Method for producing lysed cell suspensions

Abstract

The present invention relates to a novel lysed cell oil suspension having a solids content of at least 10 wt.% and a particle size Dv90 value of 100 to 300 microns. The lysed cell oil suspensions of the present invention typically have a cell lysis of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. The present invention relates to a composition comprising a lysed cell oil suspension and one or more components useful in feed. The invention also relates to a feed product comprising the composition, a method of making a feed product, a method of feeding an animal comprising feeding the animal with the feed product, and a method of sustainably producing a meat product by feeding the animal with the feed product.

Description

[0001] This application is a divisional application of an application with an application date of January 3, 2020, international application number PCT / EP2020 / 050056, national application number 202080007471.9, and invention name “Method for producing a lysed cell suspension”.

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 787,974 filed on January 3, 2019 and European Patent Application No. 19157377.3 filed on February 15, both of which are entitled “Process for Manufacturing Lysed Cell Suspension” and are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a method for producing a lysed cell suspension, in particular an oil suspension of lysed cells. The present invention also relates to a lysed cell suspension having specific properties obtainable by the method according to the present invention. Background Art

[0004] WO2018005856 describes a method for manufacturing a composition, the method comprising the steps of mixing microbial cells and another oil source to form a mixture, and lysing the microbial cells in the mixture to form a composition in the form of a dispersion.

[0005] As described in WO2018005856, the triglyceride oil produced by microorganisms and plants provides essential nutrients for consumption by higher organisms in the food chain. This triglyceride oil is composed of certain fatty acids, which are not found or produced in higher-order organisms in a relatively small amount. Of particular nutritional value in the food chain are triglyceride oils rich in polyunsaturated fatty acids (PUFA) 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 shellfish also need DHA in their diets to develop and grow normally. In addition, DHA is fed to newborn domesticated animals, such as pigs, cattle and other mammals, to improve the survival rate of piglets, calves, children and other newborn mammals.

[0006] The main source of commercial long-chain omega-3 fatty acids today is fish oil. About 1 million metric tons of fish oil are produced each year, primarily for aquaculture feed applications, terrestrial animal feeds, and human nutrition. Aquaculture is growing, but the supply of long-chain omega-3 fatty acids from wild-caught fish has not increased in line with demand. Sustained supply depends on sustainable fisheries management policies, the productivity of natural systems that are sensitive to climate change, and other factors. Many countries have strict quotas on wild-caught fish.

[0007] A particularly attractive source of long-chain omega-3 fatty acids are microorganisms, in particular yeasts, fungi and algae. These microorganisms are often provided as spray-dried or drum-dried products. However, compositions of spray-dried or drum-dried microorganisms are not always attractive for a number of reasons. Firstly, dried microbial cells containing large amounts of PUFAs may need to be classified as autothermal. This means that safety measures are required for the manufacture, storage and use of these compositions. In addition, the bulk density of the dried cell mass is low, which means that a relatively large volume is required to transport a given amount of cell mass. Furthermore, due to the relatively large surface area of ​​the dried cell mass, there is a high risk of the cells interacting with oxygen in the air, which may lead to degradation.

[0008] These problems are solved by providing an oil suspension containing lysed cells, as described in WO2018005856. It is also necessary to develop a large-scale production process that is particularly suitable for providing a suspension with a high cell load in the suspension, thereby providing a higher concentration of valuable biomaterials (e.g., DHA or other ω-3 fatty acids) in the suspension. The present invention meets these needs and other needs.

[0009] The present invention provides a method for allowing to manufacture this suspension in an economical and reproducible manner. In some embodiments, the present invention provides a high-throughput method, which is economical and further capable of introducing a large amount of cells (i.e., high cell load) in the suspension. It is advantageous to disperse a large amount of cells in oil to form a suspension comprising lysed cells, because this allows the manufacture of a suspension containing a large amount of oil, particularly DHA and / or other ω-3 fatty acids derived from microbial cells.

[0010] The invention further relates to specific cell suspensions. Advantageously, suspensions provided herein have specific particle size distribution ranges. Without wishing to be bound by any theory, it is believed that a suspension comprising a Dv90 value particle size range of 100 to 300 microns of lysed cells provides physical stability for the suspension, resulting in minimum sedimentation of particles in the suspension. This property enables the suspension to be stored for a long time, with minimum sedimentation of solids at the bottom of the storage container and without (or minimum) hard accumulation (hardpacking). It is also easier to use this suspension later, without having to input too much energy to redisperse the suspension before use. When the solid content of the suspension is relatively high in the suspension, these advantageous properties of the suspension can also be achieved. Summary of the invention

[0011] The present invention relates to a method for producing a suspension of lysed cell oil having a solid content of at least 10 wt.% and a particle size Dv90 value of 100 to 300 microns, the method comprising the following steps:

[0012] a) adding a first portion of microbial cell material to the oil,

[0013] b) subjecting the mixture of microbial cell material and oil to one or more shearing steps to achieve at least partial lysis of the cells and produce an oil suspension comprising lysed cells,

[0014] c) adding a further portion of the microbial cell material to the oil suspension comprising the lysed cells,

[0015] d) subjecting the mixture of microbial cell material and oil suspension comprising lysed cells to one or more shearing steps to achieve at least partial cell lysis and produce a further oil suspension comprising lysed cells.

[0016] In one embodiment, the water content of the microbial cell material is less than 10 wt.%.

[0017] The present invention relates to a novel lysed cell oil suspension having a solid content of at least 10 wt.% and a particle size Dv90 value of 100 to 300 microns. The lysed cell oil suspension produced by the present method generally has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% cell lysis.

[0018] The present invention relates to a composition comprising a lysed cell oil suspension and one or more components that can be used in feeds, and also relates to a feed product comprising the composition.

[0019] The invention further relates to a method for making an impregnated pellet, wherein a porous pellet is contacted with a composition according to the invention.

[0020] The invention further relates to raising animals comprising feeding the animals with a feed product according to the invention.

[0021] The present invention further relates to the sustainable production of meat products by feeding animals with a feed product according to the present invention, wherein the meat products contain increased levels of omega-3 fatty acids compared to meat products produced without feeding the animals with a composition comprising a lysed cell oil suspension.

[0022] The present invention also relates to a system comprising a low shear mixer, a high shear mixer, one or more pumps for producing a suspension comprising lysed cells, in particular an oil suspension of lysed cells.

[0023] Embodiments of the present invention provide many advantages. The present method can achieve high cell biomass loading (e.g., 70-95wt.% of the final lysed cell suspension is derived from microbial cell material) and high cell lysis degree (e.g., at least 70%, 75%, 80%, 85%, 90%, 95% or 98% cell lysis). It should be noted that it is obvious to the technician that the terms "biomass" and "cell matter" are used interchangeably in this article.

[0024] It can be seen from the particle size distribution of the lysed cell oil suspension obtained according to the method of the present invention that a high percentage of cell lysis can be achieved according to the method of the present invention. The particle size Dv90 value of the lysed cell suspension is 100 to 300 microns. In some embodiments, the particle size Dv90 value of the lysed cell suspension is at most 260 microns, more particularly at most 240 microns, more particularly at most 220 microns, and in some embodiments at most 200 microns. In some embodiments, the particle size D90 value is 110 to 250 microns, optionally 120 to 200 microns. A particle size Dv90 value of less than 300 microns is required to prevent solid particles from settling out of the suspension and hard accumulation, making them extremely difficult to resuspend without a large amount of energy input to remix the suspension. Reference Figure 6 , which shows the sedimentation behavior of suspensions with different particle size Dv90 values. On the other hand, lysed cells with too low particle size Dv90 values ​​may aggregate. It takes more energy to process aggregated lysed cells than to process unaggregated lysed cells. Therefore, it is not desirable for lysed cells to aggregate. The particle size Dv90 value is determined using a Malvern Mastersizer 3000 laser diffraction particle size analyzer. More specifically, a lysed cell oil suspension sample is dispersed in a suitable (shoutable) particle-free oil (e.g., rapeseed oil or soybean oil) and measured using a Malvern Mastersizer 3000, and calculations are applied according to the Mie theory.

[0025] The lysed cell oil suspension with higher cell biomass load provides a larger amount of valuable biomaterial (e.g., DHA) per volume unit. A larger amount of valuable biomaterial per unit volume can add higher doses of biomaterial to feed products (e.g., by coating on feed particles). The higher biomass load per unit volume also reduces transportation costs and greenhouse gas emissions. In addition, antioxidants of different types and / or amounts can be introduced into the lysed cell oil suspension (compared with stem cell biomass) to increase the oxidative stability of the suspension. The inventive method can also process a cell material intake greater than 5000kg / hour, which is the required capacity for commercial scale production systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Six different lysed cell oil suspension samples are shown as shear rate (1 / s) versus viscosity (Pa·s) at 25°C. The curve with the lowest y-axis value at the bottom is a lysed cell oil suspension test sample derived from 50wt.% microbial cell material and 50wt.% rapeseed oil. Other lysed cell suspension test samples are derived from about 80wt.% microbial cell material and about 20wt.% rapeseed oil. The particle size Dv90 value of the lower cell load (the curve at the bottom) is 168 microns. The particle size Dv90 values ​​of the higher cell load samples are 139 microns, 155 microns, 151 microns, 159 microns, and 192 microns, respectively. These five different higher cell load samples are generated under slightly different conditions (e.g., high shear frequency varies between 30Hz and 70Hz; total number of passes varies between 10 and 35).

[0027] Figure 2 A bimodal particle size distribution of a lysed cell oil suspension sample is shown.

[0028] Figure 3 A system according to one embodiment of the present invention is shown showing the recirculation of a suspension of microbial cell material and oil through a disperser 12 (i.e., a low shear mixer, such as a YTRON-ZC). The flow of the suspension is shown in dashed lines. A powder feed hopper 11 can be used to add microbial cell material to the system. Oil can be fed into the mixer 2.

[0029] Figure 4 A system according to one embodiment of the invention is shown showing the recirculation of an oil suspension containing lysed cells through a high shear mixer 13 (eg a YTRON Z) to lyse the cells in the oil. The flow of the suspension is shown in dotted lines.

[0030] Figure 5A system according to one embodiment of the invention is shown showing the dispersion of microbial cell material by a low shear mixer 12 and the lysis of cells in oil by a high shear mixer 13 in the same loop to produce a suspension. The flow of the suspension is shown in dotted lines.

[0031] Figure 6 The sedimentation of a suspension having about 20% biomass and about 80% rapeseed oil after 4 weeks of treatment with low shear, medium shear or high shear conditions is shown. DETAILED DESCRIPTION

[0032] The lysed cell oil suspension produced by the method according to the invention is therefore derived from microbial cells which are introduced into the oil and at least partially lysed, i.e. the cell walls of some or most of the cells are destroyed and the oil is released from the lysed microbial cells. The solid content of the suspension is a measure of the amount of cell material (e.g. cell fragments and intact cells with oil) in the suspension. The solid content (in wt.%) is defined as follows:

[0033] Solid content (wt.%) = 100% - water (wt.%) - free oil (wt.%).

[0034] The wt.% of water is determined by Karl Fischer titration. The Karl Fischer method is a volumetric titration method used to quantitatively determine the water content in liquid and solid samples. The titration standard Hydranal Composite 2 (a single-component reagent that already contains all reactants, including iodine, sulfur dioxide and base: imidazole and 2-methylimidazole) is used for titration. The titration medium used is a mixture of dry dichloromethane (DCM) and dry methanol (in a ratio of 75 / 25% v / v). The endpoint of the determination is indicated using bivoltammetry. That is, the potential of the polarized double platinum needle electrode is below a certain value, for example, for a Mettler Toledo V30 KF titrator with a DM143-SC electrode and a polarization current set to 24.0μA, the value is 100.0mV.

[0035] The water content in the sample can be determined by the amount of this reagent added.

[0036] The wt.% of free oil is determined as follows: the free oil present in the sample (unbound oil in intact microalgae cells) is extracted into a solvent layer, in this case hexane. The solvent / oil portion is separated from the solid portion by adding water and applying centrifugal force (at least 12000 RPM for 6 minutes). The hexane / oil partition is transferred to a tray placed in a fume hood. The hexane is evaporated at room temperature until stable, leaving only free oil, the amount of which can be determined by gravimetric analysis.

[0037] The method according to the invention makes it possible to obtain a lysed cell oil dispersion having a relatively high solid content, for example at least 10 wt.%, at least 15 wt.%, in particular at least 20 wt.%, more particularly at least 25 wt.%, even more particularly at least 28 wt.%, in some embodiments at least 30 wt.%. In some embodiments, the invention makes it possible to obtain a lysed cell oil suspension having a solid content of 15 wt.% to 45 wt.%, in particular a solid content of 20 wt.% to 40 wt.%, more particularly a solid content of 25 wt.% to 35 wt.%.

[0038] Since water may adversely affect the shelf life of the suspension, it is preferred that the final lysed cell suspension has a water content of at most 10 wt.%, particularly at most 8 wt.%, more particularly at most 6 wt.%, even more particularly at most 4 wt.%, more particularly at most 2 wt.%, even more particularly at most 1.0 wt.%. The water content is determined using the Karl Fischer titration method described above.

[0039] In certain embodiments, the microbial cell material as starting material has a water content of less than 10wt.%. Particularly, the water content of the microbial cell material is less than 8wt.%, more particularly less than 6wt.%, even more particularly less than 4wt.%, still more particularly less than 2wt.%, and in some embodiments less than 1wt.%. Particularly, the water content of the microbial cell material is between 0.5wt.% and 8wt.%, more particularly between 1wt.% and 5wt.%, even more particularly between 0.5wt.% and 3wt.%. The microbial cell material provided as the starting material of the method of the present invention can include complete and broken microbial cells. Depending on the source and optional pretreatment step, the cell material provided as starting material according to the method of the present invention generally accounts for at least 10wt.% of the whole microbial cell, particularly at least 20wt.%, more particularly at least 30wt.%, or at least 40wt.%, or at least 50wt.%, or at least 60wt.%.

[0040] Microbial cell material with a water content within this range can be obtained in a manner known in the art. For example, microbial cell material with a water content within a specified range can be obtained by subjecting a fermentation broth containing microbial cells to a drying step. The drying of microbial cells in an aqueous fermentation broth can be accomplished, for example, by first optionally dehydrating the fermentation broth (concentrating the fermentation broth) to increase the cell content of the broth. Dehydration or concentration refers to separating biomass from fermentation broth or other liquid culture media, so it is a solid-liquid separation. Therefore, during dehydration, the culture medium is removed from the biomass (for example, the fermentation broth is discharged by a filter that retains the biomass), or the biomass is otherwise removed from the culture medium. Common dehydration methods include centrifugation, filtration, evaporation, and the use of mechanical pressure. These methods can be used alone or in any combination. After the optional dehydration step, the concentrated broth now having a higher solid content can be dried by known drying methods, including but not limited to drum drying, pneumatic drying, spray drying, freeze drying, freeze drying, and other drying methods.

[0041] A drum dryer works by applying a film of fermentation broth (or dehydrated fermentation broth) to the surface of a rotating heated drum. The watery portion of the broth evaporates, leaving dry solids on the surface of the drum. The dry solids are then scraped off the drum with a knife. A pneumatic dryer captures or entrains the material to be dried in a stream of hot air. When the material is entrained in the hot air, the moisture is quickly removed. The dried material is then separated from the wet air, which is then recirculated for further drying. A spray dryer works by spraying the fermentation broth (or dehydrated fermentation broth) into a stream of hot air as a dispersion of fine droplets. The entrained material is quickly dried and formed into a dry powder. Spray drying can be done by a box dryer or a tail-end spray dryer, a fluidized bed dryer, or a moving fluidized bed dryer (e.g., Spray dryer, GEA Process Engineering, Inc.)

[0042] In one embodiment, the microbial cell is an oleaginous yeast cell, a fungal cell or a microalgae cell. In some embodiments, the oleaginous yeast includes organisms such as Yarrowia lipolytica, Rhodotorula glutinis, Cryptococcus curvatus and Lipomyces starkeyi. In other embodiments, the microalgae cell includes organisms from Thraustochytriaceae and Crypthecodiniaceae. The microbial cell may preferably be from a genus selected from:

[0043] Crypthecodinium, Ulkenia, Thraustochytrium, Aurantiochytrium and Schizochytrium. In one embodiment, Schizochytrium limacinum or Aurantiochytrium limacinum is used.

[0044] In some embodiments, the microbial cell is an oleaginous microorganism capable of producing a large amount of lipid, particularly triglyceride. In certain embodiments, the microbial cell comprises at least 30wt.%, at least 35wt.%, at least 40wt.%, at least 45wt.%, at least 50wt.%, at least 55wt.%, at least 60wt.%, at least 65wt.%, at least 70wt.%, particularly 40wt.% to 75wt.% or 45wt.% to 67wt.% lipid, particularly triglyceride, calculated by dry biomass weight.

[0045] The selection of microbial cells depends on the required biomaterial in the lysis cell oil suspension. In one embodiment, microbial cells comprising a large amount of DHA can be used. In some embodiments, the microbial cells comprise at least 15wt.%DHA, at least 20wt.%DHA, at least 25wt.%DHA, at least 30wt.%DHA, at least 35wt.%DHA, at least 40wt.%DHA, at least 45wt.%DHA or optionally 20wt.% to 50wt.% DHA, or 20wt.% to 45wt.% DHA, or 25wt.% to 35wt.% DHA, or 25wt.% to 45wt.% DHA of dry matter weight.

[0046] In one embodiment, microbial cells rich in eicosapentaenoic acid (EPA) are used alone or in combination with microbial cells rich in DHA. In other embodiments, microbial cells capable of producing both DHA and EPA can be used. In some embodiments, the ratio of DHA to EPA in microbial cells (from a single microbial source or from multiple microbial sources) can be between 100:1 and 1:100, particularly between 10:1 and 1:10, more particularly between 4:1 and 1:4. In some embodiments, one or more microbial cells producing arachidonic acid (ARA) are used alone or in combination with any microbial cells described herein.

[0047] In some embodiments, the microbial cell is not transformed with transgenic. In other embodiments, the microbial cell is genetically modified to produce one or more combinations and / or the required biological material of ratio in the microbial cell. Many of these microbial cells are commercially available (for example, ATCC deposits), or can be produced by methods known in the art. See, for example, WO2012 / 021703, US20180000130, US2012204802, US20070082008, US2003100097, WO199633263, WO2010107415 and Aquaculture 277 (2008): 109-116.

[0048] In this specification, the term "lipid" refers to any fat-soluble, lipophilic, naturally occurring molecule. See US20090093543. The term "oil" refers to a lipid substance that is liquid at 25°C and is generally unsaturated. Oil has hydrophobicity and lipophilicity and can be extracted in hexane. In oleaginous microbial cells, oil is the main component of total lipids, and oil is mainly composed of triacylglycerols. Oil is different from semisolid or solid fats, which are not liquid at a temperature of 25°C.

[0049] The term "triacylglycerol" refers to a neutral lipid composed of three fatty acyl residues esterified to a glycerol molecule. Triacylglycerols contain long-chain polyunsaturated and saturated fatty acids, as well as shorter-chain saturated and unsaturated fatty acids.

[0050] The term "neutral lipids" refers to lipids that are commonly found in cells in liposomes and that have no charged groups at cellular pH. Typically, they are completely nonpolar and have no affinity for water. Neutral lipids typically refer to monoesters, diesters, and / or triesters of glycerol with fatty acids, also known as monoacylglycerols, diacylglycerols, or triacylglycerols (collectively referred to as acylglycerols). A hydrolysis reaction must occur to release the fatty acids from the acylglycerols.

[0051] The term "total fatty acids" refers to the sum of all cellular fatty acids (or fatty acids in a sample) that can be derived into fatty acid methyl esters ("FAME") by alkaline interesterification in a given sample, which can be, for example, a biomass, an oil, a lysed cell oil suspension. Thus, total fatty acids include fatty acids from the neutral lipid fraction (including diacylglycerols, monoacylglycerols, and triacylglycerols) and from the polar lipid fraction (including, for example, the phosphatidylcholine and phosphatidylethanolamine fractions), but do not include free fatty acids.

[0052] The term "total lipid content" of a cell is a measure of total fatty acids as a percentage of cell dry weight ("DCW"), although total lipid content can be approximated as a measure of FAME as a percentage of DCW ("FAME % DCW"). Thus, total lipid content (TFA % DCW) is equivalent to, for example, milligrams of total fatty acids per 100 milligrams of DCW.

[0053] The concentration of fatty acids in the total lipids is expressed herein as a weight percent of TFAs (% TFAs), e.g., milligrams of a given fatty acid per 100 milligrams of TFAs. Unless otherwise specifically stated in the disclosure herein, reference to a given fatty acid relative to the percentage of total lipids is equivalent to the concentration of the fatty acid, expressed as % TFAs (e.g., % DHA of total lipids is equivalent to % DHA of TFAs).

[0054] Suitable oil includes the oil derived from animal, plant and oleaginous microorganism such as microalgae, fungi or yeast or its combination.In some embodiments, use the oil derived from plant material, the plant material is selected from coconut, corn, cottonseed, olive, palm, peanut, walnut, rapeseed, rapeseed, safflower, sesame, soybean, soybean oil, sunflower, flaxseed, linseed, camellia oil, shea butter or citrus oil, or one or more combinations thereof.These oils can be used in unfiltered crude form, or can be used in any improved supply form (for example, degumming and / or refining).In some embodiments, the oil used is derived from krill or fish, the fish is selected from herring, menhaden, anchovy, pilchard, sardine or mackerel, tuna or its one or more combinations thereof.

[0055] In some embodiments, oil extracted from a microbial source can be used to produce a lysed cell oil suspension. In one embodiment, the oil extracted from the microbial cells is different from the source of the microbial cells used to prepare the lysed cell oil suspension. For example, a lysed cell oil suspension can be produced by lysing Schizochytrium in oil extracted from Cryptodinium. In another embodiment, the suspension can be prepared using oil extracted from the same organism. For example, oil extracted from Schizochytrium rich in DHA can be added to the same Schizochytrium cell material to produce a lysed cell oil suspension. Such a suspension can further enrich the amount of biological material required per unit volume in the suspension, such as DHA. In some embodiments, the extracted microbial oil can provide a source of other valuable endogenous biological materials such as EPA, ARA, carotenoids, or astaxanthin.

[0056] In the method according to the invention, in one embodiment a first portion of microbial cell material having a water content of less than 10 wt.% is added to the oil.

[0057] In the method according to the present invention, microbial cell material is mixed with oil in a portioned manner, and intermediate cracking is performed in one or more shearing steps. Using a larger number of parts requires that each part adds less cell material. This can be conducive to better process control. On the other hand, using a larger number of parts leads to more process steps, which may be disadvantageous from a cost perspective. Therefore, in some embodiments, in the method according to the present invention, the total amount of cell material is added with at least 2 parts and usually with up to 50 parts. Cell material can be preferably added to the process with up to 40 parts, up to 30 parts, up to 25 parts or up to 20 parts. Cell material can be particularly preferably added with up to 15 parts, more particularly with up to 10 parts. In some embodiments, cell material can be preferably added with at least three parts.

[0058] The sizes of different parts can be the same or different. The size of each part can preferably be at least 1wt.%, more particularly at least 2wt.% of the total amount of microbial cell material added in the process. Generally speaking, the largest part size is at least 50wt.% of the microbial cell material added in the process. In some embodiments, the size of each part can vary between 5wt.% and 20wt.% or between 10wt.% and 15wt.%.

[0059] In one embodiment, the microbial cell material is added continuously to the oil. For example, the microbial cell material can be added continuously to an oil suspension containing lysed cells.

[0060] In the method according to the present invention, one or more shearing steps are performed after adding a portion of the microbial cell material. The number of shearing steps performed after adding a portion of the microbial cell material can vary within a wide range. In one embodiment, a single step is performed. In another embodiment, more than one shearing step can be performed, for example, a low shearing step combining mixing and cracking, followed by one or more higher shearing or increased shearing steps. In some embodiments, subsequent shearing steps can be performed in the same equipment by recycling, for example, by feeding the product multiple times through the same homogenizer, bead mill or rotor-stator system. In other embodiments, subsequent shearing steps can also be performed in different equipment, for example in multiple sequential rotor-stator mixers. The total number of shearing steps performed after adding a portion of the microbial cell material is generally no more than 50.

[0061] Therefore, in the method according to the invention, it is considered preferred to carry out the following steps at least once more, in particular at least twice more: adding a further portion of the microbial cell material to the oil suspension comprising lysed cells and subjecting the mixture of the microbial cell material and the oil suspension comprising lysed cells to one or more shearing steps to achieve at least partial cell lysis and produce a further oil suspension comprising lysed cells.

[0062] The method according to the invention is carried out at a temperature at which both the oil and the suspension are in the liquid phase. Although there is no specific upper limit, higher temperatures may be less attractive because they may have an adverse effect on the quality of the system. The method may preferably be carried out at a temperature below 80°C, preferably in the range of 10°C to 60°C, more particularly in the range of 15°C to 50°C, even more particularly in the range of 15°C to 45°C. The temperature referred to here is the product temperature. In some embodiments, the oil is cooled in a tank to below 25°C, below 20°C, below 15°C or between 10°C and 30°C before it is added to the microbial cell material.

[0063] In the method according to the invention, when the first portion of microbial cell material has been added to the oil, the mixture of microbial cell material and oil is subjected to a shearing step to achieve at least partial lysis of the cells in the oil.

[0064] In this specification, the term "lysis" refers to the process in which the cell wall of a microbial cell is destroyed and oil is released from the cell. The purpose of the method according to the invention is to provide a suspension in which most of the microbial cells provided to the method have been lysed. In one embodiment, at the completion of the method according to the invention, at least 70% of the cells provided to the method have been lysed, particularly at least 75%, particularly at least 80%, more particularly at least 85%, and even more particularly at least 90%. In some embodiments, at least 95% of the cells have been lysed, particularly at least 98%. In some embodiments, 80% to 98% of the cells have been lysed, particularly 85% to 98% of the cells have been lysed, more particularly 85% to 95% of the cells have been lysed.

[0065] The percentage of cell lysis can be determined as follows. The percentage of lysis can be determined qualitatively by visual inspection under a microscope by observing and counting the number of lysed and unlysed cells in the sample.

[0066] Alternatively, the percentage of cell lysis is determined by comparing the amount of free oil in the lysed cell suspension to a predetermined lipid content of the microbial cell material (eg, dried cell biomass).

[0067] The shearing step can be carried out in different methods and equipment known in the art. Examples include roll mills, homogenizers, bead mills, shear mixers such as inline mixers, waring blenders, and rotor-stator mixers. French presses or centrifuges may also be used, as may pumps and stirred vessels that provide shearing forces.

[0068] The amount of shear provided in each step will depend on the total number of shear steps in the method. The higher the total number of shear steps, the less shear must be provided in each individual step.

[0069] In one embodiment, a pressure crusher (e.g., a high pressure homogenizer) can be used to lyse cells. The pressure crusher lyses cells by pumping a mixture of cells and oil (e.g., rapeseed oil or other vegetable oils) through a restricted aperture valve. High pressure (up to 1500 bar from 50 bar) is applied and then expanded immediately through an outlet nozzle. A Niro (Niro Soavi GEA) homogenizer (or any other high pressure homogenizer) can be used. Processing biomass with a high pressure homogenizer can cause cell lysis of more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the cells by controlling pressure, outlet velocity, and other parameters.

[0070] 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 by shear forces, grinding between beads, and collisions with beads. The beads break the cells to release the cell contents. Dyno-mill ECM Ultra (CB Mills) ball mills and other commercially available bead mills can be used. Processing biomass in a ball mill can be performed by controlling, in particular, the tip speed of the shaft, the feed rate of the product through the mill, and / or the size and density of the beads, so that more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the cells undergo cell lysis.

[0071] In one embodiment, a rotor-stator mixer is applied in at least one shearing step. A rotor-stator mixer or system uses a rotating metal shaft (rotor) inside a fixed metal housing (stator). The rotation of the rotor produces a suction effect, which draws the sample into the space between the rotor and the stator, and in the small space between the rotor and the stator, due to the change in speed, the sample is subjected to the effect of shearing force. Centrifugal force then pushes the material out through the slot in the stator, and the rapid movement of the fluid caused by the rotor-stator ensures that the method is repeated when the sample is repeatedly circulated through it. In one embodiment, the shearing step is carried out in a series of rotor-stator mixers, for example 2 to 10, particularly 3 to 8, more particularly 3 to 6. In this embodiment, it can be preferred that at least one of the rotor-stator mixers has a narrower gap width than the preceding rotor-stator mixer. It can be particularly preferred that the gap width of each rotor-stator mixer is narrower than the gap width of the previous rotor-stator mixer. Processing biomass with one or more rotor-stator mixers can achieve cell lysis of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or greater than 95% of the cells by controlling, among other features, the speed, gap width, number of teeth on the rotor and / or the gap between the teeth.

[0072] The degree of shearing in the shearing step in the method according to the invention can be determined, for example, by the difference between the content of lysed cells in the cell / oil mixture provided to the shearing step and the content of lysed cells in the cell / oil mixture discharged from the shearing step. In one embodiment, at least one shearing step in the method according to the invention is performed such that 1-60%, 5-50%, 10-40% or 15-30%, 15%-25% of the cells entering the shearing step are lysed.

[0073] In one embodiment, at least one shearing step in the method of the invention is performed such that 80% of the cells entering the shearing step are lysed, particularly at least 85%, more particularly at least 90%, still more particularly at least 95%, even more particularly at least 98%. In order to achieve the desired high degree of cell lysis in the method according to the invention, it is preferred that the final shearing step in the method of the invention is performed only under specified conditions. It is within the scope of the skilled person to determine how to perform the shearing step to ensure that the desired degree of cell lysis is achieved.

[0074] It may be preferred that at least one shearing step in the method of the present invention is carried out at a higher shear than the previous shearing step. It may be preferred that the method according to the present invention comprises the following steps:

[0075] a) adding a first portion of microbial cell material to the oil,

[0076] b) subjecting the mixture of microbial cell material and oil to at least one shearing step to achieve at least partial lysis of the cells and produce an oil suspension comprising lysed cells, wherein a combined set of one or more shearing steps is performed such that 1-60%, particularly 5-50%, more particularly 10-40%, even more particularly 15-30%, even more particularly 15-25% of the cells entering the combined set of one or more shearing steps are lysed upon exiting the combined set of one or more shearing steps,

[0077] c) adding a further portion of the microbial cell material to the oil suspension comprising lysed cells and optionally repeating step a),

[0078] d) subjecting the mixture of microbial cell material and oil suspension containing lysed cells to at least one additional shearing step to achieve at least partial lysis of the cells and produce a further oil suspension containing lysed cells, wherein a combination of one or more shearing steps is performed such that at least 70% of the cells entering the combination of one or more shearing steps are lysed, at least 75% are lysed, at least 80% are lysed, in particular at least 85%, more in particular at least 90%, still more in particular at least 95%, even more in particular at least 98% are lysed.

[0079] In one embodiment, step b) is performed with one device, while step d) is performed with a different type of device. For example, step b) is performed in a device that primarily introduces microbial cells into the oil, although a certain degree of cell lysis occurs during the introduction or dispersion process. Any suitable disperser can be used. For example, it can be a low shear mixer, such as a YTRON-ZC powder disperser. On the other hand, step d) is performed with a different device that primarily lyses cells to increase the percentage of cell lysis, reduce particle size (e.g., Dv90) and reduce the viscosity of the oil suspension containing the lysed cells. Examples of suitable devices in step d) include a YTRON-Z homogenizer, which includes a rotor-stator system. Other suitable devices include powder introducers and rotor-stator systems from IKA.

[0080] In one embodiment, step b is performed in one or more shearing steps. As used herein, "shearing step" refers to a process step that causes the cell wall of the microbial cells to rupture or break. Although a powder disperser is usually used to perform step b), its low shear mixing causes a certain degree of cell lysis. After certain shearing steps, another portion of microbial cells is added to the mixture of microbial cells and oil described in step c). For example, in step b), about 30-70wt.%, especially 40-60wt.%, more especially about 50wt.% of the pre-weighed microbial cell material batch of the final lysed cell suspension can be added in 2-10 parts, especially 3-7 parts. Each part can contain about the same amount of microbial cell material or different amounts of microbial cell material. In some embodiments, after each part is added to the oil suspension containing lysed cells, the suspension can be recycled by the device without further introducing an additional portion of microbial cell material. In some embodiments, the suspension can be recycled 1 to 10 times, especially 3 to 8 times, and then another portion of microbial cell material is added.

[0081] As mentioned above, a "shearing step" refers to a process step that results in the rupture or breakage of the cell walls of the microbial cells. In particular, a shearing step is a process step that results in at least 2%, in particular at least 5%, in some embodiments at least 10% of the cells provided to the step being lysed. In the present specification, the term low shearing step generally refers to a shearing step in which 1-60%, such as 2-60%, or 5-60% or 10-60% of the cells provided to the step are lysed. The term high shearing step generally refers to a shearing step in which more than 60% of the cells provided to the step are lysed.

[0082] When the lysed cell suspension is too viscous to be pumped through during step (b), the lysed cell suspension can be pumped into a higher shear step (d) to reduce its viscosity and further lyse additional microbial cells. In one embodiment, about 5-60%, particularly 10-40%, more particularly 15-30%, even more particularly 15-25% of the microbial cells in the cell suspension are lysed after the shear step (b) and the addition of an additional portion of microbial cells (c) but before the lysed cell suspension is pumped into the higher shear device in step (d).

[0083] In one embodiment, in step (d), the lysed cell suspension is pumped to a different device having a higher shear force (e.g., a rotor-stator system) than the device used in step (b). In some embodiments, the lysed cell suspension is recirculated 1 to 50 times, particularly 2 to 30 times, more particularly 3 to 15 times, through a higher shear mixer device until the cell particle size in the suspension is reduced and the viscosity of the lysed cell suspension is reduced. Once it is visually or quantitatively determined that the viscosity of the lysed cell suspension is sufficiently reduced, optionally return to steps (b) and (c) to add the remaining or additional pre-weighed microbial cell material of the suspension.

[0084] Once the lysed cell suspension is returned to the low shear step (b), one or more additional portions of microbial cells are introduced into the lysed cell suspension. In one embodiment, at this stage, the remainder of the microbial cell material can be added in any suitable increment, such as 1-10 parts, particularly 2-8 parts, or more particularly 3-6 parts. Each portion can contain approximately the same amount of microbial cell material or different amounts of microbial cell material. After each portion is added to the oil suspension comprising the lysed cells, in one embodiment, the suspension can be recirculated by the device without further introducing an additional portion of microbial cell material. In some embodiments, the suspension can be recirculated 1 to 10 times, particularly 3 to 8 times, and then another portion of microbial cell material is added.

[0085] After the remaining portion is added (or if the cell suspension is too viscous to further mix the additional portion of microbial cells) to the lysed cell suspension, the lysed cell suspension is returned to the higher shear mixer device in step (d). In step (d), the lysed cell suspension is recirculated through the device until the desired viscosity and / or cell particle size Dv90 value is reached. In some embodiments, another lysed cell suspension is passed through the device in step (d) 1 to 50 times, particularly 3 to 30 times, more particularly 5 to 15 times.

[0086] The antioxidant may be added at any stage of the process, during any of steps (a) to (d). However, it is advantageous to add the antioxidant near the start of the process to prevent thermal degradation of the oil due to the heat of the process and any oxygen that may be introduced as a result of the process.

[0087] During any step of (a) to (d), one or more pumping steps may be added to facilitate recirculation of the suspension in a continuous system. In certain embodiments, a vacuum pumping step may be added to facilitate dispersion of microbial cell material in the oil. In certain embodiments, two or more different types of devices are connected inline in a continuous system. Instead of recirculating the cell suspension into the same device, two or more identical devices may be connected inline to recirculate the suspension through multiple devices rather than through the same device.

[0088] During any step of (a) to (d), one or more cooling steps may be added to cool the lysed cell suspension. In one embodiment, a cooling step is added after the higher shear step (d). In one embodiment, the cooling step allows the suspension to maintain its temperature at 80°C or below, at 70°C or below, at 60°C or below, or at 50°C or below, particularly at 40°C or below, more particularly at 35°C or below, and in some embodiments at 30°C or below.

[0089] The solids content of the final product (i.e., the final lysed cell suspension) is at least 10 wt.%, at least 15 wt.%, in particular at least 20 wt.%, more particularly at least 25 wt.%, even more particularly at least 28 wt.%, in some embodiments at least 30 wt.%, in some embodiments between 20 wt.% and 40 wt.%, and in some embodiments between 25 wt.% and 35 wt.%.

[0090] The particle size Dv90 value of the final lysed cells in the oil suspension is 100 to 300 microns. The Dv90 value of the final lysed cells in the oil suspension can be particularly at most 260 microns, more particularly at most 240 microns, still more particularly at most 220 microns, and in some embodiments at most 200 microns. In some embodiments, the particle size Dv90 value of the final cell suspension is between 110 microns and 250 microns, more particularly between 120 microns and 200 microns.

[0091] In some embodiments, the final lysed cells in the oil suspension have a particle size Dv50 value of less than 150 microns. The particle size Dv50 value of the final lysed cells in the oil suspension can be particularly at most 130 microns, more particularly at most 100 microns, and even more particularly at most 80 microns. In some embodiments, the particle size Dv50 value of the final cell suspension is between 20 and 150 microns, particularly between 30 microns and 100 microns, and more particularly between 40 microns and 80 microns. It is obvious to the technician that for the same particle distribution, the particle size Dv50 value is always less than the particle size Dv90 value.

[0092] In some embodiments, the method using two different types of apparatus is particularly useful in producing a lysed cell oil suspension having a high solid content and a high degree of cell lysis. For example, the method can produce a final lysed cell suspension derived from 70-95 wt.% microbial cell material and 5 wt.% to 30 wt.% added oil, particularly derived from 75-85 wt.% microbial cell material and 15-25 wt.% added oil, more particularly derived from 80 wt.% microbial cell material and 20 wt.% added oil.

[0093] In one embodiment of the method according to the invention, the microbial cell material is added at a rate of at least 1000 kg / hour. This allows commercial scale production of suspensions. The microbial cell material can preferably be added at a rate of at least 2000 kg / hour, particularly at least 3000 kg / hour, more particularly at least 4000 kg / hour, in some embodiments at least 5000 kg / hour. The maximum value of the microbial cell material addition rate is not important. As a general value, a maximum value of 20.000 kg / hour can be mentioned. Depending on commercial demand, 1000 kg / hour to 20.000 kg / hour, particularly 2000 kg / hour to 15.000 kg / hour, more particularly 3000 kg / hour to 10.000 kg / hour, or 4000 kg / hour to 8000 kg / hour, for example a microbial cell material addition rate between 5000 kg / hour to 6000 kg / hour can be preferably used.

[0094] In one embodiment, the total energy input (to the low shear mixer, high shear mixer and pump) to produce the final lysed cell suspension can be between 0.01 and 0.2 kW*Hr / kg, particularly between 0.02 and 0.18 kW*Hr / kg, more particularly between 0.03 and 0.16 Kw*Hr / kg, and also particularly between 0.04 and 0.13 kW*Hr / kg, or any number between these ranges. As used herein, kilogram weight refers to the weight of microbial cell material and added oil in the system. In one embodiment, the total energy consumption of the method (for introducing biomass into a liquid and applying shear) is between 0.050 and 0.150 kW*Hr / kg suspension, particularly between 0.060 and 0.130 kW*Hr / kg, and more particularly between 0.070 and 0.120 kW*Hr / kg. The total energy consumption of the method (for introducing the biomass into the liquid and applying shear) may be between 0.050 and 0.100 kW*Hr / kg suspension, in particular between 0.06 and 0.090 kW*Hr / kg. Alternatively, the energy consumption of the method (for introducing the biomass into the liquid and applying shear) may be between 0.090 and 0.130 kW*Hr / kg suspension, in particular between 0.100 and 0.120 kW*Hr / kg.

[0095] In one embodiment, as described above, two different types of shearing devices may be used alternately to perform the shearing step, e.g. Figure 3 and Figure 4 In another embodiment, two shearing devices can be used simultaneously in the same circuit, for example Figure 5 shown.

[0096] Although the above method describes the shearing step using two different types of devices, the method according to the present invention is not limited to using two types of devices to perform the shearing step. In some embodiments, a single type of device can be used for the shearing step to produce a lysed cell oil suspension. An example of such a method is provided in Example 3. In other embodiments, more than two (e.g., three, four, five or more) types of devices can be used for the shearing step to produce a lysed cell oil suspension.

[0097] The above method describes adding microbial cell material to oil. As mentioned above, microbial cell material includes whole cells and broken cells. Cell disruption can occur, for example, by grinding or other pre-treatment steps that cause some cell disruption.

[0098] The present invention also relates to a novel lysed cell oil suspension. The novel cell oil suspension according to the present invention has a solid content of at least 10 wt.% and a Dv90 of 100 to 300 microns. Preferably, the solid content of the lysed cell oil suspension is at least 15 wt.%, particularly at least 20 wt.%, more particularly at least 25 wt.%, even more particularly at least 28 wt.%, in some embodiments at least 30 wt.%, in some embodiments between 20 wt.% and 40 wt.%, and in some embodiments between 25 wt.% and 35 wt.%.

[0099] Because water may adversely affect the shelf life of the suspension. Preferably, the water content of the lysed cell oil suspension is at most 10 wt.%, particularly at most 8 wt.%, more particularly at most 6 wt.%, even more particularly at most 4 wt.%, still more particularly at most 2 wt.%, even more particularly at most 1.0 wt.%. According to the Karl Fischer water determination method, the water content is determined as described above. It may be preferred that the particle size Dv90 value of the lysed cell oil suspension is at most 300 microns, at most 260 microns, more particularly at most 240 microns, still more particularly at most 220 microns, and in some embodiments at most 200 microns.

[0100] In one embodiment, the particle size Dv50 value of the lysed cell oil suspension is in the range of 20-150 microns. The particle size Dv50 value of the lysed cell oil suspension may in particular be in the range of 30-120 microns. The particle size Dv50 value is determined in a Malvern Mastersizer 3000 as described for Dv90.

[0101] In one embodiment, the lysed cell oil suspension has a density of at least 0.95g / ml. In some embodiments, the density can be at least 1.00g / ml, or even at least 1.05g / ml. This relatively high density of oil composition is relevant to the relatively high solid content (for example, at least 25wt.% or at least 30wt.% solid content) of suspension. The density of adding oil, particularly vegetable oil is generally about 0.9g / ml. Therefore, with higher solid content, the density of lysed cell suspension is at least 7%-20% higher than the density of the carrier oil itself added to suspension, particularly at least 8%-16%, or at least 10% higher. If the lysed cell suspension has lower biomass load and lower solid content (for example, derived from 20wt.% microbial cell material and 80wt.% suspension adding oil), the density of the lysed cell suspension will be similar to the density of adding oil (for example, 0.92g / ml to 0.95g / ml). Higher solids content in the lysed cell suspension may further provide the advantages of providing larger amounts of valuable biomaterials, such as DHA, per volume unit and lower shipping costs of the final suspension.

[0102] For one embodiment, an exemplary relationship between the viscosity and shear rate of a suspension is shown in Figure 1 . It will be apparent to the skilled person that in this figure, Dx(90) is Dv(90) and DHA refers to microbial cell material. In this example, the suspension is derived from about 80 wt.% microbial cell material and about 20 wt.% carrier oil. In this particular example, the suspension comprises a solid content of about 32 wt.%. In some embodiments, the viscosity of the oil suspension comprising lysed cells is at most 3 Pa·s at a shear rate of 100 / s, particularly at most 2 Pa·s at a shear rate of 100 / s, and more particularly at most 1 Pa·s at a shear rate of 100 / s. At a shear rate of 100 / s, the viscosity of the suspension with a high solid content is at least 40% higher, at least 50% higher, at least 100% higher, at least 150% higher, at least 200% higher than the viscosity of a suspension with a lower cell biomass load (50 wt.% microbial cell material and 50 wt.% added oil). See Figure 1 The viscosity was measured by rheometer (Anton Paar, MCR301, spindle CC27, 25°C and 40°C, shear 1s -1 →1000s -1 , then 1000s -1 →1s -1 ) measurement. Greater shear results in lower viscosity (at 100s -1 Down).

[0103] In some embodiments, the viscosity of the lysed cell suspension (measured at 20° C.) is between 10 and 100,000 mPa·s, particularly between 100 and 50,000 mPa·s. After 60 days, the viscosity of the lysed cell suspension may be between 10 and 100,000 mPa·s, particularly between 100 and 50,000 mPa·s. Additionally or alternatively, after 143 days, the viscosity of the lysed cell suspension may be between 10 and 100,000 mPa·s, particularly between 100 and 50,000 mPa·s.

[0104] In some embodiments, the viscosity of the lysed cell suspension remains stable over time. For example, the viscosity after 30 days may deviate from the viscosity of the freshly prepared suspension by less than 10%, preferably less than 5%, and more preferably less than 3%. Additionally or alternatively, the viscosity after 60 days may deviate from the viscosity of the freshly prepared suspension by less than 10%, preferably less than 5%, and more preferably less than 3%. The viscosity after 143 days may deviate from the viscosity of the freshly prepared suspension by less than 10%, preferably less than 5%, and more preferably less than 3%.

[0105] In one embodiment, the DHA content of the lysed cell suspension is at least 10 wt.%, particularly at least 15 wt.%, more particularly at least 18 wt.%, and more particularly at least 22 wt.%, based on the total fatty acid content (in wt.%) in the lysed cell oil suspension. In some embodiments, the DHA content can be even higher, particularly when the oil also contains DHA. For example, the DHA content can be at least 30 wt.%, particularly at least 35 wt.%, more particularly at least 40 wt.%, in some embodiments between 20 wt.% and 45 wt.%, and in some embodiments between 25 wt.% and 35 wt.%.

[0106] In one embodiment, the oil suspension comprising lysed cells also comprises an antioxidant. The example of a suitable antioxidant comprises a natural or synthetic antioxidant. In some embodiments, the antioxidant is lecithin, starch, ascorbic acid, tocopherol, rosemary extract, green tea extract, ascorbyl palmitate butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), ethoxyquin or one or more combinations thereof. In some embodiments, the oil suspension comprising lysed cells does not comprise ethoxyquin as an antioxidant. Because the oil suspension comprising lysed cells has a smaller surface area than dry cell biomass, it has a longer shelf life, and the level of natural or synthetic antioxidants is lower than the level required in dry cell biomass.

[0107] In another embodiment, the lysed cell suspension is combined with one or more components to produce a composition suitable for inclusion in the feed. As used herein, the term "feed" includes any food for animals, such as terrestrial animal feed, including human food, or aquaculture feed products. Terrestrial animal feed products can be monogastric or ruminant feed products. Terrestrial animals can include cattle (such as dairy cows or beef cattle), poultry (such as tender chickens or hens). Feed can also include food for companion animals. Aquaculture feed products include feed products for fish or shellfish (such as salmon, trout or shrimp). In some embodiments, one or more components can be additional oils, which are added to the lysed cell oil suspension at the feed production site. For example, additional oil can be coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, walnut oil, rapeseed oil, canola oil, safflower oil, sesame oil, soybean oil, soybean oil, sunflower oil, linseed oil, linseed oil, camellia oil, shea butter or citrus oil, or one or more combinations thereof. Additional oil can also come from fish, krill or microorganisms. In some embodiments, extra oil can be identical with the oil introduced into the lysed cell suspension.In other embodiments, extra oil can be different from the oil introduced into the lysed cell suspension.In some embodiments, one or more components can include other additives that can be used in aquaculture or terrestrial animal feed, such as astaxanthin, carotenoid, flavonoid, lecithin, sterol, calcitriol (vitamin D), tocopherol (vitamin E) and phylloquinone and menadione (vitamin K), antibiotic, antifungal, antiparasitic and hormone.In other embodiments, one or more components can include the source of ω-3 or ω-6 fatty acid, such as DHA, EPA and / or ARA.

[0108] In some embodiments, the lysed cell oil suspension can be mixed with dry ingredients and provided as feed or food as is without further processing. In other embodiments, the lysed cell oil suspension can be mixed with wet food ingredients, which are then packaged as feed or food.

[0109] In some embodiments, the composition comprising the lysed cell oil suspension is applied to the feed particles, or is pressed or extruded together with the feed particles. In some embodiments, the particles are porous. The porous particles can be impregnated with the lysed cell oil suspension or its composition. Any suitable contact method can be used to impregnate the porous particles. These include, for example, top coating, spray coating or vacuum coating. In one embodiment, the particle size Dv90 value of the lysed cell oil suspension is no more than 20%, 10% or 5% larger than the average pore size of the porous particles. In another embodiment, the particle size Dv90 of the lysed cell oil suspension is equal to or less than the average pore size of the porous particles. The particle size Dv90 value of the lysed cell oil suspension is between 100 microns and 300 microns, optionally between 110 microns and 250 microns, optionally between 120 microns and 200 microns. In some embodiments, the lysed cell suspension or its composition can be introduced together with other feed ingredients to extrude or manufacture feed products.

[0110] Any suitable amount of the lysed cell oil suspension may be introduced into the feed product.In one embodiment, 0.5-20 wt.%, optionally 1-10 wt.%, more optionally 2 to 8 wt.% of the feed product is derived from microbial cell material.

[0111] In some implementation methods, a method of raising an animal is provided. The method of raising an animal includes feeding the animal a feed product containing a lysed cell oil suspension. The method may also include adjusting the amount of the lysed cell suspension in the feed product during different life cycles of the animal as needed. In some embodiments, an animal obtained by the method of raising an animal is provided.

[0112] In some embodiments, a method for sustainably producing meat products is provided. The method includes feeding an animal a feed product comprising a lysed cell oil suspension or its composition. Compared with the meat produced without feeding the animal a lysed cell oil suspension or its composition, the method can further provide an increased level of ω-3 fatty acids (e.g., DHA and / or EPA). In some embodiments, a meat product obtained by a method for sustainably producing a meat product is provided.

[0113] It will be clear to those skilled in the art that the various embodiments of the present invention may be combined as desired unless they are mutually exclusive.

[0114] The present invention will be illustrated by reference to the following examples but is not limited thereto or thereby.

[0115] Example

[0116] Example 1. Microbial cells

[0117] Schizochytrium cells enriched with DHA (docosahexaenoic acid) are prepared by standard heterotrophic fermentation and dried using a drum dryer. The dried cell biomass contains about 60 wt.% oil. The dried cell biomass typically contains less than 5% water content, typically less than 2% or less than 1% water. Dried Schizochytrium biomass enriched with DHA can also be AlgaPrime TM DHA (Corbion Biotech, Inc., South San Francisco, USA) was purchased commercially.

[0118] Example 2. Measurement of total fatty acids or DHA in lysed cell suspensions or cell biomass

[0119] The fatty acid composition of FAME analysis sample is analyzed by using direct transesterification. Sample (10-20mg) is directly weighed into the test tube with PTFE liner screw cap of 16X 100mm, then 200uL 20mg / mLC19:0 internal standard solution (NuChek Prep, Inc, Elysian, MN) and 2mL 5% sulfuric acid methanol solution containing 0.5% BHT are added. The tube is covered and placed in the dry bath maintained at 75 ℃ for 3.5 hours, then vortexed and ultrasonicated twice in the ultrasonicator bath heated to 75 ℃ during the transesterification process intermittently. Take out sample tube from dry block, once after being cooled to room temperature, 2mL10% tripotassium phosphate and 2mL heptane are added in tube. Sample tube is reversed and stirred violently, then centrifuged at 1600rpm for 2 minutes to provide two different layers. The appropriate portion of the upper layer was transferred to a sample vial or vial insert containing sodium sulfate (anhydrous) lining the bottom for analysis by GC-FID. The FAMEs were quantified using the empirical relative response factor (ERRF) for each FAME determined relative to the C19:0 internal standard following the guidelines for acceptable ERRF standards outlined in AOCS method Ce 1i-07.

[0120] Example 3. Production of Lysed Cell Oil Suspensions Using a Single Type of High Shear Mixer

[0121] The lysed cell oil suspension is produced by lysing the dry Schizochytrium biomass described in Example 1 in a vegetable oil (e.g., rapeseed oil) using a single piece of equipment. In this example, a high shear mixer is used to add, wet, disperse, and lyse the cell biomass in the oil under vacuum. The cell biomass (approximately 80% by weight of the final product) is added continuously through a single piece of equipment. Rapeseed oil (approximately 20% by weight of the final product) is added to the feed tank. The oil is pumped from the bottom of the tank and injected into the equipment. Cell biomass from a separate hopper is introduced into the equipment at the same time. The oil and cell biomass mixture is pumped back to the top of the tank. This process continues uninterrupted until all pre-weighed microbial cells are introduced into the oil. At this point, the oil and cell biomass mixture is continuously recirculated through the same piece of equipment until maximum lysis (e.g., 80-90% cell lysis) and minimum Dv90 particle size are achieved (3 complete turnovers for the entire batch / tank). During this process, the speed of the shear mixer varied between about 2500 and 3600 revolutions per minute. Continuing the process did not result in more additional lysis or additional reduction in particle size. The particle size Dv90 value was less than 300 microns. The particle size Dv50 value was less than 150 microns. The solid content of the resulting lysed cell suspension produced by this method was about 32 wt.%. The water content of the lysed cell oil suspension was less than 5 wt.%, typically less than 1 wt.%.

[0122] Example 4. Production of Lysed Cell Oil Suspensions Using Two Different Types of Shear Mixers

[0123] The lysed cell oil suspension was produced by mixing the dried microbial cells of Example 1 with a vegetable oil (e.g., rapeseed oil) using two different devices: a YTRON-ZC powder dispersion system (i.e., a low shear mixer) and a YTRON-Z high shear mixer (also known as an inline homogenizer) (YTRON Process Technology GmbH & Co. KG, Germany). Figure 3 The vacuum pump 6 shown in the figure applies vacuum) is mainly used to introduce or disperse microbial cells (in powder form) into the oil. After the microbial cells are dispersed in the oil, the mixture is pumped into a high shear mixer, which is mainly used to lyse the cells and reduce their particle size. The YTRON-Z is equipped with a rotor / stator group. Parameters such as slot width, distance between shear slots, number of rotor / stator groups, rotational speed and flow rate and / or energy input are changed to produce the desired dispersion or lysis effect. The pump is used to call the circulation flow, so that the circulation flow can be set independently of other equipment in the circulation loop (e.g., YTRON-Z or YTRON-ZC). The resulting lysed cell suspension is cooled on a heat exchanger and recirculated on a high shear mixer to achieve the desired particle size and viscosity.

[0124] In the first stage of the production of lysed cell suspension (in Figure 3 In the present invention, the microalgae cells of the present invention are mixed with the microalgae of the present invention, and the microalgae of the present invention are mixed with the microalgae of the present invention. The microalgae of the present invention is mixed with the microalgae of the present invention, and the microalgae of the present invention is ... After each portion of microalgae cells was introduced into the mixture, the mixture was passed through a low shear mixer 1 to 10 times before another portion of microalgae cells was introduced into the mixture. By the time approximately 60 kg of microalgae flour had been introduced into the 30 kg of oil, approximately 20% cell lysis was observed in the cell and oil mixture.

[0125] The cell and oil mixture with partial cell lysis is then fed into a high shear mixer to further lyse the cells to reduce the viscosity of the mixture so that additional portions of microalgae cells can be added to the partially lysed cell suspension. Figure 4 The partially lysed cell suspension was passed through the high shear mixer approximately 10 times, during which variable energy input was provided. After the viscosity of the lysed cell suspension was reduced to allow for the addition of an additional portion of microalgae cells, the further lysed cell suspension was returned to the process tank so that it could be passed through the low shear mixer together with an additional portion of microalgae cells.

[0126] After the high shear mixer step and before the lysed cell suspension is returned to the low shear mixer for further introduction of an additional portion of microalgae cells, the cell suspension is passed through a heat exchanger where the temperature is maintained below 50°C. The high shear mixer can increase the temperature of the suspension, which can affect the stability of the valuable biological material in the suspension. One or more pumps are also added to the entire system to call for a recirculation flow and set the recirculation flow independently of other equipment in the recirculation loop. Adding a vacuum pump also helps remove bubbles to improve the oxidative stability of the lysed cell suspension.

[0127] In case return to low shear mixer, remaining microalgae cell portioning is added in the oil suspension of further cracked cell.In a test, the remainder of microalgae cell is added with 4-5 increments, so that a whole batch of pre-weighed microalgae cells are introduced in the oil suspension of further cracked cell.After adding every portion of microalgae cells, before adding next portion of microalgae cells, make the oil suspension of further cracked cell pass through low shear mixer 1 to 10 times.After all pre-weighed microalgae cells are introduced in the oil suspension of further cracked cell, make it return to high shear mixer with further cracked cell to reduce viscosity and cell particle size of suspension.

[0128] In the high shear mixer, the suspension of further lysed cells was passed through the high shear mixer multiple times to reduce the viscosity of the suspension and the cell particle size. In one experiment, the suspension of lysed cells was recirculated to the high shear mixer approximately 25 times until the desired particle distribution size and viscosity were achieved.

[0129] Several experiments were conducted using a similar process as described above, and using a higher cell loading in the lysed cell suspension (i.e., 80 wt.% microalgae cell material and 20 wt.% oil), with a cell suspension viscosity of about 1.05 g / ml. The particle size Dv90 values ​​of the final lysed cell suspension typically ranged from 139 microns to 192 microns between the different experiments. The particle size Dv50 values ​​of the final lysed cell suspension typically ranged from 53 microns to 77 microns between the different experiments. The particle size distribution in the lysed cell oil suspension was a bimodal curve with two peaks. See Figure 2 The particle size Dv50 value for the smaller of the two peaks is in the range of 3 to 6 microns, and the particle size Dv50 value for the larger of the two peaks is in the range of 80 to 200 microns.

[0130] The degree of cell lysis in the suspension ranged from 80% to 98%.

[0131] In one experiment, the total energy input (to the low shear mixer, high shear mixer and pump) to produce the final lysed cell suspension was about 0.1198 kW*Hr / kg. In the context of total energy input, kilogram weight refers to the weight of both biomass and oil in the system.

[0132] An exemplary system for producing an oil suspension comprising lysed cells is Figure 3-5As shown, a powder hopper 11 is provided to add microbial cell material to the system. Oil is provided to container 2 with mixer 1 with the aid of vacuum provided by pump 6. Disperser 12 (low shear mixer, such as YTRON-ZC) is used to introduce and disperse microbial cell material into carrier oil, which is usually added to container 2 with mixer 1. Pump 10 can help cause material recycling in the entire system. A cooler 14 can be added after, for example, a high shear mixer 13 (such as YTRON-Z) to dissipate heat generated by the high shear mixer and other equipment. An inert gas (such as nitrogen or argon) 7 can be purged into the product container 15 to reduce oxidation of the suspension. A vacuum pump 6 can be added to help introduce microbial cell material into the carrier oil and remove any bubbles. Valve 5 can be used for sampling. Valve 8 can be used to control the flow of material and change the process configuration. Temperature sensor 3 and pressure sensor 4 can also be added to the system.

[0133] Example 5. Production of oil suspension containing lysed cells using two different types of shears simultaneously

[0134] In this example, rather than alternately recirculating a mixture of microbial cell material and oil through a YTRON-Zc powder dispersion system and a YTRON-Z high shear mixer, both pieces of equipment are used simultaneously in the same loop to produce an oil suspension containing lysed cells, such as Figure 5 As shown. In this example, the pre-weighed microalgae cell material (137.6 kg) from Example 1 was divided into nine equal parts. These parts were added to 30 kg of carrier oil in an incremental manner over a process of about 21 minutes while being recirculated by a YTRON-Zc powder dispersion system and a YRON-Z high shear mixer. After all pre-weighed microalgae cell material was added to the carrier oil, the mixture was continued to be recirculated by these two pieces of equipment for another 43 minutes until an acceptable particle size distribution and cell lysis were reached. In this example, at least 75% cell lysis was achieved. The total energy input (low shear mixer, high shear mixer and pump) to generate the final lysed cell suspension was approximately 0.1496 kW*Hr / kg.

[0135] WO2018005856 (and its US counterpart application US20180000130) and all other publications cited in this application are incorporated herein by reference for all purposes.

[0136] It should be understood that the embodiments and implementations described herein are not intended to limit the scope of the present invention. Various modifications, alternative configurations and equivalents may be adopted without departing from the scope of the appended claims. In addition, without departing from the scope of the present invention, one or more features or steps of any embodiment may be combined in any way with any other features or steps of any other embodiment example.

Claims

1. A lysed cell oil suspension having a solid content of at least 10 wt.% and a particle size Dv90 value of 100 to 300 μm, the cell suspension comprising lysed cell material and oil, the solid content of the cell suspension preferably being at least 15 wt.%, particularly at least 20 wt.%, more particularly at least 25 wt.%, even more particularly at least 30 wt.%. The lysed cell suspension according to claim 1 , further comprising an antioxidant.

3. The lysed cell suspension according to claim 1 or 2, wherein the DHA content is at least 10% DHA, in particular at least 18% DHA, more in particular at least 22% DHA, at least 30wt.% DHA, at least 35wt.% DHA or at least 45wt.% DHA; or wherein the DHA content is 20wt.% to 45wt.% DHA, in particular 25wt.% to 35wt.% DHA.

4. The lysed cell suspension according to any one of claims 1 to 3, wherein the water content of the lysed cell suspension is at most 10 wt.%, particularly at most 8 wt.%, more particularly at most 6 wt.%, even more particularly at most 4 wt.%, still more particularly at most 2 wt.%, most particularly at most 1.0 wt.%.

5. A composition comprising the lysed cell suspension according to any one of claims 1 to 4, further comprising one or more components of a feed or food, wherein the one or more components are optionally additional oils.

6. A method for making impregnated particles, wherein the porous particles are contacted with a lysed cell suspension according to any one of claims 1 to 4, the porous particles are preferably formulated as aquaculture feed particles, the method optionally further comprising coating the porous particles with the composition under vacuum, wherein the particle size Dv90 value of the lysed cell suspension is 150 microns to 250 microns.

7. A feed product comprising the lysed cell suspension of any one of claims 1 to 4, the composition of claim 5 or the product of the method of claim 6, wherein the feed product is optionally an aquaculture feed or other animal feed, and preferably, 0.5 wt.% to 20 wt.%, optionally 1 wt.% to 10 wt.%, optionally 2 wt.% to 8 wt.% of the feed product is derived from microbial cell material.

8. A method for raising animals, comprising feeding the feed product according to claim 7 to the animals.

9. A method for sustainably producing a meat product by feeding an animal the feed product of claim 7, wherein the meat product comprises an increased level of omega-3 fatty acids compared to a meat product produced without feeding the animal a composition comprising a lysed cell suspension.

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