WO2025036970A1

WO2025036970A1 - Single cell protein products with high digestibility for use as aquafeed

Info

Publication number: WO2025036970A1
Application number: PCT/EP2024/073001
Authority: WO
Inventors: Carol Andrea ROA-ENGEL; Andre VENTE
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2023-08-16
Filing date: 2024-08-15
Publication date: 2025-02-20
Anticipated expiration: 2026-02-16
Also published as: WO2025036970A8

Abstract

The present invention relates to a composition comprising yeast single cell protein (SCP) product with a degree of hydrolysis of at least 12%, preferably at least 16%, more preferably at more preferably least 20%, more preferably at least 24%, most preferably at least 28%. Preferably, the yeast single cell protein product is used as a feed ingredient in aquaculture. The yeast single cell protein product is advantageously digestible in contrast to other proteins sources for aquaculture, such as fish meal.

Description

Single Cell Protein Products With High Digestibility For Use As Aquafeed

I. FIELD OF THE INVENTION

[0001] The present invention relates to a composition comprising a yeast single cell protein (SCP) product with a degree of hydrolysis of at least 12%, preferably at least 16%, more preferably at more preferably least 20%, more preferably at least 24%, most preferably at least 28%. Preferably, the yeast single cell protein product is used as a feed in aquaculture.

II. BACKGROUND

[0002] The world's population is growing and so is the demand for food, including the demand for seafood. Aquaculture requires considerable amounts of protein for ensuring optimal growth of the aquatic animals being cultured animals, with a major source of protein currently consisting of fish meal. However, the use of fish meal can be problematic for a variety of reasons - it is expensive in contrast to other protein sources, it means that harvested fish are often used to make fish meal to feed to other fish thus reducing net yield for human consumption, and thereby also contributes to overfishing and depletion of wild fish populations. Seafood has been experiencing an increasing demand as it is considered to confer health benefits when included in a regular diet. To meet this demand, aquaculture provides currently almost half of the world's fish for human consumption. Also, the culture of fish and crustaceans for example is becoming increasingly intensive and resource consuming. Thus, breeders, and hence the animal-protein derived protein production industry, including the aquaculture industry, need new protein sources to keep growing at high speed. Alternative plant proteins, such as soy protein, are more sustainable but are poorly digested by aquatic organisms. There is thus a need to produce sustainable, nutrient dense, yet readily digestible proteins for use in aquaculture.

[0003] Besides plant- and animal-derived protein, a further source of proteins has been identified, namely microbial-derived proteins. Said single cell proteins (SCPs) can be produced using fungi, algae and/or bacteria that offer the ability to be produced through large scale culturing at comparatively low cost.

[0004] Hence, there is still a need to have at hand an aquafeed that comprises protein from an alternative protein source and thus, ideally protein that is neither animal- nor plant-derived, and that provides adequate nutrients, such as unsaturated fatty acids, to ensure animal growth and which can be produced in a sustainable and cost-effective manner. III. SUMMARY

[0005] Thus, the present invention aims at providing a composition comprising yeast single cell protein (SCP) product with a degree of hydrolysis of at least 12%, preferably at least 16%, more preferably at least 20%, more preferably at least 24%, most preferably at least 28%, thus providing a nutritionally rich alternative protein source that can supplement or even replace currently used aquafeeds.

[0006] Preferably, in said composition said yeast single cell protein (SCP) product is used as a feed ingredient in aquaculture.

[0007] Preferably, in said composition said yeast single cell protein (SCP) product is used as a feed ingredient in aquaculture, wherein said aquaculture comprises the growing and harvesting of fish, crustaceans, or molluscs.

[0008] Preferably, in said composition said yeast single cell protein (SCP) product is prepared as an aquafeed for use as a feed.

[0009] Preferably, in said composition said yeast single cell protein (SCP) product comprises between about 10% to 8%, 10% to 6%, 10% to 4%, 8% to 6%, 8% to 4%, 6% to 4% (w/w) crude lipids per gram of dry yeast SCP product.

[0010] Preferably, said crude lipids comprise saturated fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids.

[0011] Preferably in said composition said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16: 1 , C18: 1 , C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.0.

[0012] Preferably in said composition said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16: 1 , C18: 1 , C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.1 , preferably greater than 1.2, more preferably greater than 1.5, most preferably greater than 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0.

[0013] Preferably, in said composition C16:0 fatty acids and C18:0 fatty acids are present in an amount of no more than 0.85% (w/w) per gram of dry yeast SCP product, preferably 0.53% (w/w) per gram of dry yeast SCP product. [0014] Preferably, wherein said mono-unsaturated fatty acids are C16:1 fatty acids and C18:1 fatty acids.

[0015] Preferably, wherein said C16:1 fatty acids and C18:1 fatty acids are present in an amount of at least about 2.3% (w/w) per gram of dry yeast SCP product, preferably 2.8% (w/w) per gram of dry yeast SCP product.

[0016] Preferably, wherein said poly-unsaturated fatty acids are C18:2 and C18:3 fatty acids.

[0017] Preferably, wherein said C18:2 fatty acids and C18:3 fatty acids are present in an amount of at least about 1 .0% (w/w) per gram of dry yeast SCP product, preferably 2.0% (w/w) per gram of dry yeast SCP product.

[0018] Preferably, in said composition wherein said yeast SCP product further comprises crude protein.

[0019] Preferably, in said composition wherein said yeast SCP product further comprises carbohydrates.

[0020] Preferably, in said composition wherein said yeast SCP product further comprises crude protein, carbohydrates and crude lipids.

[0021] Preferably, in said composition said yeast single cell protein (SCP) product comprises 35%, 40%, 45%, or 50% or more (w/w) crude protein per gram of dry yeast SCP product.

[0022] Preferably, in said composition said yeast SCP product comprises at least about 14% (w/w) essential amino acids per gram of dry yeast SCP product.

[0023] Preferably, in said composition said yeast SCP product comprises at least about 18% (w/w) essential amino acids per gram of dry yeast SCP product.

[0024] Preferably, in said composition said yeast single cell protein (SCP) product comprises 40%, 30%, 25% or 20% or less (w/w) total carbohydrates per gram of dry yeast SCP product.

[0025] Preferably, in said composition said yeast single cell protein (SCP) product comprises crude lipids to carbohydrates in a ratio of 0.6 to 1 : 2 to 5. [0026] Preferably, in said composition said yeast single cell protein (SCP) product comprises crude lipids to crude protein in a ratio of 0.6 to 1 : 3.5 to 6.

[0027] Preferably, in said composition said yeast single cell protein (SCP) product comprises crude lipids, carbohydrates and crude protein in a ratio of 0.6 to 1 crude lipids : 2 to 5 carbohydrates : 3.5 to 6 crude protein.

[0028] Preferably, in said composition said yeast single cell protein (SCP) product comprises about 6% or less (w/w) nucleic acid content per gram of dry yeast SCP product.

[0029] Preferably, in said composition said yeast single cell protein (SCP) product comprises about 5% or less (w/w) nucleic acid content per gram of dry yeast SCP product.

[0030] Preferably, in said composition said yeast single cell protein (SCP) product comprises about 4% or less (w/w) nucleic acid content per gram of dry yeast SCP product.

[0031] Preferably, in said composition said nucleic acid content comprises nucleosides and/or nucleotides.

[0032] Preferably, in said composition said nucleosides are ribonucleosides and/or deoxyribonucleosides.

[0033] Preferably, in said composition wherein said nucleotides are ribonucleotides and/or deoxyribonucleotides.

[0034] Preferably, in said composition said yeast SCP product is obtained from cultivating a Saccharomycetales yeast.

[0035] Preferably, wherein the Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.

[0036] Preferably, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.

[0037] Preferably, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504.

[0038] Preferably, wherein said Saccharomycetales yeast is cultivated by feeding ethanol, glucose or molasses as feedstock, with ethanol being preferred.

[0039] Preferably, in said composition the growth rate of said Saccharomycetales yeast is below 0.2 h’¹, preferably below 0.15 h’¹, more preferably below 0.1 h’¹, more preferably below 0.05 h’¹, more preferably the growth rate is between 0.02 and 0.05 h’¹, more preferably between 0.025 and 0.05 h’¹, most preferably between 0.025 and 0.045 h’¹.

[0040] Preferably, in said composition the feed rate is 0.179 - 0.536 g_ethanoi/gbiomass/h, more preferably 0.179 - 0.469 g_ethanoi/gbiomass/h.

[0041] Preferably, in said composition the temperature for cultivating said Saccharomycetales yeast is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.

[0042] Preferably, in said composition the pH for cultivating said Saccharomycetales yeast is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.

[0043] The composition of any one of the preceding claims, wherein said composition is further supplemented with lipids.

IV. DETAILED DESCRIPTION

[0044] The technical problem is solved by the subject-matter as defined in the claims, described in the description, and illustrated in the Figures. [0045] The composition of the invention is a yeast single cell protein (SCP) product that comprises a protein with high levels of digestibility, as demonstrated through a high degree of hydrolysis (DH), quantified by the degree of breakdown of protein content when said protein was exposed to artificial gastrointestinal fluids. It is preferred that this yeast SCP product is therefore used as a feed ingredient in aquaculture, providing an economically and environmentally viable alternative to fishmeal whilst offering improved digestibility for aquatic organisms when contrasted to other alternatives to fishmeal, such as soy protein.

[0046] Interestingly, it was found that the yeast SCP product of the present invention had a higher level of digestibility in comparison to both fish meal and soy protein concentrate when tested in its raw state and performed comparably with fish meal when tested as processed pellets comprising yeast SCP product (Table 2).

[0047] In the context of the present invention, “degree of hydrolysis” refers to the proportion of cleaved peptide bonds in a protein hydrolysate.

[0048] In the context of the present invention, “digestibility” of a protein refers to the fraction of protein that is available for absorption after it is ingested.

[0049] In the context of the present invention, the degree of hydrolysis (DH) may be quantified by any appropriate method known to those skilled in the art. Examples include, but are not limited to, SDS-PAGE, pH-stat, the osmometric method, and the SN-TCA method.

[0050] In the context of the present invention, “aquaculture” refers to the controlled cultivation of aquatic animals in either artificial or natural bodies of water. Animals suitable for aquaculture cultivation include, but are not limited to, fish, crustaceans, and molluscs.

[0051] In the context of the present invention, the term “”w/w” is intended to be understood as "weight by weight" and thus refers to the proportion of a particular substance within a mixture, as measured by weight or mass.

[0052] In the context of the present invention, the crude lipid content may be quantified by any appropriate method known to those skilled in the art. Examples include thin-layer chromatography, solid-phase extraction, or normal-phase LC and separation of particular classes of lipids into individual molecular species by high-performance liquid chromatography (HPLC)-ultraviolet detector or evaporative light-scattering detector.

[0053] In the context of the present invention, the term "crude lipids" preferably refers to the crude mixture of fat-soluble material present in a sample. This material is often extracted using diethyl ether or petroleum ether and includes both true lipids and, as non-limiting examples, fat soluble vitamins, chlorophylls etc.

[0054] Preferably, according to the present invention the composition comprising yeast SCP product comprises about 10% or less (w/w) crude lipids per gram of dry yeast SCP product, about 8% or less (w/w) crude lipids per gram of dry yeast SCP product, about 6% or less (w/w) crude lipids per gram of dry yeast SCP product. Thus, it is preferred that said crude lipids comprise, for example, about 10% to 8%, 10% to 6%, 10% to 4%, 8% to 6%, 8% to 4%, 6% to 4% (w/w) crude lipids per gram of dry yeast SCP product.

[0055] Also, it is preferred that said crude lipids may comprise saturated fatty acids, monounsaturated fatty acids and poly-unsaturated fatty acids.

[0056] Preferably, according to the present invention the saturated fatty acids of the yeast SCP product comprise C16:0 and C18:0 fatty acids, wherein preferably the C16:0 fatty acids and C18:0 fatty acids are present in an amount of no more than at least about 0.85% (w/w) per gram of dry yeast SCP product, preferably 0.53% (w/w) per gram of dry yeast SCP product.

[0057] Preferably, according to the present invention the mono-unsaturated fatty acids of the yeast SCP product comprise C16:1 and C18:1 fatty acids, wherein preferably the C16:1 fatty acids and C18:1 fatty acids are present in an amount of no more than at least 2.3% per gram of dry yeast SCP product, preferably about 2.8% (w/w) per gram of dry yeast SCP product.

[0058] Preferably, according to the present invention the poly-unsaturated fatty acids of the yeast SCP product comprise C16:2 and C18:2 fatty acids, wherein preferably the C16:2 fatty acids and C18:2 fatty acids are present in an amount of no more than at least about 1.0 % (w/w) per gram of dry yeast SCP product, preferably 2.0% (w/w) per gram of dry yeast SCP product.

[0059] Additionally, it was also surprisingly found that yeast cells, when grown with e.g. ethanol as a carbon source, produce a yeast SCP product with high levels of mono- and polyunsaturated fatty acids. This increased the ratio of unsaturated to saturated fatty acids present in the yeast SCP product. Thus, it is preferred that the ratio of unsaturated fatty acids, preferably C16:1 , C18:1 , C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, should be greater or equal to 1.0, preferably greater than 1.1 , more preferably greater than 1.2, more preferably greater than 1.5, most preferably greater than 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, or 8.0. [0060] In the context of the present invention, the term “unsaturated fatty acid” preferably refers to a fatty acid in which there is at least one double bond within the fatty acid chain. A fatty acid is “monounsaturated” if it contains a single double bond within the fatty acid chain and “polyunsaturated” if it contains more than one double bond within the fatty acid chain.

[0061] Preferably, according to the present invention the composition comprising yeast SCP product comprises 35%, 40%, 45%, or 50% or more (w/w) crude protein per gram of dry yeast SCP product.

[0062] Preferably, according to the present invention the composition comprising yeast SCP product comprises at least about 14% (w/w) essential amino acids per gram of dry yeast SCP product, more preferably at least about 18% (w/w) essential amino acids per gram of dry yeast SCP product.

[0063] In the context of the present invention, the amino acid content may be quantified by any appropriate method known to those skilled in the art. Examples include, but are not limited to, ion exchange chromatography, capillary electrophoresis, high pressure liquid chromatography and gas chromatography (GC).

[0064] In the context of the present invention, the term "essential amino acid" preferably refers to amino acids that cannot be synthesized by an animal from metabolic intermediates. Thus, such amino acids have to be supplied from an exogenous diet as they are required, e.g., for growth. Although variations may be possible, e.g., depending on the metabolic state of an animal, in general the following nine amino acids are considered essential: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine. Notably, in terms of nutrition, said nine essential amino acids are obtainable by a single complete protein containing all the essential amino acids. Such complete proteins can be derived from animalbased sources of nutrition, whereas plant-based foods represent commonly a source for essential amino acids in the form of incomplete proteins.

[0065] Preferably, according to the present invention the composition comprising yeast SCP product comprises 40%, 30%, 25% or 20% or less (w/w) total carbohydrates per gram of dry yeast SCP product.

[0066] Preferably, according to the present invention the composition comprising yeast SCP product comprises crude lipids to carbohydrates in a ratio of 0.6 to 1 : 2 to 5 and/or comprises crude lipids to crude protein in a ratio of 0.6 to 1 : 3.5 to 6. [0067] In particular, the present invention relates to a composition comprising yeast single cell protein (SCP) product comprising at most about 6% (w/w) nucleic acid content per gram of dry yeast SCP product, about 5% (w/w) nucleic acid content per gram of dry yeast SCP product, about 4% (w/w) nucleic acid content per gram of dry yeast SCP product.

[0068] Preferably said nucleic acid content comprises nucleosides and/or nucleotides, wherein it is preferred that said nucleosides are ribonucleosides and/or deoxyribonucleosides, and wherein it is preferred that said nucleotides are ribonucleotides and/or deoxyribonucleotides.

[0069] In the context of the present invention, the nucleic acid content may be quantified by any appropriate method known to those skilled in the art. Examples include quantitative PCR (qPCR), digital PCR (dPCR), and UV spectrophotometry.

[0070] In the context of the present invention, the term “single cell protein”, optionally abbreviated herein also as “SCP”, refers to a protein obtained by and/or derived from a (unicellular) microorganism. Thus, an SCP may refer to a protein purified and/or isolated from a microorganism’s cell culture for example. Alternatively or additionally, SCPs may refer to microbial proteins are the dead dried cells of microorganisms. Hence, an “single cell protein product” or “SCP product” may or may not comprise one or more selected from the group of intact (unicellular) microorganism cells, disrupted (unicellular) microorganism cells, isolated proteins obtained by one or more (unicellular) microorganism(s), isolated proteins derived by one or more (unicellular) microorganism(s), purified proteins obtained by one or more (unicellular) microorganism(s), and purified proteins derived by one or more (unicellular) microorganism(s). While an (unicellular) microorganism may relate to a bacterium, a fungus like yeast and/or an algae, said (unicellular) microorganism is yeast according to the present invention. SCP products from yeast offer the advantage of providing comparatively high protein contents, while at the same time said products can be produced on industrial scale at comparatively low cost, independent from seasonal effects and with comparatively low harvesting efforts. Thus, yeast SCP products are highly advantageous.

[0071] In the context of the present invention, the term “yeast” refers to a eukaryotic, unicellular microorganism classified as a member of the fungus kingdom that mostly reproduce asexually by mitosis with asymmetric division processes also being known as budding. Further herein, said term preferably relates to yeast cells, which can be grown under artificial and/or lab conditions, e.g. as in vitro culture conditions, and in particular under standard laboratory conditions. Said term preferably also embraces yeast cells of a single type that have been grown in the laboratory for several generations and thus, said term preferably embraces also potential mutants of a yeast cell and/or strain. Herein, yeast is preferably Saccharomycetales yeast The term “mutant” means permanent (epi-) genetic modification(s) of genetic material, i.e. nucleic acids, caused, for example, naturally or by physical means or chemical compounds/substances/agents such as EMS. Said modifications include point mutations, transitions, transversions, deletion/insertion/addition of one or more bases within a nucleic acid/gene/chromosome thereby optionally modifying the nucleic acid/gene/chromosome which can cause, inter alia, phenotypic effects like varying protein per dry matter (%) (w/w). Furthermore, such modification(s) may be induced by methods known to the person skilled in the art. The skilled person is also aware of suitable methods to select cells in view of one or more favorable and/or desired phenotypic trait(s) like an increase of protein per dry matter (%) (w/w) and/or utilization of ethanol or molasses as carbon source.

[0072] Preferably, the Saccharomycetales yeast cells are not genetically engineered. Thus, the aquafeed according to the present invention comprises up to 20% (w/w) or up to 10% (w/w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are preferably not genetically engineered. This is advantageous to ensure that the SCP product comprised in the animal feed comprises only well-defined and/or well characterized yeast cells but no mutated an thus, potentially undefined and/or uncharacterized yeast cells. This is also advantageous for a constant SCP product quality. Herein, the term “genetic engineering” is used in its broadest sense for methods known to the person skilled in the art to modify desired nucleic acids in vitro and in vivo , e.g. by targeted mutagenesis and/or recombinant DNA technology. Accordingly, said methods may comprise cloning, sequencing and transformation of recombinant nucleic acids, and appropriate vectors, primers, enzymes, host cells and the like are known by the skilled artisan. Preferably, genetically engineered cells are genetically engineered in view of high protein per dry matter (%), suitable essential amino acid composition, efficient ethanol or molasses usage as carbon source and the like in the context of the present invention.

[0073] A “yeast cell” is a cell of a yeast, preferably a cell of a yeast as described herein. Also included by said term are parts of a yeast cell. Such “parts” might refer to substances derived from a yeast cell. For example, when disrupted yeast cells are used, there might be yeast protein as well as yeast cell wall material.

[0074] In the context of the present invention, the term “Saccharomycetales" refers to the order Saccharomycetales within the phylum Ascomycota. Members of Saccharomycetales are also known and sometimes referred to as budding yeasts. [0075] SCP product producer may vary in their ability to use and/or utilize ethanol as carbon source for SCP production. Thus, the yeast SCP product preferably comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are Saccharomycetales yeast cells from one or more Saccharomycetales yeast genera, species and/or strains that are capable of using ethanol as carbon source. For example, the Saccharomycetales yeast cells may be Saccharomycetales yeast cells from one or more genera selected from the group consisting of Cyberlindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Pichia and Saccharomyces.

[0076] More specifically, the yeast SCP product comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus, preferably wherein said Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504.

[0077] Thus, it is particularly preferred that the composition comprising yeast single cell protein (SCP) product contains comprises 35%, 40%, 45%, or 50% or more (w/w) crude protein per gram of dry yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein Saccharomycetales yeast cells are yeast cells from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces. This is advantageous as yeast cells from said genera are capable of growing on a culture medium comprising ethanol as a carbon source.

[0078] When cultivating the SCP product of the present invention, it is preferred that the Saccharomycetales yeast is cultivated by feeding ethanol as feedstock.

[0079] During the cultivation, the ethanol concentration inside the fermenter itself is always close to zero (<0.1 g/L), as any ethanol that is fed is virtually immediately consumed by the yeast. The main parameter of the fermentation process, that determines the growth rate, is the ethanol feed rate. This feed rate has to be started low, as the biomass concentration at the start of fermentation is low and is subsequently increased exponentially to match the exponential growth rate of the biomass. At a certain point during the fermentation, this exponentially increasing feed rate is fixed to a constant feed rate, as the supply of oxygen in the fermenter becomes limiting. The maximum feed rate is limited by two main parameters in the fermentation process: 1) the biomass concentration, and 2) the oxygen transfer capacity. We found the ethanol feed rate should be defined by the substrate uptake rate of the organism (the qs) calculated by the amount of substrate that is consumed (g_ethanoi) per amount of biomass in the fermenter (gbiomass) per hour, of 0.179 - 0.536 g_ethanoi/gbiomass/h, more preferably 0.179 - 0.469 gethanol/gbiomass/h.

[0080] Interestingly, it was found that the biomass yield decreases with higher temperatures, causing the protein yield (the amount of protein produced per gram of ethanol or molasses) to decrease with increasing temperatures. Preferably, the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.

[0081] It was further found that protein content does change with the pH level. Protein content is increased at lower pH levels. The biomass yield however drops significantly at pH levels of 3.5 and lower. Preferably, the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.0, more preferably between 3.5 and 4.5.

[0082] The method for producing biomass may further comprise a step of recovering the biomass from the aerobic fermentation by suitable methods known in the art. Recovering biomass may comprise centrifugation or filtration.

[0083] The method for producing biomass may further comprise a step of drying the biomass by suitable methods known in the art. Drying biomass may comprise convective/direct drying technologies (like spray drying, fluidized bed) or contact/indirect technologies (like drum drying, vacuum drying, falling film) or supercritical drying (using superheated steam) or natural air/sun drying or even freeze drying.

[0084] In the context of the present invention, the term “aquafeed” (e.g., fish feed) refers to a feed for aquatic species. In case of the animal feed being a feed for aquatic species said species are preferably selected from fish, crustaceans, or molluscs. Thus, the animal feed may be a feed for fish and/or crustaceans and/or molluscs. When the animal feed is a feed for crustaceans, said crustaceans are preferably shrimps. Additionally, or alternatively, in case the animal feed is a feed for fish, said fish may preferably be warm water fish or cold water fish. When said fish are warm water fish, said fish are preferably selected from the group consisting of catfish, tilapia, seabream, seabass, and carp. Preferably, said fish are cold water fish with said fish being selected from cod, salmon, or rainbow trout. It is particularly preferred that the animal feed according to the present invention is a feed for shrimps, salmon and/or rainbow trout. Of note, it is preferred that the uses of the present invention are non-therapeutic. Indeed, the improvement of the weight of an animal like of aquatic species is deemed to be associated and/or to contribute to, e.g. health or growth of the animal, which is by way of common sense non-therapeutic.

[0085] Accordingly, it is preferred in the context of the uses of the present invention that the aquatic animal is healthy.

[0086] It is to be noted that in case of any definition given herein, the respective definition of a term, phrase, and/or abbreviation applies vice versa throughout the specification. Furthermore, all definition given herein are intended to encompass all grammatical forms.

[0087] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure.

[0088] The present invention may also be summarized in the following items:

ITEMS

1. A composition comprising a yeast single cell protein (SCP) product with a degree of hydrolysis of at least 12%, preferably at least 16%, more preferably least 20%, more preferably at least 24%, most preferably at least 28%.

2. The composition of item 1 , wherein said yeast single cell protein (SCP) product is used as a feed ingredient in aquaculture.

3. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) is used as a feed ingredient in aquaculture, wherein said aquaculture comprises the growing and harvesting of fish, crustaceans, or molluscs.

4. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product is used to prepare an aquafeed for use as a feed. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises between about 10% to 8%, 10% to 6%, 10% to 4%, 8% to 6%, 8% to 4%, 6% to 4% (w/w) crude lipids per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein crude lipids comprise saturated fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16:1, C18:1, C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.0. The composition of item 7, wherein said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16:1, C18:1, C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.1 , preferably greater than 1.2, more preferably greater than 1.5, most preferably greater than 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0. The composition of any one of items 6 to 8, wherein said saturated fatty acids are C16:0 and C18:0 fatty acids. The composition of item 9, wherein C16:0 fatty acids and C18:0 fatty acids are present in an amount of no more than 0.85% (w/w) per gram of dry yeast SCP product, preferably 0.53% (w/w) per gram of dry yeast SCP product. The composition of item 6, wherein said mono-unsaturated fatty acids are C16:1 fatty acids and C18:1 fatty acids. The composition of item 11 , wherein said C16:1 fatty acids and C18:1 fatty acids are present in an amount of at least about 2.3% (w/w) per gram of dry yeast SCP product, preferably 2.8% (w/w) per gram of dry yeast SCP product. The composition of item 6, wherein said poly-unsaturated fatty acids are C18:2 and C18:3 fatty acids. The composition of item 13, wherein said C18:2 fatty acids and C18:3 fatty acids are present in an amount of at least about 1.0% (w/w) per gram of dry yeast SCP product, preferably 2.0% (w/w) per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast SCP product further comprises crude protein. The composition of any one of the preceding items, wherein said yeast SCP product further comprises carbohydrates. The composition of any one of the preceding items, wherein said yeast SCP product further comprises crude protein, carbohydrates and crude lipids. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises 35%, 40%, 45%, or 50% or more (w/w) crude protein per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast SCP product comprises at least about 14% (w/w) essential amino acids per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast SCP product comprises at least about 18% (w/w) essential amino acids per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises 40%, 30%, 25% or 20% or less (w/w) total carbohydrates per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises crude lipids to carbohydrates in a ratio of 0.6 to 1 : 2 to 5. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises crude lipids to crude protein in a ratio of 0.6 to 1 : 3.5 to 6. The composition of any one of the preceding items, wherein said yeast single cell protein (SCP) product comprises crude lipids, carbohydrates and crude protein in a ratio of 0.6 to 1 crude lipids : 2 to 5 carbohydrates : 3.5 to 6 crude protein. The composition of any one of the preceding items, wherein said yeast SCP product comprises about 6% or less (w/w) nucleic acid content per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast SCP product comprises about 5% or less (w/w) nucleic acid content per gram of dry yeast SCP product. The composition of any one of the preceding items, wherein said yeast SCP product comprises about 4% or less (w/w) nucleic acid content per gram of dry yeast SCP product. The composition of any one of items 25 to 27, wherein said nucleic acid content comprises nucleosides and/or nucleotides. The composition of item 28, wherein said nucleosides are ribonucleosides and/or deoxyribonucleosides. The composition of item 28, wherein said nucleotides are ribonucleotides and/or deoxyribonucleotides. The composition of any one of the preceding items, wherein said yeast SCP product is obtained from cultivating a Saccharomycetales yeast. The composition of item 31 , wherein the Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces. The composition of item 31 or 32, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus. The composition of any one of items 31 to 33, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM- BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504. The composition of any one of items 31 to 34, wherein said Saccharomycetales yeast is cultivated by feeding ethanol as feedstock. The composition of any one of items 31 to 35, wherein the growth rate of said Saccharomycetales yeast is below 0.2 h’¹, preferably below 0.15 h’¹, more preferably below 0.1 h’¹, more preferably below 0.05 h’¹, more preferably the growth rate is between 0.02 and 0.05 h’¹, more preferably between 0.025 and 0.05 h’¹, most preferably between 0.025 and 0.045 h’¹.

37. The composition of any one of items 31 to 36, wherein the feed rate is 0.179 - 0.536 g_ethanoi/gbiomass/h, more preferably 0.179 - 0.469 g_ethanoi/gbiomass/h.

38. The composition of any one of items 31 to 37 wherein the temperature for cultivating said Saccharomycetales yeast is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.

39. The composition of any one of items 31 to 38, wherein the pH for cultivating said Saccharomycetales yeast is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.

40. The composition of any one of the preceding items, wherein said composition is further supplemented with lipids.

[0089] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0090] Unless otherwise stated, the following terms used in this document, including the description and items, have the definitions given below.

[0091] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0092] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention. [0093] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0094] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.

[0095] Throughout this specification and the items which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

[0096] When used herein “consisting of" excludes any element, step, or ingredient not specified in the item element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the item.

[0097] In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms.

[0098] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the items.

[0099] Other embodiments are within the following items. In addition, where features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will recognize that the present invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0100] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

V. BRIEF DESCRIPTION OF THE TABELS AND FIGURES [0101] Table 1 shows a table quantifying the content of the yeast SCP of the present invention, when grown with ethanol as carbon source. Shown are general parameters and broad categories, such as total carbohydrates, crude lipids, and crude proteins, along with a breakdown of specific amino acid and lipid content.

[0102] Table 2 shows a table quantifying the degree of hydrolysis of the yeast SCP of the present invention, contrasted with the same yeast SCP prepared as aquafeed, with soy protein concentrate, and with fish meal. The degree of hydrolysis is shown for each protein or protein preparation.

[0103] Figure 1 shows a graph quantifying the final weight of fish that were fed aquafeed prepared from described SCPs (SCP1 and SCP2) of the present invention, in contrast to the final weight of fish that were fed with a control feed containing fish meal as its primary protein constituent. Weights (g) were measured after 84 days of feeding with each specified food.

VI. EXAMPLES

[0104] Example 1 : SCP product content

[0105] A single cell protein (SCP) product was generated using Saccharomycetales yeast cells using ethanol or molasses as carbon source. As an example, Cyberlindnera jadinii was used. Feed rate, growth rate and pH of the culture medium was chosen pursuant to the teaching of the present application. The nutritional content of said SCP product was quantified, primarily as weight per total weight of dry product or % w/w of (dry) product. Content quantified includes total crude lipid content, total RNA/DNA content, amino acids, and fatty acids. These data indicate that the crude lipid content in the sample of SCP product was 6.0% as a percentage of dry product by weight. All measured values are shown in Table 1 , below.

Table 1

[0106] Example 2: Digestibility of proteins quantified by degree of enzymatic hydrolysis

[0107] Single cell protein (SCP) products (prepared as either powder or pellets), soy protein concentrate, and fish meal were assessed for degree of hydrolysis (DH) using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) in order to quantify their digestibility. SCP products were prepared from both Kluyveromyces lactis ( . lactis) and from Cyberlindnera jadinii (C. jadinii). Simulated gastric fluid (SGF) was prepared by dissolving 0.1g of sodium chloride and 10mg of pepsin in a final volume of 50 mL water, adjusted to pH 2 with 0.5M HCI. Trypsin stock solution was prepared by dissolving 20 mg Trypsin and 20 mg Chymo-Trypsin in 2 ml Milli-Q water. Simulated intestinal fluid (SIF) was prepared by bringing the remaining SGF solution containing the sample to pH 7 with NaOH 0.2M and adding 200uL Trypsin stock solution. After digestion, the Kjeldahl technique was used to determine total nitrogen content and primary amine content was also quantified. Degree of hydrolysis was then calculated:

DH (%) = h/h_tot x 100 wherein h is the primary NH2 per g of protein (mmol) and h_tot is the primary NH2 theoretically available per g of protein. The measured values for degree of hydrolysis (DH) for all samples analysed are shown below, in Table 2. SCPs were analysed either alone (SCP1 , SCP2) or when added to a standard aquafeed preparation as either 5%, 10% or 20% of the final product by mass. Fish meal and soy protein concentrate references show a lower degree of hydrolysis compared to both DSM SCPs. Table 2

[0108] Example 3: Final body weight of fish (g) fed with SCP1, SCP2, or a control diet with no added SCP.

[0109] Fish were fed one of seven diets - either a control diet containing a high percentage of fish meal or the control diet adapted to contain SCP1 present at either 5%, 10% or 20% or SCP2 present at either 5%, 10% or 20%. All 7 meals contained the same total crude protein and crude lipid content. Fish body weight was measured after 84 days. Fish fed with SCP1 had a final body weight of between +0.81 and -2.02% of control-fed fish. Fish fed with SCP2 had a final body weight of between +3.13 to -4.40% when compared to control-fed fish. These data indicate that SCP1 and SCP2 are well tolerated by fish and produce comparable growth rates as compared with control diet; see also Figure 1.

Claims

2. The composition of claim 1 , wherein said yeast single cell protein (SCP) product is used as a feed ingredient in aquaculture.

3. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) is used as a feed ingredient in aquaculture, wherein said aquaculture comprises the growing and harvesting of fish, crustaceans, or molluscs.

4. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises between about 10% to 8%, 10% to 6%, 10% to 4%, 8% to 6%, 8% to 4%, 6% to 4% (w/w) crude lipids per gram of dry yeast SCP product.

5. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16:1, C18:1, C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.0.

6. The composition of claim 6, wherein said yeast single cell protein (SCP) product comprises unsaturated fatty acids, preferably C16:1, C18:1, C18:2 and C18:3, and saturated fatty acids, preferably C16:0 and C18:0, in a ratio equal to or greater than 1.1 , preferably greater than 1.2, more preferably greater than 1.5, most preferably greater than 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 or 8.0.

7. The composition of any one of claims 5 to 6, wherein said saturated fatty acids are C16:0 and C18:0 fatty acids.

8. The composition of claim 9, wherein C16:0 fatty acids and C18:0 fatty acids are present in an amount of no more than 0.85% (w/w) per gram of dry yeast SCP product, preferably 0.53% (w/w) per gram of dry yeast SCP product.

9. The composition of claim 5, wherein said C16:1 fatty acids and C18:1 fatty acids are present in an amount of at least about 2.3% (w/w) per gram of dry yeast SCP product, preferably 2.8% (w/w) per gram of dry yeast SCP product.

10. The composition of claim 5, wherein said poly-unsaturated fatty acids are C18:2 and C18:3 fatty acids.

11. The composition of claim 1043, wherein said C18:2 fatty acids and C18:3 fatty acids are present in an amount of at least about 1.0% (w/w) per gram of dry yeast SCP product, preferably 2.0% (w/w) per gram of dry yeast SCP product.

12. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises 35%, 40%, 45%, or 50% or more (w/w) crude protein per gram of dry yeast SCP product.

13. The composition of any one of the preceding claims, wherein said yeast SCP product comprises at least about 14% (w/w) essential amino acids per gram of dry yeast SCP product.

14. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises 40%, 30%, 25% or 20% or less (w/w) total carbohydrates per gram of dry yeast SCP product.

15. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises crude lipids to carbohydrates in a ratio of 0.6 to 1 : 2 to 5.

16. The composition of any one of the preceding claims, wherein said yeast single cell protein (SCP) product comprises crude lipids to crude protein in a ratio of 0.6 to 1 : 3.5 to 6.

17. The composition of any one of the preceding claims, wherein said yeast SCP product comprises about 6% or less (w/w) nucleic acid content per gram of dry yeast SCP product.

18. The composition of any one of the preceding claims, wherein said yeast SCP product is obtained from cultivating a Saccharomycetales yeast.

19. The composition of claim 18, wherein said Saccharomycetales yeast is cultivated by feeding ethanol as feedstock.