Feed ingredient comprising lysed microbial cells

Abstract

The present disclosure relates to feed ingredients and formulated feeds, methods for their manufacture, and their use in nutritional applications such as aquaculture, terrestrial animal feed, and human nutrition. The feed ingredient composition comprises lysed microbial cells having a small aspect ratio and a triglyceride oil.

Description

[0001] This application is a divisional application of an application with an international application number of PCT / US2017 / 040087, a national application number of 201780036267.8, and an invention name of “Feed ingredient containing lysed microbial cells” filed on June 29, 2017.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 357,829, filed on July 1, 2016, and entitled “FEED INGREDIENTS CONTAINING LYSED MICROBIAL CELLS,” and to U.S. Provisional Patent Application No. 62 / 408,630, filed on October 14, 2016, and entitled “FEED INGREDIENTS CONTAINING OXIDATIVELY STABLE UNLYSED AND LYSED MICROBIAL CELLS,” and to U.S. Provisional Patent Application No. 15 / 636,506, filed on June 28, 2017, and entitled “FEED INGREDIENTS CONTAINING LYSED MICROBIAL CELLS,” under 35 U.S.C. §120. CELLS), each of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to a feed ingredient composition, a transport or storage container, a method for preparing a feed ingredient composition, a method for preparing a formulated feed and a formulated feed. Background Art

[0005] Triglyceride oils produced by microorganisms and plants provide essential nutrients for consumption by higher organisms in the food chain. These triglyceride oils are composed of certain fatty acids that are not found or are produced in lower amounts in higher organisms.

[0006] Of particular nutritional importance in the food chain are triglyceride oils containing high levels of polyunsaturated fatty acids (PUFAs) produced by microorganisms and plants. Polyunsaturated fatty acids include long-chain ω-3 fatty acids, such as docosahexaenoic acid (DHA). DHA is an important component in human nutrition, especially for infants. Aquatic animals (such as fish and aquatic shellfish) also need DHA for proper development and growth in their diets. In addition, feeding DHA to newborn livestock such as pigs, cows and other mammals has increased the survival rate of piglets, calves, kid goats and other newborn mammals.

[0007] Today, the primary source of commercial long-chain omega-3 fatty acids is fish oil. Approximately 1 million metric tons of fish oil are produced annually, primarily for aquaculture feed applications, terrestrial animal feed, and human nutrition. Aquaculture is growing, but the availability of long-chain omega-3 fatty acids from wild-caught fish has not increased in line with demand. Continued availability depends on sustainable fisheries management policies, the productivity of natural systems sensitive to climate change, and other factors. Many countries have strict quotas for wild-caught fish. Summary of the Invention

[0008] In one embodiment, a feed ingredient composition is provided comprising a dispersion of lysed microbial cells in triglyceride oil, wherein:

[0009] a) 5% to 90% by weight of the composition are lysed cells, and

[0010] b) 10% to 90% by weight of the composition is triglyceride oil,

[0011] wherein the triglyceride oil comprises oil from lysed cells and oil from another organism.

[0012] In some embodiments, the triglyceride oil has a fatty acid profile of 10-70% docosahexaenoic acid (DHA) by weight of fatty acids, 15-65% DHA by weight of fatty acids, 20-60% DHA by weight of fatty acids, 25-55% DHA by weight of fatty acids, 30-55% DHA by weight of fatty acids, or 40-55% DHA by weight of fatty acids.

[0013] In some embodiments, DHA is 4%-45%, 4%-40%, 4%-35%, 4%-30%, 4%-25% by weight of the composition. In other embodiments, DHA is 4%-20%, 4%-15%, 5%-15%, 5%-12%, 5%-10%, 5%-7%, 5%-8%, 6%-8%, or 6%-7% by weight of the composition.

[0014] In some embodiments, the compositions provided herein comprise 5-15%, 5-10%, 10-15%, 10-25%, 15-20%, or 20-25%, 30-40%, 30-50%, 30-60%, 30-70%, 30-80%, 30-90% lysed microbial cells by weight.

[0015] In some embodiments, the compositions provided herein further comprise less than 20%, 15%, 10%, 5%, 3%, or 1% by weight of unlyzed microbial cells or wherein the composition contains no unlyzed microbial cells. In some embodiments, the composition contains no unlyzed microbial cells.

[0016] In some embodiments, the oil from the lysed microbial cells has a fatty acid profile of 40-70%, 40-50%, 40-50%, 45-55%, or 50-55% DHA by weight of fatty acids.

[0017] In some embodiments, the lysed cells have an aspect ratio of less than 1:1. In other embodiments, the lysed cells have an aspect ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the lysed cells have an aspect ratio of 1:2-1.5, 1:2-1:4, or 1:2-1:3. In one embodiment, the lysed cells have an aspect ratio between 1:1 and 1:5, between 1:5 and 1:10, between 1:10 and 1:15, or between 1:15 and 1:20.

[0018] In some embodiments, the lysed cells have a median particle size of from 1 micron to 20 microns, 1 micron to 18 microns, 1 micron to 15 microns, 1 micron to 12 microns, 1 micron to 10 microns, 1 micron to 9 microns, 1 micron to 8 microns, 1 micron to 7 microns, 1 micron to 6 microns, 1 micron to 5 microns, 1 micron to 4 microns, 1 micron to 3 microns, 5 microns to 100 microns, 5 microns to 90 microns, 5 microns to 80 microns, 5 microns to 80 microns, 5 microns to 70 microns, 5 microns to 60 microns, 5 microns to 50 microns, 5 microns to 40 microns, 5 microns to 30 microns, 5 microns to 20 microns, or 5 microns to 10 microns.

[0019] In some embodiments, greater than half of the lysed cells remain suspended in the composition without settling for at least one week.

[0020] In certain embodiments, the oil from another organism is an oil from fish, plant, oleaginous microorganism, or a combination thereof. These oils include those extracted and separated from another organism. In certain embodiments, the another microorganism is a microalgae, fungus, or yeast.

[0021] In some embodiments, the oil from a plant is coconut, corn, cottonseed, olive, palm, peanut, rapeseed, canola, safflower, sesame, soybean, soybean oil, nut oil, camelina oil, or citrus oil, or one or more combinations thereof.

[0022] In some embodiments, the oil from a fish is from a guppy, a grass carp, a herring, a pilchard, a sardine, or a mackerel or one or more combinations thereof.

[0023] In some embodiments, the microbial cell is from the family Thaustochytriacae.

[0024] In some embodiments, the microbial cell is from a genus selected from the group consisting of Crypthecodinium, Blastocystis, Thraustochytrium, and Schizochytrium.

[0025] In some embodiments, the microbial cell is adapted to grow under low-chlorine conditions.

[0026] In some embodiments, the composition provided herein further comprises lecithin.

[0027] In some embodiments, the composition provided herein further comprises a dietary supplement such as a micronutrient. Dietary supplements include astaxanthin, carotenoids, flavonoids, sterols, calcitriol (vitamin D), tocopherols (vitamin E), and phylloquinone and menaquinone (vitamin K). Other dietary supplements include antibiotics, antifungals, antiparasitics, and hormones. Astaxanthin, carotenoids, and other dietary supplements can be added in the form of microbial biomass. For example, yeast, bacteria, fungi, microalgae, or other microorganisms that produce astaxanthin, carotenoids, and other micronutrients can be added to the composition.

[0028] In some embodiments, the composition provided herein further comprises an antioxidant. In other embodiments, the antioxidant is a natural antioxidant, lecithin, starch, ascorbic acid, tocopherols, rosemary extract, green tea extract, ascorbyl palmitate, butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), ethoxyquin, or one or more combinations thereof.

[0029] In some embodiments, a shipping or storage container comprising the composition provided herein is provided. In other embodiments, the container is a 55-gallon drum or tote.

[0030] In some embodiments, a method of making the food ingredient composition provided herein is provided, the method comprising:

[0031] a) blending the microbial cell with the oil from another organism to form a blend;

[0032] b) lysing the microbial cells in the blend to form the composition as a dispersion.

[0033] In some embodiments, a method of preparing a formulated feed is provided, the method comprising contacting a composition provided herein with an edible food.

[0034] In some embodiments, a formulated feed comprising a composition provided herein and an edible food is provided.

[0035] In some embodiments, an edible food is coated with the composition. In other embodiments, the edible food is coated with the composition under vacuum.

[0036] In some embodiments, the edible food is an aquaculture or animal feed. In other embodiments, the edible food is a salmon feed. In other embodiments, the edible food comprises fish or animal byproducts or combinations thereof.

[0037] In some embodiments, the formulated feed is in the form of pellets.

[0038] In some embodiments, the formulated feed contains at least 1%, 1.5%, or 2% omega-3 fatty acids.

[0039] In some embodiments, the composition provided herein represents at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% by weight of the formulated feed.

[0040] In some embodiments, the slurry of the present invention is no longer pyrophoric. Microbial cells containing high amounts of PUFAs are oxidatively unstable. Storage and transportation of microbial cells containing high amounts of PUFAs can be problematic because of their tendency to be pyrophoric. A dispersion of lysed microbial cells in triglyceride oil does not become pyrophoric when subjected to temperatures between 50°C and 150°C. In some embodiments, combustion of the composition or dispersion of lysed microbial feed ingredient does not occur for at least 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours when the composition or feed formulation is subjected to 80°C. The slurry of the present invention is less costly to ship and insure because it is not pyrophoric.

[0041] The slurries of the present invention also minimize the volume required to transport a given amount of microbial cells. Uncracked microbial cells have a low bulk density. The dispersion of cracked cells and triglyceride oil significantly increases the bulk density of the solid (eliminating the air spaces between the aggregates), allowing almost twice as much biomass to fit into the same space; thus resulting in reduced shipping and storage costs. In addition, the dispersions of the present invention are more easily isolated from oxygen exposure. Drying biomass with nitrogen purging oversized bags is difficult and requires large amounts of nitrogen and is difficult to achieve low levels of residual oxygen. In contrast, purging a small headspace in a tote containing the slurries of the present invention is easily accomplished and has far less nitrogen.

[0042] The compositions or feed ingredients provided herein comprise at least one or more antioxidants selected from the group consisting of lecithin, starch, ascorbic acid, tocopherol, rosemary extract, green tea extract, ascorbyl palmitate, BHT, TBHQ, and PWL. In some embodiments, the two or more antioxidants delay or inhibit combustion of biomass when subjected to temperatures between 50° C. and 150° C. In some embodiments, combustion of the composition or feed ingredient does not occur for at least 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours when the composition or feed formulation is subjected to 80° C.

[0043] In some embodiments, a composition is provided comprising microalgae cells of the genus Crypthecodinium, Chytridium, Thraustochytrium, or Schizochytrium and two or more antioxidants selected from the group consisting of lecithin, starch, ascorbic acid, tocopherol, rosemary extract, green tea extract, ascorbyl palmitate, BHT, TBHQ, and PWL. When the composition or feed formulation is subjected to 80° C., the microalgae cells of the composition do not burn for at least 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, or 20 hours.

[0044] In some embodiments, a method of increasing body weight in an animal is provided, comprising feeding the animal a feed coated with a dispersion of lysed microbial cells in triglyceride oil, wherein the lysed cells have an aspect ratio of less than 1:1.

[0045] A dispersion of lysed microbial cells in triglyceride oil contains from 5% to 90% lysed cells and between 10% and 95% triglyceride oil by weight, wherein the triglyceride oil comprises oil from the lysed microbial cells and oil from another organism. For example, a slurry made with 50 g of Schizochytrium cells containing 50% lipid and 50 grams of canola oil would have a total of 75 g of oil (50 g canola oil and 25 g Schizochytrium oil).

[0046] In some embodiments, a method for increasing the thermal growth coefficient (TGC), feed intake (FI), or feed efficiency (FE) of an animal is provided. The method comprises feeding the animal a feed coated with a dispersion of lysed microbial cells in a triglyceride oil, wherein the lysed cells have an aspect ratio of less than 1:1. The method can increase the TGC, FI, and FE of the animal.

[0047] In some embodiments, a method for reducing the amount of docosahexaenoic acid in animal feces is provided, wherein the method comprises feeding the animal a feed coated with a dispersion of lysed microbial cells in triglyceride oil, wherein the lysed cells have an aspect ratio of less than 1:1.

[0048] In some embodiments, a method of increasing protein deposition (PD), lipid deposition (LD), docosahexaenoic acid deposition (DHAD), or eicosapentaenoic acid (EPAD) in an animal is provided. The method comprises feeding the animal a feed coated with a dispersion of lysed microbial cells in a triglyceride oil, wherein the lysed cells have an aspect ratio of less than 1:1. The method can increase the animal's PD, LD, DHAD, or EPAD. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shown are the bulk density percentages as a function of particle size (μm) for the canola oil and biomass mixture of Example 2 and the dispersion resulting from subsequent homogenization or bead milling of the mixture.

[0050] Figure 2 Micrographs of the mixture and the dispersion of Example 2 are shown.

[0051] Figure 3 The viscosity as a function of shear rate is shown for the mixture and the Example 2 dispersion.

[0052] Figure 4 The appearance of the mixture and the Example 2 dispersion after 1 week is shown.

[0053] Figure 5 OxiPress values ​​(hours) for the biomass and / or antioxidants of Example 3 are shown.

[0054] Figure 6 The relationship between dietary DHA levels and DHA deposition efficiency (eDHAD) for Example 5 is shown.

[0055] Figure 7 Shown are the changes in DHA content of salmon fillets (skinless) during the study period. Each data point represents the average of two samples, and each sample consisted of fillets from three fish pooled in each tank. DETAILED DESCRIPTION

[0056] definition

[0057] "Aspect ratio" refers to the ratio of the width to the height of a lysed or unlysed cell.

[0058] "Dietary additives" are ingredients added to feed ingredients to provide micronutrients or other compounds to improve the yield or quality of animal products. Dietary additives are typically oil-soluble compounds, but can be water-soluble compounds. Dietary additives include, but are not limited to, astaxanthin, carotenoids, flavonoids, sterols, calcitriol (vitamin D), tocopherol (vitamin E), and phylloquinone and menadione (vitamin K). Astaxanthin is used in aquaculture to enhance the color of products such as salmon and trout. Other dietary additives include antibiotics, antifungals, and antiparasitic drugs for protecting animal health, as well as hormones to increase the growth rate and size of animals. The feed ingredient composition may further include yeast, bacteria, fungi, microalgae, or other microorganisms that produce astaxanthin, carotenoids, and other micronutrients.

[0059] "Dispersion" refers to a solid in oil mixture in which the solid is visible to the naked eye without the aid of magnification.

[0060] " fatty acid profile " is the distribution of acyl groups in the triglyceride of oil, and does not relate to the attachment with the glycerol main chain. Fatty acid profile is typically by being converted into fatty acid methyl ester (FAME), then carries out gas chromatography (GC) analysis with flame ionization detection (FID) and determines. Fatty acid profile can be expressed as one or more percentages of fatty acid in the total fatty acid signal determined by the area under the curve of this fatty acid. FAME-GC-FID measures the weight percent that is similar to fatty acid.

[0061] "Fatty acid" in the context of triglyceride oils refers to the fatty acyl portion of the triglyceride. Thus, it should be understood that the fatty acyl groups of triglycerides can be described in terms of the carboxylic acids produced when the triglycerides are hydrolyzed or saponified.

[0062] "Feed ingredient" means a substance added to other ingredients or foods to make or modify food.

[0063] “Feed efficiency” (FE) is the increase in body weight (mass) of an animal per amount of feed consumed.

[0064] “Feed intake” (FI) is the amount of feed consumed by an animal during a defined period of time.

[0065] “Food,” “edible food,” “feed,” “formulated feed,” and “finished food” refer to a product having some nutritional value suitable for uptake by a living organism.

[0066] “Lipid deposition” (LD) is the increase in lipid content of an animal in milligrams per degree day (mg° / day). Lipid deposition efficiency (eLD) is the percentage of lipid fed to an animal that ends up as the lipid content of the animal. “Docosahexaenoic acid deposition” (DHAD) is the increase in DHA content of an animal in milligrams per degree day (mg° / day). “Docosahexaenoic acid deposition” efficiency (eDHAD) is the percentage of docosahexaenoic acid fed to an animal that ends up as the DHA content of the animal. “Eicosapentaenoic acid deposition” (EPAD) is the increase in EPA content of an animal in milligrams per degree day (mg° / day). “Eicosapentaenoic acid deposition” efficiency (eEPAD) is the percentage of eicosapentaenoic acid fed to an animal that ends up as the lipid content of the animal.

[0067] “Low-chloride” refers to growth conditions in which the amount of chloride is lower than the salinity of a marine environment.

[0068] “Lyse,” “Lysing,” “lysis” means disrupting or breaking apart the cell wall or membrane of a cell sufficient to release at least some of the intracellular contents.

[0069] “Lysed” cells or “broken” cells are those in which the cell wall and / or membrane has been broken. Upon lysis, the cell contents, including the triglyceride oil, are partially or completely released from the cell. Upon disruption of the cell wall and / or membrane, some portion of the intracellular contents, including the triglyceride oil, can remain within the disrupted cell wall or membrane.

[0070] “Microbial cell” refers to a unicellular microorganism. Unicellular microorganisms include eukaryotic microbial organisms. Microbial organisms include those capable of photosynthesis as well as heterotrophs capable of living on fixed carbon sources only.

[0071] “Oil-producing” cells, microbes, or microorganisms are capable of producing at least 20% lipid by dry cell weight, either naturally or by recombinant or classical strain improvement. Oil-producing cells and microbes include those such as microalgae, fungi, and yeasts.

[0072] "Protein deposition" (PD) is the increase in the protein content of an animal in degree days (mg°C / day). Protein deposition efficiency (ePD) is the percentage of protein fed to an animal that ends up being the animal's protein content.

[0073] A "slurry" or "microalgae slurry" is a dispersion of lysed microbial cells in a triglyceride oil. The slurry contains lysed microalgae cells having an aspect ratio of less than 1:1. The slurry can be top-coated, vacuum-coated, or spray-coated onto the solid feed. The solid feed can be prepared by pelleting, extrusion, molding, or by using other known methods for preparing solids.

[0074] The "thermal growth coefficient" (TGC) is a mathematical coefficient that describes the growth of aquatic species, accounting for variations in growth patterns that occur during the life stages of an animal.

[0075] A "triglyceride molecule" refers to a single triglyceride composed of three fatty acids attached as esters to a glycerol backbone. A "triglyceride oil" refers to a collection of different triglyceride molecules that differ in the nature and proportions of the different fatty acids and how the different fatty acids are attached to the glycerol backbone relative to each other.

[0076] In some embodiments, the microbial cells contain a DHA-rich triglyceride oil. A commercial source of DHA-rich oil is obtained from a species from the genus Schizochytrium, where sp. indicates that the species is unidentified. Such cells can be prepared by heterotrophic fermentation, as described by Barclay in US Pat. Nos. 5,130,242, 5,340,742, and 5,340,594.

[0077] Most commercial processes for producing DHA cells involve the use of defined culture media, industrial aerobic fermentation vessels, defined operating parameters (such as pH / temperature / salt levels), and double drum dryers to produce a characteristic fine flaky powder.

[0078] Seawater contains about 0.55M chloride. Chloride ions cause corrosion of stainless steel equipment in industrial environments. It is advantageous to minimize the amount of chloride in the fermentation medium and other liquids used in cultivation and processing to minimize corrosion. Provided herein are marine organisms that have been adapted to grow under conditions where the chloride concentration is less than 0.55M. Low chloride conditions of the present invention are 300mM to 500mM chloride, 100mM to 300mM chloride, 50mM to 100mM chloride, 1mM to 75mM chloride, 1mM to 50mM chloride, 1mM to 40mM chloride, 1mM to 30mM chloride, 1mM to 20mM chloride, 1mM to 15mM chloride, 1mM to 10mM chloride, 0.5mM to 10mM chloride, 0.5mM to 7.5mM chloride, or 0.5mM to 5mM chloride.

[0079] By first optionally making fermentation liquid dehydration (concentrating fermentation liquid), the drying of microbial cells in aqueous fermentation liquid can be achieved to increase the cell content of fermentation liquid. Dehydration or concentration refers to separation of biomass from fermentation liquid or other liquid media, and solid-liquid separation is also the same. Therefore, during dehydration, substratum is removed from biomass (for example, by draining fermentation liquid through a filter retaining biomass), or otherwise biomass is removed from substratum. The common method of dehydration comprises centrifugation, filtration and use mechanical pressure. These methods can be used alone or in any combination.

[0080] Following the optional dehydration step, the concentrate, now having a higher solids content, can be dried by known drying methods including, but not limited to, drum drying, pneumatic drying, spray drying, freeze drying, and other drying methods.

[0081] The drum dryer operates by applying a thin film of fermentation liquid (or dehydrated fermentation liquid) to the surface of a rotating heated drum. The aqueous portion of the fermentation liquid evaporates, leaving a dry solid on the drum surface. The dry solid is then scraped off the drum with a knife. The pneumatic dryer draws or inhales the material to be dried in a stream of hot air. When the material is inhaled in the hot air, moisture is quickly removed. The dried material is then separated from the moist air, and the moist air is then recycled for additional drying. The spray dryer operates by spraying the fermentation liquid (or dehydrated fermentation liquid) in a dispersion of fine droplets into a heated air stream. The inhaled material dries quickly and forms a dry powder. Spray drying can be accomplished by a box dryer, or a high-profile spray dryer, a fluidized bed dryer, or a mobile fluidized bed dryer (e.g., Spray dryer, GEA Process Engineering, Inc.

[0082] The dried powder can be mixed with oils extracted from other organisms. The oil can be a pure oil that is substantially free of solid materials. The oil can be, for example, an oil extracted from a plant oil, a fish oil, or a combination thereof. The oil is primarily a triglyceride oil. Fish oils rich in DHA include oil from wild anchovies in South America and herring from the Northern Hemisphere. The oil mixed with the dried powder can also include oil extracted from a thraustochytrid fungus. In some cases, the oil is primarily a plant oil and contains less than 50%, 40%, 30%, 20%, or 10% fish oil.

[0083] The mixed powder and oil can be ground or other methods used to lyse the microbial cells. Lysing the cells allows the triglyceride oil from the cells to be released into the feed ingredient composition. Mechanical lysis can be performed by various well-known methods (such as by roller mills, homogenizers or bead milling).

[0084] The degree of cell destruction can be determined by microscopic analysis. The percentage of lysed cells can be determined by observing and counting the number of lysed and unlysed cells after cell lysis. In specific embodiments, the lysed cells of the present invention are greater than 50%, 60%, 70%, 80%, 90% or 95% lysed.

[0085] The cracking or destruction of cells can be accomplished by mechanical, enzymatic, chemical, viral, electrical, ultrasonic, osmotic or other methods. Pressure breaker (such as high pressure homogenizer) can be used for cracking cells. Pressure breaker is by pumping the mixture of cells and oil (such as canola oil or other vegetable oils) through the restricted orifice valve cracking cell with cracking cells. Apply high pressure (from 50 bar to 1500 bar), then expand immediately by the nozzle leaving. Niro (Niro SoaviGEA) homogenizer (or any other high pressure homogenizer) or other commercially available homogenizers can be used for processing cells into particles with a length of 5 microns to 500 microns. The aspect ratio of the cracked cells is reduced to 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 from the value of about 1:1 of the uncracked cells. Treatment of biomass with a high pressure homogenizer can produce cell lysis exceeding 50%, 60%, 70%, 80%, 90%, 95% or greater than 95% of the cells by controlling pressure, outlet velocity and other parameters.

[0086] Alternatively, a ball mill (also referred to as a bead mill) can be used. In a ball mill, cells are stirred in a suspension with small abrasive particles (such as beads). Cells are broken due to shear force, grinding between beads, and collision with beads. The beads destroy cells to release cell contents. Dyno-mill ECM Ultra (CB Mills) ball mills and other commercially available bead mills can be used. Cells can also be destroyed by shear force, such as using blending (such as using high speed or Waring blender as an example), French press (French press) or even centrifugal to destroy cells. Shear mixers can be used for cell lysis, including linear high shear mixers and bulk high shear mixers.

[0087] The aspect ratio of the lysed cells is less than 1:1. In another embodiment, the aspect ratio is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. The aspect ratio of the unlysed cells is about 1:1. One advantage of lysed cells is that they have an aspect ratio of less than 1:1. A smaller aspect ratio is advantageous because when the lysed cell / oil slurry is used in industrial equipment, the lysed cell / oil slurry prevents or minimizes clogging of the equipment, including the orifice used to spray the lysed cell / oil slurry onto the extruded feed.

[0088] The feed ingredient composition can then be formulated with other feed ingredients to produce a formulated feed. In some embodiments, the feed ingredient composition containing the lysed cells is applied to a compressed or extruded feed, such as by spraying. The compressed or extruded feed can be high in protein, such as those derived from fish and / or animal by-products. The formulated feed can also include additional additives to improve flavor, stability, and shelf life.

[0089] Formulated feeds can be used in aquaculture to feed farmed fish or shellfish. Farmed fish include carnivorous fish. In some embodiments, the farmed fish or shellfish are salmon, eels, crustaceans, marine fish, freshwater fish, tilapia, or eels. In other embodiments, the fish or shellfish are sea bass, sea bream (seabrim), yellowtail, grouper, barramundi, or shrimp.

[0090] Examples

[0091] Example 1. Food ingredients

[0092] DHA-enriched Schizochytrium cells (biomass) prepared by standard heterotrophic fermentation were dried and blended with canola oil using a standard impeller mixer at loading levels of 10%, 20%, and 30% by weight. The biomass in the oil blend was then processed through a high-pressure homogenizer or bead mill to produce a dispersion of lysed algal cells in the oil. For lysis in the high-pressure homogenizer, a pressure of 200 to 1200 bar was used, sufficient to lyse the cells.

[0093] Example 2. Analysis of mixtures and dispersions prepared from the mixtures

[0094] A 20% by weight blend of biomass blended with the canola oil of Example 1 was prepared and analyzed by laser diffraction particle size analysis on a Malvern Mastersizer 3000. The mixture was also homogenized at 400 bar and bead milled, and the resulting dispersion was also analyzed. Figure 1 It is shown that the bead milled dispersion has a lower average particle size than the homogenized dispersion, and both have a lower average particle size than the masterbatch.

[0095] exist Figure 2 Smaller sizes can also be seen in the micrographs of the mixture (magnification 400x). The Dv50 value (distribution value 50% or less) of the mixture was 228 μm, while the homogenized dispersion had a Dv50 of 15.3 μm and the bead milled dispersion had a Dv50 of 3.8 μm, indicating that the dispersion had better characteristics for use in the nebulizer.

[0096] Measured using a Malvern Kinexus professional rheometer Figure 3 It was found that at any given shear rate, the bead milled dispersion had a lower viscosity than the homogenized dispersion (which in turn was lower than the mixture).

[0097] The mixture and both dispersions were allowed to settle and their appearance after one week was shown in Figure 4 The bead milled dispersion was found to remain essentially in suspension. The mixture showed the greatest degree of sedimentation.

[0098] Example 3. Oxidative Stability Analysis

[0099] The dried biomass of Example 1 was combined with various antioxidants using a hand blender. The dried biomass with and without antioxidants was analyzed using an Oxipress at approximately 80° C. Natural and synthetic antioxidants were used and included L-α-phosphatidylcholine (soy, Calbiochem #429415), starch (corn, Argo), ascorbic acid (Spectrum Chemical Co.), Paradigm Ox white liquid (PWL-gallate, Kemin Health), RPT40 (rosemary, tocopherol, ascorbyl palmitate, Kemin Health), NaturFort LGR105 (green tea and rosemary extract, Kemin Health), Rendox CQ (TBHQ, Kemin Health), Rendox EQ (ethoxyquin, Kemin Health), BHT (crystallized in ethanol, Kemin Health), and Covi-ox T-30P (mixed tocopherols). Figure 5 Surprising and unexpected oxidative stability of certain biomass-containing lecithins is demonstrated.

[0100] When prepared with the antioxidant RPT40 (4,000 ppm) and lecithin (1%), the oxidative stability of the biomass at 80°C was approximately 22 hours. This was surprising and unexpected, as the oxidative stability of the biomass was approximately 16 hours when formulated with 4,000 ppm RPT40 alone, and approximately 1.5 hours when formulated with 1% lecithin alone. When the biomass was formulated with RPT40 and lecithin, the stability was 22 hours, far exceeding the predicted stability of 17.5 hours.

[0101] Example 4. Feed preparation

[0102] The slurry of Example 1 was top coated onto the extruded feed pellets using vacuum coating equipment. The resulting pellets contained 20% by weight of the dispersion of Example 1.

[0103] Example 5. Salmon breeding study

[0104] Four aquaculture diets were formulated to contain 2.0% DHA and a DHA to EPA ratio between 2.1 and 2.5. Table 1 below discloses the ingredients and amounts of each ingredient for diets A, B, C, and D. Table 2 below discloses the nutritional content of the four diets. Diet A (control) did not contain Schizochytrium biomass but used standard fish oil as a topcoat as the primary dietary source of DHA (along with fish meal containing low levels of DHA). Diet B contained un-cracked Schizochytrium biomass. For Diet B, after feed extrusion, the pellets were top-coated with canola oil and herring oil. Diet C contained bead-milled Schizochytrium biomass, which was mixed with canola oil and vacuum-coated onto extruded salmon feed along with the other oils shown in Table 1. Diet D contained Schizochytrium biomass, which was homogenized at 400 bar and mixed with canola oil and vacuum-coated onto extruded salmon feed along with the other oils shown in Table 1. The final DHA level was lower than expected, at 1.5% instead of 2%. The discrepancy between the calculated and observed dietary DHA content may be due to the lower than usual DHA level in one of the fish oils. Table 2 shows the nutritional content of diets A, B, C, and D. Herring oil was purchased, and typical fatty acid content of herring oil can be found in NRC (2011). The fish oil tuna 70 DHA ingredient was commercially available from Edwards International Inc.

[0105] Table 1: Feed formulations for experimental diets (calculated)

[0106]

[0107]

[0108] Table 2: Calculated nutrient composition of the experimental diets

[0109]

[0110]

[0111] Table 3 shows a comparison between the calculated (Cal.) and analyzed nutritional compositions of the experimental diets. In Table 3, AVC and NJFL refer to the laboratories that performed the analyses. The analyzed crude protein in the experimental diets was 2%-4% higher than the calculated values. In contrast, the analyzed dietary crude lipids, DHA, and EPA were all lower than the calculated values. Slight differences were also observed between the laboratories.

[0112] Discrepancies between calculated and analyzed nutrients can be explained by ingredient combinations that sometimes vary from batch to batch, as well as by ingredient combinations that differ from the compositions specified in the formulation software and uncertainties in the analytical methods.

[0113] Table 3: Comparison between calculated (Cal.) and analyzed nutritional compositions

[0114]

[0115] Rearing conditions: Saltwater, in a recirculating aquaculture system equipped with eight 850-liter circular tanks. Water was maintained at 13.7°C ± 1.3°C and dissolved oxygen saturation >80%. Each experimental diet was randomly assigned to two tanks.

[0116] Sample Collection: Fillets were prepared from nine fish (pooled) at the start, and three fish from each tank on days 28, 56, 84, and 112. Fillets corresponded to Trim E (skinless, trimmed (no belly fat), fins removed) as described on http: / / primanor.com / salmon-fillet-trim-guide / . The only exception was that pin bones were not removed. Pooled fecal material was collected from each treatment group on days 84 and 112.

[0117] Laboratory analysis: Diet (n = 4), feces (n = 8), and fillets of three fish per tank were pooled for protein, lipids, ash, dry matter, and total fatty acid composition. Chemical analyses were performed as follows: dry matter at 105°C for 16 hours (AOAC 930.15), ash at 550°C under nitrogen for at least 6 hours (AOAC 990.03; using a 78 elemental analyzer LECO FP528, St. Joseph, Michigan, USA; crude protein = N × 6.25), and lipids (Bligh & Dyer, 1959). Total fatty acid content of diet and fillets was determined according to McNiven et al. (2011). Briefly, fatty acid methyl esters (FAMEs) were prepared according to the procedure of Sukhija and Palmquist (1988) and analyzed on a Hewlett Packard 5890 gas-liquid chromatograph equipped with a 7673 series autosampler and syringe, an Agilent DB23 fused silica capillary column (30 m × 0.53 mm id × 0.5 m film thickness), an FID detector, and integrated with Agilent Chemstation software (Agilent Technologies Canada Ltd., Mississauga, Canada). The operating conditions were: on-column injection; oven temperature, 70°C for 0.5 min, then 10°C / min to 170°C and hold for 3 min, 5°C / min to 210°C and hold for 6 min, and 25°C / min to 230°C and hold for 4.2 min; detector temperature, 250°C; hydrogen as carrier gas, and nitrogen as make-up gas. Nonadecanoic acid was added as an internal standard, and chromatographic peaks were identified using FAME standards (Nu-Chek-Prep, Elysium, MN, USA; Matreya, Pleasant Pass, PA, USA). Results are reported as mg / 100 mg total fatty acids using published correction factors (Ackman, 2002).

[0118] Computational and statistical analysis:

[0119] Growth rate was calculated using the thermal growth coefficient (TGC):

[0120]

[0121] Where W f and W0 are the final and initial body weights of the fish in g, n (=1, 2, ...) is the number of days recorded from W0, T i (℃) is the average daily water temperature.

[0122] Results from proximate and fatty acid analyses were used to describe the rate of nutrient deposition using the following equation (Dumas et al., 2007):

[0123]

[0124] Among them D j is the deposition rate of nutrient j [mg(℃·d) -1 ], F j and I j are the final and initial whole body weights of nutrient j (mg) at the end and beginning of 84 days, respectively, and n represents the coverage from F j to I j The number of days in the period, T i (℃) is t i The average daily water temperature for the day, with the product being expressed in degree-days.

[0125] In turn, the rate of nutrient deposition was used to estimate the efficiency of nutrient deposition using the following equation modified from Dumas et al. (2007):

[0126]

[0127] Among them, eD j It is a nutrient j Efficiency of deposition (%) [mg (fish) -1 ], IN j (mg) is t i Daily intake of nutrients j.

[0128] One-way ANOVA and Tukey's multiple comparison test were used with The results were analyzed using SAS version 12.0.1 (SAS Institute Inc., Cary, NC, USA).

[0129] 4.0 Results and Discussion: No statistical differences were observed between treatments for initial body weight (P = 0.590), final body weight (P = 0.483), total gastrointestinal calorie count (TGC) (P = 0.387), feed intake (P = 0.588), and feed efficiency (P = 0.484) (Table 4). In addition, dietary treatment did not significantly affect feed intake and feed efficiency (P values ​​varied between 0.44 and 0.90). Growth of salmonids fed diets C (AP-BM) and D (AP-H400) was superior to that of the control group and B (AP). The TGC values ​​observed in this study are comparable to other studies conducted with the same salmonid species (e.g., Wolters et al., 2009). These results demonstrate the sustained growth performance of AlgaPrime included in the salmonid diet and similar feed conversion rates to fish oil.

[0130] Table 4 shows the initial body weight (IBW) and final body weight (FBW) of Atlantic salmon fed diets without AlgaPrime (control), with AlgaPrime powder (AP), with bead-milled AlgaPrime (AP-BM), and with AlgaPrime homogenized at 400 bar (AP-H400). Data are means (standard deviations); means in columns without a common superscript are significantly different (p < 0.05) based on Tukey's test (no superscript indicates no difference). Final body weight of diets B, C, and D when compared to salmon fed the control diet (without AlgaPrime). The thermal growth coefficient increased from 0.176 to 0.193, p < 0.05.

[0131] Table 4

[0132]

[0133] DHA utilization and deposition: The differences observed between treatments in the concentrations of protein (P = 0.626), lipids (P = 0.185), DHA (P = 0.699) and EPA (P = 0.256) in Atlantic salmon fillets in this study are shown in Table 5. Unexpectedly, the highest levels of DHA in fillets were obtained with AlgaPrime homogenized at 400 bar. Fillet composition was not significantly affected by the dietary treatments in this study (Table 5). The protein content in Atlantic salmon fillets (FP) was consistent with the literature (e.g. et al., 1993). However, more variations in lipids and fatty acids were observed. The lipid content (FL) of fillets was lower than (1993) and Acharya (2011), but still within the range reported by the FAO (http: / / www.fao.org / wairdocs / tan / x5916e / x5916e01.htm). Compared to Atlantic salmon fillets from Chile and Canada purchased at local grocery stores, the DHA (FDHA) and EPA (FEPA) contents in this study were nearly 5x and 2x higher, respectively (Appendix 2). In contrast, DHA+EPA levels in Atlantic salmon purchased at local grocery stores were at least 2x higher than in our studies (e.g., Kousoulaki et al., 2015; Sprague et al., 2016). The DHA and EPA contents of fillets in the Kousoulaki et al. (2016) study were comparable to those in the present study, at 0.7% and 0.1%, respectively. These differences may be explained, at least in part, by the fatty acid content of the dietary lipid source and the trimming of the fillets, which can influence lipid and fatty acid results.

[0134] Table 5 shows the protein and lipid content of salmon fillets. Initial and final fillet protein (FP), lipid (FL), docosahexaenoic acid (FDHA), and eicosapentaenoic acid (FEPA) of Atlantic salmon fed diets without AlgaPrime (control), with AlgaPrime powder (AP), with bead-milled AlgaPrime (AP-BM), and with AlgaPrime homogenized at 400 bar (AP-H400). Data are means (standard deviation); means in columns without a common superscript are significantly different based on Tukey's test. Initial samples are salmon fillets before the start of the feeding study (treatments).

[0135] Table 5:

[0136]

[0137] Fecal DHA Content: Fecal DHA content was determined. With the exception of diet B (AP), fecal DHA content was approximately 75X lower than that of the diets, suggesting that most dietary DHA was absorbed by the digestive tract of salmon fed these other diets (Table 6). Fecal DHA content from salmon fed diet B (AP) was significantly higher (P < 0.05) than fecal DHA content from the other treatments. It is possible that DHA availability from AP meal is lower compared to that from the more processed AP, but this hypothesis needs to be verified. Although we did not estimate digestibility coefficients in this study, we have previously demonstrated that DHA digestibility from AP is as high as approximately 95% in a trial (Dumas, 2016).

[0138] Table 6 shows the docosahexaenoic acid (DHA) content in the diet and feces of Atlantic salmon fed diets containing no AlgaPrime (control), containing AlgaPrime powder (AP), containing bead-milled AlgaPrime (AP-BM), and containing AlgaPrime homogenized at 400 bar (AP-H400). Data are means (standard deviations); means in columns without a common superscript are significantly different based on the Tukey test (no superscript indicates no difference). The amount of DHA in feces of salmon fed diet B was different, p < 0.05. Therefore, DHA provided to salmon in the form of a lysed algal slurry resulted in better absorption by salmon than when provided to salmon as uncleaved algal biomass incorporated into dry feed.

[0139] Table 6

[0140]

[0141] 1 No repetition of the diet was performed.

[0142] Salmon fillets fed diets B and C increased DHA levels more rapidly in the first month compared to other treatments Figure 1 ). At day 28, the DHA content of salmon fillets fed diet D was 0.27% and 0.45% in samples 1 and 2, respectively. The 0.27% sample can be considered an outlier. In this case, the DHA content in the fillets was the same between diet D and the control. Overall, the highest DHA levels were reached between one and two months of the study and levels remained relatively similar for salmon fed AP containing diets thereafter. See Figure 7

[0143] Protein, DHA and EPA deposition: The rate of protein, DHA and EPA deposition in Atlantic salmon fillets was increased when compared to fish fed fish oil (Table 7). The p values for the rate of protein, DHA and EPA deposition were P=0.417, P=0.639 and P=0.197, respectively. The highest rate of DHA deposition was observed with AlgaPrime that was homogenized at 400 bar, which is consistent with the highest DHA content reported in salmon fillets fed the same treatment (Table 5 above). The lowest DHA deposition was recorded in the case of diet B (AP), which also resulted in the highest DHA content in feces, as reported in Table 6. It can therefore be reasonably concluded that DHA from AP powder (diet B) was less available. Lipid deposition (LD) in salmon fillets fed AlgaPrime powder (diet B) was lower than the control (fish oil) and AlgaPrime treated at 400 bar (Table 7). This difference can be explained by a lower lipid digestibility in AlgaPrime powder compared to homogenized AlgaPrime.

[0144] The deposition rates of protein (PD), lipid (LD), docosahexaenoic acid (DHAD) or eicosapentaenoic acid (EPAD) in fillets (skinless, trimmed) of Atlantic salmon fed diets without AlgaPrime (control), with AlgaPrime diet (AP), with AlgaPrime bead mill (AP-BM), and with AlgaPrime homogenized at 400 bar (AP-H400) are shown in Table 7. Data are means (standard deviations); means in columns without a common superscript are significantly different based on Tukey's test (no superscript indicates no difference). Both homogenized and non-homogenized AlgaPrime increased protein deposition in salmon. In addition, the amount of docosahexaenoic acid and eicosapentaenoic acid deposition increased with homogenized algal biomass. Lipid deposition (LD) in salmon fillets fed AlgaPrime meal (Diet B) was significantly lower (P=0.024) than the control (fish oil) and AlgaPrime treated at 400 bar (Table 5). This difference can be explained by the lower lipid digestibility in AlgaPrime meal compared to homogenized AlgaPrime. Therefore, including algal cells in the salmon diet as a slurry coated on extruded particles significantly increased lipid deposition when compared to direct inclusion of algal cells in aquaculture feeds.

[0145] Table 7

[0146]

[0147] AlgaPrime and fish oil resulted in a higher efficiency of protein (P = 0.397), lipid (P = 0.205), DHA (P = 0.239), and EPA (P = 0.371) deposition (Table 8). The efficiency of DHA deposition (eDHAD) is inversely proportional to dietary DHA content / availability (Roselund et al., 2016; Kousoulaki et al., 2016). The results published herein confirm this observation ( Figure 6 ). Therefore, low eDHAD values ​​can be explained by high levels of available DHA in the diet and do not necessarily mean that one source of DHA is superior to another. The efficiency of nutrient deposition was calculated using the laboratory results of the AVC reported in Table 3.

[0148] Table 8 shows the efficiency of protein (ePD), lipid (eLD), docosahexaenoic acid (eDHAD), or eicosapentaenoic acid (eEPAD) deposition in fillets (skinless, trimmed) of Atlantic salmon fed diets without AlgaPrime (control), with AlgaPrime meal (AP), with AlgaPrime bead mill (AP-BM), and with AlgaPrime homogenized at 400 bar (AP-H400). Data are means (standard deviations); means in columns without a common superscript are significantly different based on Tukey's test (no superscript indicates no difference). Both homogenized and non-homogenized AlgaPrime increased the efficiency of protein deposition. The efficiency of lipid deposition increased with homogenized algal biomass. In addition, the efficiency of eicosapentaenoic acid deposition increased with homogenized algal biomass.

[0149] Table 8

[0150] 7

[0152] Example 6. Low-chlorine-tolerant strains of Schizochytrium

[0153] The presence of high levels of chlorides poses corrosion challenges to industrial equipment, including fermenters, piping, pumps, and other units. Reduction of chlorides offers the benefit of lower corrosion potential in equipment used to cultivate and process DHA-rich biomass.

[0154] The Schizochytrium sp. culture is continuously grown in reduced chloride medium until the population evolves and is able to achieve growth similar to that of the parent strain in the original full chloride medium. Isolates of the evolved population are screened to identify strains that are adapted to culture in medium with reduced chloride levels.

[0155] Schizochytrium strain S9026 was used as a source culture for the evolution of low chloride tolerance. Growth in these cultures was monitored as optical density (OD) by measuring absorbance at 750 nm. S9026 cells (1.5 mL) cryopreserved at -80°C with 20% (w / v) glycerol were thawed at ambient temperature and used to inoculate 50 mL of O3SF25 medium (Tables 9 and 10) containing 8.4 mM chloride in a 250 mL baffled flask. The cell culture was then incubated on a rotary shaker at 28°C and 200 rpm with a 2-inch stroke for 24 hours until the OD reached 0.00. 750The OD values ​​ranged from 3 to 6. 5% of the resulting primary seed culture was used to inoculate 50 ml of modified O3SF25 medium containing 0.25 mM chloride (97% chloride reduction) in a 250 mL baffled flask. The reduced chloride culture was incubated on a rotary shaker at 28°C and 200 rpm until its final OD 750 Reach 3-6. In contrast to the primary culture of S9026 that reached the target growth within 24 hours, the reduced chloride culture required 4-5 days to reach the target growth, indicating that S9026 requires chloride. The resulting cell culture was serially subcultured 4 times in a 250 mL baffled flask into a modified O3SF25 medium with 0.25 mM chloride until its cell growth rate was similar to that of its parent strain S9026 in O3SF25 medium. If a 1%-5% v / v inoculum entering a modified O3SF25 medium containing 0.25 mM Cl is able to reach an OD of 3-6 within 24 hours, the growth rates are estimated to be similar. The final subculture (denoted as S9026-MF5-1-0.25 mM Cl) was plated on GYPS agar, and single colonies were screened for lipid production.

[0156] Table 9. Composition of defined or modified O3SF25 medium

[0157]

[0158]

[0159] Table 10. Composition of DAS3 vitamin stock solution

[0160]

[0161] Table 11. Composition of C-Trace 7 (Trace Metal Stock Solution)

[0162]

[0163] 80 isolates picked from S9026-MF5-1-0.25mM Cl were screened in 50mL bioreactor tubes containing 10mL of modified O3SF25 medium containing 0.25mM chloride as described below. Each isolate was inoculated into 0.8mL of modified O3SF25 medium (0.25mM Cl) in a 96-well block culture format and then incubated on a multitron shaker at 28°C and 900rpm for 20-24 hours to prepare a primary seed culture. The resulting primary seed culture was inoculated into 9.6mL of modified O3SF25 medium (0.5mM chloride) in a 50mL bioreactor tube at 4% (v / v). The production bioreactor tubes were incubated on a rotary shaker at 28°C and 200rpm for 3 days and then harvested for lipid determination. The parent strain S9026 was also cultured in parallel in O3SF25 (8.4mM Cl) as a control.

[0164] From the initial screening, two isolates with good lipid productivity and DHA content in lipids were identified and then cryopreserved as S9179 and S9180, respectively. After cryopreservation, it was confirmed that S9179 and S9180 provided lipid production and DHA content in lipids comparable to the parent strain in reduced chloride medium (modified O3SF25 with 0.5 mM Cl).

[0165] S9179 and S9180 were also evaluated in a 3-L laboratory scale fermentor (Applikon, The Netherlands) using 350 mM NH4, N / P16, which was comparable to that used for the S9026 evaluation. To cultivate S9026, the seed stage typically included cultivation in O3SF25 (8.4 mM Cl) in a flask and inoculated into a production medium containing 10-11 mM Cl at 10% v / v. To evaluate S9179 and S9180, the seed flask stage was cultivated in modified O3SF25 (0.25 mM Cl), and the production medium was modified to reach 1 mM Cl (Table 12). To promote sucrose hydrolysis, 1.5 mL / L of 20 g / L Maxvert invertase solution (filter sterilized) was added at the start of the fermentation.

[0166] The cultures were incubated at 28°C with an aeration rate of 1.0 vvm. Sterilized VHP glycerol (70% w / w) was used as carbon feedstock and a DO response algorithm (pulsed feed on demand to <10 g / L total sugar per injection) was used for feeding. The pH was automatically maintained at 5.1 ± 0.2 by the addition of acid (16% w / w H2SO4) and base (28% w / w ammonium hydroxide followed by 10% w / w NaOH). The dissolved oxygen (DO) level was maintained at >20% of air saturation by automatic control of agitation (500-1250 rpm) followed by pure oxygen supplementation (0-100%). Ammonium sulfate (50 mM NH4+batch) and ammonium hydroxide (300 mM NH4+) were used as nitrogen sources. The broth was sampled as needed to determine DCW, lipid titer and fatty acid composition, total solids, and residual sugar concentration in the broth.

[0167] Table 12. Composition of the determined fed-batch fermentation media in the fermentor

[0168]

[0169]

[0170] Both strains S9179 and S9180 showed comparable strain performance in terms of biomass and lipid production / yield, DHA content in the lipids, and DHA yield / production in the fermentor.

[0171] While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as can be applied to the essential features hereinbefore set forth.

Claims

1. A feed ingredient composition comprising a dispersion of lysed microbial cells in a triglyceride oil, wherein: a. 5% to 90% by weight of the composition are lysed cells, and b. 10%-95% by weight of the composition is triglyceride oil, wherein the triglyceride oil comprises oil from lysed cells and oil from another organism, wherein the oil from the other organism is an oil from a plant, and wherein the microbial cells are from the family Thraustochytriacae.

2. The composition of claim 1 , wherein the triglyceride oil has a fatty acid profile of 10-50% docosahexaenoic acid (DHA) by weight of fatty acids, preferably wherein the DHA is 4-15%, 5-12%, 5-10%, 5-7%, 5-8%, 6-8%, or 6-7% by weight of the composition.

3. The composition of claim 1 or 2, comprising 5%-15%, 5%-10%, 10%-15%, 10%-25%, 15%-20%, or 20%-25% by weight of lysed microbial cells.

4. The composition of claim 1, further comprising less than 20%, 15%, 10%, 5%, 3%, or 1% by weight of the composition of unlyzed microbial cells.

5. The composition of any one of claims 1 to 4, wherein the oil from the microbial cells has a fatty acid profile of 40%-70%, 40%-60%, 40%-50%, 45%-55%, or 50%-55% DHA by weight of the fatty acids, as determined by FAME-GC-FID measurement.

6. The composition of any one of claims 1 to 5, wherein the lysed cells have a median particle size of from 5 microns to 100 microns.

7. The composition of any one of claims 1 to 6, wherein the plant-derived oil is coconut, corn, cottonseed, olive, palm, peanut, rapeseed, canola, safflower, sesame, soybean, soybean oil, nut oil, camelina oil, or citrus oil, or one or more combinations thereof.

8. The composition of any one of claims 1 to 7, wherein the microbial cells are from a genus selected from the group consisting of: Chytrid, Thraustochytrid, and Schizochytrium genera.

9. A composition as claimed in any one of claims 1 to 8, wherein the composition comprises 4-45% DHA by weight of the composition, wherein the oil from the lysed microbial cells has a fatty acid profile of 40%-70% DHA by weight of the fatty acids as determined by FAME-GC-FID measurement.

10. A shipping or storage container comprising the composition according to any one of claims 1 to 9, preferably wherein the container is 2.1×10 2 L (55 gallon) drums or totes.

11. A method for preparing the composition according to any one of claims 1 to 9, comprising: a) blending microbial cells with oil from another organism to form a blend; as well as b) lysing the microbial cells in the blend to form the composition as a dispersion.

12. A method of preparing a formulated feed comprising contacting a composition according to any one of claims 1 to 9 with an edible food.

13. The method of claim 12, wherein the edible food is coated with the composition, preferably, wherein the edible food is coated with the composition under vacuum.

14. The method of claim 12 or 13, wherein the edible food is an aquaculture feed or an animal feed, preferably, wherein the edible food is a salmon feed.

15. The method of any one of claims 12 to 14, wherein the formulated feed is in pelleted form.

16. The method of any one of claims 12 to 15, wherein the formulated feed contains at least 1%, 1.5%, or 2% omega-3 fatty acids.

17. The method of any one of claims 12 to 16, wherein the composition represents at least 10%, 15%, 20%, 25%, 30%, 35% or 40% by weight of the formulated feed.

18. A formulated feed comprising the composition of any one of claims 1 to 9 and an edible food, preferably wherein the edible food is an aquaculture feed or an animal feed.

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