Compositions made of agricultural waste for the production of microbial biomass for use in animal feeding

Abstract

A method of producing a carbon source useful for culturing microorganisms is provided as well as sterile compositions comprising a reducing sugar extract of agricultural waste. In addition, there is provided a method of producing a biomass of a microorganism of interest using the sterile composition. The microbial preparation may be comprised in an animal feed to be used in a method of increasing body weight of an animal, especially when the animal is of an aquatic species.

Description

[0001] COMPOSITIONS MADE OF AGRICULTURAL WASTE FOR THE PRODUCTION OF MICROBIAL BIOMASS FOR USE IN ANIMAL FEEDING

[0002] RELATED APPLICATIONS:

[0003] This application claims the benefit of priority from U. S. Provisional Patent Application No.

[0004] 63 / 713,644 filed on October 30, 2024, which is hereby incorporated in its entirety.

[0005] SEQUENCE LISTING STATEMENT

[0006] The XML file, entitled 105308. xml, created on October 28, 2025, comprising 315,392 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0007] FIELD AND BACKGROUND OF THE INVENTION

[0008] The present invention, in some embodiments thereof, relates to compositions made of agricultural waste for the production of microbial biomass for use in animal feeding.

[0009] The world faces today two major challenges facing in the agricultural sector, these are demand for high quality animal feed and creation of sustainable processes for waste elimination. However, most popular compound of animal feed, fish meal, that has balanced ratio of essential amino acids is not sustainable product. Fish meal production is limited by overfishing, which depletes fish stocks; environmental concerns related to habitat destruction; high energy and resource consumption; fluctuating market prices; and the challenge of managing waste and byproducts. Additionally, there are regulatory hurdles and sustainability pressures that can complicate operations.

[0010] These disadvantages are fueling the search for cheaper and sustainable alternative essential amino acid sources such as the use of dried bacteria, yeast, algae and insect cells.

[0011] One of the most promising alternative amino acid sources is the use of single cell organisms, such as bacteria, yeast and algae to produce amino acid rich substitutes (Koukoumaki et al., 2024). While the use of single cell factories for production of nutritional compounds has many advantages, the creation of scalable, efficient and cost-effective industrial processes still faces many challenges.

[0012] Yeast has several advantages, including fast generation times, efficient nutrient utilization, a balanced amino acid composition and the presence of health promoting B-complex vitamins and polysaccharides (Agboola et al., 2021). Yeasts are suitable for use in animal feed and were shown to confer additional health benefits when fed to various fish species (Cherdthong et al., 2019). Currently, large scale production of yeast nutritional biomass utilizes a small number of species, namely Saccharomyces cerevisiae and Cyberlindnera jadinii (also known as Candida utilis) (Ritala et al., 2017). While these species have been used in the food industry for many decades, they do not necessarily represent the most efficient and cost-effective way to produce yeast biomass rich in essential amino acid. Many studies have explored the potential of producing yeast biomass, emphasizing that amino acid quantity and quality are highly dependent on species and substrate identity, fermentation parameters and downstream processing (Koukoumaki et al., 2024).

[0013] P. kudriavzevii is a prevalent yeast known for its involvement in traditional fermented foods, including wine and other alcoholic beverages, cheese, fermented cereals and coffee and cocoa bean processing. Interestingly, the aromatic profiles of the fermented products are improved by the presence of P. kudriavzevii which contributes to the formation of various flavor enhancing compounds (Chu et al., 2023). P. kudriavzevii has also been explored as a prebiotic due to its antimicrobial activity (Chelliah et al., 2016) as well as its ability to degrade mycotoxins (Intanoo et al., 2018). P. kudriavzevii is innately stress resistant with the ability to grow quickly under acidic, high salt and high temperature conditions (Pongcharoen, 2022). Thermotolerance is particularly beneficial in industrial fermentations as it greatly reduces cooling costs and allows for simultaneous enzyme hydrolysis and cell growth (Choudhary et al., 2016). While a small number of previous studies have explored the use of P. kudriavzevii for amino acid rich biomass production (Boundy-Mills K, et al., 2018), biomass yield was eventually quite low (<10 g / L dry weigh).

[0014] One of the major tenets of a circular economy is creating value using materials that would otherwise be discarded. A significant advantage of microbial biomass production is the ability to use agricultural and industrial waste as substrates for microbe growth (Spalvins K. et al., 2018), reducing production costs and increasing environmental sustainability. Carbon sources for microbial biomass production have been a focus of intense research and consist of two main alternatives, the use of hydrocarbons found in crude oil and natural gas or the use of agricultural waste (Goldberg, 1985). Agricultural waste can be further grouped based on its composition into waste rich in simple sugars (mono and disaccharides), starch, structural polysaccharides or amino acids / lipids (Spalvins K. et al., 2018). Wastes rich in simple sugars (fruit waste) are ideal as they require little pretreatment and can be used directly in fermentation (Sekoai et al., 2024).

[0015] In addition to carbon, yeast requires sources of nitrogen, additional trace elements (such as phosphate) and essential vitamins to sustain growth. The nitrogen needed for cell growth and amino acid production can also be supplied from agricultural waste as opposed to directly from inorganic chemicals. One of the most promising nitrogen sources is corn steep liquor (CSL), which routinely exhibits comparable results to more expensive substrates, such as yeast extract (Wu et al., 2018). Another common waste used in microbial biomass production is milk whey, a by-product of the dairy industry that is generated globally in staggering amounts (Spalvins K. et al., 2018). Whey is typically considered as a carbon source due to the presence of lactose sugar, however whey also contains other essential nutrients such as calcium, phosphate and various vitamins important for growth (Zandona et al., 2021).

[0016] To date, the promise of using yeast biomass as an amino acid source for animal feed has been hindered by the difficulty of creating an efficient, scalable and cost-effective process.

[0017] Additional background art includes: https: / / www(dot)fda(dot)gov / media / 148294 / download;

[0018] WO2021158927 mentions the inclusion of viable P. kudriavzevii among other yeast, as a probiotic component, in animal feed especially of ruminants;

[0019] CN 110769687 mentions the inclusion of P. kudriavzevii among other yeast, as killer yeast.

[0020] SUMMARY OF THE INVENTION

[0021] According to an aspect of some embodiments of the present invention there is provided a method of producing a carbon source useful for culturing microorganisms, the method comprising:

[0022] (a) providing agricultural waste;

[0023] (b) extracting the agricultural waste so as to obtain at least one extract which comprises reducing sugars of the agricultural waste;

[0024] (c) sterilizing the at least one extract so as to obtain a sterile extract or extracts.

[0025] According to some embodiments of the invention, the method further comprises determining a level of the reducing sugars in the extract and / or the sterile extract.

[0026] According to some embodiments of the invention, the sterilizing is by heating.

[0027] According to some embodiments of the invention, the agricultural waste comprises a level of reducing sugars above a predetermined threshold prior to the extracting.

[0028] According to some embodiments of the invention, the agricultural waste is selected from the group consisting of fruit waste, molasses, fruit pomace, sugarcane bagasse juice, whey, ripe banana peels, vegetable waste, sweet potato residues and juice pressings or residues from sugar-rich crops, and sugar beet pulp.

[0029] According to some embodiments of the invention, the fruit waste comprises dates, optionally wherein the dates are overripe or damaged.

[0030] According to some embodiments of the invention, the fruit waste comprises dates with pits. According to some embodiments of the invention, the fruit waste comprises dates without pits. According to some embodiments of the invention, the fruit waste comprises dates with and without pits.

[0031] According to some embodiments of the invention, the agricultural waste comprises a level of reducing sugars below a predetermined threshold prior to the extracting.

[0032] According to some embodiments of the invention, the method comprises treating the agricultural waste to breakdown cellulose into reducing sugars.

[0033] According to some embodiments of the invention, the treating comprises enzymatic hydrolysis, acid hydrolysis or a combination thereof.

[0034] According to some embodiments of the invention, the agricultural waste is primarily lignocellulosic.

[0035] According to some embodiments of the invention, the agricultural waste is selected from the group consisting of straw, corn stover, sugarcane bagasse, wood chips, rice husks, wheat bran, cottonseed hulls, peanut shells, coconut husks, coffee pulp, coffee husks, and palm kernel cake.

[0036] According to some embodiments of the invention, the extracting is by water extraction. According to some embodiments of the invention, the water extraction is by boiling the agricultural waste in water and centrifuging to collect a supernatant which comprises the reducing sugars, the supernatant being a first extract, optionally wherein the boiling and centrifuging is repeated 2-5 times, such that extracts resultant of the repeating comprise decreasing amounts of reducing sugars.

[0037] According to an aspect of some embodiments of the present invention there is provided a sterile composition comprising a reducing sugar extract of agricultural waste, optionally comprising salt, nitrogen source, micro elements, macro elements and / or an anti-foaming agent.

[0038] According to some embodiments of the invention, the nitrogen source is corn steep liquor. According to some embodiments of the invention, the microelements are selected from the group consisting of magnesium, iron and phosphorous.

[0039] According to some embodiments of the invention, the salt is selected from the group consisting of magnesium sulfate, sodium chloride, ammonium sulfate, monopotassium phosphate and calcium chloride.

[0040] According to some embodiments of the invention, the salt is selected from the group consisting of magnesium sulfate and sodium chloride.

[0041] According to some embodiments of the invention, the agricultural waste is selected from the group consisting of fruit waste, molasses, fruit pomace, sugarcane bagasse juice, whey, ripe banana peels, vegetable waste, sweet potato residues and juice pressings or residues from sugar-rich crops, and sugar beet pulp. According to some embodiments of the invention, the fruit waste comprises dates, optionally wherein the dates are overripe or damaged.

[0042] According to some embodiments of the invention, the agricultural waste is selected from the group consisting of straw, corn stover, sugarcane bagasse, wood chips, rice husks, wheat bran, cottonseed hulls, peanut shells, coconut husks, coffee pulp, coffee husks, and palm kernel cake.

[0043] According to some embodiments of the invention, the composition as described herein is obtainable according to the method described herein.

[0044] According to some embodiments of the invention, a concentration of reducing sugars in the extract is 2 % to 20 %.

[0045] According to some embodiments of the invention, a concentration of reducing sugars in the extract is 2 % to 15 %.

[0046] According to some embodiments of the invention, a concentration of reducing sugars in the extract is 5 % to 8 %.

[0047] According to some embodiments of the invention, a concentration of reducing sugars in the extract is 10 % to 18 %.

[0048] According to an aspect of some embodiments of the present invention there is provided a method of producing a biomass of a microorganism of interest, the method comprising culturing the microorganism under conditions which allow expansion of the microorganism and wherein the conditions comprise the composition described herein as a carbon source.

[0049] According to some embodiments of the invention, the composition is the sole carbon source in the method.

[0050] According to some embodiments of the invention, the conditions comprise reducing sugars concentration of 0.5-2.5 %.

[0051] According to some embodiments of the invention, the conditions comprise about 10 % dissolved oxygen.

[0052] According to some embodiments of the invention, the conditions comprise pH of about 4-6, e.g., 5.

[0053] According to some embodiments of the invention, the conditions comprise batch fermentation, fed fermentation and / or continuous fermentation.

[0054] According to some embodiments of the invention, the method further comprises harvesting the microorganism.

[0055] According to some embodiments of the invention, the conditions ensure culturing of no more than 3 species of microorganisms. According to some embodiments of the invention, the method further comprises monitoring during the culturing at least one of: reducing sugar concentration, amino nitrogen concentration, pH and oxygen level.

[0056] According to an aspect of some embodiments of the present invention there is provided a microbial preparation obtainable according to the method as described herein.

[0057] According to some embodiments of the invention, the microorganism is viable.

[0058] According to some embodiments of the invention, the microorganism is non- viable.

[0059] According to some embodiments of the invention, the microorganism is intact.

[0060] According to some embodiments of the invention, the microbial preparation is dry.

[0061] According to some embodiments of the invention, the microbial preparation is liquid. According to some embodiments of the invention, the microorganism is selected from the group consisting of yeast, fungi, bacteria and algae.

[0062] According to some embodiments of the invention, the yeast is of the genus Pichia.

[0063] According to some embodiments of the invention, the yeast is of the species Pichia kudriavzevii.

[0064] According to some embodiments of the invention, the yeast is of the strain PY1 P. kudriavzevii comprising a genomic signature of SEQ ID Nos. 1-8.

[0065] According to some embodiments of the invention, the yeast has been deposited under the Budapest Treaty in the ATCC® Patent Depository under PTA- 127830.

[0066] According to some embodiments of the invention, the microbial culture comprises all essential amino acids (e.g., amino acid profile as in Table 1), lipids and vitamins.

[0067] According to an aspect of some embodiments of the present invention there is provided an animal feed comprising the microbial preparation as described herein.

[0068] According to some embodiments of the invention, the feed is for culturing an aquatic species.

[0069] According to some embodiments of the invention, the aquatic species is selected from the group consisting of crustaceans and fish.

[0070] According to some embodiments of the invention, the fish is carnivorous.

[0071] According to some embodiments of the invention, the carnivorous fish comprises Lates calcarifer.

[0072] According to some embodiments of the invention, individuals of the fish are at a synchronized developmental stage.

[0073] According to some embodiments of the invention, the the synchronized developmental stage is fingerling. According to some embodiments of the invention, a concentration of the microbial preparation in the animal feed is 2-40 %, e.g., 20-40 % w / w.

[0074] According to some embodiments of the invention, the feed is in a formulation selected from the group consisting of pellet, granule, powder, crumble, block, liquid feed, and meal.

[0075] According to an aspect of some embodiments of the present invention there is provided the feed further comprises at least one of an energy source, protein source, fat and / or oil, vitamin, mineral, fiber and a non-nutritional additive (e.g., probiotics, enzymes, antioxidants, preservatives, coloring or flavoring agents) in addition to the microbial preparation.

[0076] According to an aspect of some embodiments of the present invention there is provided the protein source comprises fishmeal.

[0077] According to an aspect of some embodiments of the present invention there is provided a weight ratio between the microbial preparation and the fishmeal is at least 1:9.

[0078] According to an aspect of some embodiments of the present invention there is provided a microbial preparation comprising a yeast strain as deposited under the Budapest Treaty in the ATCC® Patent Depository under PTA-127830.

[0079] According to an aspect of some embodiments of the present invention there is provided a method of increasing body weight of an animal comprising feeding the animal with an effective amount of the feed as described herein.

[0080] According to an aspect of some embodiments of the present invention there is provided a method of growing an animal species, comprising feeding the animal with an effective amount of the feed as described herein.

[0081] According to an aspect of some embodiments of the present invention there is provided a method of supporting survival of an animal comprising feeding the animal with an effective amount of the feed as described herein.

[0082] According to an aspect of some embodiments of the present invention there is provided the animal is an aquatic species.

[0083] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0084] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0085] In the drawings:

[0086] FIG. 1 shows biomass accumulation during fed batch cultivation of Pichia kudriavzevii PY1 strain on yeast extract and corn steep liquor.

[0087] FIG. 2 is a bar graph showing an average percentage of weight gain for the control and 10%, 20%, 30%, 40%, 50% replacement of fish meal in feed formulation. Significant difference between groups and control is marked with an asterix.

[0088] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0089] The present invention, in some embodiments thereof, relates to compositions made of agricultural waste for the production of microbial biomass for use in animal feeding.

[0090] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0091] There is a growing need for an alternative animal feed with high level and balanced amino composition in biomass to supplement animal dietary demands. One of the most promising alternative amino acid sources is yeast biomass.

[0092] Whilst reducing embodiments of the invention to practice and, the present inventors were successful in producing large-scale, industrial biomass of a particular yeast strain by growing on a combination of agricultural wastes.

[0093] As is illustrated hereinbelow and in the Examples section which follows, the present inventors developed an efficient process to produce yeast biomass that can be used as animal feed. The present inventors found that P. kudriavzevii PY1, is a highly advantageous yeast strain for industrial fermentation. Efficient growth of this strain provides a solution for the elimination of agricultural waste. The fermentation process allows the accumulation of high dry weight content over time, thereby increasing productivity. By combining the benefits of P. kudriavzevii strain, growth media composition and fermentation process, the present inventors have created a scalable, sustainable and cost-effective solution to alternative biomass with high amino acid content production that can be harnessed towards the fermentation of other microbial species.

[0094] As shown, the process uses wasted date fruit as the main carbon source. Dates are produced in large amounts and a significant percentage are rejected for human consumption because of low quality or damage. Wasted dates have high sugar content (~66 %) which mainly consists of the monosaccharides glucose and fructose, making them an excellent substrate for yeast growth. An adaptive process that depends on quantifying and maintaining reducing sugar concentrations during fermentation was developed, allowing utilization of a wide range of agricultural waste and by-products containing simple sugars can be used to efficiently obtain amino acid rich biomass.

[0095] In addition to the general advantages of the embodiments described herein, the present inventors found that the combination of P. kudriavzevii PY 1 strain and the fermentation process of some embodiments of the invention ensure high biomass yields (>30 g / L), elevated protein content (>55% of total protein) and amino acid content (>50 % crude amino acid). Comparison of the amino-acid profile of Pichia kudriavzevii PY 1 (ProtYeast™) biomass with fish meal shows enrichment in most essential amino acids, except for methionine and arginine. This composition supports at least the partial substitution of fish meal in animal feed formulations. Thus, according to an aspect of the invention there is provided a method of producing a carbon source useful for culturing microorganisms, the method comprising:

[0096] (a) providing agricultural waste;

[0097] (b) extracting said agricultural waste so as to obtain at least one extract which comprises reducing sugars of said agricultural waste;

[0098] (c) sterilizing said at least one extract so as to obtain a sterile extract or extracts.

[0099] As used herein the phrase “carbon source” refers to an organic carbon-containing compound that can be used by a microorganism in a fermentation procedure to increase biomass.

[0100] According to a specific embodiment, the carbon source is a carbohydrate.

[0101] According to a specific embodiment, the carbon source is a reducing carbohydrate.

[0102] As used herein “microorganism” or “ microorganisms” refer to a single cell eukaryotic or prokaryotic microorganism. The microorganism can be naturally occurring, lab generated, genetically modified or non-genetically modified.

[0103] Examples include, but are not limited to a bacterium, a fungus like yeast and / or an algae. According to a specific embodiment, the microorganism is yeast.

[0104] As used herein, 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. According to a specific embodiment, the yeast is a Saccharomycetales yeast. According to a specific embodiment, the Saccharomycetales yeast cells are yeast cells from the genus Pichia, Cyberlindnera, Kluyveromyces, Wickerhamomyces or Yarrowia. Exemplary strains include, but are not limited to Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, 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, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.

[0105] According to a specific embodiment, the yeast is of the genus Pichia. Examples include, but are not limited to Pichia kudriavzevii, Pichia pastoris, Pichia fermentans, Pichia anomala and Pichia kluyveri.

[0106] According to a specific embodiment, exemplary strains of Pichia include but are not limited to Pichia pastoris GS115, Pichia pastoris KM71, Pichia pastoris X33, Pichia kudriavzevii CBS 5147, Pichia kudriavzevii NRRL Y- 12745, Pichia fermentans CBS 187, Pichia fermentans NRRL Y-1442, Pichia anomala K, Pichia anomala WRL-076, Pichia kluyveri DSM 70815, and Pichia kluyveri T02 are notable strains used in various industrial applications.

[0107] According to a specific embodiment, the Pichia is of the species Pichia kudriavzevii (formerly Candida krusei ), also referred to herein as “P. kudriavzevii.

[0108] Exemplary strains of P. kudriavzevii include, but are not limited to Pichia kudriavzevii AC 1 (OP678979.1), Pichia kudriavzevii B-NC-13-OZ23 (KJ794697.1), Pichia kudriavzevii 11 Page 10 / 20 (KR259307.1), Pichia kudriavzevii YZ4 (EU394711.1), Pichia kudriavzevii PEX-11 (MW990004.1), Pichia kudriavzevii 15 (KR259308.1), Pichia kudriavzevii LI (EF126365.1), Pichia kudriavzevii feni48 (KM234455.1). Other exemplary strains include, but are not limited to Pichia kudriavzevii CBS 5147, Pichia kudriavzevii NRRL Y- 12745, Pichia kudriavzevii ATCC 6258.

[0109] According to a specific embodiment, the yeast is the strain ProtYeast™ PY 1 P. kudriavzevii comprising a genomic signature of SEQ ID Nos. 1-8. According to a specific embodiment, the strain has been deposited on October 31st, 2024 under the Budapest Treaty in the ATCC® Patent Depository P.O. Box 1549 Manassas, VA20110 USA under PTA- 127830.

[0110] PY 1 is a distinctive strain which can be identified using the signatory sequences which are listed in Table E below.

[0111] The method according to this aspect of the invention makes use of agricultural waste. As used herein, the phrase “agricultural waste” refers to byproducts and residues generated during the cultivation, harvesting, extracting, and storage of agricultural crops, as well as parts of crops that are damaged, overripe, or otherwise unsuitable for sale or consumption. This waste includes non-food parts of plants, such as stalks, husks, and leaves, as well as edible parts that are discarded due to quality or quantity (overproduction, non-consumption) issues.

[0112] The selection of the agricultural waste depends on the ability to extract reducing sugars therefrom (which are the main carbon source according to the present teachings.

[0113] Thus, the agricultural waste can be agricultural waste which comprises a level of reducing sugars above a predetermined threshold prior to extracting (e.g., above 5 % w / w). This, allows extracting the reducing sugars without additional processing of the plant, portion or tissue thereof.

[0114] Accordingly, the agricultural waste can be selected from the group consisting of fruit waste, molasses, fruit pomace (e.g., skins, pulp, seeds, and stems of the fruit), sugarcane bagasse juice, whey, ripe banana peels, vegetable waste, sweet potato residues and juice pressings or residues from sugar-rich crops, and sugar beet pulp.

[0115] Examples of plants and specifically fruit that can be used according to this embodiment of the invention include, but are not limited to, dates which contain about 63-70 g of reducing sugars per 100 grams, figs about 16-26 g per 100 grams, grapes about 15-23 g per 100 grams, mangoes about 14-17 g per 100 grams, cherries about 13-15 g per 100 grams, pineapples about 9-14 g per 100 grams, bananas about 12-14 g per 100 grams, pears about 9-12 g per 100 grams, apples about 8-12 g per 100 grams, oranges about 7-9 g per 100 grams, and watermelons about 6-9 g per 100 grams. These values can vary based on factors such as ripeness and fruit variety.

[0116] According to a specific embodiment, the reducing sugars are selected from the group consisting of glucose, xylose, arabinose, galactose, mannose, and fructose.

[0117] According to a specific embodiment, the reducing sugars are selected from the group consisting of glucose, fructose, and xylose.

[0118] According to a specific embodiment, the reducing sugars are selected from the group consisting of glucose and fructose.

[0119] According to a specific embodiment, the level of reducing sugars is determined at the beginning of the method, prior to the extracting and optionally during or after the extracting. Methods of determining reducing sugar content are well known in the art, e.g., Fehling’s method for reducing sugars.

[0120] Thus, determining a level of said reducing sugars in the extract and / or the sterile extract. According to a specific embodiment, the fruit waste comprises dates. According to a specific embodiment, the dates are overripe or damaged. According to another embodiment, the agricultural waste comprises a level of reducing sugars below a predetermined threshold prior to said extracting (e.g., below 5 % w / w).

[0121] In such cases, the method may comprise treating said agricultural waste to breakdown cellulose into reducing sugars prior to the extracting.

[0122] In this embodiment, the agricultural waste initially contains a low level of reducing sugars, which are crucial for fermentation processes like yeast biomass production. Agricultural waste, particularly lignocellulosic biomass, often consists of complex carbohydrates, including cellulose and hemicellulose, which need to be broken down into simpler sugars before further processing. The treatment of agricultural waste involves breaking down cellulose, a polysaccharide composed of glucose units, into these reducing sugars. This breakdown increases the availability of fermentable sugars, optimizing the fermentation process.

[0123] According to a specific embodiment, treating comprises enzymatic hydrolysis, acid hydrolysis or a combination thereof.

[0124] Enzymatic hydrolysis involves the use of enzymes, such as cellulases, to catalyze the breakdown of cellulose into simpler sugars like glucose. This method is often preferred for its specificity and efficiency under mild conditions. Several enzymes are involved in this process, including endoglucanases, exoglucanases, and P-glucosidases, which sequentially break down the cellulose structure into glucose units, each of which is contemplated herein and may be considered as separate embodiments.

[0125] Conversely, acid hydrolysis uses strong acids, such as sulfuric acid, to break the glycosidic bonds in cellulose, converting it into reducing sugars.

[0126] According to a specific embodiment, acid hydrolysis is performed using either concentrated or dilute acids. In concentrated acid hydrolysis, strong acids (e.g., 70-90 % by weight) such as sulfuric acid (H2SO4) or hydrochloric acid (HC1) are used at high concentrations to dissolve the biomass, breaking down cellulose into glucose rapidly. Dilute acid hydrolysis, in contrast, uses lower acid concentrations (e.g., 1-10 % by weight) at elevated temperatures (e.g., 150-230°C) and pressures. Both methods require neutralization steps following the hydrolysis to remove residual acids before the sugars can be fermented.

[0127] A combination of both enzymatic and acid hydrolysis can be employed to optimize sugar release while minimizing the downsides of each individual method. For example, a mild acid pretreatment can weaken the biomass structure, making it more accessible to enzymes during subsequent enzymatic hydrolysis.

[0128] According to a specific embodiment, the agricultural waste is primarily lignocellulosic, typically containing a mixture of cellulose, hemicellulose, and lignin. According to a specific embodiment, the agricultural waste is selected from the group consisting of straw, corn stover, sugarcane bagasse, wood chips, rice husks, wheat bran, cottonseed hulls, peanut shells, coconut husks, coffee pulp, coffee husks, and palm kernel cake.

[0129] As mentioned, the agricultural waste is subjected to an extraction process which is repeated at least once or more to get a series of extracts in which the reducing sugar content is decreased in the later rounds of extraction.

[0130] According to a specific embodiment, extracting is by water extraction.

[0131] According to a specific embodiment, water extraction is performed by boiling the agricultural waste in water and centrifuging to collect a supernatant which comprises the reducing sugars (may be considered as Extract 1), the supernatant being a first extract, optionally wherein the boiling and centrifuging is repeated 2-5 times, such that extracts resultant of the repeating comprise decreasing amounts of reducing sugars.

[0132] Specific conditions are described hereinbelow: for instance 5-20 min boiling time (e.g., 10 min), 3000-5000 x g (e.g., 4000 x g). Another option for separation can be by filtration.

[0133] Thus, for example, the extract from the first repeat may be considered Extract 2. The extract from the second repeat may be considered Extract 3. The extract from the third repeat may be considered Extract 4. The extract from the fourth repeat may be considered Extract 5.

[0134] The volume of water to biomass is typically 2: 1-5: 1, e.g., 3:1 (v / w).

[0135] According to a specific embodiment, when referring to biomass it is the initial biomass weight.

[0136] According to a specific embodiment, extract 1 comprises about 16 % (w / v) reducing sugars.

[0137] According to a specific embodiment, extract 2 comprises about 5.5 % (w / v) reducing sugars.

[0138] According to a specific embodiment, extract 3 comprises about 2 % (w / v) reducing sugars. Once the extract(s) are at hand they may be pooled or maintained individually.

[0139] As mentioned, following extraction the extracts are subjected to sterilization.

[0140] According to a specific embodiment, sterilization is by heating (e.g., autoclave).

[0141] Other methods of sterilization which may be used include, but are not limited to, filtration, chemical sterilization, radiation, gas sterilization, ultrasonic sterilization, cold sterilization with plasma.

[0142] The sterile composition of reducing sugars (also referred to herein as “reducing sugar extract of agricultural waste”) can be included in a sterile medium for producing a biomass of microorganisms of interest (such as described above, e.g., P. kudriavzevii, e.g., ProtYeast™ PY1 such as deposited at the ATCC® under PTA- 127830).

[0143] Thus, according to an aspect of the invention there is provided a sterile composition comprising a reducing sugar extract of agricultural waste, optionally comprising salt, nitrogen source, micro elements, macro elements and / or an anti-foaming agent.

[0144] As used herein “sterile composition” refers to a medium composition (e.g., liquid or powder) which comprises the waste derived extract(s) as carbon source. A base medium typically includes a carbon source, nitrogen sources, e.g., peptone, yeast extract, ammonium salts, or amino acids. Also may be included are mineral salts or microelements, such as magnesium sulfate, potassium phosphate, sodium chloride, and calcium chloride, provide essential ions and cofactors for enzyme activity. Water may be used as a solvent to dissolve nutrients and facilitate transport within the medium. Buffering agents, such as potassium phosphate, sodium citrate, HEPES, or MOPS, are included to maintain the pH of the medium within an optimal range for yeast growth, typically around 4.5 to 6.5. Growth factors, like inositol and adenine, are typically added for membrane synthesis and nucleotide production, while biotin supports fatty acid synthesis. Vitamins, such as biotin, thiamine (vitamin Bl), pantothenic acid (vitamin B5), and pyridoxine (vitamin B6) may be included.

[0145] Typical composition is provided in Table 4.

[0146] According to a specific embodiment, the nitrogen source comprises or consists of corn steep liquor.

[0147] According to a specific embodiment, the microelements are selected from the group consisting of magnesium, iron and phosphorous.

[0148] According to a specific embodiment, the salt is selected from the group consisting of magnesium sulfate, sodium chloride, ammonium sulfate, monopotassium phosphate and calcium chloride.

[0149] According to a specific embodiment, the salt is selected from the group consisting of magnesium sulfate and sodium chloride.

[0150] The skilled artisan would know which concentrations of each of the components described herein to select.

[0151] According to a specific embodiment, the carbon source consists of the waste extract described herein (without additional carbon sources, sugars, lipids or alcohols from sources outside the extracted waste). It can be a single extract or a combination of extracts dependent on the concentration of sugars which is desired. For instance, a combination of extract 2 and extract 3 can be used at the beginning of cultivation (starter medium). Conversely, a combination of extract 1 and extract 2 can be used for the feeding medium (in fed batch culture). It is desired to avoid diluting the extracts prior to adding them to the medium, therefore the desired reducing sugars content will be achieved by selecting a single or a number of extracts which together achieve the ultimate reducing sugar concentration in the medium.

[0152] According to a specific embodiment, the concentration of reducing sugars in the extract is 2 % to 20 %.

[0153] According to a specific embodiment, the concentration of reducing sugars in said extract is 2 % to 15 %.

[0154] According to a specific embodiment, the concentration of reducing sugars in said extract is 5 % to 8 %.

[0155] According to a specific embodiment, the concentration of reducing sugars in said extract is 10 % to 18 %.

[0156] According to a specific embodiment, the concentration of reducing sugars in said extract is about 2-20 %, 2-18 %, 2-16 %, 2-12 %, 2-10 %, 2-8 %, 2-6 %, 2-4 %, 2-18 %, 2-18 %, 4-18 %, 6-18 %, 8-18 %, 10-18 %, 12-18 %, 14-18 %, 16-18 %, 6-18 %, 6-16 %, 6-14 %, 6-12 %, 6-10 %, 6-8 %.

[0157] According to a specific embodiment, the concentration of reducing sugars in the extract is about 14-18 %.

[0158] According to a specific embodiment, the concentration of reducing sugars in the extract is about 4.5-6.5 %.

[0159] According to a specific embodiment, the concentration of reducing sugars in the extract is about 1-3 %.

[0160] According to a specific embodiment, the sterile composition is obtainable or obtained by the method described herein.

[0161] The sterile composition described herein can be used in microbial cultivation, either for lab procedures, e.g., research or preferably in large scale settings.

[0162] Thus, according to an aspect of the invention, there is provided a method of producing a biomass of a microorganism of interest, the method comprising culturing the microorganism under conditions which allow expansion of the microorganism and wherein the conditions comprise the sterile composition as a carbon source.

[0163] According to a specific embodiment, the composition is the sole carbon source in the method. According to a specific embodiment, the conditions comprise reducing sugars concentration of 0.5-2.5 %. Thus for instance the starter medium may comprise 1.5-2.5 %, e.g., 2 % reducing sugars w / v). The feeding medium may comprise 8-14 e.g., 12 % reducing sugars w / v.

[0164] As used herein “starter medium” refers to the initial medium used to grow the microorganisms when they are first inoculated into the bioreactor or fermenter (see for example Table 2 below).

[0165] As used herein “feeding medium” refers to the medium that is added incrementally during a fed-batch process or continuously in a continuous culture (see for example Table 3 below).

[0166] According to a specific embodiment, the conditions comprise about 10 % dissolved oxygen. According to a specific embodiment, the conditions comprise pH of about 4-6, e.g., 5. According to a specific embodiment, the conditions comprise batch fermentation, fed fermentation (fed batch) and / or continuous fermentation.

[0167] As used herein “large scale” refers to culture setting of at least 2000 liters.

[0168] Large-scale microbial culture refers to the industrial-scale cultivation of microorganisms. The scale of these operations typically involves bioreactors or fermenters with volumes ranging from several hundred liters to thousands of liters, depending on the process (e.g., up to 100,0000 liters, e.g., 2000-100,000 liters). Large-scale culture methods can be categorized into three primary modes: batch, fed-batch, and continuous. In batch culture, the bioreactor is filled with a fixed volume of culture medium, and microorganisms are allowed to grow until nutrients are depleted or inhibitory byproducts accumulate. This method is typically used for smaller-scale production or for processes where product formation is closely linked to cell growth. Batch cultures often have volumes ranging from 100 liters to thousands of liters (e.g., up to 2000 liters), depending on the scale of production. In fed-batch culture, nutrients are added incrementally to the culture as the microorganisms grow. This approach allows for better control over nutrient availability, preventing substrate inhibition and prolonging the growth phase of the microorganisms. By feeding the culture with nutrients over time, it is possible to increase cell density and overall productivity. Fed-batch is often used in large-scale processes, with volumes ranging from 500 liters to 20,000 liters or more. Continuous culture involves the constant addition of fresh nutrients to the bioreactor and the simultaneous removal of spent culture, allowing the process to maintain microorganisms in a steady-state growth phase. This method enables longterm production and is used for processes requiring constant product output over extended periods. The steady-state nature of continuous cultures allows for high productivity over time but requires precise control of the growth environment to prevent contamination and maintain optimal conditions. According to a specific embodiment, the conditions ensure culturing of no more than 3 species of microorganisms.

[0169] According to a specific embodiment, the method further comprises comprising monitoring during the culturing at least one of: reducing sugar concentration, amino nitrogen concentration, pH and oxygen level.

[0170] In a culturing process, reducing sugar concentration, amino nitrogen concentration, pH, and oxygen level are typically monitored using various methods known in the art to optimize microbial growth and fermentation. Reducing sugar concentration can be measured using chemical assays such as the DNS (Dinitro salicylic Acid) method or Benedict’s test, which detect sugars by their ability to reduce reagents, or by more advanced methods like high-performance liquid chromatography (HPLC) for precise sugar analysis. Amino nitrogen concentration is commonly monitored using the ninhydrin assay or formol titration, which measure free amino groups, while Kjeldahl analysis can provide an estimate of total nitrogen content. pH is monitored continuously using pH electrodes or probes inserted into the culture vessel, which give real-time feedback and can be connected to systems that automatically adjust pH levels. Oxygen levels, specifically dissolved oxygen, are measured using dissolved oxygen probes such as Clark-type electrodes or optical oxygen sensors. These probes provide continuous measurements of oxygen in the medium, allowing adjustments to be made to aeration or mixing to maintain the desired oxygen concentration.

[0171] According to a specific embodiment, amino nitrogen concentration is not below about 0.2 %, while starting with about 0.35 %), pH is about 4.5-6, and oxygen level is about 10 %.

[0172] According to a specific embodiment, the culture is pure i.e., comprises a single species, however also culture which comprise no more than 3 species of microorganisms are contemplates herein (e.g., 2 or 3).

[0173] Once a sufficient amount of biomass is obtained the microorganisms are typically harversted. Harvesting microorganisms from a culture can be done through various methods known in the art, such as centrifugation, filtration, flocculation, sedimentation, membrane separation, and cell lysis.

[0174] Thus, according to another aspect of the invention there is provided a microbial preparation obtainable according to the method as described herein.

[0175] According to a specific embodiment, the microbial preparation comprises no more than 3 species of microoirganisms, e.g., 1, 2 or 3.

[0176] According to a specific embodiment, the microorganism in the microbial preparation is viable (above 50 %). According to a specific embodiment, the microorganism in the microbial preparation is non-viable (above 50 %).

[0177] According to a specific embodiment, the microorganism in the microbial preparation is intact (less than 10 % of the biomass comprises lysed cells).

[0178] According to a specific embodiment, the microorganism in the microbial preparation is lysed (less than 10 % of the biomass comprises intact cells).

[0179] Accursing to a specific embodiment, the microbial preparation is formulated in a dry formulation.

[0180] Accursing to a specific embodiment, the microbial preparation is formulated in a liquid formulation.

[0181] Accursing to a specific embodiment, the microbial preparation is formulated in a solid formulation.

[0182] Accursing to a specific embodiment, the microbial preparation is formulated in a semi- solid formulation.

[0183] According to a specific embodiment, the microbial preparation comprises all essential amino acids (e.g., amino acid profile as in Table A), vitamins (Table B), elemants (Table C) and lipids (Table D). Table E provides sequences which can be used as markers for ProtYeast™ PY1 P. kudriavzevii.

[0184] Table A- amino add composition (determined by HPLC) and protein level (determined by AOAC 981.10 method)

[0185] Notes Results Units Test

[0186] 1 Amino Acids

[0187] 0.80 % Cysteic acid

[0188] 5.59 % Aspartic acid and Asparagine

[0189] 0.92 % Methionine sulfon

[0190] 3.05 % Threonine

[0191] 2.70 % Serine

[0192] 6.72 % Glutamic acid and Glutamine

[0193] 2.19 % Proline

[0194] 2.50 % Glycine

[0195]

[0196] 3.47 % Alanine

[0197] - 2.79 % Valine

[0198] 2.60 % Isoleucine

[0199] 3.76 % Leucine

[0200] 2.06 % Tyrosine

[0201] 2.20 % Phenylalanine

[0202] 4.77 % Lysine

[0203] 1.26 % Histidine

[0204] 2.59 % Arginine

[0205] 50.0 % Total

[0206] 2 59 % Protein

[0207]

[0208] 1. The amino acid tryptophan was not tested because it is destroyed by hydrolysis.

[0209] 2. Protein - Factor for calculating protein - 6.52

[0210] Table B-Vitamin content in PY1 biomass

[0211] Pichia kudriavzevii PY1 Fish meal

[0212] A (mg / kg) 5.9 0

[0213] E (mg / kg) 10 5.6

[0214] C (mg / kg) 28 0

[0215] Bl (mg / kg) 26 0.2

[0216] B2 (mg / kg) 16 7.9

[0217] B3 (mg / kg) 536 121

[0218] B4 (mg / kg) 287 4138

[0219] B5 (mg / kg) 222 13

[0220] B6 (mg / kg) 30 4.6

[0221] B7 (mkg / kg) 70 200

[0222] B9 (mg / kg) 25 0.3

[0223] B12 (mkg / kg) 1280 338

[0224]

[0225] Table C-Elemental composition of PY1 biomass

[0226] Ca, Co, Fe, K, Mg, Mn, Na, Se, Zn, P, mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg mg / kg PY1 1037.7 0.05 244.5 7251 120.3 13.72 353.8 0 132.7 25601

[0227]

[0228] Table D -Fatty acid composition of PY1 biomass (% of total fatty acids)

[0229] Fatty acid Value

[0230] C4:0 0 C6:0 0 C8:0 0 C10:0 0.04 Cll:0 0 C12:0 0.06 C13:0 0 C 14:0 0.23 C 14:1 n5-c9 0 C 15:0 0.3 C 15:1 n6-c9 iso 13 0 C 16:0 11.85 C 16:1 n7-c9 9.54 C 17:0 0.1 C 17:1 n8-c9 1.25 C 18:0 1.61 C 18:1 n9t 0.2 C 18:1 n9c 42.58 C 18:2 n6t-t9, tl2 0 C 18:2 n6c 27.44 C 18:3 n6-c6, c9, cl2 0.14 C 18:3 n3-c9, cl2, cl5 3.85 C 20:0 0 C 20:l 0.33 C 21:0 0 C 20:2 c-11, 14 0.24 C 20:3 n6c-8, 11, 14 0 C 20:4 n6c-5, c8,cll, cl4 0 C 22:0 0 C 22:l n9c-13 0 C 20:5 n3-5, 8,11,14,17 0 C 23:0 0 C 22:2 n6c-13, 16 0 C 24:0 0 C 24:l 0.18 C 22:6 n3c-4,7,10,13,16,19 0

[0231]

[0232] Table E -signatory nucleic acid sequences characteristic of ProtYeast™ PY1 P. kudriavzevii

[0233] | ID | Length | Description | Query Cover | Per Ident | Accession | 11780 (SEQ ID Pichia kudriavzevii strain NRRL YB-373 NODE_8486_length_671_cov_81 240809, whole genome NO: 1) 555 shotgun sequence 68% 10000% JASUTW010008051 1

[0234] Pichia kudriavzevii strain NG7 NODE_8_length_194196_cov_18 1154, whole genome shotgun sequence 100% 9964% NWTR01000008.1 Pichia kudriavzevii strain PB2809B ctg_89, whole genome shotgun sequence 75% 9959% JAMFQN01OOOOO88 I Pichia kudriavzevii strain PM40-I5 ctg_259, whole genome shotgun sequence 91 % 98 25% JAMAIV010000259 1 Pichia kudriavzevii strain X2809A ctg_79, whole genome shotgun sequence 91% 9799% JAMAIR010000079 1 Pichia kudriavzevii strain M7004B ctg_35, whole genome shotgun sequence 100% 97 87% JAMAIW010000035.1 Pichia kudriavzevii strain NRRL YB-431 NODE_6309_length_775_cov_34.487654, whole genome shotgun sequence 76% 97 16% JASUTX010006230 1 Pichia kudriavzevii strain Y114-04A ctg_1167, whole genome shotgun sequence 91% 96 88% JAXQKA010001167.1 Pichia kudriavzevii strain Bl 14-03 ctg_793, whole genome shotgun sequence 91% 96 88% JAXQHL010000792.1 Pichia kudriavzevii strain Y5A ctg_19, whole genome shotgun sequence 100% 9623% JAMFQM010000019.1 33695 (SEQ ID Pichia kudriavzevii strain NRRL YB-373 NODE_7912_length_698_cov_65 809107, whole genome NO: 2) 1425 shotgun sequence 48% 10000% JASUTW010007541 1

[0235] Pichia kudriavzevii strain NRRL YB-615 NODE_5371_length_593_cov_ 14.446352, whole genome

[0236]

[0237] shotgun sequence 41% 10000% JASUUA010005371.1 Pichia kudriavzevii strain NRRL YB-431 NODE_4625_length_890_cov_20.301442. whole genome shotgun sequence 21% 10000% JASUTX010004577 1 Pichia kudriavzevii strain NRRL YB-431 NODE_4393_length_911_cov_22.269133, whole genome shotgun sequence 20% 10000% JASUTX010004349 1 Pichia kudriavzevii strain SD108 contig03383, whole genome shotgun sequence 12% 10000% JQFKO 1000746 1 Pichia kudriavzevii strain NRRL YB-592 NODE 286 length 6566 cov 36.130766. whole genome shotgun sequence 100% 9993% J A SUTYO 10000286 1 Pichia kudriavzevii strain X2809A ctg_ 11, whole genome shotgun sequence 96% 9992% JAMA1R010000011 1 Pichia kudriavzevii strain B7027 ctg_14, whole genome shotgun sequence 81 % 99 88% JAMATNO 10000014 1 MAG TPA_asm: Pichia kudriavzevii isolate CTeuk-1743 NODE_508_length_36505_cov_25.756488, whole genome shotgun sequence 100% 9986% DAGWRI010000116.1 Pichia kudriavzevii strain 129 BOH78_Sc016, whole genome shotgun sequence 100% 9979% MQVM01000016 1 Pichia kudriavzevii NBRC1664 DNA, 1664ST70, whole genome shotgun sequence 100% 9979% BHFP01000070.1 Pichia kudriavzevii strain M2002 ctg_l 10. whole genome shotgun sequence 88% 9976% JAMA1L010000110.1 18340 (SEQ ID NO: 3) 5781 Pichia kudriavzevii strain KMBL5774 contig637. whole genome shotgun sequence 97% 8628% MPBH01000605 1

[0238] Pichia kudriavzevii strain NCYC 2658 ()0()000F_arrow, whole genome shotgun sequence 100% 8486% J ARGSBO 10000005 1 Pichia kudriavzevii strain KCKM 1001 contig7, whole genome shotgun sequence 100% 9025% JATXMTO 10000007 1 Pichia kudriavzevii strain Nunik contigl_MBY1358, whole genome shotgun sequence 100% 8740% J AJCTIO 10000001.1 Pichia kudriavzevii strain HJ2 contig_6, whole genome shotgun sequence 100% 8797% JAQKPW010000006.1 Pichia kudriavzevii NBRC1664 DNA, 1664ST06, whole genome shotgun sequence 100% 8959% BHFP01000006.1

[0239]

[0240] Pichia kudriavzevii strain Ckrusei653 scaffold00008. whole genome shotgun sequence 100% 8905% NHMM01000008 1 Pichia kudriavzevii strain SD108 scaffoldOOOlO, whole genome shotgun sequence 100% 9040% JQFK01000010.1 Pichia kudriavzevii strain NRRL YB-615 NODE_42_length_9079_cov_12.148570, whole genome shotgun

[0241] sequence 83% 98 36% JASUUA010000042.1 Pichia kudriavzevii strain 129 BOH78_Sc002, whole genome shotgun sequence 100% 85 87% MQVM01000002 1 Pichia kudriavzevii strain Y 1 104A ctg_31, whole genome shotgun sequence 89% 9482% JAM 1M0100000 1 1 Pichia kudriavzevii strain Y114-04A ctg_9. whole genome shotgun sequence 78% 97 81% JAXQKA010000009.1 Pichia kudriavzevii strain M7004B ctg_2, whole genome shotgun sequence 87% 96 15% JAMATW010000002 1 Pichia kudriavzevii isolate B143 Pichia-kudriavzevii_B143_contig-0722, whole genome shotgun sequence 89% 92 17% JACRSC010000722 1 10195

[0242] (SEQ ID

[0243] NO: 4) 1281 Pichia kudriavzevii strain Nuruk contigl_MBY1358. whole genome shotgun sequence 100% 9977% JAJCTIO 10000001.1

[0244] Pichia kudriavzevii strain KCKM 1001 contig8. whole genome shotgun sequence 100% 9969% J A1XM1010000008 1 Pichia kudriavzevii strain B7027 ctg_27, whole genome shotgun sequence 100% 9927% J AMAIN 010000027 1 Pichia kudriavzevii strain Ckrusei653 scaffold00006. whole genome shotgun sequence 100% 99 15% NHMM01000006 1 Pichia kudriavzevii strain Ml 110A ctg_47. whole genome shotgun sequence 100% 98 41% JAM A1Y010000047 1 Pichia kudriavzevii strain SD108 scaffold00026, whole genome shotgun sequence 100% 9960% JQFK01000026.1 Pichia kudriavzevii strain M2002 ctg_122, whole genome shotgun sequence 100% 9960% JAMAIL010000122.1 Pichia kudriavzevii strain F2020 ctg_192, whole genome shotgun sequence 100% 9960% JAMATK01000 192 1 Pichia kudriavzevii NBRC1664 DNA, 1664ST06, whole genome shotgun sequence 100% 9960% BHFPO 1000006.1 Pichia kudriavzevii strain NG7 NODE_21_length_155712_cov_19.096, whole genome shotgun sequence 100% 99 34% NWTR01000021.1 Pichia kudriavzevii strain Y1104A ctg_37, whole genome shotgun sequence 100% 9920% J AMAIM010000037 1 Pichia kudriavzevii strain Y114-04A ctg_457, whole genome shotgun sequence 100% 9942% JAXQKA010000457.1 Pichia kudriavzevii strain NCYC 2658 000000F_arrow, whole genome shotgun sequence 100% 9942% JARGSB010000005 1 Pichia kudriavzevii strain PM40-15 ctg_21, whole genome shotgun sequence 100% 9942% JAMA1V010000021 1 Pichia kudriavzevii strain D108-B21 ctg_20, whole genome shotgun sequence 100% 9942% JAMA1LI010000020 1 Pichia kudriavzevii strain S40-07 ctg_10, whole genome shotgun sequence 100% 9942% JAMA1T010000010.1 30250 (SEQ ID Pichia kudriavzevii strain NRRL Y- 10940 NODE_1 179_length_2793_cov_9.632033, whole genome NO: 5) 807 shotgun sequence 100% 9938% JASUTZO 10001 179.1

[0245] Pichia kudriavzevii strain NRRL Y-5396 scf7180000023365, whole genome shotgun sequence 100% 8900% PPIQ02000993.1 Pichia kudriavzevii strain 129 BOH78_Sc014, whole genome shotgun sequence 100% 8900% MQVM01000014 1 Pichia kudriavzevii strain SCHI0131. M.3 NODE_872_length_3830_cov_8.977884, whole genome shotgun sequence 100% 8900% JASKDR010000867 1 Pichia kudriavzevii strain S40-07 ctg_2. whole genome shotgun sequence 100% 8900% JAMAIT010000002.1 Pichia kudriavzevii strain P40-11 ctg_2. whole genome shotgun sequence 100% 8900% J AMA1Q010000002 1 Pichia kudriavzevii strain M2002 ctg_390, whole genome shotgun sequence 100% 8900% JAMA1L010000390.1 Pichia kudriavzevii strain NRRL Y-5396 xfScOOOOOOO, whole genome shotgun sequence 100% 8900% JAKTRB010000136 1 Pichia kudriavzevii NBRC1279 DNA, 1279ST69, whole genome shotgun sequence 100% 8900% BHFOOl 000069 1 Pichia kudriavzevii strain F2008 ctg_7, whole genome shotgun sequence 100% 88 88% J AMA1O010000007 1 Pichia kudriavzevii strain Y 1104A ctg_2, whole genome shotgun sequence 100% 88 88% JAMAIM010000002 1 Pichia kudriavzevii strain Ckrusei653 scaftbld00005. whole genome shotgun sequence 100% 88 39% NHMM01000005 1 Pichia kudriavzevii strain NG7 NODE_2_length_279745_cov_20 ()32, whole genome shotgun sequence 100% 88 27% NWTR01000002 1 38240 (SEQ ID Pichia kudriavzevii strain NRRL Y-10940 NODE_1214_length_2713_cov_12.622583, whole genome NO: 6) 1434 shotgun sequence 100% 9986% JASUTZ010001214.1

[0246]

[0247] Pichia kudriavzevii strain NRRL Y-5396 scf718OOOOO2333O. whole genome shotgun sequence 100% 99 79% PP1Q02000958.1 Pichia kudriavzevii strain NRRL YB-592 NODE_412_length_5375_cov_39.853659. whole genome

[0248] shotgun sequence 100% 9979% JASUTY010000412 1 Pichia kudriavzevii strain NCYC 2658 000004F_arrow, whole genome shotgun sequence 100% 9979% JARGSB010000007 1 Pichia kudriavzevii strain F2008 ctg_535, whole genome shotgun sequence 100% 9979% JAMAI0010000535 1 Pichia kudriavzevii strain F2020 ctg_485, whole genome shotgun sequence 100% 9979% TAMA1K010000485 1 Pichia kudriavzevii strain NRRL Y-5396 Sc0000006, whole genome shotgun sequence 100% 9979% J AKTRB010000007 1 Pichia kudriavzevii strain ASCUSDY21 NODE_82_length_45476_cov_646031_ID_49839, whole genome

[0249] shotgun sequence 100%; 9979%> JABFNE010000081 1 Pichia kudriavzevii NBRC1279 DNA, 1279ST1 1, whole genome shotgun sequence 100% 99 79% BHFO 1000 11.1 Pichia kudriavzevii DNA, contig: contig346, strain: NBRC 1279, whole genome shotgun sequence 100% 99 79% BBOI01000346.1 Pichia kudriavzevii strain NG7 NODE_27_length_140337_cov_19.0926, whole genome shotgun sequence 100% 99 72% NWTR01000027.1 Pichia kudriavzevii strain 129 BOH78_Sc007, whole genome shotgun sequence 100% 99 72% MQVM01000007 1 Pichia kudriavzevii strain Y114-04A ctg_5O. whole genome shotgun sequence 100% 99 72% JAXQKA010000050.1 26225

[0250] (SEQ ID

[0251] NO: 7) 408 Pichia kudriavzevii strain NCYC 2658 000000F_arrow, whole genome shotgun sequence 100% 9976% JARGSB010000005 1

[0252] Pichia kudriavzevii strain D108-B21 ctg_20, whole genome shotgun sequence 100% 9976% JAMAIU010000020 1 Pichia kudriavzevii strain Nuruk contig 1 _MBY 1358, whole genome shotgun sequence 100% 99 76% TAJCT1010000001 1 Pichia kudriavzevii strain NG7 NODE_21_length_155712_cov_19.()96, whole genome shotgun sequence 100% 9951%; NWTR01000021.1 Pichia kudriavzevii strain Ckrusei653 scaffold00006, whole genome shotgun sequence 100%; 9951% NHMM01000006 1 Pichia kudriavzevii strain 129 BOH78_Sc041, whole genome shotgun sequence 100%; 9951% MQVM01000041 1 Pichia kudriavzevii strain SD108 scaffold00026, whole genome shotgun sequence 100%; 9951% JQFK01000026.1

[0253]

[0254] Pichia kudriavzevii strain PB2809B ctg_29, whole genome shotgun sequence 100%> 9951% JAMFQN010000029.1 Pichia kudriavzevii strain X2809A ctg_22, whole genome shotgun sequence 100% 9951% J AMA1R010000022 1 Pichia kudriavzevii strain F4008B ctg_23, whole genome shotgun sequence 100% 9951% JAMA1PO1OOOOO23.1 Pichia kudriavzevii strain B7027 ctg_27, whole genome shotgun sequence 100% 9951% J AMAIN 010000027 1 Pichia kudriavzevii strain Y1104A ctg_37, whole genome shotgun sequence 100% 9951% J AMA1M010000037 1 15045

[0255] (SEQ ID

[0256] NO: 8) 3275 Pichia kudriavzevii strain Y5A ctg_17, whole genome shotgun sequence 100% 9890% JAMFQMO 10000017.1

[0257] Pichia kudriavzevii strain D108-B21 ctg_67, whole genome shotgun sequence 100% 98 90% J AMAIUO 10000067 1 Pichia kudriavzevii strain Ckrusei653 scaffold00008, whole genome shotgun sequence 100% 98 75% NHMMO 1000008 1 Pichia kudriavzevii strain SD108 scaffold00013, whole genome shotgun sequence 100% 98 72% JQFK01000013.1 Pichia kudriavzevii strain NCYC 2658 000000F_arrow, whole genome shotgun sequence 100% 98 72% JARGSB010000005 1 Pichia kudriavzevii strain F2020 ctg_9, whole genome shotgun sequence 100% 98 72% J AMA1K010000009 1 Pichia kudriavzevii strain NRRL Y-5396 scf7180000023360. whole genome shotgun sequence 100% 98 69% PP1Q02000988.1 Pichia kudriavzevii strain HJ2 contig_6. whole genome shotgun sequence 100% 98 69% JAQKPW010000006.1 Pichia kudriavzevii strain NRRL Y-5396 Sc(X)()0044, whole genome shotgun sequence 100% 98 69% JAKTRB010000045 1 Pichia kudriavzevii strain Nuruk contigl_MBY 1358, whole genome shotgun sequence 100% 98 69% J AJCT1010000001.1 Pichia kudriavzevii NBRC1279 DNA, 1279ST53, whole genome shotgun sequence 100% 98 69% BHF001000053.1 Pichia kudriavzevii DNA, contig: contig461, strain: NBRC 1279, whole genome shotgun sequence 100% 98 69% BBOTO 1000461 1 Pichia kudriavzevii strain Y4012 ctg_3, whole genome shotgun sequence 100% 98 66% JAMAIX0I0000003 1 Pichia kudriavzevii strain Y 1 104A ctg_84, whole genome shotgun sequence 100% 98 66% JAMATM 1 000084 1 Blast was done by putative CDS sequences against Pichia kudravazeii (taxid:4909) whole-genome shotgun contigs (wgs) at National Center of Biotechnology information of National Library of Medicine (https: / / blast(dot)ncbi(dot)nlm(dot)nih(dot)gov / )

[0258] Zheng Zhang, Scott Schwartz. Lukas Wagner, and Webb Miller (2000), " A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(l-2):203-14 The genomic sequences of ProtYeast™ PY1 P. kudriavzevii comprising a genomic signature of SEQ ID Nos. are covered by SEQ ID NO: 9-72 or at least 99 %, 99.9 %, 99.999 % or 99.9999 % identical sequences thereto, as determined by Blast.

[0259] The microbial preparation can be used in various applications, such as requiring alternative lipid, vitamins and protein sources.

[0260] Thus, according to an aspect of the invention there is provided an animal feed comprising the microbial preparation as described herein.

[0261] As shown in Example 3 of the Examples section which follows, the present inventors have shown that the contemplated feed can be used in feeding animals (e.g., aquatic animals) in a safe manner and can support growth and survival of animals such as fish. The animal according to some embodiments of the invention is a domesticated animal e.g., a pig, cow, or sheep. Alternatively, the animal is preferably an aquatic species and thus, an animal of an aquatic species.

[0262] According to a specific embodiment, the method or use is 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.

[0263] Accordingly, it is preferred in the context of the uses of the present invention that the animal is healthy.

[0264] Accordingly, the feed is used for culturing an aquatic species.

[0265] According to a specific embodiment the aquatic species is selected from the group consisting of crustaceans and fish.

[0266] According to some embodiments, the aquatic species is a farmed fish or crustaceans (e.g. shrimp or prawns), and in particular, any one of the group consisting of Shrimp, Prawns, Crabs, Lobsters and Crayfishes. In some specific embodiments the farmed crustacean is a shrimp or prawn. Suitable shrimps or prawns can be selected from the group consisting of Litopanaeus vannamei, Panaeus monodon, Penaeus japonicas and Macrobrachium rosenbergii.

[0267] The term “farmed crustacean” or “farmed fish” as used herein refers to a fish or crustacean which is either strictly or partially aquatic (i.e. living at least a portion of the organism’s life cycle in water), and which is cultivated (i.e. grown) by man, in an aquaculture environment. Finfish (e.g. flounder and whiting), marine crustaceans (e.g. prawns, shrimp, lobsters and crabs) and marine mollusks (e.g. oysters and abalone) can be cultured in seawater. Fresh water aquaculture is suitable for fresh water species, including fish (e.g. tilapia, trout), crustaceans (e.g. crayfish) and fresh water mollusks (e.g. clams). Some species are particularly suited for culture in brackish water (carp, catfish).

[0268] According to a specific embodiment, the aquatic animal is a marine fish or crustacean.

[0269] According to a specific embodiment, the aquatic species is a diadromous fish or crustacean.

[0270] According to a specific embodiment, the aquatic species is a freshwater fish or crustacean.

[0271] According to a specific embodiment, the aquatic species is carnivore (e.g., carnivore fish).

[0272] According to a specific embodiment, the aquatic species is herbivore (e.g., herbivore fish).

[0273] According to a specific embodiment, the aquatic species is omnivore (e.g., omnivore fish).

[0274] According to a specific embodiment, the fish is a finfish.

[0275] According to a specific embodiment, the fish are fish of the salmonid group, for example, cherry salmon (Oncorhynchus masou), Chinook salmon (Oncorhynchus tshawytscha), chum salmon (Oncorhynchus keta), coho salmon (Oncorhynchus kisutch), pink salmon (Oncorhynchus gorbuscha), sockeye salmon (Oncorhynchus nerka) and Atlantic salmon (Salmo salar). Other fish of interest for aquaculture include, but are not limited to, various trout, as well as whitefish such as tilapia (including various species of Oreochromis, Sarotherodon, and Tilapia), grouper (subfamily Epinephelinae), sea bass, sea bream, catfish (order Siluriformes), bigeye tuna (Thunnus obesus), carp (family Cyprimidae) and cod (genus Gadus). Other fish species that may be used according to the present teachings are provided hereinbelow (Table 1). Table 1: List of animal species used in aquaculture

[0276] Common Scientific Common Common Scientific Common Scientific Scientific name Common name Scientific name

[0277] name name name name name name name Siberian Acipenser Piaractus Ictiobus Common Centropomus Puntius Paco Bigmouth buffalo Java barb

[0278] sturgeon baeri mesopotamicus cyprinellus snook undecimalis javanicus Acipenser Black Ichthyoelephas

[0279] Sterlet sturgeon Ictalurus melas Bocachico Barramundi Lares calcarifer Roach Ratlins ruthenus bullhead humeralis

[0280] Acipenser Channel Jctahirus Prochilodus

[0281] Starry sturgeon Bocachico Nile perch Lares niloticus bench Tinea stellatus catfish punctatus reticulatus

[0282] Misgurnus Acipenser Chrysichthys Maccullochella

[0283] White sturgeon Bagrid catfish Dorada B rye on moorei Murray cod Pond loach anguillicaudatu transmontanus nigrodigitatiis peeli

[0284] s Colossoma Golden Macquaria Climbing Anabas Beluga Huso Weis catfish Siluris glanis Cachama

[0285] macropomum perch ambigua perch testudineus Arapaima Piaractus Gilthead

[0286] Arapaima Pangas catfish Pangasius Cachama blanca Sparus aurata Snakehead Channa argus gigas brachypomus seabream

[0287] African Jleterotis Sciaenops

[0288] Striped catfish Pangasius sutchi Striped bass Morone saxatilis Red drum Turbot Psetta maxima bonytongue niloticus ocellatus

[0289] Clarias Dicentrarchus Aequidens Salvelinus European eel Anguilla Mudfish European seabass Green terror Lake trout

[0290] anguillaris labrax rivulatus namaycush Anguilla Philippine Hong Kong Epinephelus Blackbelt Cichlasoma

[0291] Japanese eel Clarias hatrachus Atlantic cod Gadus morhua japonica catfish grouper akaara cichlid maculicauda

[0292]

[0293] Anguilla Hong Kong Epinephelus Jaguar Ciehlasoma Odontesthes American eel Clarias fuscus Areolate grouper Pejerrey

[0294] rostrata catfish areolatus guapote tnanaguense bonariensis North African Clarias Epinephelus Mexican Ciehlasoma Monopterus Milkfish Chanos Greasy grouper Lai

[0295] catfish gariepinus tauvina moj arra urophthalmus albus Freshwater Ahramis Bighead Clarias Spotted Plectropomus Etroplus Snakeskin Trichogaster Pearlspot

[0296] bream brama catfish macrocephalus coralgrouper maculatus suratensis gourami pectoralis Three

[0297] Heterobranchus Oreochromis Indonesian Channa Asp Aspius African catfish Silver perch Bidyanus spotted

[0298] bidorsalis andersonii snakehead micropeltes tilapia

[0299] Heterobranchus Largemouth black Micropterus Oreochromis Bastaid Paralichthys Catla Catla Samp a Blue tilapia

[0300] longifilis bass salmoides aureus halibut olivaceus South

[0301] Carassius Longfin Oreochromis Goldlined Goldfish American Rhamdia sapo European perch Perea fluviatilis Siganus guttatus auratus tilapia macrochir spinefoot

[0302] catfish

[0303] Hoplosternum Stizostedion Mozambique Oreochromis Marbled Siganus Crucian carp Carassius Atipa Pike-perch

[0304] lift o rale lucioperca tilapia mossambicus spinefoot rivulatus Cirrhinus Pomatomus Oreochromis Southern Thunnus Mud carp Northern pike Esox Indus Bluefish Nile tilapia

[0305] molitorella saltatrix niloticus bluefin tuna maccoyii Cirrhinus Plecoglossus Oreochromis Northern Thunnus Mrigal carp Ayu sweetfish Greater amberjack Seriola dumerili Tilapia

[0306] mrigala altivelis spilurus bluefin tuna thynnus Ctenopharyng Japanese Seriola Oreochromis Kissing Helostoma Grass carp Vendace Coregonus alhula Wami tilapia

[0307] odon idellus amberjack quinque radiata urolepis gourami temmincki

[0308]

[0309] Cyprinns Coregonus Snubnose Trachinotus Blackchin Sarotherodon Spotted Channa Common carp Whitefish

[0310] carpio lavaretus pompano blochii tilapia melanotheron snakehead punctatus Hypophthahni Oncorhynchus Trachinotus Tilapia

[0311] Silver carp Pink salmon Florida pompano Tilapia Common sole Solea vulgaris chthys molitrix gorbuscha Carolinas guineensis

[0312] Hypophthalmi Oncorhynchus Palometa Trachinotus Redbreast Lebranche Bighead carp Chum salmon Tilapia rendalli Mugil liza chthys nobilis keta pompano goodei tilapia mullet

[0313] Oncorhynchus Japanese jack Trachurus Redbelly Pacific fat Donnitator Orangefin labeo Labeo calbasu Coho salmon Tilapia zillii

[0314] kisutch mackerel japonicus tilapia sleeper latifrons Oncorhynchus Mangrove red Lutjanus Golden grey Oxyeleotris Roho labeo Labeo rohita Masu salmon Liza aurata Marble goby

[0315] mason snapper argentimaculatus mullet marmorata Leptobarbus Oncorhynchus Yellowtail Ocyurus Largescale White-spotted Siganus Hoven's carp Rainbow trout Liza macrolepis

[0316] hoeveni mykiss snapper chrysurus mullet spinefoot canaliculatus Megalohrama Sockeye Oncorhynchus A canthopagrus Gold-spot Osphronemus Wuchang bream Dark seabream Liza parsia Giant gourami amblycephala salmon nerka schlegeli mullet goramy Mylopharyngo Chinook Oncorhynchus Thinlip grey Striped

[0317] Black carp White seabream Diplodus sargus Liza ramada Channa striata don piceus salmon tshawytscha mullet snakehead Notemigonus Atlantic Crimson Leaping

[0318] Golden shiner Salmo salar Evynnis japonica Liza saliens

[0319] crysoleucas salmon seabream mullet

[0320] Osteochilus

[0321] Nilem carp Sea trout Sa Imo trutta Red seabream Pagrus major Tade mullet Liza tade

[0322] hasselti

[0323] White amur Parahramis Flathead

[0324] Arctic char Salvelinus alpinus Red porgy Pagrus Mugil cephalus bream pekinensis grey mullet

[0325]

[0326] Puntius Salvelinus Goldlined Rhabdosargus

[0327] Thai silver barb Brook trout White mullet Mugil curemu gonionotus foritinalis seabream sarba

[0328]

[0329] Source: FAO corporate document repository, List of animal species used in aquaculture Contemplated are both lower-value staple food fish species [e.g., freshwater fish such as carp, tilapia and catfish] and higher-value cash crop species for luxury or niche markets [e.g., mainly marine and diadromous species such as shrimp, salmon, trout, yellowtail, seabass, seabream and grouper]).

[0330] According to a specific embodiment, the species is Gilthead seabream (GHB), rainbow trout or Steelhead Trout.

[0331] According to a specific embodiment, the fish is at a developmental stage between larvae and adult or broadstock stage.

[0332] According to a specific embodiment, the fish is in at least a juvenile developmental stage. According to a specific embodiment, the fish is in at least a juvenile developmental stage e.g., nursing stage, juvenile stage, fingerling stage, fry stage, post larvae stage, dependent on the species.

[0333] According to a specific embodiment, the fish is up to grower / growout / parr / smolt / adult developmental stage or the broodstock stage. The names and stages are different between species of fish and also crustaceans.

[0334] According to a specific embodiment, a majority of the fish in the aquaculture are at least 0.5 gr.

[0335] According to a specific embodiment, a majority of the fish in the aquaculture are at least 1 gr- According to a specific embodiment, a majority of the fish in the aquaculture are at least 1-5 gr.

[0336] According to a specific embodiment, a majority of the fish in the aquaculture are at least 2 gr- According to a specific embodiment, a majority of the fish in the aquaculture are at least 2.5 gr.

[0337] According to a specific embodiment, a majority of the fish in the aquaculture are at least 3 gr- According to other specific embodiments, the aquaculture comprises a plurality of species (polyculture, e.g., 2, 3, 4) having compatible growth conditions requirements. For example the following combinations are known to be co-cultured: tilapia and carp; tilapia and grey mullet.

[0338] As used herein, the term “feed” or “aquafeed” relates to a manufactured or artificial diet (i.e., formulated feeds) to supplement or to replace natural feeds in the aquaculture industry. These prepared foods are most commonly formulated as flake, pellet or tablet form.

[0339] According to a specific embodiment, the feed is agglomerated, granulated or extruded. According to some embodiments of the invention the feed is in a formulation selected from the group consisting of pellet, granule, powder, crumble, block, liquid feed, and meal.

[0340] These formulated feeds are composed of several ingredients in various proportions complementing each other to form a nutritionally complete diet for the species or a functional feed such as a medical diet, broadstock diet or finishing diet.

[0341] While some emphasis is provided herein on marine species, the skilled artisan would know how to use for other animals.

[0342] Feeds are typically composed of micro and macro components. In general, all components, which are used at levels of more than 1%, are considered as macro components. Feed ingredients used at levels of less than 1% are micro components. Both macro and micro ingredients are subdivided into components with nutritional functions and technical functions. Components with technical functions improve the physical quality of the aquaculture feed composition or its appearance.

[0343] Macro components with nutritional functions provide aquatic animals with protein and energy required for growth and performance. The feed (e.g., for fish) should ideally provide the animal with: 1) fats, which serve as a source of fatty acids for energy (especially for heart and skeletal muscles); and, 2) amino acids, which serve as building blocks of proteins. Fats also assist in vitamin absorption; for example, vitamins A, D, E and K are fat-soluble or can only be digested, absorbed, and transported in conjunction with fats. Carbohydrates, are also often included in the feed compositions, although carbohydrates are not a superior energy source for fish over protein or fat. Carbohydrates are typically provided in a range of 5-50 % of the composition by weight. Minerals and vitamins are also typically included as micro components as well as others.

[0344] Thus, according to a specific embodiment, the feed comprises lysine, methionine, lipids, biotin, choline, niacin, ascorbic acid, inositol, pantothenic acid, folic acid, pyridoxine, riboflavin, thiamin, vitamin A, vitamin B 12, vitamin D, vitamin E, vitamin K, calcium, phosphorus, potassium, sodium, magnesium, manganese, aluminum, iodine, cobalt, zinc, iron, selenium, or combinations of same.

[0345] Fats are typically provided via incorporation of fish meals (which contain a minor amount of fish oil) and fish oils into the feed compositions. Extracted oils that may be used in feeds include fish oils (e.g., from the oily fish menhaden, anchovy, herring, capelin and cod liver), and vegetable oil (e.g., from soybeans, rapeseeds, sunflower seeds and flax seeds). Typically, fish oil is the preferred oil, because it contains the long chain omega-3 polyunsaturated fatty acids (PUFAs), EPA and DHA; in contrast, vegetable oils do not provide a source of EPA and / or DHA. These PUFAs are needed for growth and health of most aquaculture products. A typical feed will comprise from about 5-30 % or 15-30% of oil (e.g., fish, vegetable, etc.), measured as a weight percent of the aquaculture feed composition.

[0346] According to a specific embodiment, the feed comprises a minimum of 5 % total lipids and / or 5-50 % carbohydrates.

[0347] As used herein “protein” refers to proteins, peptides and / or amino acids. According to a specific embodiment, the protein in the feed is a single cell protein which can be provided as the sole protein source or in addition to other protein sources known in the art such as fish meal. When referring to quantitative assessment of protein and amino-acid content a no more than about 10 % variation between analytical methods / assays is contemplated (e.g., no more than 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %) and by no more than about 10 % between experiments.

[0348] According to a specific embodiment, the concentration of the microbial preparation in the animal feed is 2-40 % w / w (e.g., 20-40 %, 20-30 % such as for aquaculture).

[0349] . As used herein, the term “single cell protein”, optionally abbreviated herein also as “SCP”, refers to a protein obtained by and / or derived from 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 microorganism cells, disrupted microorganism cells, isolated proteins obtained by one or more microorganism) s), isolated proteins derived by one or more microorganism(s), purified proteins obtained by one or more microorganism(s), and purified proteins derived by one or more microorganism(s).

[0350] As mentioned, additional sources of proteins may be used (but not solely).

[0351] According to a specific embodiment, the protein is a protein preparation such as a meal. As used herein, the term "feed meal" refers to a protein-rich feed component derived from cereals, plants, animals or fish. Feed meals may be provided in comminuted and / or dried form.

[0352] According to a specific embodiment, meals may be selected from fish meal, chicken meal, soybean meal, hydrolyzed feather meal, blood meal, meat and bone meal. According to a specific embodiment, the feed meal is fish meal or chicken meal.

[0353] As used herein, "fish meal" refers to meal produced by the boiling of landed fish and other aquatic animal species (either caught or produced), separating out water and oil (e.g. by use of a press), and then drying. Normally fish meal is dried to a moisture content of less than or equal to about 10%, and then the fish meal is distributed at room temperature. Many fish species may be used as the raw material of fish meal, such as horse mackerel, true sardine, various other sardines, mackerel, herring, capelin smelt, sand eel, various types of codfish, and Antarctic krill. Fish meal is widely used as the main source of dietary protein for most commercially farmed fish, in part because fish meal provides a balanced amount of the essential amino acids.

[0354] According to a specific embodiment, the feed includes other protein sources for example, plant protein, animal protein such as fish meal or chicken meal as a component (e.g., not more than 50 %, 40 %, 30 %, 26 %, 25 %, 20 %, 15 %, 10 % w / w of the feed).

[0355] According to a specific embodiment, the feed includes chicken meal as a component. According to a specific embodiment, the feed includes soy meal as a component.

[0356] According to other embodiments, the protein is derived from surimi, ground fish meat, krill, gelatin, collagen, gluten, egg albumen.

[0357] According to a specific embodiment, the feed may comprise at least one of energy sources, protein sources, fats or oils, vitamins, minerals, fiber and a non-nutritional additive (e.g., probiotics, enzymes, antioxidants, preservatives, coloring or flavoring agents).

[0358] According to yet a further aspect of the invention there is provided a method of increasing body weight of an animal comprising feeding the animal with an effective amount of the feed as described herein.

[0359] According to still a further aspect there is provided a method of growing an animal species, comprising feeding the animal with an effective amount of the feed as described herein.

[0360] According to still a further aspect there is provided a method of supporting survival of an animal comprising feeding the animal with an effective amount of the feed as described herein.

[0361] As used herein “feeding” refers to the act of providing an animal e.g., aquaculture with a man-made composition that is formulated for feeding a species of interest.

[0362] As used herein “growing” refers to promoting development and / or weight gain of individuals in a culture

[0363] As used herein “an aquaculture” or “aquaculturing” refers to cultivation of aquatic populations (e.g., freshwater, saltwater aquatic animals e.g., saltwater, brackish water) under controlled conditions.

[0364] As used herein “increasing” is as compared to the same feed without the microbial preparation, given to the same population (same species and developmental stage) of cultivated animals in terms of growth conditions, feed composition, feeding regimen, also referred to as “control”. According to a specific embodiment, increasing is in a statistically significant manner.

[0365] According to some embodiments, “control” comprises optimal growth conditions.

[0366] According to some embodiments, increasing is with respect to the effect of other sources of protein such as fishmeal or chicken meal.

[0367] According to some embodiments, increasing is with respect to the beginning of culturing (or preceding developmental stages during culturing. According to a specific embodiment, increasing is by at least 10 %, 20 %, 30 %, 40 %, 50 %, 70 %, 80 %, 1 fold, 2 fold, 3 fold, 5 fold or 10 fold as compared to a control.

[0368] According to a specific embodiment, the concentration of the microbial preparation in various animal feeds is listed below (percent of inactivated yeast which can be intact or lysed). % is weight percent.

[0369] According to some embodiments the concentration of the microbial preparation in various animal feeds such as aquatic animal feed is any one of about 10-70 %, 10-60 %, 10-50 %, 10-40%, 20-70 %, 20-60%, 20-50 %,

[0370] Poultry 5-10 % NRC (National Research Council)

[0371] Swine 3-7 % NRC (Nutrient Requirements of Swine)

[0372] Aquaculture Global Aquaculture Alliance Report

[0373] 20-40 %

[0374] Ruminants 2-5 % NRC (Nutrient Requirements of Ruminants)

[0375] Pet Food 5-10 % Feed Manufacturer Guidelines (e.g., Cargill)

[0376] As used herein “supporting survival” refers to the ability of an effective amount of the microbial preparation to keep at least about the same (or increased) survival rate compared to that of a culture of the same species under the same developmental stage and optimal growth conditions.

[0377] Hence the present teachings provides for compositions and methods for cultivation of yeas. Also provided is use of the cultivated yeast as a protein source for growing, maintaining, supporting survival, of various types of farmed animals, such as farmed fish.

[0378] Any of the yeast, feed or food containing same can be packaged in dedicated articles of manufacture. Alternatively, the yeast can be used as an active ingredient in the production of feed or food.

[0379] As used herein the term “about” refers to ± 10 %.

[0380] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0381] The term “consisting of’ means “including and limited to”.

[0382] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0383] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0384] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0385] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0386] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0387] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0388] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0389] EXAMPLES

[0390] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0391] EXAMPLE 1

[0392] Batch cultivation

[0393] Dates extraction process

[0394] Fermentation media included extract from whole dates, corn steep liquor and supplemented salts magnesium sulfate and sodium chloride. For extracting of dates prior inclusion to the media, with or without pitsz, grinded for homogenization and boiled in water for 15 minutes at a w / v ratio of 1:3. Date pulp was then centrifuged at high speed and the supernatant removed (also referred to herein as “extract 1”). Then an equal water portion was added to the sediment and mixture was boiled for another 15 minutes and pulp was separated by centrifugation (also referred to herein as “extract 2”). Another portion of water at w / v ratio 1:3 to initial dates weight was added and boiling separation steps were repeated (also referred to herein as “extract 2”). Reducing sugar content in collected supernatants was quantified throughout the extraction process and the resultant solution was sterilized.

[0395] Corn steep liquor process

[0396] Press filtration 1 µm pore size was used to remove insoluble substances from corn steep liquor and free amino nitrogen in corn steep liquor was quantified.

[0397] Table 2. Composition of starter medium - medium composition in the fermenter for starting fermentation (w / v).

[0398] Component Concentration %

[0399] Date sugar solution (extracts 2 and 3) Diluted to 2 % reducing sugars

[0400] Corn steep liquor Diluted to -0.35 % (w / v) of Amine Nitrogen

[0401]

[0402] Ammonium Sulphate 0.2 (2 g / 1)

[0403] Monopotassium phosphate 0.2 (2 g / 1)

[0404] Magnesium Sulfate 0.1 (1 g / 1)

[0405] Sodium chloride 0.021 (0.21 g / 1)

[0406] Calcium chloride 0.02 (0.2 g / 1)

[0407] Defoamer - Sofexil 0.03

[0408] Water To final volume

[0409]

[0410] Table 3. Feeding media for maintaining fermentation (fed batch).

[0411] Sugar solution from date pretreatment undiluted (extract 1 and / or extract 2) 12 %

[0412] Ammonium Sulphate 0.2 (2 g / 1)

[0413] Magnesium Sulfate 0.1 (1 g / 1)

[0414] Sodium chloride 0.021 (0.21 g / 1)

[0415] Calcium chloride 0.02 (0.2 g / 1)

[0416] Defoamer - Sofexil 0.03

[0417]

[0418] Table 4. Medium for flasks cultivation of seeding culture

[0419] Component Concentration %

[0420] Glucose 2

[0421] Ammonium sulphate 0.5

[0422] Monopotassium phosphate 0.1

[0423] Magnesium Sulfate 0.05

[0424] Sodium chloride 0.02

[0425] Calcium chloride 0.02

[0426] Yeast extract 0.1

[0427] Water To final volume

[0428]

[0429] Fermentation parameters

[0430] A fed-batch fermentation was used to maximize yield and productivity. Inoculum was grown in standardized media in flasks, inoculum size was 2.5 % and fermenter initial volume was set to 2 / 3 of the final working volume (7 lit). After approximately 8 hours of growth, Reducing Sugars in the fermenter was tested every 2 hours and Feeding media was added to keep Reducing Sugars at 1 % level. Fermentation and feed media temperature was kept at 40 °C. pH was controlled and kept at 5, formation of foam was controlled by the addition of antifoam. Air supply and agitator speed were adjusted to ensure 10 % dissolved oxygen.

[0431] Downstream extracting Harvested volume with cultivation media and grown biomass was processed to produce an amino acid rich product. Harvested volume was heat treated at 85 °C for at least 30 minutes for cell inactivation. Then cells were separated from the media by centrifugation. For excessive salt removal, cells were washed once with X3 volumes of water and re-separation. Biomass was then dried to <10% moisture content resulting in the final product.

[0432] EXAMPLE 2

[0433] Fed-Batch cultivation

[0434] For high quality yeast biomass production, fermentation parameters are needed to guarantee high cell titers, dry weight content and biomass amino acid content. Industrial fermentation is typically categorized into three types, batch, fed batch and continuous. Batch fermentation consists of inoculating cells into the final volume of media and harvesting when the culture reaches optimal conditions. In fed batch fermentation, a smaller volume of media is inoculated, and additional media is added over time followed by harvesting of the final volume. By adjusting the feed rate, nutrient concentrations are kept at optimal levels leading to higher cell densities and productivity. During continuous fermentation new media is added while an equivalent volume of media and cells are removed. During continuous fermentation, cells are kept in optimal condition for long periods of time, while constant cell harvesting increases productivity substantially. The process, according to some embodiments of the invention starts with a fed batch regime culminating with a high cell density culture, followed by a continuous fermentation regime that allows for long fermentation times and increased cell harvesting per unit time. Precision feeding can be achieved by addition of carbon and nitrogen independently, and hence: optimal sugar concentration and carbon-to-nitrogen ratio are maintained.

[0435] The fermentation media included a 2 % sugar solution from wasted dates, corn steep liquor and additional salts. The process started with an inoculum size of at least 2.5 % pre-grown in flasks on standard media. After cells have consumed most available sugar (reducing sugar content approached 0.2%), feeding was commenced with a 10 % date sugar solution (extracts 2 and 3), keeping reducing sugar content between 0.7%-l%. Free amino nitrogen monitoring (e.g., by formol titration - results are given by w / v units) dictated when additional media components were added to the fermenter. The specialized process led to consistently high dry weight content over time (Figure 1).

[0436] The protein content of ProtYeast™ was compared between dates and molasse and compared to also to Fish Meal. The protein content was determined by Kjeldahl analysis. The results are shown in Table 5 below. Table 5- Example of Protein and Amino Acid composition of dry biomass of P. kudriavzevii PY 1 strain, cultivated on Dates extract of on Beet Molasses and comparision with Fish Meal.

[0437] PY1, Beet

[0438] PY1, Dates Fish Meal

[0439] molasses

[0440] Protein

[0441] 61 63 73

[0442] content, %

[0443] AA composition of protein, %

[0444] Ala 7.0 7.3 7.0

[0445] Arg 4.4 4.7 6.7

[0446] Asn 5.3 TBD 4.0

[0447] Asp 8.6 12.3 6.0

[0448] Cys 2.5 2.4 0.9

[0449] Gin 11.0 TBD 5.4

[0450] Gin 10.3 15.4 8.3

[0451] Gly 4.0 5.1 9.1

[0452] His 2.3 2.2 2.1

[0453] lie 4.4 4.9 4.5

[0454] Leu 6.3 7.8 7.2

[0455] Lys 8.7 10.0 7.3

[0456] Met 1.5 1.7 2.8

[0457] Phe 3.4 4.4 3.8

[0458] Pro 3.2 4.4 5.9

[0459] Ser 4.5 4.8 3.9

[0460] Trp 1.0 0.9 1.0

[0461] Thr 4.5 6.2 5.7

[0462] Tyr 3.0 0.9 3.3

[0463] Vai 4.3 4.8 5.2

[0464]

[0465] Importantly, the amino acid profile of P. kudriavzevii PY1 biomass substantially aligns with that of fish meal, apart from methionine. Accordingly, PY1 biomass can replace a significant portion of fish meal in animal feeds, optionally with methionine supplementation to achieve target nutritional specifications.

[0466] Example 3

[0467] Fish feeding Fish and experimental diets

[0468] The experimental fish tested were fingerlings of the Asian sea bass Lates calcarifer also known as Barramundi at an average weight of ± 2.8 grams. They were purchased from Maagan Michael hatchery, Israel. The fish were acclimatized to the laboratory conditions for 12 days prior to the onset of the experiment. The Asian sea bass is a predatory fish that requires a high level of protein in its diet. The control diet had 45 % protein and 12 % fat and contained as a source of protein 26 % fishmeal and other proteins as listed in Table 6.

[0469] Yeast biomass preparation

[0470] The five experimental diets contained graded levels of fishmeal replacement: 10 %, 20 % 30 % 40 % and 50 % replacement of the fishmeal with the PY1 P. kudriavzevii dry yeast biomass (comprising ProtYeast™ PY1 P. kudriavzevii, hereinafter ProtYeast™). The whole mixture was extruded to 2 mm pellets

[0471] Pellets were produced from a complex diet matrix containing (percent by weight of finished diet): wheat 21.0; wheat gluten meal 6.5; com gluten 16.0; soybean meal (48 % CP) 21.0; mixed oil 9.0; vitamin-mineral premix 0.4; anti-mold agent 0.1; and a variable fishmeal / yeast fraction totaling 26.0 %. The control diet contained fishmeal (65 % CP) at 19.0 % of the diet with no yeast. Experimental diets replaced fishmeal with Pichia kudriavzevii PY1 yeast biomass at 10, 20, 30, 40, or 50 % of the fishmeal fraction (w / w), with the total fishmeal + yeast kept constant at 26.0 %.

[0472] All dry ingredients were ground to <1.0 mm (preferably ~0.5 mm) in a hammer mill and dry-mixed for 5 min in a ribbon mixer. The vitamin-mineral premix and anti-mold were added during the final minute of dry mixing. The mash was then conditioned by the gradual addition of hot water and / or low-pressure steam to reach a mash moisture of 25-30 % and an internal temperature of 80-90 °C, followed by 1-3 min residence to initiate starch gelatinization and protein pasting. A portion of the oil (1-2 % of the diet) was included at this stage solely for lubrication; the remainder was applied post-drying as described below.

[0473] Conditioned mash was formed on a small single-screw pellet press fitted with a 2.0 mm die (die L / D 8-12). Extrudate temperature at die exit was maintained at 65-90 °C. Strands were cut to 2-4 mm length and transferred immediately to drying trays. Drying was performed in a forced-air oven at 60-80 °C until pellet moisture was <10 %. While still warm, pellets were tumble-coated with the remaining oil to a total oil inclusion of 9.0 % w / w.

[0474] Quality control included: (i) particle size verification of ground meals (sieve analysis, target D90 <1.0 mm); (ii) pellet dimensions (2.0 ± 0.2 mm diameter; 2-4 mm length); (iii) floatability (>95 % of pellets floating after 5 min in freshwater at 25-28 °C); (iv) water stability (>60 min without visible breakup under static conditions); and (v) final moisture (<10 % w / w). When yeast inclusion levels >30 % of the fishmeal fraction were used, minor process adjustments (within the ranges above) were optionally applied to maintain float and durability: increasing binder / starch contribution by 1-2 % (e.g., additional wheat flour), targeting the upper end of conditioning moisture (28-30 %), and / or extending drying time to achieve the specified hardness and stability.

[0475] Prior to the beginning of the growth experiments the different diets were given to the fish in order to ascertain that the fish readily consume the diets containing the yeast product. Observations confirmed that the fish consume the experimental diets and do not reject them.

[0476] Table 6- Composition of fish feed. The diets were administered as a 2mm floating pellet Raw material % in diet Vender

[0477] Wheat 21 Tahanot Kemach Israeliot

[0478] Wheat gluten meal 6.5 Pa Dutch Shoppes of Virginia

[0479] Corn gluten 16 Foodcom Creatine / MSM

[0480] Soybean meal (48%) 21 Myprotein

[0481] Fishmeal (65%) Various Peruvian

[0482] ProtYeast™ Various Depository (ATCC®)

[0483] Mixed oil 9 AuSable Brand

[0484] Vitamin& Mineral premix 0.4 Shangqiu Xianghe Pharmaceutical

[0485] Anti molds 0.1 LorAnn

[0486] Nutrients (%)

[0487] Protein 45

[0488] Fat 12

[0489] Ash 6

[0490] Fiber 1.8

[0491] Calcium 1.2

[0492] Total phosphorus 0.8

[0493]

[0494] Experimental setup

[0495] An experiment was designed to examine the partial substitution of the fishmeal Ingredient in a carnivorous fish diet with varying levels of dry yeast (ProtYeast™). The experiment was carried out in a closed facility located in the Aquaculture Department of the Agricultural Research Organization the Volcani Center. The fish were raised in an experimental system where the water is recycled through special biofilters. Initial average weight of the fingerling was about 2.8 grams. The fish that were introduced into the experimental system were as uniform as possible and underwent acclimatization to the experimental system for 12 days before the experiment began. The experimental tanks are of a uniform size of 250 liters each and were stocked with 20 fish in each tank. Each six containers are connected to a 350-liter biological filter, through which the rearing water is recycled in a closed system. The experiment was conducted in 18 containers (3 units of 6 containers) in which 6 feeding treatments were tested, including substitution of fish meal with different levels of ProtYeast™ at a substitution level of 10 %, 20 %, 30 %, 40 % and 50 %, compared to a control in which the fishmeal component was not replaced at all. Each treatment had 3 replicates. Throughout the experiment, the temperature in the rearing tanks was between 25-27 °C that is considered optimal growth temperature for these fish. Water quality parameters (oxygen, ammonia and nitrite levels) were also at optimal levels. Oxygen level was at saturation, ammonia was below 0.05 PPM and nitrite levels were below 0.5 PPM. These low levels are mainly due to the fact that the biofiltering system was operating well and the overall load of the fish in the tanks was relatively low.

[0496] Ongoing routine maintenance

[0497] Every morning - 7 days a week throughout the experiment, all the experimental tanks were checked to make sure that there is a proper flow of air and water. Dead fish (if any) were removed and recorded, and the temperature was measured and recorded using a minimum / maximum thermometer. Every morning, experimental food was taken out of the refrigerator and the exact amount of food was weighed for each tank according to the total weight of the fish in it. The amount of food that was given daily to each tank, of the various treatments, was 8 % of the weight of the fish in the tank during the initial week and throughout the rest of the experiment was adjusted to 5 % of the fish weight. The food was administered in two feedings - half of the amount in the morning and the other half in the afternoon. Once every few days, the level of ammonia / nitrite was measured in each set of 6 tanks and a siphon of the bottom of the tanks was performed to prevent the accumulation of food and feces on the bottom. The effluent water from the system was discharged into the municipal sewage system.

[0498] Once every two weeks, all the fish in each of the tanks were weighed and counted, and the amount of food given to the fish in each of the experimental tanks was updated accordingly. During the experiment, weight gain and survival were tested throughout the 6 weeks of the experiment.

[0499] Results

[0500] Throughout the experiment the temperature in the rearing tanks was between 25-27 °C that is considered optimal growth temperature for these fish. Water quality parameters (oxygen, ammonia and nitrite levels) were also at optimal levels. The precent growth of the fish, as measured every fortnight, is presented in the following tables. In can be clearly seen that already during the initial 2 weeks of growth the treatments that had the fishmeal ingredient replaced by graded levels of the ProtYeast™ product grew better than the control diet that had no replacement. This result was significant (P<0.05) for the 40 %, 50% treatments compared to the control diet with no replacement. However, in all groups growth was not hampered by the ProtYeast™ substitute.

[0501] Table 7. Data for average feed gain for the control and 10 %, 20 %, 30 %, 40 %, 50 % replacement of fish meal in feed formulation (also shown in Figure 2), significant difference, compared to control group is indicated by an asterix.

[0502] 0 Week 2 Week 4 Week 6

[0503] control 0 74 203 456 10% 0 86 235 476 20% 0 70 227 489 30% 0 66 206 469 40% 0 89* 222* 478 50% 0 99* 248* 504*

[0504] Survival rate

[0505] There was no significant difference in terms of survival between the different groups tested.

[0506] Conclusions

[0507] Throughout the experiment the growth results show unequivocally that the replacement of fishmeal with ProtYeast™ resulted in either better growth of the fish or no hampering of the fish growth.

[0508] The results of this study clearly show that the ProtYeast™ product can successfully replace fishmeal in the diet of fish (e.g., carnivore fish, e.g., Lates calcarife ). It seems that the addition of high levels of ProtYeast™ to the diet of the fish improved growth of the fish in a statistically significant manner.

[0509] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety. REFERENCES

[0510] (other references are cited in the application)

[0511] Agboola, J. O., Overland, M., Skrede, A., & Hansen, J. 0. (2021). Yeast as major protein-rich ingredient in aquafeeds: A review of the implications for aquaculture production. Reviews in Aquaculture, 13(2), 949-970. https: / / doi(dot)org / 10.1111 / raq.l2507

[0512] Ashraf, Z., & Hamidi-Esfahani, Z. (2011). Date and Date Processing: A Review. Food Reviews International, 27(2), 101-133. https: / / doi(dot)org / 10.1080 / 87559129.2010.535231

[0513] Boundy-Mills K, Karuna N, Garay LA, Lopez JM, Yee C, Hitomi A, Nishi AK, Enriquez LL, Roberts C, Block DE, Jeoh T. Conversion of cassava leaf to bioavailable, high-protein yeast cell biomass. J Sci Food Agric. 2019 Apr;99(6):3034-3044. doi: 10.1002 / jsfa.9517. Epub 2019 Jan 28. PMID: 30488472.

[0514] Chelliah, R., Ramakrishnan, S. R., Prabhu, P. R., & Antony, U. (2016). Evaluation of antimicrobial activity and probiotic properties of wild-strain Pichia kudriavzevii isolated from frozen idli batter. Yeast, 33(8), 385-401. https: / / doi(dot)org / 10.1002 / yea.3181

[0515] Cherdthong, A., Sumadong, P., Foiklang, S., Milintawisamai, N., Wanapat, M., Chanjula, P., Gunun, N., & Gunun, P. (2019). Effect of post-fermentative yeast biomass as a substitute for soybean meal on feed utilization and rumen ecology in Thai native beef cattle. Journal of Animal and Feed Sciences. https: / / doi(dot)org / 10.22358 / jafs / l 10992 / 2019

[0516] Choudhary, J., Singh, S., & Nain, L. (2016). Thermotolerant fermenting yeasts for simultaneous saccharification fermentation of lignocellulosic biomass. Electronic Journal of Biotechnology, 21, 82-92. https: / / doi(dot)org / 10.1016 / j.ejbt.2016.02.007

[0517] Chu, Y., Li, M., Jin, J., Dong, X., Xu, K., Jin, L., Qiao, Y., & Ji, H. (2023). Advances in the Application of the Non-Conventional Yeast Pichia kudriavzevii in Food and Biotechnology Industries. Journal of Fungi, 9(2), 170. https: / / doi(dot)org / 10.3390 / jof9020170

[0518] Goldberg, I. (1985). Single cell protein. Springer- Verlag A., Hakkinen, S. T., Toivari, M., & Wiebe, M. G. (2017). Single Cell Protein — State-of-the-Art, Industrial Landscape and Patents 2001-2016. Frontiers in Microbiology, 8, 2009. https: / / doi(dot)org / 10.3389 / fmicb(dot)2017.02009

[0519] Lapena, D., Kosa, G., Hansen, L. D. et al. Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken byproducts. Microb Cell Fact 19, 19 (2020). https: / / doi.org / 10.1186 / sl2934-020-1287-6

[0520] Pongcharoen, P. (2022). The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes. Letters in Applied Microbiology, 75(1), 36-44. https: / / doi(dot)org / 10.1111 / lam.13703

[0521] Ritala, Intanoo, M., Kongkeitkajorn, M. B., Pattarajinda, V., Bernard, J. K., Callaway, T. R., Suriyasathaporn, W., & Phasuk, Y. (2018). Isolation and screening of aflatoxin-detoxifying yeast and bacteria from ruminal fluids to reduce aflatoxin B 1 contamination in dairy cattle feed. Journal of Applied Microbiology, 125(6), 1603-1613. https: / / doi(dot)org / 10.1111 / jam.l4060

[0522] Sekoai, P. T., Roets-Dlamini, Y., O’Brien, F., Ramchuran, S., & Chunilall, V. (2024). Valorization of Food Waste into Single-Cell Protein: An Innovative Technological Strategy for Sustainable Protein Production. Microorganisms, 12(1), 166. https: / / doi(dot)org / 10.3390 / microorganisms 12010166

[0523] Sorour, M., Ramadan, B., Mehanni, A., & Abdelsabour, N. (2021). Single cell protein production using low quality fruits of some dates. Archives of Agriculture Sciences Journal, 0(0), 115-126. https: / / doi(dot)org / 10.21608 / aasj.2021.65566.1056

[0524] Spalvins, K., Ivanovs, K., & Blumberga, D. (2018). Single cell protein production from waste biomass: Review of various agricultural by-products [PDF]. 413.7Kb. https: / / doi(dot)org / 10.15159 / AR.18.129

[0525] Wu, J., Hu, J., Zhao, S., He, M., Hu, G., Ge, X., & Peng, N. (2018). Single-cell Protein and Xylitol Production by a Novel Yeast Strain Candida intermedia FL023 from Lignocellulosic Hydrolysates and Xylose. Applied Biochemistry and Biotechnology, 185(1), 163-178. https: / / doi(dot)org / 10.1007 / s 12010-017-2644-8 Zandona, E., Blazic, M., & Rezek Jambrak, A. (2021). Whey Utilisation: Sustainable Uses and Environmental Approach. Food Technology and Biotechnology, 59(2), 147-161. https: / / doi(dot)org / 10.17113 / ftb.59.02.21.6968

Claims

WHAT IS CLAIMED IS:

1. A microbial preparation comprising a yeast strain as deposited under the Budapest Treaty in the ATCC® Patent Depository under PTA-127830.

2. A method of producing a carbon source useful for culturing microorganisms, the method comprising:(a) providing agricultural waste;(b) extracting said agricultural waste so as to obtain at least one extract which comprises reducing sugars of said agricultural waste;(c) sterilizing said at least one extract so as to obtain a sterile extract or extracts.

3. The method of claim 2, further comprising determining a level of said reducing sugars in said extract and / or said sterile extract.

4. The method of claim 2 or 3, wherein said sterilizing is by heating.

5. The method of any one of claims 2-4, wherein said agricultural waste comprises a level of reducing sugars above a predetermined threshold prior to said extracting.

6. The method of claim 5, wherein said agricultural waste is selected from the group consisting of fruit waste, molasses, fruit pomace, sugarcane bagasse juice, whey, ripe banana peels, vegetable waste, sweet potato residues and juice pressings or residues from sugar-rich crops, and sugar beet pulp.

7. The method of claim 6, wherein said fruit waste comprises dates, optionally wherein said dates are overripe or damaged.

8. The method of any one of claims 2-4, wherein said agricultural waste comprises a level of reducing sugars below a predetermined threshold prior to said extracting.

9. The method of claim 8, wherein said method comprises treating said agricultural waste to breakdown cellulose into reducing sugars.

10. The method of claim 9, wherein said treating comprises enzymatic hydrolysis, acid hydrolysis or a combination thereof.

11. The method of any one of claims 8-10, wherein said agricultural waste is primarily lignocellulosic.

12. The method of claim 11, wherein said agricultural waste is selected from the group consisting of straw, corn stover, sugarcane bagasse, wood chips, rice husks, wheat bran, cottonseed hulls, peanut shells, coconut husks, coffee pulp, coffee husks, and palm kernel cake.

13. The method of any one of claims 2-12, wherein said extracting is by water extraction.

14. The method of claim 13, wherein said water extraction is by boiling said agricultural waste in water and centrifuging to collect a supernatant which comprises said reducing sugars, said supernatant being a first extract, optionally wherein said boiling and centrifuging is repeated 2-5 times, such that extracts resultant of said repeating comprise decreasing amounts of reducing sugars.

15. A sterile composition comprising a reducing sugar extract of agricultural waste, optionally comprising salt, nitrogen source, micro elements, macro elements and / or an antifoaming agent.

16. The sterile composition of claim 15, wherein said nitrogen source is corn steep liquor.

17. The sterile composition of claim 15, wherein said microelements are selected from the group consisting of magnesium, iron and phosphorous.

18. The composition of any one of claims 15-17, wherein said salt is selected from the group consisting of magnesium sulfate, sodium chloride, ammonium sulfate, monopotassium phosphate and calcium chloride.

19. The composition of any one of claims 15-18, wherein said salt is selected from the group consisting of magnesium sulfate and sodium chloride.

20. The composition of any one of claims 15-19, wherein said agricultural waste is selected from the group consisting of fruit waste, molasses, fruit pomace, sugarcane bagasse juice,whey, ripe banana peels, vegetable waste, sweet potato residues and juice pressings or residues from sugar-rich crops, and sugar beet pulp.

21. The composition of any one of claims 15-20, wherein said fruit waste comprises dates, optionally wherein said dates are overripe or damaged.

22. The composition of any one of claims 15-19, wherein said agricultural waste is selected from the group consisting of straw, corn stover, sugarcane bagasse, wood chips, rice husks, wheat bran, cottonseed hulls, peanut shells, coconut husks, coffee pulp, coffee husks, and palm kernel cake.

23. The composition of any one of claims 15-22 obtainable according to the method of any one of claims 2-14.

24. The composition of any one of claims 15-23, wherein a concentration of reducing sugars in said extract is 2 % to 20 %.

25. The composition of any one of claims 15-23, wherein a concentration of reducing sugars in said extract is 2 % to 15 %.

26. The composition of any one of claims 15-23, wherein a concentration of reducing sugars in said extract is 5 % to 8 %.

27. The composition of any one of claims 15-23, wherein a concentration of reducing sugars in said extract is 10 % to 18 %.

28. A method of producing a biomass of a microorganism of interest, the method comprising culturing said microorganism under conditions which allow expansion of said microorganism and wherein said conditions comprise the composition of any one of claims 15-27 as a carbon source.

29. The method of claim 28, wherein the composition is the sole carbon source in the method.

30. The method of any one of claims 28-29, wherein said conditions comprise reducing sugars concentration of 0.5-2.5 %.

31. The method of any one of claims 28-29, wherein said conditions comprise about 10 % dissolved oxygen.

32. The method of any one of claims 28-31, wherein said conditions comprise pH of about 4-6, e.g., 5.

33. The method of any one of claims 28-32, wherein said conditions comprise batch fermentation, fed fermentation and / or continuous fermentation.

34. The method of any one of claims 28-33, further comprising harvesting the microorganism.

35. The method of any one of claims 28-34, wherein said conditions ensure culturing of no more than 3 species of microorganisms.

36. The method of any one of claims 28-35, further comprising monitoring during said culturing at least one of: reducing sugar concentration, amino nitrogen concentration, pH and oxygen level.

37. A microbial preparation obtainable according to the method of any one of claims 34-36.

38. The microbial preparation of claim 37, wherein said microorganism is viable.

39. The microbial preparation of any one of claims 37-38, wherein said microorganism is non-viable.

40. The microbial preparation of any one of claims 37-39, wherein said microorganism is intact.

41. The preparation of any one of claims 37-40, wherein said microorganism is selected from the group consisting of yeast, fungi, bacteria and algae.

42. The preparation of any one of claims 28-40, wherein said yeast is of the genus Pichia.

43. The preparation of any one of claims 28-40, wherein said yeast is of the species Pichia kudriavzevii.

44. The preparation of any one of claims 28-40, wherein said yeast is of the strain of PY1 P. kudriavzevii which comprises a genomic signature of SEQ ID Nos. 1-8.

45. The preparation of any one of claims 28-40, wherein said yeast is deposited under the Budapest Treaty in the ATCC® Patent Depository under PTA-127830.

46. An animal feed comprising the microbial preparation of any one of claims 1, 37-45.

47. A method of increasing body weight of an animal comprising feeding the animal with an effective amount of the feed of claim 46.

48. A method of growing an animal species, comprising feeding the animal with an effective amount of the feed of claim 46.

49. A method of supporting survival of an animal comprising feeding the animal with an effective amount of claim 46.

50. The method of any one of claims 47-49, wherein said animal is an aquatic species.

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