possible culture time or increase the yield, among others. The advantage of feeding during cultivation is that it allows to achieve higher product quantities overall. Under specific growth conditions, the microorganisms and / or cells constantly double and therefore follow an exponential growth curve. Therefore, in certain embodiments the feed rate may be increased exponentially as well. Generally, the substrate is pumped from the supply bottle (or a feed tank) into the culture vessel, for example through a silicone tube (or a sterilizable piping). The user can either manually set the feed at any time (linear, exponential, pulse-wise), or add nutrients when specific conditions are met, such as when a certain biomass concentration is reached or when a nutrient is depleted. The fed-batch process offers a wide range of control strategies and is also suitable for highly specialized applications. However, it may increase the processing time and potentially leads to inhibition through the accumulation of toxic by-products. At high cell density, limitation through limited oxygen transfer from the gas phase may also occur. Preferably, in the method of the present invention the submerged fermentation is operated as a continuous process. After a batch growth phase, an equilibrium is established with respect to a particular component (also called steady state). Under these conditions, as much fresh culture medium is added, as it is removed (chemostat). These bioprocesses are referred to as continuous cultures, and are particularly suitable when an excess of nutrients would result in inhibition due to e.g. acid or ethanol build up or excessive heating. Other advantages of this method include reduced product inhibition and an improved space-time yield. When medium is removed, cells are harvested, which is why the inflow and outflow rates must be less than the doubling time of the microorganisms. Alternatively, the cells can be retained in a wide variety of ways (for example, in a spin filter), which is called perfusion. In a continuous process, the space-time yield of the bioreactor can be even further improved compared to that of a fed- batch process. However, the long cultivation period also increases the risk of contamination and longterm changes in the cultures. The three most common types of continuous culture are chemostat (The rate of addition of a single growth-limiting substrate controls cell multiplication), turbidostat (an indirect measurement of cell numbers - turbidity or optical density -which needs an additional sensor but is driven by real-time feedback, controls addition and removal of liquid), and perfusion (this type of continuous bioprocessing mode is based on either retaining the cells in the bioreactor or recycling the cells back to the bioreactor; fresh medium is provided and cell-free supernatant gets removed at the same rate).

[0089] Once cultivating is complete, the biomass is harvested according to the methods known to the skilled person. Harvesting the biomass may include separation and / or washing. The state-of-the art separation techniques employ tangential flow filtration or industrial (nozzle or centrifugal) separators. The biomass obtainable as described herein has a dry mass between 5% and 25% (as defined in weight to weight ratio expressed as percentage, %w / w).

[0090] As mentioned, the invention is not limited to microbial biomass, but also extends to extracts and / or hydrolysates thereof. These can be obtained from the microbial biomass, such as bacterial biomass as described herein, according to any method that are apparent and well known to the skilled person.

[0091] In certain embodiments, mechanical disruption methods like bead mill or PEF are used, particularly if the at least one microorganism strain(s) is a gram-negative bacteria. Chemical methods for breaking down the cells can also be used within the scope of the present invention. It is noted that the method of lysing the cells as discussed herein may be adapted based on the microorganism. For example, when gramnegative bacterium / bacteria are used, they are easier to lyse than gram-positive bacteria, e.g., Geobacillus strains or Bacillus megaterium. Further compared to yeast extracts or plant cells, bacteria have a thinner cell wall that is easier to break during the lysis process (it is particularly true for gramnegative bacteria, like Vibrio natriegens). It is noted that lysis of such cells is more efficient than that of yeast or plant cells.

[0092] Chemical method for breaking down the cells include, but are not limited to, treatment with salt(s), basic reagent(s) detergent(s) and / or surfactant(s). It is noted that chemical methods, in particular those involving basic reagent(s), also referred to as alkali reagent(s), may lead to partial degradation of nucleic acid, for example to degradation of RNA and formation of 2’-ribonucleotides and 3’-ribonucleotides.

[0093] It is further noted herein that upon heat pre-treatment followed by enzymatic treatment with protease(s) the cell wall remains in the reaction mixture, and can be separated. The separation can be performed by the means of mechanical and / or physical separation. Without wishing to be bound by the theory, it is understood herein that heat pre-treatment followed by enzymatic treatment with protease(s) leads to cell lysis. It is nevertheless preferred not to perform the separation of the cell wall remains, as it would lead to the removal of peptidoglycans from the composition.

[0094] The biomass extract can be subjected to hydrolysis. It is conceivable to the skilled person, that the step of hydrolysis is done to hydrolyse specific biopolymers or all biopolymers. Examples are the hydrolysis of protein into amino acids and / or peptides, or hydrolysis of nucleic acid such as, RNA into nucleotides. To this end, any method known to the skilled person can be used. For instance, treatment of said biomass or extracts at different pH and / or temperatures, or by adding specific hydrolytic enzymes, can be done to this end.

[0095] Itis conceivable to the skilled person that the feed composition of the present invention, which necessarily comprises a bacterial biomass or an extract or hydrolysate thereof, wherein the biomass comprises at least 25%w / w protein with respect to the dry mass, at least 5% w / w nucleic acid with respect to the dry mass and optionally at least 3% w / w peptidoglycans in respect to the dry mass, is obtainable in a process of mixing said biomass with a feed ingredient(s), thereby obtaining a feed composition.

[0096] Thus, as encompassed by the present invention, the feed composition preferably comprises up to 30% w / w of the biomass or an extract or hydrolysate thereof (preferably biomass). Accordingly, the feed composition may comprise 0,04, 0,08, 0, 1 , 0,25, 0,5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 % w / w of the biomass or an extract or hydrolysate thereof (preferably biomass) of the present invention. Preferably, the feed composition comprises between 0.5% w / w and 10% w / w of the biomass or extract or hydrolysate thereof, more preferably 2 and 8% w / w of the biomass or extract or hydrolysate thereof. More preferably, the feed composition comprises between 2 and 6% w / w of the biomass or an extract or hydrolysate thereof. Even more preferably, the feed composition comprises between 2 and 5% w / w of the biomass or an extract or hydrolysate thereof. It is to be understood that provided %w / w values herein are provided with respect to all the ingredients of the feed compositions, before addition of water. Accordingly, unless specifically indicated to the contrary, provided values are not normalized with respect to the dry mass of said biomass (or an extract or hydrolysate thereof).

[0097] In a specific embodiment, wherein an extract or hydrolysate of the biomass is used, preferably the feed composition comprises between 0,02 and 1 % w / w of said extractor hydrolysate, more preferably between 0,03 and 0,9 % w / w of said extract or hydrolysate, even more preferably between 0,04 and 0,08% of said extract or hydrolysate.

[0098] As understood herein, the skilled person is capable of formulating the animal feed. As noted herein, the feed composition is to be a suitable feed composition for fish, crustacean and cephalopod. Thus, the skilled person knows how to use the standard feed ingredients like fish meal, soybean meal, pea protein, soy protein concentrate, blood meal, feather meal, wheat gluten meal, poultry meal, corn gluten meal, fish oil, tuna oil, rapeseed oil, wheat flour when formulating a feed composition. As understood herein, the ingredients are finely ground to a similar particle size and undergo dry mixing, followed by the wet mixing, wherein typically the moisture content of 45 to 50% (understood as weight %) are mixed.

[0099] Accordingly, the form of the feed of the present invention is not meant to be particularly limiting and any form of the feed is considered encompassed by the present invention. Thus, the feed composition of the present invention may be provided as a dry meal, a liquid blend, a briquette, a pellet, a crumble or other similar feed form. In particular, it is preferred that the feed composition is provided as a briquette, a pellet or a crumble. More preferably, the feed composition of the invention is provided as a pellet.

[0100] Further, specific examples of making the feed composition according to the invention are provided herein. Thus, the skilled person is aware that specific animal feed of the invention can be prepared by following the recipes provided in the Examples section, or per analogy to the recipes provided in the Examples section.

[0101] In one embodiment, the present invention refers to the feed composition of the present invention for use as functional food in a therapeutic application. In other words, the feed composition of the present invention can be administered to animals to treat and / or prevent a disease.

[0102] As understood herein, a functional food is a food that has an additional function (often one related to health promotion or disease prevention), beyond its nutritional value. Functional foods may be obtainable from existing foods, for example by adding new ingredients or more of existing ingredients. Functional foods may be designed to have physiological benefits and / or reduce the risk of chronic disease beyond basic nutritional functions, and may be similar in appearance to conventional food and consumed as part of a regular diet.

[0103] As used herein, the term “treatment” (or "treating”) in relation to a disease or disorder refers to the management and care of a patient / subject for the purpose of combating the disease or disorder, such as to reverse, alleviate, inhibit or delay the disease or disorder, or one or more symptoms of such disease or disorder. The “treatment” may be curative, ameliorating or palliative. It is particularly preferred that treatment refers to the treatment of symptoms, in particular to managing, reducing or alleviating the symptoms of the diseases.

[0104] As used herein, the term “prevention” (or “prevent”) in relation to a disease or disorder refers to the administration of a composition for the purpose of preventing the onset of symptoms of the disease or disorder, alleviating such symptoms, or eliminating the disease or disorder.

[0105] The feed composition of the present invention is particularly useful in treating or preventing an infectious disease in fish, crustacean and / or cephalopod. Accordingly, in one embodiment, the present invention provides the feed composition of the present invention for use in treating or preventing an infectious disease in fish, crustacean and / or cephalopod.

[0106] Likewise, in one embodiment, the present invention relates to use of the feed composition of the present invention in the manufacture of functional food for treating or preventing an infectious disease in fish, crustacean and / or cephalopod. In another embodiment, the present invention relates to a method of treating an infectious disease in fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof, with the feed composition of the present invention. It is to be understood that therapeutically effective amount of feed is to be provided.

[0107] As understood herein, an infectious disease is a disease caused by an infection with another organism. Such organism may be bacterium, virus, or protozoa. Thus, the infectious disease, as referred to herein, may be a viral infectious disease, a bacterial infectious disease, or a protozoan infectious disease.

[0108] Accordingly, the feed composition of the present invention is preferably antiviral feed composition, antibacterial feed composition or antiprotozoan feed composition. Thus, the feed composition of the present invention is preferably suitable for treating or preventing a bacterial infectious disease or antiprotozoan infectious disease. Further preferably, the feed composition of the present invention is preferably not an antifungal feed composition. Accordingly, the feed composition of the present invention is preferably not suitable for treating or preventing a fungal infectious disease.

[0109] Viral infectious diseases such as diseases caused by an infection with White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV), and Yellow Head Virus (YHV) have caused devastating losses worldwide. It is particularly preferred in the context of the present invention that the infectious disease is a viral infectious disease caused by White Spot Syndrome Virus.

[0110] The infectious disease as referred to herein may also be a protozoan infectious disease. In different aquacultures, the diseases caused by Enterocytozoon hepatopenaei (EHP) are known to lead to significant losses. Accordingly, the infectious disease may also be a protozoan infectious disease caused by Enterocytozoon hepatopenaei (EHP). Preferably, according to the present invention, the infectious disease may also be a protozoan infectious disease caused by Enterocytozoon hepatopenaei (EHP).

[0111] The feed composition of the present invention can also be effectively used against bacterial infectious diseases. For example, a bacterial infectious disease may be a bacterial infectious disease caused by Vibrio parahaemolyticus. As understood herein, the fish, crustacean and / or cephalopod is preferably a fish or a crustacean. More preferably, the fish, crustacean and / or cephalopod is preferably a crustacean. Preferably, the crustacean is selected from Palaemon carinicauda, Penaeus vanammei, Penaeus monodom, Penaeus stylirostris and Penaeus japonicus. More preferably, the crustacean is Penaeus vanammei.

[0112] However, in one embodiment, the fish, crustacean and / or cephalopod is a fish. Preferably, the fish is of the genus Salmo or of the genus Oncorhynchus. Accordingly, it is preferred that the fish belongs to salmon or trout family.

[0113] In the context of particularly preferred disease to be treated, i.e., infectious disease of a crustacean, such as Penaeus vanammei, by Enterocytozoon hepatopenaei (EHP), the symptoms include worse immune parameters, compromising the overall health of shrimp; higher feed conversion ratio; reduced growth rates; uneven size distribution. These in turn lead to lower nutritional value of EHP-infected shrimp. Accordingly, the EHP infection in the shrimp leads to loss of weight of the animals as one of major symptoms of the infectious disease. However, if crustacean is fed with a feed of the present invention, this symptom appears to be significantly alleviated, and the shrimp keep gaining weight.

[0114] In one embodiment, the present invention relates to the feed composition of the present invention for use in a method of stimulating immune system of fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof with the feed composition of the present invention. It is to be understood that stimulation of the immune system improves response of the animals to potential challenges, like viral, bacterial or protozoan infections.

[0115] Feeding the fish, crustacean or cephalopod is a routine task for the skilled person, which may encompass contacting said fish, said crustacean or said cephalopod with the feed composition, possibly by distributing said feed composition to a water tank in which said fish, crustacean or cephalopod is kept. Said feed is eaten / consumed by marine organism, i.e. is provided to it perorally, through gastrointestinal tract.

[0116] The feed composition of the present invention, upon feeding / administration to fish, crustacean or cephalopod, increases the resistance of said organism against a challenge through microorganisms that cause infectious diseases. Accordingly, in one embodiment, the present invention relates to a method of stimulating immune system of fish, crustacean and / or cephalopod, the method comprising feeding a fish, a crustacean and / or a cephalopod in the need thereof with the feed composition of the present invention. As per analogy, the present invention relates to the feed composition of the present invention for use in stimulating immune response of fish, crustacean and / or cephalopod.

[0117] The feed composition has been further demonstrated to cause an increase in body weight, and also in rate of the body weight gain, in fish, crustacean or cephalopod, upon being fed to said animals. Thus, in one embodiment, the present invention relates to use of the feed composition of the present invention for increasing the growth rate of fish, crustacean and / or cephalopod. Likewise, the present invention relates to a method of increasing the growth rate of fish, crustacean or cephalopod, the method comprising the step of administering an effective amount of feed composition of the invention to fish, crustacean or cephalopod to be treated.

[0118] As understood herein, if reference is made to growth rate of a fish, crustacean or cephalopod, the reference is preferably made to the rate of gaining weight by the animals. Preferably, an average weight of animals in a tank is followed in order to measure the parameter.

[0119] The application is further illustrated in the following examples, which are not meant to be construed as limiting the scope of the invention in any way. Said scope is defined by the hereto appended set of claims.

[0120] Examples

[0121] Preparative Example - biomass / SCP preparation

[0122] To produce the bacterial biomass and the extracts or lysates afterwards, V. natriegens and / or P. flexa were pre-cultured and cultured in a medium with glucose at a concentration between 2 and 5 g / L and yeast extract at a concentration of 1 g / L. The medium for preculture and culture contains the elements shown in Table 1 . at a concentration of at least the one defined as “minimum value” and at a maximum of the one defined as “maximum value”. The elements are in a form which enables bioavailability of the element, such as, but not limited to mineral, ionic or inorganic.

[0123] Table 1 . Element composition of medium for preculture and culture

[0124] For the preculture phase, 50 ml of the preculture medium were prepared and inoculated with the strain from a cryovial in a shake flask. Shake flasks are placed in an incubator set at 37°C and 275 rpm. When an OD600 value between 1 .5 and 2 is reached, the preculture is used to inoculate the bioreactor with the culture medium to which it was added a carbon source (e.g. beet molasses) for a concentration of at least 20 g L-1.

[0125] The fermenter was run at a constant temperature of 37°C and pH was regulated to 7 during the fermentation course. Similarly, the air supply and stirring speed were set 1 .5 WM and 400 rpm at start. After fermentation, the broth was cooled down directly to 10-15°C and biomass was separated by centrifugation (4500 rpm, 10 min, 4°C).

[0126] For production of the hydrolysate, mechanical lysis followed by enzymatic treatment was performed as follows, unless indicated to the contrary. For mechanical lysis, the biomass was resuspended in water to obtain a final dry mass between 5 and 15% that was subsequently homogenized at 1500 bar. The extract was subjected to enzymatic treatments to convert the nucleic acid from the previous extract into nucleotides. To do so, 0.4% Nuclease was added to the suspension that was subsequently incubated at 65°C for 16 hours.

[0127] Both biomass and hydrolysate are dried until achieving a powder consistency, using standard methods such as spray drying.

[0128] The nucleotide content (preferably herein understood as nucleotide, nucleoside and nucleobase content, more preferably as nucleotide and nucleoside content) in the produced extracts was determined by ionpair reverse phase high performance liquid chromatography (IPRP-LC) using Agilent 1100 system followed by diode array detection (DAD) at 254 nm. The separation was performed using the Synergy Hydrocolumn as stationary phase and a mobile phase consisting of a mixture of 50 mM phosphate buffer (pH 5.8) and methanol at a flow rate of 0.4 mL / min and a temperature of 20°C. To achieve proper separation of analytes the composition of the mobile phase was varied during the run according to following gradient: 0 to 3 min with 50% methanol; 3 to 12 min with 0-50% methanol and finally 12 to 13.5 min 50% Methanol.

[0129] For the protein content, the nitrogen content in the samples was determined using the Kjeldahl method (ASU method L 06.00-7 (2014 / 08)) and later converted to protein content by multiplying with the factor 6.25, typically applied in the food industry and finally subtracting nucleic acid content as it also contains nitrogen. The total protein content could also be verified with the analysis of the digested amino acid content.

[0130] The amount of peptidoglycan can be determined according to the protocol from Ryan and Joseph, (2017). Digestion of Peptidoglycan and Analysis of Soluble Fragments, Bio-protocol 7 (15). In short, a known amount of cell pellets are suspended in phosphate buffer and boiled with SDS for 30 min. The insoluble peptidoglycans are then recovered by centrifugation and washed by adding phosphate buffer, resuspending and centrifuging to remove SDS. This washing step is repeated until there is no more SDS, which can be checked by the absence of foam. A treatment with a-amylase and pronase is subsequently done to remove high-molecular weight glycogen and peptidoglycan-associated proteins. An additional treatment with hydrochloric acid is necessary in case one wants to determine the peptidoglycan analysis of gram-positive bacteria. Quantification can be done by either drying the resulting pellet, or by measuring absorption at either 206 nm or 254 nm against a peptidoglycan standard curve of, for instance, Micrococcus lysodeikticus.

[0131] Example 1 : Growth Performance and Survival of Whiteleg Shrimp (Penaeus vannamei) fed with complete feed with Single Cell Proteins and derivatives

[0132] Experimental diets:

[0133] Whiteleg Shrimp (Penaeus vannamei) were fed five experimental diets and one control diet. The Control Diet (Diet 1) had standard fish feed ingredients such as fish meal, plant protein source ingredients (e.g., soybean meal, pea protein, wheat gluten meal), fish oil, plant base carbohydrate source ingredients (eg: wheat flour) and choline chloride. The experimental diets contained the same ingredients as the Control Diet and further included the biomass compositions of any of claims 1 to 3. Diet 2 included 2% inclusion (w / w %) of SCP 1 , Diet 3 included 5% inclusion (w / w %) of SCP1 , Diet 4 included 0.25% of SCP2, Diet 5 included 2% of SCP2, Diet 6 included 5% of SCP2. (Table 1) Table 1 . Main feed ingredients of the test diets in example 1 .

[0134] Experimental design:

[0135] The growth out tests were conducted at the ShrimpVet Demonstration Farm located in Can Gio, Ho Chi Minh City, Vietnam. The trial lasted for 30 days including 2 days of acclimation and 28 days of feeding and was composed of 6 groups, 1 Control and 5 treatment groups. Among those, 3 groups including Group 1 (Control), Group 3, and Group 6 were designated for digestibility analyses. Chromium oxide (Cr2O3) was used as an inert marker in these diets.

[0136] Specific pathogen-free (SPF) shrimp (P. vannamei), with an average body weight of 5.27 ± 0.78 g / shrimp were stocked, at a density of 50 shrimp / tank. The trials were conducted in fiberglass tanks in a recirculating aquaculture system (RAS). The feed was provided in the form of pellets. Shrimp were fed 4 times a day. The feeding amount was dependent on shrimp’s feeding demand, health status and feeding behavior, with the amounts of daily feed intake adjusted based on the quantity of uneaten feed from the previous meal. Growth performance and survival was calculated after 30 days of culture (Table 2).

[0137] Survival rate and Growth performance indices calculation

[0138] The growth performance results were calculated by the following formulae.

[0139] 1 . Survival rate; SR (%) = NfNi x 100

[0140] 2. Average daily growth; ADG; (g / day) = MGWDOC

[0141] 3. Mean weight gain; MWG (g) = Final mean weight - Initial mean weight

[0142] Where N represents the number of shrimp, Ni represents the initial number of shrimp, and Nf represents the final number of shrimp. MGW and DOC are the abbreviations for Mean Body Weight and Days of Culture. Data analysis

[0143] Values of survival rate and growth performance parameters were analyzed for normal distribution using standard techniques in the field. All the tests were carried at the significance level of 5% (p<0.05), and analyzed results were presented as mean ± standard deviation, followed by superscripts to show the significance or insignificance, where one-way analysis of variance (ANOVA) followed by the DUNCAN’s test was used to analyze the significant values (p < 0.05) among the groups.

[0144] Results

[0145] According to Table 2 the growth performance parameters were similar between all groups. Group 2, in which there was a 5% inclusion of SCP 1 , showed the best performance among the groups. At the end of the trial the average survival rates of the shrimp from G1 , G2, G3 and G5 was similar and above 85%. The best average final SRs was observed in G6 with 100% SR on average.

[0146] Table 2 The growth performance parameters

[0147] Values are presented as mean + standard deviation. Different letters on the same column indicate significant differences (P < 0.05).

[0148] Conclusions

[0149] The inclusion of 5% of SCP2 into the shrimp feed significantly increased the survival rate to 100%. Although there were no significant differences in weight gain between test groups and control group, in all groups excepted group 4 the mean weight gain and the average daily gain were higher than in the control group. Example 2: EHP challenge

[0150] Experimental diets:

[0151] Whiteleg Shrimp (Penaeus vanname / ) were fed with one control diet and four experimental diets. The Control Diet (Diet 1) had standard fish feed ingredients such as fish meal, plant protein source ingredients (eg. soybean meal, pea protein, wheat gluten meal), fish oil, plant base carbohydrate source ingredients (eg: wheat flour) and choline chloride. The experimental diets contained the same ingredients as the control diet and further included the biomass compositions of any of claims 1 to 3. In particular, Diet 2’ included 0.04% inclusion (w / w %) of hydrolysate of SCP 1 on top of Control Diet, Diet 3’ included 0.08% inclusion (w / w %) of hydrolysate of SCP 1 on top of Control Diet, Diet 4’ included 5% of SCP1 (same as Diet 3 in Example 1) and Diet 5’ included 5% of SCP2 (same as Diet 6 in Example 1). The general composition of Diet 1 , Diet 4’ and Diet 5’ can be found in Table 1 .

[0152] Experimental design:

[0153] The EHP challenge tests were conducted at the ShrimpVet Laboratory located in Ho Chi Minh City, Vietnam. The trial was set up as a completely randomized design (CRD) and lasted for 51 days including 2 days of acclimation, 14 days pre-challenge, 7 days of EHP Per os challenge, and 28 days of postchallenge. It was composed of 5 groups, 1 Control and 4 treatment groups. Specific pathogen-free (SPF) shrimp (P. vannamei), with an average body weight of 1.36 g / shrimp were stocked, at a density of 50 shrimp / tank. The trials were conducted in 350L tanks in a static system. The feed was provided in the form of pellets 4 times a day. The feeding amount was dependent on shrimp’s feeding demand, health status and feeding behavior, with the amounts of daily feed intake adjusted based on the quantity of uneaten feed from the previous meal.

[0154] During the 7 days of EHP per os challenge, EHP-contaminated feed was fed little by little to make sure EHP was delivered to the shrimp. EHP contaminated feed was prepared using the feeces of contaminated shrimp. Growth performance (Table 3) and survival rate (Figure 2) were calculated at different time points in the trial and after the 51 days.

[0155] Survival rate and Growth performance

[0156] Dead shrimps were removed from the tanks when observed._At the end of the trial, shrimp from each tank were harvested and counted to determine the final survivors. Survival rate and Growth performance indices calculation were made as previously described in Example 1 Statistical analysis

[0157] The values of survival rate and mortality rate of shrimp were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan test. All significant tests shall be at P < 0.05 levels. Results were presented as mean ± standard deviation.

[0158] Results

[0159] Table 3: Growth performance parameters after 51 days of culture.

[0160] Values are presented as mean + standard deviation. Different letters on the same column indicate significant differences (P < 0.05).

[0161] Conclusions

[0162] As shown in Figure 2, at the end of the trial (after 28 days of post-challenge), the survival rates of shrimp in Group 3, Group 4, and Group 5 were significantly higher than those of Group 1 (Control group), indicating that the feed composition of these groups provided protection against infection.

[0163] At the end of the trial, Group 2, Group 3, Group 4, and Group 5 achieved significantly higher mean weight gain (WG) and average daily gain (ADG) compared to Group 1) (Control Group). Among the treatments, Group 4 (SCP1_5%) exhibited the best overall performance, with the highest mean weight gain (9.73 g) and ADG (0.19 g / day). Dietary supplementation with SCP1_5% (Group 4) and SCP2_5% (Group 5) resulted in the most efficient feed conversion ratio (FCR) of approximately 1.0, indicating superior feed utilization compared to the control and other treatments.

Claims

New PCT-Patent Application based on EP 24 186 691.2MicroHarvest GmbHVossius Ref.: AJ2400 PCT BSCLAIMS1 . A feed composition for fish, crustaceans and / or cephalopods, comprising a bacterial biomass or an extract or hydrolysate thereof, wherein the biomass comprises at least 25%w / w protein with respect to the dry mass and at least 5% w / w nucleic acid with respect to the dry mass.

2. The feed composition of claim 1 , where the bacterial biomass comprises at least 3% w / w peptidoglycans with respect to the dry mass.

3. The feed composition according to claim 1 or 2, where the biomass comprises bacterial cells, in particular gram positive or gram negative cells.

4. The feed composition according to claim 1 to 3, wherein the biomass comprises at least one of the following bacterial strains: Priestia spp (such as Priestia flexa), Vibrio spp (such as Vibrio natriegens), Bacillus spp (such as Bacillus stearothermophilus) and Geobacillus spp (such as Geobacillus LC300).

5. The feed composition according to claims 1 to 4, wherein the microorganism strains are characterized by a growth rate of at least 0.4 h1, preferably at least 0.9 h1, more preferably at least 1.2 h-\6. The feed composition according to any one of claims 1 to 5, wherein the feed comprises between 2 and 5% w / w of the biomass or between 0.02% to 2% w / w of an extract or the hydrolysate thereof with respect to the ingredients before addition of water.

7. The feed composition according to any one of claims 1 to 6, wherein the microbial biomass or the extract or hydrolysate thereof is microbial biomass.

8. The feed composition according to any one of claims 1 to 7, wherein at least 99% cells in the biomass are dead, preferably wherein at least 99.8% cells in the biomass are dead, more preferably wherein at least 99.9% cells in the biomass are dead, even more preferably wherein substantially all cells in the biomass are dead.

9. The feed composition according to any one of claims 1 to 8, wherein the biomass is Vibrio natriegens biomass.

10. The feed composition according to any one of claims 1 to 8, wherein the biomass is Priestia flexa biomass.11 . The feed composition according to any one of claims 1 to 10, wherein the biomass comprises at least 55%w / w protein with respect to the dry mass and / or at least 15% w / w nucleic acid with respect to the dry mass.

12. The feed composition of any one of claims 1 to 11 for use in treating or preventing an infectious disease in fish, crustacean and / or cephalopod.

13. The feed composition for use of claim 12, when the infectious disease is a viral infectious disease, a bacterial infectious disease or protozoan infectious disease.

14. The feed composition for use of claim 13, wherein the infectious disease is a protozoan infectious disease caused by Enterocytozoon hepatopenaei (EHP).

15. The feed composition for use of claim 13, wherein the infectious disease is a bacterial infectious disease caused by Vibrio parahaemolyticus.

16. The feed composition for use of claim 13, wherein the infectious disease is a viral infectious disease caused by White Spot Syndrome Virus.

17. The feed composition for use of any one of claims 12 to 16, for use in treating or preventing an infectious disease in fish, wherein the fish is of the genus Salmo or of the genus Oncorhynchus.

18. The feed composition for use of any one of claims 12 to 16, for use in treating or preventing an infectious disease in crustacean, wherein the crustacean is selected from Palaemon carinicauda, Penaeus vanammei, Penaeus monodom, Penaeus stylirostris and Penaeus japonicus, preferably wherein the crustacean is Penaeus vanammei.

19. Use of the feed composition of any one of claims 1 to 11 for increasing the growth rate of fish, crustacean and / or cephalopod.

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