Sustainable biomass production

Abstract

The present invention relates to a method for culturing a microorganism capable of using ethanol as a starting material, preferably a yeast of the Saccharomyces cerevisiae. The present invention relates to a method for producing a biomass having an increased protein content, in particular a single-cell protein, wherein a yeast single-cell protein product comprises yeast cells of the Saccharomyces cerevisiae; and an animal feed comprising such biomass.

Description

Technical Field

[0001] This invention relates to a method for culturing microorganisms capable of utilizing ethanol as a raw material. It also relates to a method for producing biomass, particularly single-cell protein, wherein the yeast single-cell protein product comprises yeast cells; and an animal feed comprising such biomass. Background Technology

[0002] The world's growing population is driving increasing demand for food, including meat, dairy, and seafood. Animal husbandry requires significant amounts of protein to ensure optimal growth and fattening, and currently, the primary protein source in traditional farming is plants. However, plant-based proteins are rarely converted into animal-based proteins. Furthermore, the production of plant-based proteins is associated with potential ethical conflicts between food and feed production. Therefore, farmers and the resulting animal protein-derived protein production industry require new protein sources to sustain rapid growth. However, while moving away from traditional protein sources, it is crucial to ensure animal health and welfare and optimize their growth. Thus, alternative protein sources are needed in animal husbandry to meet current and anticipated consumption levels for meat, dairy, and seafood. In addition, new protein sources are receiving increasing attention for human consumption.

[0003] In addition to plant and animal-derived proteins, another protein source, namely microbial-derived proteins, has been identified. These single-cell proteins (SCPs) can be produced using fungi, algae, and / or bacteria that can be cultured on a large scale at relatively low cost. However, the production of SCP products still faces several challenges, such as the availability and sustainability of raw materials (e.g., waste-based materials). Therefore, feed processors, farmers, and food producers still need alternative solutions for protein production to deliver low-carbon footprint diets while ensuring the environmentally friendly use of Earth's resources.

[0004] Therefore, there remains a need for animal feed that contains protein from alternative protein sources and is therefore neither animal-derived nor plant-derived. The industry is exploring the use of renewable energy and renewable raw materials to produce organic chemicals from waste materials. Ideally, these renewable raw materials could be used to produce protein from alternative protein sources, but this presents a challenge. Summary of the Invention

[0005] Therefore, one object of the present invention is to provide a more sustainable route for preparing high-protein (microbial) biomass or SCP from ethanol feedstock. Specifically, one object is to provide a method for preparing such high-protein (microbial) biomass, wherein the single-cell protein product comprises isocitrate lyase and / or malate synthase. The present invention also aims to provide an animal feed comprising such single-cell protein products. Thus, the growing demand for alternative feed solutions can be met while ensuring sustainability. Specifically, the present invention relates to a method for culturing microorganisms capable of producing at least 60% protein, preferably at least 65% protein, more preferably at least 70%, and most preferably at least 75% protein, comprising the steps of: (i) supplying microorganisms to a reactor, (ii) feeding EtOH as a feedstock, (iii) controlling the feed rate, (iv) controlling the temperature, (v) controlling the pH, and (vi) controlling the growth rate.

[0006] Preferably, the growth rate is at least 0.02 h. -1 Preferably at least 0.025 h -1 More preferably at least 0.03 h -1 More preferably at least 0.04 h -1 More preferably at least 0.05 h -1 More preferably at least 0.06 h -1 More preferably at least 0.07h -1 More preferably at least 0.08 h -1 More preferably at least 0.09 h -1 More preferably at least 0.1 h -1 More preferably at least 0.15 h -1 More preferably at least 0.16 h -1 More preferably at least 0.17 h -1 More preferably at least 0.18 h -1 More preferably at least 0.19 h -1 More preferably at least 0.2 h -1 More preferably at least 0.21 h -1 The most preferred option is at least 0.22 h. -1 .

[0007] Preferably, the feed rate is 0.179 - 0.536 g. 乙醇 / g 生物质 / h, more preferably 0.179 - 0.469 g 乙醇 / g 生物质 / h.

[0008] Preferably, the temperature is maintained between 30°C and 40°C, more preferably between 30°C and 38°C, more preferably between 30°C and 36°C, and most preferably between 30°C and 34°C.

[0009] Preferably, the pH is maintained between 3.5 and 5.5, more preferably between 3.5 and 5.0, and most preferably between 3.5 and 4.5.

[0010] Preferably, the method further includes step (vii) drying the biomass.

[0011] The present invention also relates to a yeast single-cell protein product, wherein the yeast single-cell protein product comprises at least 60% protein by weight of stem cells, preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / or malate synthase.

[0012] Preferably, the microorganism is yeast from the order Yeastae.

[0013] Preferably, the yeast in the order Yeastales is from the genus *Ceberlindna* (…). Cyberlindnera ), Yeast ( Saccharomyces Kluyveromyces ( ) Kluyveromyces ), Wickham yeast ( Wickerhamomyces ), Pichia pastoris ( Pichia ) or Yersinia genus ( Yarrowia ( ), preferably yeasts from the genera *Cerberindnae*, *Saccharomyces*, *Kluyveromyces*, or *Wickhamia*.

[0014] Preferably, the yeast in the order Yeast is *Saccharomyces cerevisiae* (Jedin Seberlindnae). Cyberlindnera jadinii ), brewer's yeast ( Saccharomyces cerevisiae Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), abnormal Wickham yeast ( Wickerhamomyces anomalus ), abnormal Pichia pastoris ( Pichia anomala ) or Yersinia lipophila ( Yarrowia lipolytica (More preferably, yeasts derived from *Gerdinium sacchariformis*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Wickhamia suspensa*.)

[0015] Preferably, the yeast is derived from *Saccharomyces cerevisiae* ATCC 26387, *Saccharomyces cerevisiae* FERM-BP1656, *Saccharomyces cerevisiae* CBS621, *Saccharomyces cerevisiae* CBS841, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* AQFM-009, *Saccharomyces cerevisiae* AQFM-035, *Saccharomyces cerevisiae* AQFM-036, *Saccharomyces cerevisiae* AQFM-037, *Saccharomyces cerevisiae* AQFM-038, *Saccharomyces cerevisiae* AQFM-039, *Saccharomyces cerevisiae* AQFM-041, *Saccharomyces cerevisiae* AQFM-017, etc. Mother AQFM-042, Jading Saccharomyces cerevisiae AQFM-043, Jading Saccharomyces cerevisiae AQFM-044, Jading Saccharomyces cerevisiae AQFM-046, Jading Saccharomyces cerevisiae AQFM-048, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-049, Jading Saccharomyces cerevisiae AQFM-050, Jading Saccharomyces cerevisiae AQFM-051, Jading Saccharomyces cerevisiae AQFM-052, Jading Saccharomyces cerevisiae AQFM-053, Jading Saccharomyces cerevisiae AQFM-054, Jading Saccharomyces cerevisiae AQFM-055, Aberrant Wickham Saccharomyces IFO 569. *Saccharomyces cerevisiae* CBS1980, *Saccharomyces cerevisiae* ATCC 9950, *Kluyveromyces lactis* CBS 2896, *Saccharomyces cerevisiae* CBS2576, or *Yersinia lipolytica* CBS 7504, preferably derived from *Saccharomyces cerevisiae* ATCC 26387, *Saccharomyces cerevisiae* FERM-BP1656, *Saccharomyces cerevisiae* CBS621, *Saccharomyces cerevisiae* CBS841, *Saccharomyces cerevisiae* GHP1, or *Saccharomyces cerevisiae* CEN.PK113-7D, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-009, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-035, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-036, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-037, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-038, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-039, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-041, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-017, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-042, J.D. Seberlindner's Saccharomyces cerevisiae AQFM-043, J.D. Seberlindner's Saccharomyces cerevisiae The following yeasts are used: AQFM-044, AQFM-046, AQFM-048, AQFM-017, AQFM-049, AQFM-050, AQFM-051, AQFM-052, AQFM-053, AQFM-054, AQFM-055, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* CBS 1980, *Saccharomyces cerevisiae* ATCC 9950, or *Kluyveromyces lactis* CBS 2896.

[0016] Specifically, the present invention relates to an animal feed comprising up to 20% (w / w), preferably up to 10% (w / w) of yeast single-cell protein (SCP) product, wherein the yeast single-cell protein product comprises at least 60% protein by weight of stem cells, preferably at least 65% protein, more preferably at least 70%, and most preferably at least 75% protein, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / or malate synthase.

[0017] Preferably, the yeast single-cell protein product comprises yeast cells fed with ethanol.

[0018] Preferably, the yeast cells in the order Yeast are from the genera *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Kluyveromyces spp.*, *Wickham's yeast*, *Pichia pastoris*, or *Yersinia*. More preferably, they are from the genera *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Kluyveromyces spp.*, or *Wickham's yeast*. Preferably, the yeast cells in the order Yeast are from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Wickham's yeast aberrant*, *Pichia pastoris*, or *Yersinia lipolytica*. More preferably, they are from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Wickham's yeast aberrant*. Preferably, the yeast cells in the order Yeast are or are derived from *Saccharomyces cerevisiae* ATCC. 26387, Jading Saccharomyces cerevisiae FERM-BP1656, Jading Saccharomyces cerevisiae CBS621, Jading Saccharomyces cerevisiae CBS841, Jading Saccharomyces cerevisiae AQFM-009, Jading Saccharomyces cerevisiae AQFM-035, Jading Saccharomyces cerevisiae AQFM-036, Jading Saccharomyces cerevisiae AQFM-037, Jading Saccharomyces cerevisiae AQFM-038, Jading Saccharomyces cerevisiae AQFM-039, Jading Saccharomyces cerevisiae AQFM-041, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-042, Jading Saccharomyces cerevisiae... *Saccharomyces cerevisiae* AQFM-043, *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* CBS 1980, *Yersinia sebifera* ATCC 9950, *Kluyveromyces lactis* CBS 2896, *Saccharomyces cerevisiae* CBS 2576, or *Yersinia lipophila* CBS 7504, preferably from *Yersinia sebifera* ATCC. 26387, Jading Saccharomyces cerevisiae FERM-BP1656, Jading Saccharomyces cerevisiae CBS621, Jading Saccharomyces cerevisiae CBS841, Jading Saccharomyces cerevisiae AQFM-009, Jading Saccharomyces cerevisiae AQFM-035, Jading Saccharomyces cerevisiae AQFM-036, Jading Saccharomyces cerevisiae AQFM-037, Jading Saccharomyces cerevisiae AQFM-038, Jading Saccharomyces cerevisiae AQFM-039, Jading Saccharomyces cerevisiae AQFM-041, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-042, Jading Saccharomyces cerevisiae AQFM-0 43. *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* CBS1980, *Saccharomyces cerevisiae* ATCC 9950, or *Kluyveromyces lactis* CBS 2896.

[0019] Preferably, the yeast cells are capable of producing at least 60% protein per gram of dry weight of the yeast cells using ethanol as a carbon source, more preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein.

[0020] Preferably, the yeast SCP product contains all essential amino acids.

[0021] Preferably, the yeast cells of the order Yeast are not genetically engineered.

[0022] Preferably, the yeast SCP product contains at least 60% protein per gram of yeast cells, more preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein.

[0023] Preferably, the yeast SCP product comprises dried yeast cells, preferably wherein the dried yeast cells are whole or broken or a mixture of whole and broken cells.

[0024] Preferably, the animal feed also contains i) less than 25% (w / w) of plant-based protein products, ii) an additional 5% (w / w) or less of fishmeal, iii) no fishmeal, and / or iv) 1% to 25% (w / w) of oil.

[0025] Preferably, the animal feed is feed for poultry, pigs, horses, camels, cattle, sheep or companion animals.

[0026] Preferably, the animal feed is feed for aquatic species, wherein the aquatic species is preferably selected from crustaceans or fish, wherein the crustaceans are preferably shrimp, and / or wherein the fish are preferably warm-water or cold-water fish, wherein the warm-water fish are preferably selected from catfish, tilapia, sea bream, sea bass or carp, and / or wherein the cold-water fish are preferably selected from cod, salmon or rainbow trout.

[0027] The present invention also relates to the use of animal feed according to the invention for feeding animals.

[0028] The present invention also relates to the use of animal feed according to the invention for increasing animal weight.

[0029] Preferably, the animal is poultry, pig, horse, camel, cow, sheep, or companion animal, or the animal is an aquatic species, preferably the aquatic species is selected from crustaceans or fish, preferably the crustacean is shrimp, and / or preferably the fish is warm-water fish or cold-water fish, preferably the warm-water fish is selected from catfish, tilapia, sea bream, sea bass, or carp, and / or preferably the cold-water fish is selected from cod, salmon, or rainbow trout. Detailed Implementation

[0030] The technical problem is solved by the subject matter as defined in the claims, described in the specification, illustrated in the embodiments, and illustrated in the drawings.

[0031] The method of the present invention is an aerobic fermentation for producing biomass. This aerobic fermentation in the method for producing biomass includes culturing microorganisms, namely yeast. The microorganisms in the aerobic fermentation use ethanol fed into the aerobic fermentation as a raw material to produce biomass.

[0032] It has been found that yeast cells can produce relatively high protein content when grown using ethanol as a carbon source (see [link]). Figure 1Furthermore, surprisingly, it was found that increasing the growth rate at the end of the fermentation process significantly increased the protein production by yeast cells (see [link to original text]). Figure 2 Therefore, yeast cells of this order, especially when grown on ethanol, appear suitable as a sustainable single-cell protein product, such as a protein source, intended to at least partially, and preferably completely, replace animal-derived protein sources in animal feed, such as fishmeal. Yeast cells of this order are preferably derived from the genera *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Kluyveromyces spp.*, or *Wickhamia spp.*, and more preferably from *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Saccharomyces spp.*, *Kluyveromyces lactis*, or *Wickhamia spp.*

[0033] To regulate the growth rate of microbial cells at the end of fermentation and thus increase the protein content in biomass, fermentation must be terminated at an appropriate growth rate. During cultivation, the ethanol concentration inside the fermenter is consistently kept close to zero (<0.1 g / L) because almost all ethanol fed into the fermenter is immediately consumed by the yeast. The primary parameter determining the growth rate during fermentation is the ethanol feed rate. This feed rate must start low because the biomass concentration is low at the beginning of fermentation and then increases exponentially to match the exponential growth rate of the biomass. At some point in the fermentation process, when the oxygen supply in the fermenter becomes a limiting factor, this exponentially increasing feed rate is fixed as a constant feed rate. The maximum feed rate is limited by two main parameters in the fermentation process: 1) biomass concentration and 2) oxygen transfer capacity. We found that the ethanol feed rate should be determined by the organism's substrate uptake rate (q... S The rate is limited to 0.179–0.536 g. 乙醇 / g 生物质 / h, more preferably 0.179 - 0.469 g 乙醇 / g 生物质 / h of biomass per unit volume (g) in the fermenter per hour 生物质 The amount of substrate consumed (g) 乙醇 ) was calculated.

[0034] Interestingly, it was found that biomass yield decreases at higher temperatures, which in turn leads to a decrease in protein yield (the amount of protein produced per gram of ethanol) with increasing temperature (see [link to relevant documentation]). Figure 3 Preferably, the temperature is maintained between 30°C and 40°C, more preferably between 30°C and 38°C, more preferably between 30°C and 36°C, and most preferably between 30°C and 34°C.

[0035] It was also found that protein content does indeed vary with pH level. Protein content increases at lower pH levels. However, at pH levels of 3.5 and lower, biomass yield decreases significantly. Preferably, the pH is maintained between 3.5 and 5.5, more preferably between 3.5 and 5.0, and even more preferably between 3.5 and 4.5 (see [link to study]). Figure 4 ).

[0036] Surprisingly, we found high levels of the enzymes isocitrate lyase and malate synthase in the biomass when fed with ethanol feedstock (see [link to article]). Figure 5 and Figure 6 ).

[0037] Methods for producing biomass may also include the step of recovering biomass from aerobic fermentation by suitable methods known in the art. Biomass recovery may include centrifugation or filtration.

[0038] Methods for producing biomass may also include the step of drying the biomass by suitable methods known in the art. Drying biomass may include convection / direct drying techniques (such as spray drying, fluidized bed drying) or contact / indirect techniques (such as drum drying, vacuum drying, falling film drying) or supercritical drying (using superheated steam) or natural air drying / sun drying or even freeze drying.

[0039] Therefore, when yeast cells of this order are used as single-cell protein products in animal feed, animal feed containing up to 20% (w / w) yeast single-cell protein product has been found to be well consumed by animals, particularly aquatic species such as fish or crustaceans. Furthermore, when yeast cells of this order are used as single-cell protein products in animal feed, animal feed containing up to 10% (w / w) yeast single-cell protein product has been found to have a beneficial effect on weight gain in animals, particularly aquatic species such as fish or crustaceans. Additionally, animal feed containing up to 20% (w / w) or 10% (w / w) yeast single-cell protein product can advantageously completely replace animal-derived proteins such as fishmeal.

[0040] In the context of this invention, the term "animal" means any animal other than a human. Examples of animals are non-ruminant and ruminant animals. Ruminant animals include, for example, animals such as horses, sheep, goats, cattle (e.g., beef cattle, cows, dairy cows, and calves), deer, yaks, camels, llamas, and kangaroos. Non-ruminant animals include monogastric animals, including but not limited to companion animals (including but not limited to cats and dogs), pigs or swine (including but not limited to piglets, growing pigs, and sows); poultry, such as turkeys, ducks, quails, guinea fowl, geese, pigeons (including squabs), and chickens (including but not limited to broiler chickens, chicks, and laying hens); horses (including but not limited to hot-blooded horses, cold-blooded horses, and warm-blooded horses); crustaceans (including but not limited to shrimp and prawns); and fish, including but not limited to warm-water fish and cold-water fish, and therefore, fish including but not limited to amberjack and arapaima. apaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, crappie, dorada, drum, eel, goby, goldfish, gourami Grouper, guapote, halibut, Java fish, labeo, lai, loach, mackerel, milkfish, silver perch, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, sea bream, shiner The following fish are included: sleeper, blackfish, snapper, snookerfish, sole, spinefoot, sturgeon, sunfish, sweetfish, bream, terror, tilapia, trout (preferably rainbow trout), tuna, turbot, vendace, walleye, and whitefish. Among these, the preferred warm-water fish are catfish, tilapia, sea bream, sea bass, or carp, and / or the preferred cold-water fish are cod, salmon, or rainbow trout.

[0041] In the context of this invention, the microbial biomass is yeast and therefore the terms "single-cell protein product" and "yeast single-cell protein product" are used interchangeably.

[0042] In the context of this invention, protein yield is defined as the amount of protein produced per gram of ethanol. A common method for determining protein content, as stated in US3151038, is by analyzing total nitrogen using the Kjeldahl method and then multiplying by 6.25 (a standard factor according to accepted practice). Hawk, Philip B., Oser, Bernard L., and Summerson, William H., 1947, Practical Physiological Chemistry, 12th edition, The Blakiston Company, Philadelphia, and Toronto, state on pages 213 and 214 that the commonly used factor for calculating protein from nitrogen content is 6.25 and is based on the assumption that the protein contains an average of 16% nitrogen.

[0043] In the context of this invention, protein content is defined as the amount of protein in dry matter biomass.

[0044] It is important to note that any animals mentioned herein are preferably not wild animals. Therefore, the animals are preferably farm animals and / or livestock. In the case of aquatic species, the animals are preferably aquaculture animals. In this document, the term "aquaculture" refers to aqua-farming and therefore the cultivation of aquatic species, such as fish or crustaceans, in various environments, including but not limited to tanks, lakes, ponds, or any other natural or artificial bodies of water suitable for the breeding, hatching, rearing, and harvesting of aquatic species.

[0045] In the context of this invention, the term "animal feed" (e.g., fish feed) refers to any compound, formulation, or mixture suitable or intended for consumption by animals (e.g., fish). Animal feed for monogastric animals typically contains concentrates along with vitamins, minerals, enzymes, directly fed microorganisms, amino acids, and / or other feed ingredients (such as in the form of premixes), while animal feed for ruminants typically contains forage (including roughage and silage) and may also contain concentrates along with vitamins, minerals, enzymes, directly fed microorganisms, amino acids, and / or other feed ingredients (such as in the form of premixes). Animal feed additives (e.g., fish feed additives) are formulated enzyme products that may also contain, for example, vitamins, minerals, enzymes, amino acids, preservatives, and / or antibiotics; i.e., premixes. Animal feed additives / premixes are typically mixed with concentrates and / or forage (such as plant proteins, legumes, or other plant materials) in feed processing plants. Furthermore, animal feed is typically fed to monogastric animals as pelleted feed.

[0046] In the context of this invention, the term "single-cell protein" (also optionally abbreviated herein as "SCP") refers to a protein obtained from and / or derived from a (single-celled) microorganism. Thus, SCP can refer to, for example, a protein purified and / or isolated from a cell culture of a microorganism. Alternatively or additionally, SCP can refer to the dead, dried cells of a microorganism. Therefore, a "single-cell protein product" or "SCP product" may or may not contain one or more of the following: whole (single-celled) microbial cells, broken (single-celled) microbial cells, isolated proteins obtained from one or more (single-celled) microorganisms, isolated proteins derived from one or more (single-celled) microorganisms, purified proteins obtained from one or more (single-celled) microorganisms, and purified proteins derived from one or more (single-celled) microorganisms. While (single-celled) microorganisms may refer to bacteria, fungi (such as yeast), and / or algae, according to this invention, the (single-celled) microorganism is yeast. SCP products from yeast have the advantage of providing relatively high protein content, while the products can be produced on an industrial scale at relatively low cost, unaffected by seasonality, and with relatively low harvesting workload. Therefore, yeast SCP products are highly advantageous.

[0047] In the context of this invention, the term "yeast" refers to a eukaryotic single-celled microorganism classified as a member of the kingdom Fungi, which reproduces asexually primarily through mitosis. Furthermore, the term preferably refers to yeast cells that can be grown under artificial and / or laboratory conditions, for example, under in vitro culture conditions, particularly under standard laboratory conditions. The term preferably also includes a single type of yeast cell that has been grown for several generations in the laboratory, and therefore, the term preferably also includes potential mutants of yeast cells and / or strains. In this document, yeast is preferably yeast from the order Sacchariformes.

[0048] "Yeast cell" is a yeast cell, preferably a yeast cell as described herein.

[0049] In the context of this invention, the term "yeast order" ( Saccharomycetales ")" refers to the phylum Ascomycota ( Ascomycota The order Yeasts is a suborder of yeasts. Members of the order Yeasts are also known and sometimes referred to as budding yeasts.

[0050] In the context of this invention, the term "w / w" is intended to be understood as "weight ratio" and therefore refers to the proportion of a particular substance in a mixture as measured by weight or mass.

[0051] SCP product manufacturers may vary in their ability to use and / or utilize ethanol as a carbon source to produce SCP. Therefore, yeast SCP products preferably contain yeast cells from the order Sacchariformes, wherein the yeast cells are yeast cells from one or more genera, species, and / or strains of yeast capable of using ethanol as a carbon source. For example, yeast cells may be yeast cells from one or more genera selected from the group consisting of *Sacchariformes*, *Kluyveromyces*, *Wickham's yeast*, *Yersinia*, *Pichia pastoris*, and *Sacchariformes*.

[0052] More specifically, the yeast SCP product comprises yeast cells of the order Saccharidae, wherein the yeast cells are preferably selected from the group consisting of Pichia pastoris, Yersinia lipolytica, Wickham's yeast, J.C. Seberlindnerella vaginalis, Saccharomyces cerevisiae, and / or Kluyveromyces lactis. Preferably, the strain is an improved strain.

[0053] Therefore, it is particularly preferred that the animal feed according to the invention contains up to 20% (w / w) or up to 10% (w / w) of a yeast SCP product, wherein the yeast SCP product comprises yeast cells of the order Saccharidae, and wherein said yeast cells are from the genera *Wickhamia*, *Cyperus*, *Saccharidae*, *Kluyveromyces*, *Yersinia*, and / or *Pichia*, preferably from the genera *Cyperus*, *Saccharidae*, *Kluyveromyces*, and / or *Wickhamia*. This is advantageous because yeast cells from said genera can be grown on a culture medium containing ethanol as a carbon source, as further illustrated herein in the examples (see, for example...). Figure 1 ).

[0054] Preferably, the animal feed according to the invention comprises up to 20% (w / w) or up to 10% (w / w) of yeast SCP product, wherein the yeast SCP product comprises yeast cells of the order Saccharidae, and wherein said yeast cells are preferably derived from *G. sacchariformis*, *Sacchariformis*, *Kluyveromyces lactis*, *W. aberrantia*, *Pichia aberrantia*, and / or *Yersinia lipolytica*, more preferably from *W. aberrantia*, *G. sacchariformis*, *Sacchariformis*, and / or *Kluyveromyces lactis*. This is particularly advantageous because the SCP product from said species can have, for example, 60% or more protein per unit dry matter, preferably protein, more preferably 70% or more, most preferably 75% or more protein, and can completely replace animal-derived proteins, such as fishmeal, in the animal feed according to the invention.

[0055] Preferably, the animal feed according to the invention comprises up to 20% (w / w) or up to 10% (w / w) of a yeast SCP product, wherein the yeast SCP product comprises yeast cells of the order Saccharidae, and wherein the yeast cells are derived from and / or from *G. sabinatum* ATCC. 26387, Jading Saccharomyces cerevisiae FERM-BP1656, Jading Saccharomyces cerevisiae CBS621, Jading Saccharomyces cerevisiae CBS841, Jading Saccharomyces cerevisiae AQFM-009, Jading Saccharomyces cerevisiae AQFM-035, Jading Saccharomyces cerevisiae AQFM-036, Jading Saccharomyces cerevisiae AQFM-037, Jading Saccharomyces cerevisiae AQFM-038, Jading Saccharomyces cerevisiae AQFM-039, Jading Saccharomyces cerevisiae AQFM-041, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-042, Jading Saccharomyces cerevisiae... *Saccharomyces cerevisiae* AQFM-043, *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* CBS 1980, *Yersinia sebifera* ATCC 9950, *Kluyveromyces lactis* CBS 2896, *Saccharomyces cerevisiae* CBS 2576 and / or *Yersinia lipolytica* CBS 7504, preferably from *Yersinia sebifera* ATCC. 26387, Jading Saccharomyces cerevisiae FERM-BP1656, Jading Saccharomyces cerevisiae CBS621, Jading Saccharomyces cerevisiae CBS841, Jading Saccharomyces cerevisiae AQFM-009, Jading Saccharomyces cerevisiae AQFM-035, Jading Saccharomyces cerevisiae AQFM-036, Jading Saccharomyces cerevisiae AQFM-037, Jading Saccharomyces cerevisiae AQFM-038, Jading Saccharomyces cerevisiae AQFM-039, Jading Saccharomyces cerevisiae AQFM-041, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-042, Jading Saccharomyces cerevisiae... *Saccharomyces bolindensis* AQFM-043, *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Wickhamia lanceolata* CBS 1980, *Grindelwald's yeast* ATCC9950, and / or *Kluyveromyces lactis* CBS 2896.

[0056] In the context of this invention, the term "derived from" preferably refers to yeast cells originally obtained from and therefore derived from a given yeast strain. Such derived cells may differ from the given yeast strain due to naturally occurring and / or artificially introduced alterations (such as gene mutations), but preferably have characteristics similar to cells from the yeast strain from which they originate. Such similar characteristics preferably include the ability to produce 60% or more protein per gram of dry weight of yeast cells, preferably protein, more preferably 70% or more, and most preferably 75% or more protein, using ethanol as a carbon source. This ability can be easily tested by a person skilled in the art by culturing yeast cells with ethanol as a carbon source, thereby testing a range of ethanol concentrations as a carbon source. Therefore, cells derived from a given strain preferably, at the genomic level, have 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with a corresponding strain that can be considered as a reference. Therefore, the derived cells may have at least, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding reference, preferably at the genomic level.

[0057] As used herein, the term “sequence identity” or “identity” refers to a sequence property that measures the similarity or relationship between sequences. As used in this disclosure, “sequence identity” or “identity” means the percentage of identical residues in pairs relative to the number of residues in the longer of the two sequences after (homologous) alignment of the sequences of nucleotides and / or amino acids with the corresponding sequences under discussion. Sequence identity is determined by dividing the number of identical nucleotide and amino acid residues, respectively, by their total number and multiplying the result by 100. Those skilled in the art will recognize available computer programs, such as BLAST (Altschul et al., 1997), BLAST2 (Altschul et al., 1990), FASTA (Pearson and Lipman, 1988), GAP (Needleman and Wunsch, 1970), Smith-Waterman (Smith and Waterman, 1981), and the Wisconsin GCG Package, for determining sequence identity using standard parameters. The percentage of sequence identity can be determined, for example, using the BLASTP program (version 2.2.5, November 16, 2002) (Altschul et al., 1997), which calculates the percentage of “positive” (homologous amino acids) out of the total number of amino acids selected for alignment.

[0058] Therefore, the "sequence identity percentage (%)" relative to the cells and / or strains described herein is preferably defined at the nucleic acid level and is thus defined as the percentage of nucleotides in the candidate sequence that are identical to those in the reference sequence after sequence alignment and, where necessary, the introduction of vacancies, and without regard to any conserved substitutions as part of sequence identity, to achieve the maximum sequence identity percentage. Alignment used to determine the nucleotide sequence identity percentage can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. This also applies to amino acid sequences, with only necessary modifications.

[0059] For example, the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2: 482-489 provides a suitable alignment for nucleic acid sequences. This algorithm can be applied to amino acid sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff (ed.), 5 Supplement 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6): 6745-6763. An exemplary implementation of this algorithm for determining the percentage of sequence identity is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters used for this method are described in the *Wisconsin Sequence Analysis Package Program Manual*, 8th edition (1995) (available from Genetics Computer Group, Madison, Wis.). In the context of this invention, a preferred method for determining the percentage of identity is to use the MPSRCH package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (MountainView, Calif). From this package, the Smith-Waterman algorithm can be employed, where the scoring table uses default parameters (e.g., a gap opening penalty of 12, a gap extension penalty of 1, and 6 gaps). Based on the generated data, the "match" value reflects "sequence identity." Other suitable procedures for calculating the percentage identity or similarity between sequences are generally known in the art; for example, another alignment procedure is BLAST, run using default parameters.

[0060] In the context of this invention, the following illustrative examples may be considered. When an obtained yeast strain (which may initially be a formally preserved yeast strain) is cultured and propagated for several generations under, for example, laboratory conditions, the resulting yeast cells may be genetically identical to the preserved genetic material of the original yeast strain. In this case, these cells are "the" cells derived from the preserved strain and have 100% sequence identity with the preserved material at the genomic level. However, some or even all cells may exhibit a degree of genetic and / or epigenetic variation, for example, due to one or more mutations. In this case, the cells under study are "derived" from the initial strain from which their genetic material and / or cells were preserved. Such mutations may occur naturally during cell culture and propagation. Alternatively or additionally, although preferably, such mutations may be artificially introduced, for example, through genetic engineering. Thus, compared to the original yeast strain from which the cells are derived, the derived cells may exhibit variations, for example, in terms of the percentage of protein in dry matter (%) (w / w), the amount of essential amino acids, and / or the composition of essential amino acids. The resulting yeast cells can also be called mutants compared to the cells and / or strains from which they are derived.

[0061] In this document, the term "genetic engineering" is used in its broadest sense for methods known to those skilled in the art for modifying desired nucleic acids in vitro and in vivo, such as by targeted mutagenesis and / or recombinant DNA techniques. Therefore, such methods may include the cloning, sequencing, and transformation of recombinant nucleic acids, and suitable vectors, primers, enzymes, host cells, etc., are known to those skilled in the art. In the context of this invention, preferably, the genetic engineering of the cells involves aspects such as a high percentage (%) of protein in dry matter, a suitable essential amino acid composition, and efficient use of ethanol as a carbon source.

[0062] Furthermore, in this document, the terms "mutation" and "mutant" refer to permanent (epimal) genetic modifications of genetic material (i.e., nucleic acids), which are caused, for example, by nature or by physical means or by chemical compounds / substances / reagents (such as EMS). These modifications include point mutations, transitions, transversions, deletions / insertions / additions of one or more bases in nucleic acids / genes / chromosomes, thereby optionally modifying the nucleic acids / genes / chromosomes, which in particular can lead to phenotypic effects, such as changes in the percentage of protein in dry matter (%) (w / w). Moreover, such modifications can be induced by methods known to those skilled in the art. Those skilled in the art also understand suitable methods for selecting cells based on one or more advantageous and / or desired phenotypic traits, such as an increase in the percentage of protein in dry matter (%) (w / w) and / or the use of ethanol as a carbon source.

[0063] Preferably, the yeast cells are unengineered. This is advantageous for ensuring that the SCP product included in animal feed contains only well-defined and / or well-characterized yeast cells, without mutant cells, and thus avoids potentially undefined and / or uncharacterized yeast cells. It also helps maintain consistent SCP product quality.

[0064] Alternatively or preferably, the yeast SCP product included in the animal feed according to the invention contains all essential amino acids. This is advantageous because animal feed containing a yeast SCP product with all essential amino acids can completely replace currently used animal and / or plant-based protein sources in animal feed. Therefore, the SCP product can represent an alternative protein source that requires no supplementation in terms of essential amino acids.

[0065] In the context of this invention, the term "essential amino acid" preferably refers to amino acids that animals cannot synthesize from metabolic intermediates. Therefore, such amino acids must be supplied from an exogenous diet, as they are required, for example, for growth. While variations may exist, such as depending on the metabolic state of the animal, the following nine amino acids are generally considered essential: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine. It is noteworthy that, nutritionally, these nine essential amino acids can be obtained from a single complete protein containing all the essential amino acids. Such complete proteins can be derived from animal nutritional sources, while plant-based foods typically represent a source of essential amino acids in the form of incomplete proteins.

[0066] The animal feed according to the invention is preferably feed for poultry, horses, camels, pigs, cattle (such as beef or dairy cattle), sheep, or companion animals (such as cats or dogs). Alternatively or additionally, the animal feed is preferably feed for aquatic species. When the animal feed is feed for aquatic species, the species is preferably selected from crustaceans or fish. Thus, the animal feed can be feed for crustaceans and / or fish. When the animal feed is feed for crustaceans, the crustaceans are preferably shrimp. Additionally or alternatively, when the animal feed is feed for fish, the fish may preferably be warm-water fish or cold-water fish. When the fish are warm-water fish, the fish are preferably selected from the group consisting of catfish, tilapia, sea bream, sea bass, and carp. Preferably, the fish are cold-water fish, wherein the fish are selected from cod, salmon, or rainbow trout. Particularly preferred is that the animal feed according to the invention is feed for shrimp, salmon, and / or rainbow trout.

[0067] It should be noted that, in the case of any definition given herein, the corresponding definitions of terms, phrases, and / or abbreviations apply in reverse throughout the specification. Furthermore, all definitions given herein are intended to cover all grammatical forms.

[0068] Further objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon viewing the following embodiments and accompanying drawings, which are not intended to be limiting. Therefore, it should be understood that although this disclosure is embodied by way of exemplary embodiments and optional features, modifications and variations thereof may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of this disclosure. Attached Figure Description

[0069] Figure 1 This diagram shows yeast single-cell protein products from different yeast strains grown with ethanol as the carbon source. The diagram shows the percentage (%) (w / w) of protein in the dry matter of the obtained SCP product for each yeast strain (x-axis) from a total of eight genera (y-axis).

[0070] Figure 2 The graph shows the percentage of protein in dry matter (%) (w / w) and the growth rate (h) of the modified strain of *G. jetting-Seberlindnerella vaginalis* AQFM-009, which was grown using ethanol as a raw material. -1 The relationship between ).

[0071] Figure 3 The right side shows the protein content (%DM) in the biomass of Jedinger Cyberlindnerella vaginalis CBS621 and Saccharomyces cerevisiae CEN.PK113-7D, which are produced from ethanol, compared to glucose.

[0072] Figure 3 The protein yields of fermentation using Jeddings cerebral filamentana FERM-BP1656 at different temperatures are shown.

[0073] Figure 4 This shows the protein content in biomass fermented using Jeddings cerebrindnerella vaginalis FERM-BP1656 at different pH levels. 。

[0074] Figure 5 The study shows the malate synthase content in proteins of *J. cerevisiae* FERM-BP1656 and *Saccharomyces cerevisiae* GHP1, which are derived from ethanol, compared to glucose.

[0075] Figure 6 The content of isocitrate lyase in proteins of *J. gerbinafine* FERM-BP1656 and *Saccharomyces cerevisiae* GHP1, which are derived from ethanol, is shown compared to glucose.

[0076] Figure 7 The figures show the percentage of protein in dry matter (w / w) and the growth rate (h) of the modified strains *Saccharomyces cerevisiae* AQFM-009, AQFM-035, AQFM-036, AQFM-037, and AQFM-038, produced from ethanol. -1 The relationship between ).

[0077] Figure 8 The figures show the percentage of protein in dry matter (w / w) and the growth rate (h) of the modified strains *Saccharomyces cerevisiae* AQFM-039, AQFM-041, AQFM-017, AQFM-042, and AQFM-043, which were produced using ethanol as a raw material. -1 The relationship between ).

[0078] Figure 9 The figures show the percentage of protein in dry matter (w / w) and the growth rate (h) of the modified strains *Saccharomyces cerevisiae* AQFM-044, AQFM-046, AQFM-048, AQFM-017, and AQFM-049, which were produced using ethanol as a raw material. -1 The relationship between ).

[0079] Figure 9 The following table shows the percentage of protein in dry matter (w / w) and growth rate (h) of the modified strains *Saccharomyces cerevisiae* AQFM-050, AQFM-051, AQFM-052, AQFM-053, AQFM-054, and AQFM-055, produced using ethanol as a raw material. -1 The relationship between ).

[0080] Example Example 1: Production of single-cell protein (SCP) from *Giardia lamblia* sacchariformis. Yeast strain J. Seberlindnerella vaginalis FERM-BP1656 was placed in a shake flask (100 ml) at 32°C and 280°C. Incubate at rpm for 24 h. The shake-flask medium is based on Verduyn (Verduyn et al., 1992), and its overview is shown in Table 1. From 10 ml of material was taken from the shake flask and inoculated into a 250 ml inoculum fermenter, yielding an initial weight of 100 g. The culture in this fermenter... The culture medium composition is described in Table 1. During fermentation, the pH was maintained at 5.0 by adding 10% (w / w) ammonia. The temperature was controlled at... 30℃. Air flow rate controlled at 0.25 nL / min. Dissolved oxygen concentration controlled at 20% by stirring. In the inoculum fermenter. After all glucose has been consumed, an ethanol feed rate of 0.615 g / h is started. This feed rate is then increased at 0.1 h / h. -1 of The index grows exponentially. After reaching a feed rate of 3.0 g / h, the feed rate is kept constant at this value. The feed rate is increased by 10%. (w / w) ethanol composition. The culture was run for 72 h, after which the biomass was harvested for inoculation of the main fermenter. At the end of the fermentation, A biomass concentration of 32.35 g dry weight / kg was obtained.

[0081] 0.6 g of dry matter, or approximately 19 ml of culture, was inoculated into a 250 ml master fermenter to obtain an initial weight of 150 g. The culture medium composition for this fermenter is described in Table 1. During fermentation, the pH was maintained at 5.0 by adding 10% (w / w) ammonia. The temperature was maintained at 30°C. The air flow rate was maintained at 0.25 nL / min. The dissolved oxygen concentration was maintained at 20% by stirring. The ethanol feed rate was started at 2.119 g / h after inoculation. This feed rate was subsequently increased at 0.2 h / h. -1 The index grows exponentially. After reaching a feed rate of 7.86 g / h, the feed rate is kept constant at this value. The feed consists of 10% (w / w) ethanol. The ethanol feed rate range can also be expressed as 0.035 g. S / g X / h to 0.450 g 乙醇 / g 生物质 / h of q S Range. The oxygen uptake rate (OUR) achieved at this feed rate was 190 mmol / kg / h. After 48 h of culture, a biomass concentration of 38.46 g dry weight / kg was obtained, with a total hydrolyzed amino acid concentration (excluding cysteine ​​and tryptophan) of g / g. 生物质 Furthermore, the protein content (N*6.25) determined by the Kjeldahl method was 57.4%. During fermentation, samples were collected at t = 6 h, t = 23 h, t = 30 h, and t = 48 h, and the dry matter content, Kjeldahl protein content, RNA content, amino acid content, polysaccharide content, and residual ethanol concentration of the samples were subsequently analyzed.

[0082] Table 1. Composition of culture media in pre-culture shake flasks, inoculum fermenters, and main fermenters.

[0083] Description of proteomics methods: Prior to proteomics analysis, the biomass concentration of the samples was normalized based on DM measurements, followed by lysis by adding lysis buffer (PreOmics) and incubating at 95°C for 20 min. Cell lysates were further processed by reduction, alkylation, and digestion with trypsin. Samples were technically reproducible using liquid chromatography-tandem mass spectrometry (LC-MS / MS) via a Vanquish UHPLC system connected to an Orbitrap Exploris480 MS (Thermo Fisher Scientific). Peptides were separated by reversed-phase chromatography on an ACQUITY UPLC CSH C18 column (Waters), 130 Å, 1.7 µm, 2.1 mm × 100 mm analytical column, from 5% B to 40% B over 20 min, using a gradient of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Data-independent acquisition (DIA) was performed with a full MS scan resolution of 60,000, ranging from 350 to 1,200 m / z, followed by high-energy collision-induced dissociation activation (HCD) MS / MS with an isolation width of 17 m / z, covering 400 to 1000 m / z, and a resolution of 15,000. Proteins from the *C. jadinii* or *S. cerevisiae* databases were analyzed using Spectronaut (Biognosys version 17). Label-free quantification was performed using the top three peptides measured for each protein. Retention time calibration was performed based on nonlinear regression, and normalization was set to the total peptide amount.

[0084] When feeding ethanol feedstock, high levels of enzymes such as isocitrate lyase and malate synthase (see [reference needed]) can be found in the biomass at levels exceeding 0.1%. Figure 5 and Figure 6 ).

[0085] Example 2: Strain strain improvement; how to generate improved strains, such as J.C. Seberlindnerella vaginalis AQFM-009 Classic strains were improved from the *Saccharomyces cerevisiae* strain FERM-BP 1656 from EP 0299078 A1 and *Saccharomyces cerevisiae* strain CBS 841 from the Westerdijk Fungal Biodiversity Institute, The Netherlands.

[0086] The strain was grown overnight in 100 ml of YEPhD containing 10 g / L yeast extract + 20 g / L BBL Phytone peptone, 20 g / L glucose, and 1% penicillin-streptomycin solution (L0022-100, Biowest) and incubated at 30°C and 280 rpm until 1.2 > 3.0 OD. 600 Cells were washed twice and diluted to 1.0 OD in 0.05 M phosphate buffer, pH 6.8. 600 For each mutagenic condition, 4 ml of 1x10⁻⁶ solution was used. 7 Transfer cells / ml to a 100 ml shake flask.

[0087] 4-Nitroquinoline 1-oxide (NQO) and MilliQ water were added to a total volume of 5 ml, with final NQO concentrations of 0 µg / ml, 0.1 µg / ml, 0.2 µg / ml, 0.4 µg / ml, 0.6 µg / ml, 0.8 µg / ml, and 1.0 µg / ml. In parallel, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and MilliQ water were added to a total volume of 5 ml, with final NTG concentrations of 0 µg / ml, 10 µg / ml, 20 µg / ml, 30 µg / ml, 40 µg / ml, 50 µg / ml, 60 µg / ml, and 70 µg / ml. These mixtures were incubated at 25°C for 30 min with gentle agitation in a water bath. Mutagenesis was terminated by adding 1 ml of 50% (w / v) sodium thiosulfate solution and incubating for 5 min. The mutated cells were washed twice with YEPhD medium by centrifugation at 4000 g for 10 min and removal of the supernatant. The cells were recovered in 10 ml of YEPhD and incubated at 25 °C and 100 rpm for one hour. The mutated cells were aliquoted into 0.5 ml portions, mixed with 0.5 ml of 20% glycerol, and stored at -80 °C. Finally, the dilutions of the mutant batches were plated onto YEPhD agar plates to determine viability.

[0088] The mutants were inoculated onto YEPhD 2% agar plates and incubated for four days. Single colonies were picked and grown in 96-well plates (Thermo Scientific, Denmark) containing 150 µl / well of YEPhD agar and incubated at 30°C for 3 days. The mutants were then transferred from the 96-well YEPhD agar plates to 96-well plates (Thermo Scientific, Denmark) containing 150 µl of YEPhD and covered with a breathable sealing film (Greiner bio-one, Frickenhausen, Germany) using a disposable QREP 96 replicator (Molecular Devices, USA). After incubating overnight in a Microton shaking incubator (Infors AG, Bottmingen, Switzerland) at (30°C, 750 rpm, 550 rpm, 80% humidity), 10 µl of the culture was transferred to a semi-deep well plate (HDWP, 96 wells, 1.2 ml) containing 400 µl of SMD-urea medium (Luttik MA, J Bacteriol 2000; 182:7007–7013) and 2% (w / w) ethanol (instead of glucose) and incubated for 24 hours (30°C, 750 rpm, 80% humidity).

[0089] Samples were taken from each well to measure cell density and protein concentration. A 20 µl sample was diluted (1:10) with physiological saline (0.85% NaCl) to a final volume of 200 µl, and cell density was measured at 600 nm in a 96-well flat-bottom NUNC A / S (Thermo Scientific). A second sample (66 µl) was mixed with 134 µl of Y-PER (Yeast Protein Extraction Reagent, 78991, Thermo Scientific), sealed with an aluminum seal, and mixed for 30 min in a Microton shaking incubator (750 rpm, 30°C, 80% RH). After incubation, the plate was centrifuged (2750 rcf) for 5 min. Subsequently, 100 µl of the supernatant was transferred to a black polystyrene 96-well microplate (catalog number 655090, Greiner-bio-one). Finally, using 1.5 g / L BSA solution as a standard, protein (tryptophan) content was measured at wavelengths of 295–350 nm (excitation-emission). The protein-to-cell density ratio was calculated to select mutants with increased protein content.

[0090] Subsequently, the strains selected through the initial screening were cultured in shake flasks. Pre-cultures were generated in 24-well deep-well MTP plates (Axygen deep-well plates) containing 3 ml of YEPhD in each well. The pre-cultures were incubated for 3 days in an INFORS shaker set at 30°C, 550 rpm, and 80% humidity and sealed with a breathable sealing film (Greiner Bio-One, 676051). After incubation, 30 µl of sample material was transferred to 400 µl of fresh SMD-urea medium with 2% w / w ethanol in a 24-well deep-well MTP plate for a second round of pre-culture (3 ml, 30°C, 550 rpm, and 80% humidity) and incubated for 24 hours. 1 ml of culture was transferred from this second culture to a 500 ml shake flask containing 100 ml of SMD-urea medium with 2% w / w ethanol and incubated at 30°C and 250 rpm for 40 hours.

[0091] Aliquot 100 ml of culture into two 50 ml Greiner tubes and centrifuge at 5°C, 5,000 g for 5 minutes. Wash the cell pellet with 25 ml MilliQ (8°C) and combine it into a pre-weighed 50 ml Greiner tube. After washing, centrifuge the tube again (5°C, 5,000 g for 5 minutes). Finally, discard the supernatant and weigh the tube (wet weight). Store the cell pellet at -20°C for determining dry weight and amino acid content. The dry weight of the strain was determined by measuring the biomass weight after lyophilizing the intact sample (100 ml).

[0092] The amino acid was determined using the following method: DBC-SLD-A-02773_AMINO ACIDS AFTER HYDROLYSISUSING ACCQ-TAG METHOD (UPLC) Example 3: SCP Product Generation Single-cell protein products were generated using yeast cells from different genera and orders of yeasts, with ethanol as the carbon source. More specifically, *Saccharomyces cerevisiae*, *Kluyveromyces*, *Wickham's yeast*, *Yersinia*, *Saccharomyces*, *Pichia*, and *Ogata* were studied. Ogateae ) and Botrytis cinerea ( Blastobotrys The strain of SCP. The percentage (%) of protein (g) as dry matter (w / w) of the SCP product obtained in the examples. Figure 1 The text specifies SCP products obtained using yeast cells from the following yeast orders: Yeastra lipolytica, Kluyveromyces martensii (…). Kluyveromyces marxianus ), Botrytis cinerea ( Blastobotrys adeninivorans ), Polymorphic Osaka yeast ( Ogateae polymorpha), abnormal Pichia pastoris, Pichia pastoris ( Pichia pastoris The yeast cells were *Saccharomyces cerevisiae*, *Wickhamia spp.*, *J. seldombekia*, and *Kluyveromyces lactis*. As shown, given the observed (up to) 41% (w / w) or more of protein (g) per unit dry matter (g) (given as a percentage of dry matter (%)), the relatively best-performing yeast cells were those from the order *Wickhamia spp.*, *Kluyveromyces lactis*, and *J. seldombekia*.

[0093] *** This invention can also be summarized as follows: 1. A method for culturing microorganisms capable of producing at least 60% protein, preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein, comprising the following steps: (i) Supplying microorganisms to the reactor (ii) Ethanol as feedstock (iii) Controlling the feed rate (iv) Temperature control (v) Control pH (vi) Control the growth rate.

[0094] 2. The method according to claim 1, wherein the growth rate is at least 0.02 h. -1 Preferably at least 0.025 h -1 More preferably at least 0.03 h -1 More preferably at least 0.04 h -1 More preferably at least 0.05 h -1 More preferably at least 0.06 h -1 More preferably at least 0.07 h -1 More preferably at least 0.08 h -1 More preferably at least 0.09 h -1 More preferably at least 0.1 h -1 More preferably at least 0.15 h -1 More preferably at least 0.16 h -1 More preferably at least 0.17 h -1 More preferably at least 0.18 h -1 More preferably at least 0.19 h -1 More preferably at least 0.2 h -1 More preferably at least 0.21 h -1 The most preferred option is at least 0.22 h. -1 .

[0095] 3. The method according to claim 1 or 2, wherein the feed rate is 0.179 - 0.536 g. 乙醇 / g 生物质 / h, more preferably 0.179 - 0.469 g 乙醇 / g 生物质 / h.

[0096] 4. The method according to any one of claims 1 to 3, wherein the temperature is maintained between 30°C and 40°C, preferably between 30°C and 38°C, more preferably between 30°C and 36°C, and most preferably between 30°C and 34°C.

[0097] 5. The method according to any one of claims 1 to 4, wherein the pH is maintained between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.

[0098] 6. The method according to any one of claims 1 to 5, further comprising the step of recovering the biomass.

[0099] 7. The method according to any one of claims 1 to 6, further comprising the step of drying the biomass.

[0100] 8. The method according to any one of claims 1 to 7, wherein the microorganism is yeast.

[0101] 9. The method according to any one of claims 1 to 8, wherein the yeast is a yeast from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, *Wickham's yeast*, *Pichia pastoris*, or *Yersinia*, preferably from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, or *Wickham's yeast*.

[0102] 10. The method according to any one of claims 1 to 9, wherein the yeast is a yeast derived from *Gerdinium sacchariformis*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Wickhamia sacchariformis*, *Pichia pastoris*, or *Yersinia lipolytica*, preferably from *Gerdinium sacchariformis*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Wickhamia sacchariformis*.

[0103] 11. The method according to any one of claims 1 to 9, wherein the yeast is derived from *Saccharomyces cerevisiae* ATCC 26387, *Saccharomyces cerevisiae* FERM-BP1656, or *Saccharomyces cerevisiae* CBS621. 、J.D. Lindnerella vaginalis CBS841, J.D. Lindnerella vaginalis AQFM-009, J.D. Lindnerella vaginalis AQFM-035, J.D. Lindnerella vaginalis AQFM-036, J.D. Lindnerella vaginalis AQFM-037, J.D. Lindnerella vaginalis AQFM-038, J.D. Lindnerella vaginalis AQFM-039, J.D. Lindnerella vaginalis AQFM-041, J.D. Lindnerella vaginalis AQFM-017, J.D. Lindnerella vaginalis AQFM-042, J.D. Lindnerella vaginalis AQFM-043, J.D. Lindnerella vaginalis AQFM-043, J.D. Lindnerella vaginalis AQFM-0441, J.D. Lindnerella vaginalis AQFM-017, J.D. Lindnerella vaginalis AQFM-042, J.D. Lindnerella vaginalis AQFM-043, J.D. Lindnerella vaginalis AQFM-0441, J.D. Lindnerella vaginalis AQFM-0441, J.D. Lindnerella vaginalis AQFM-0442, J.D. Lindnerella vaginalis AQFM-0443 ...2, J.D. Lindnerella vaginalis AQFM-0443, J.D. Lindnerella vaginalis AQFM-0 Saccharomyces bolindensis AQFM-044, Saccharomyces cerevisiae AQFM-046, Saccharomyces cerevisiae AQFM-048, Saccharomyces cerevisiae AQFM-017, Saccharomyces cerevisiae AQFM-049, Saccharomyces cerevisiae AQFM-050, Saccharomyces cerevisiae AQFM-051, Saccharomyces cerevisiae AQFM-052, Saccharomyces cerevisiae AQFM-053, Saccharomyces cerevisiae AQFM-054, Saccharomyces cerevisiae AQFM-055, Saccharomyces cerevisiae (Bacillus spp.) GHP1 Saccharomyces cerevisiae CEN.PK113-7D, Saccharomyces cerevisiae IFO 569, Saccharomyces cerevisiae CBS 1980, J.J. Seberlindnerella vaginalis ATCC 9950, Kluyveromyces lactis CBS 2896, Saccharomyces cerevisiae CBS 2576, or Yersinia lipolytica CBS 7504, preferably from J.J. Seberlindnerella vaginalis ATCC 26387, J.J. Seberlindnerella vaginalis FERM-BP1656, or J.J. Seberlindnerella vaginalis CBS621. 、J.D. Lindnerella vaginalis CBS841, J.D. Lindnerella vaginalis AQFM-009, J.D. Lindnerella vaginalis AQFM-035, J.D. Lindnerella vaginalis AQFM-036, J.D. Lindnerella vaginalis AQFM-037, J.D. Lindnerella vaginalis AQFM-038, J.D. Lindnerella vaginalis AQFM-039, J.D. Lindnerella vaginalis AQFM-041, J.D. Lindnerella vaginalis AQFM-017, J.D. Lindnerella vaginalis AQFM-042, J.D. Lindnerella vaginalis AQFM-043, J.D. Lindnerella vaginalis... *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* Lindt-046, *Saccharomyces cerevisiae* Lindt-048, *Saccharomyces cerevisiae* Lindt-017, *Saccharomyces cerevisiae* Lindt-049, *Saccharomyces cerevisiae* Lindt-050, *Saccharomyces cerevisiae* Lindt-051, *Saccharomyces cerevisiae* Lindt-052, *Saccharomyces cerevisiae* Lindt-053, *Saccharomyces cerevisiae* Lindt-054, *Saccharomyces cerevisiae* Lindt-055, *Saccharomyces cerevisiae* IFO 569, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Saccharomyces cerevisiae CBS 1980, Saccharomyces cerevisiae ATCC 9950 or Kluyveromyces lactis CBS 2896.

[0104] 12. A yeast single-cell protein product, wherein the yeast single-cell protein product comprises at least 60% protein by weight of stem cells, preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / or malate synthase.

[0105] 13. The yeast single-cell protein product according to claim 12, wherein the yeast is yeast from the order Saccharidae.

[0106] 14. The yeast single-cell protein product according to claim 13, wherein the yeast is a yeast from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, *Wickham's yeast*, *Pichia pastoris*, or *Yersinia*, preferably from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, or *Wickham's yeast*.

[0107] 15. An animal feed comprising up to 20% (w / w) of a yeast single-cell protein product as claimed in any of the preceding claims.

[0108] *** Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the invention.

[0109] Unless otherwise stated, the following terms used in this document, including the specification and claims, have the definitions given below.

[0110] It should be noted that, unless the context clearly indicates otherwise, the singular forms “an,” “a,” and “described” as used herein include multiple indicators. Thus, for example, a reference to “reagent” includes one or more such different reagents, and a reference to “the method” includes a reference to equivalent steps and methods known to those skilled in the art, which may modify or replace the methods described herein.

[0111] Unless otherwise stated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine many equivalents of the particular embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered by the invention.

[0112] The term “and / or” as used anywhere in this document includes the meaning of “and,” “or,” and “all or any other combination of elements connected by the term.”

[0113] As used herein, the term “about” or “approximately” means within 20% of a given value or range, preferably within 10%, and more preferably within 5%. However, it also includes specific numbers, for example, about 20 includes 20.

[0114] Throughout this specification and the following claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” and “comprising” shall be understood to imply inclusion of the stated integers or steps or groups of integers or steps, but not to exclude any other integers or steps or groups of integers or steps. When used herein, the term “comprising” may be replaced by the terms “containing” or “including,” or sometimes by the term “having.”

[0115] When used herein, "consisting of" excludes any element, step, or component not specified in the elements of the claim. When used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the essential and novel features of the claim.

[0116] In all cases in this article, any one of the terms “comprising,” “substantially consisting of,” and “consisting of” may be replaced by any one of the other two terms.

[0117] It should be understood that the present invention is not limited to the specific methods, schemes, materials, reagents, and substances described herein and therefore may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be defined solely by the claims.

[0118] Other embodiments are defined within the scope of the following claims. Furthermore, in the context of describing features or aspects of the invention according to the Markush Group, those skilled in the art will recognize that the invention is therefore also described according to any single member or subgroup of the Markush Group.

[0119] All publications (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.) referenced throughout this specification are hereby incorporated in their entirety by reference. Nothing herein shall be construed as an admission that the present invention is not entitled to such disclosure due to prior art. In the event of any conflict or inconsistency between the referenced material and this specification, this specification shall prevail over any such material.

Claims

1. A method for culturing microorganisms capable of producing at least 60% protein, preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein, comprising the following steps: (i) Supplying microorganisms to the reactor (ii) Ethanol as feedstock (iii) Controlling the feed rate (iv) Temperature control (v) Control pH (vi) Control the growth rate.

2. The method according to claim 1, wherein the growth rate is at least 0.02 h. -1 Preferably at least 0.025h -1 More preferably at least 0.03 h -1 More preferably at least 0.04 h -1 More preferably at least 0.05 h -1 More preferably at least 0.06 h -1 More preferably at least 0.07 h -1 More preferably at least 0.08 h -1 More preferably at least 0.09 h -1 More preferably at least 0.1 h -1 More preferably at least 0.15 h -1 More preferably at least 0.16 h -1 More preferably at least 0.17 h -1 More preferably at least 0.18 h -1 More preferably at least 0.19 h -1 More preferably at least 0.2 h -1 More preferably at least 0.21 h -1 The most preferred option is at least 0.22 h. -1 .

3. The method according to claim 1 or 2, wherein the feed rate is 0.179 - 0.536 g. 乙醇 / g 生物质 / h, more preferably 0.179 - 0.469 g 乙醇 / g 生物质 / h.

4. The method according to any one of claims 1 to 3, wherein the temperature is maintained between 30°C and 40°C, preferably between 30°C and 38°C, more preferably between 30°C and 36°C, and most preferably between 30°C and 34°C.

5. The method according to any one of claims 1 to 4, wherein the pH is maintained between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.

5.

6. The method according to any one of claims 1 to 5, further comprising the step of recovering the biomass.

7. The method according to any one of claims 1 to 6, further comprising the step of drying the biomass.

8. The method according to any one of claims 1 to 7, wherein the microorganism is yeast.

9. The method according to any one of claims 1 to 8, wherein the yeast is a yeast from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, *Wickham's yeast*, *Pichia pastoris*, or *Yersinia*, preferably from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, or *Wickham's yeast*.

10. The method according to any one of claims 1 to 9, wherein the yeast is a yeast derived from *Gerdinium sacchariformis*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Wickhamia sacchariformis*, *Pichia pastoris*, or *Yersinia lipolytica*, preferably from *Gerdinium sacchariformis*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Wickhamia sacchariformis*.

11. The method according to any one of claims 1 to 9, wherein the yeast is derived from *Saccharomyces cerevisiae* ATCC 26387, *Saccharomyces cerevisiae* FERM-BP1656, *Saccharomyces cerevisiae* CBS621, *Saccharomyces cerevisiae* CBS841, *Saccharomyces cerevisiae* AQFM-009, *Saccharomyces cerevisiae* AQFM-035, *Saccharomyces cerevisiae* AQFM-036, *Saccharomyces cerevisiae* AQFM-037, *Saccharomyces cerevisiae* AQFM-038, *Saccharomyces cerevisiae* AQFM-039, *Saccharomyces cerevisiae* AQFM-041, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-042, etc. *Saccharomyces cerevisiae* AQFM-043, *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* IFO 569, *Saccharomyces cerevisiae* CBS 1980, *Yersinia sebifera* ATCC 9950, *Kluyveromyces lactis* CBS 2896, *Saccharomyces cerevisiae* CBS 2576, or *Yersinia lipophila* CBS 7504, preferably from *Yersinia sebifera* ATCC. 26387, Jading Saccharomyces cerevisiae FERM-BP1656, Jading Saccharomyces cerevisiae CBS621, Jading Saccharomyces cerevisiae CBS841, Jading Saccharomyces cerevisiae AQFM-009, Jading Saccharomyces cerevisiae AQFM-035, Jading Saccharomyces cerevisiae AQFM-036, Jading Saccharomyces cerevisiae AQFM-037, Jading Saccharomyces cerevisiae AQFM-038, Jading Saccharomyces cerevisiae AQFM-039, Jading Saccharomyces cerevisiae AQFM-041, Jading Saccharomyces cerevisiae AQFM-017, Jading Saccharomyces cerevisiae AQFM-042, Jading Saccharomyces cerevisiae... *Saccharomyces bolindensis* AQFM-043, *Saccharomyces cerevisiae* AQFM-044, *Saccharomyces cerevisiae* AQFM-046, *Saccharomyces cerevisiae* AQFM-048, *Saccharomyces cerevisiae* AQFM-017, *Saccharomyces cerevisiae* AQFM-049, *Saccharomyces cerevisiae* AQFM-050, *Saccharomyces cerevisiae* AQFM-051, *Saccharomyces cerevisiae* AQFM-052, *Saccharomyces cerevisiae* AQFM-053, *Saccharomyces cerevisiae* AQFM-054, *Saccharomyces cerevisiae* AQFM-055, *Saccharomyces cerevisiae* IFO 569. Yeasts including *Saccharomyces cerevisiae* GHP1, *Saccharomyces cerevisiae* CEN.PK113-7D, *Saccharomyces cerevisiae* CBS 1980, *Saccharomyces cerevisiae* ATCC 9950, or *Kluyveromyces lactis* CBS 2896.

12. A yeast single-cell protein product, wherein the yeast single-cell protein product comprises at least 60% protein by weight of stem cells, preferably at least 65% protein, more preferably at least 70% protein, and most preferably at least 75% protein, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / or malate synthase.

13. The yeast single-cell protein product according to claim 12, wherein the yeast is yeast from the order Saccharidae.

14. The yeast single-cell protein product according to claim 13, wherein the yeast is a yeast from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, *Wickham's yeast*, *Pichia pastoris*, or *Yersinia*, preferably from the genera *Cerberlindnerella*, *Saccharomyces*, *Kluyveromyces*, or *Wickham's yeast*.

15. An animal feed comprising up to 20% (w / w) of a yeast single-cell protein product as claimed in any of the preceding claims.

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