Microbial cell product, method for obtaining microbial cell product and use of microbial cell product

Abstract

The present invention relates to a method of preparing a microbial cell product, the method comprising: providing an aqueous suspension comprising microbial cells; subjecting said suspension to mechanical cell dissociation at a temperature in the range of 15 DEG C to 35 DEG C, preferably at a pH in the range of 9 to 11, to obtain an aqueous suspension comprising dissociated microbial cells; separating the suspension to provide an extract rich in small cell debris and an extract rich in large cell debris; and combining at least a portion of the extracts to provide the microbial cell product. The invention further relates to a microbial cell product obtained or obtainable by said method. The invention further relates to the use of said products in food products, for example as a replacement for egg white; in animal food; and / or use in cosmetic formulations.

Description

Technical Field

[0001] This invention relates to a method for obtaining microbial cell products. The invention further relates to microbial cell products obtained by or obtainable by said method. The invention further relates to the use of said microbial cell products. Background Technology

[0002] Eggs are a versatile and virtually ubiquitous food and food component. They are highly valued for a variety of reasons. Not only do they provide high nutritional content, but they are also an essential component of a wide range of foods, including but not limited to: bread, cakes, cookies, custards, soufflés, muffins, scones, biscuits, pasta, sauces, gravy, and ice cream.

[0003] Egg whites make up about two-thirds of an egg's weight. They are primarily composed of about 90% water, with about 10% of the protein (including albumin, mucin, and globulin) dissolved in it. Unlike the lipid-rich yolk, egg whites contain almost no fat and less than 1% carbohydrates. Egg whites contain about 56% of the protein in an egg. Egg whites have many uses in food (such as meringues and mousses) and many other applications (such as in the preparation of vaccines, such as flu vaccines).

[0004] Consuming eggs has many disadvantages. For example, eggs contain high levels of cholesterol and saturated fat, which increase the risk of cardiovascular disease and obesity. Food allergies or other dietary restrictions may prevent other consumers who would otherwise benefit from the high nutritional content and enjoy egg products from doing so. For instance, it is estimated that 1% to 2% of young children are allergic to eggs. A significant number of people follow voluntary dietary restrictions, such as vegetarianism, and others may abstain from eggs to avoid animal exploitation or for religious or other reasons. Furthermore, the industrial-scale production of eggs is associated with the industrial farming of chickens, which incurs high costs, such as those related to farmers' food health and safety restrictions, high transportation costs, and the costs of feeding and confining laying hens. In addition, industrialized chicken farming has negative environmental impacts and raises several important humanitarian issues.

[0005] In addition, eggs have a limited shelf life and pose a risk of carrying infectious pathogens, such as Salmonella, E. coli, and other pathogens that may endanger public health.

[0006] Numerous attempts have been made to create egg substitutes (whole egg substitutes or egg white substitutes) that encapsulate the desired characteristics of a natural egg while minimizing its unwanted features. Egg substitutes aim to achieve binding, moisturizing, emulsifying, and / or leavening properties similar to those of an egg or egg white.

[0007] There are many home-cooking-based substitutes, such as mashed bananas, applesauce, chickpea water (aquafaba), or flaxseed to replace eggs in baking; baking powder / baking soda mixtures to provide leavening; and flour / water mixtures to provide binding and leavening.

[0008] Furthermore, industrially produced plant-based egg substitutes are known from, for example, WO2013067453A1, which discloses legumes or peas as starting materials. WO2017102535A1 discloses a method for obtaining a gluten-free natural rapeseed protein isolate containing <10 ppm gliadin, which can be used in any human nutritional food application, including as a foaming agent to replace egg white. WO2016077457A1 discloses a method for producing an alternative egg-free egg white protein, comprising recombinant expression of two or more egg white proteins; and mixing the two or more egg white proteins. WO2010045368A2 discloses a food composition comprising microalgal biomass, intact microalgal cells, and / or microalgal oil in combination with one or more other edible ingredients.

[0009] The above-mentioned whole egg and egg white substitutes only partially possess the desired egg white properties, which allow for the wide use of the composition in (food) products.

[0010] Therefore, there is a need for improved egg white substitutes. There is also a need for egg-free compositions that can be used as binders, humectants, emulsifiers, and / or oil / water retainers. Summary of the Invention

[0011] The object of this invention is to provide egg-free compositions that can be used as binders, gelling agents, thickeners, foaming agents, humectants, emulsifiers, and / or oil / water retainers. A further object of embodiments of the invention is to provide improved compositions for use in food, such as as an egg white substitute; for use in animal feed, such as pellet, dry / powder, semi-wet, or wet feed formulations; and / or for use in cosmetic formulations, such as solutions, creams, lotions, suspensions, ointments / pastes, powders, and gels.

[0012] The present invention relates, in a first aspect, to a method for preparing microbial cell products. The method comprises: i) providing an aqueous suspension containing microbial cells; ii) subjecting the suspension to mechanical cellular degradation to obtain an aqueous suspension containing decomposed microbial cells; iii) separating the suspension to provide an extract rich in small cell debris and an extract rich in large cell debris; and v) combining at least a portion of each extract to provide a microbial cell product.

[0013] The inventors have discovered that microbial cell products with properties different from those of a simple suspension can be produced by separating a suspension into two distinct extracts and then recombining the extracts. Furthermore, it is believed that the final properties of the microbial cell product can be altered by changing one or both of the decomposition step and the ratio of the recombined extracts. This allows for fine-tuning of the product to make it more or less suitable for a specific application. Additional "fine-tuning" steps may include incubating the composition for a period of time between certain steps of the method; for example, before the extracts are recombined.

[0014] This article describes the further details of this fine-tuning and other steps of the method in more detail.

[0015] The present invention also relates to microbial cell products that can be produced in this manner, and to food products comprising such microbial cell products. Other features and aspects of the invention will become apparent from the detailed description.

[0016] In another aspect, the present invention relates to a method for preparing microbial cell products. The method of the present invention comprises: i) providing an aqueous suspension containing microbial cells; and ii) subjecting the suspension to mechanical cytokinesis to obtain an aqueous suspension containing decomposed microbial cells as the microbial cell product. The mechanical cytokinesis according to this method occurs in a temperature range of 1°C to 45°C.

[0017] In another respect, the present invention relates to microbial cell products that can be obtained directly or by means of the present invention.

[0018] In another respect, the present invention relates to extracts rich in small cell fragments and extracts rich in large cell fragments that can be obtained by the method according to the invention.

[0019] On the other hand, the present invention relates to the use of microbial cell products according to the present invention as emulsifiers, foaming agents, adhesives, leavening agents, thickeners, humectants, glues, browning agents, clarifying agents, gelling agents, crystallization control agents, humectants, tenderizing agents, aeration agents, structure modifiers, coagulants, coating agents, colorants, gloss agents, flavoring agents, refrigerants, heat-insulating agents, texture modifiers, pH buffers, shelf-life extenders, fat substitutes, meat fillers, preservatives, antimicrobial agents, food spoilage inhibitors, malic lactic acid fermentation inhibitors, texture modifiers, egg substitutes, or any combination thereof.

[0020] In a further aspect, the present invention relates to the use of microbial cell products according to the invention in edible egg-free emulsions, egg analogues, egg-free scrambled eggs, egg-free pies, egg-free pound cakes, egg-free angel food cakes, egg-white meat substitutes, egg-free meat substitutes, yolk-free cakes, egg-free and dairy-free cream cheese, egg-free pasta dough, egg-free custard, egg-free ice cream, or dairy-free milk.

[0021] The corresponding embodiments disclosed for this method are also applicable to the microbial cell products according to the present invention, the extracts rich in small cell fragments according to the present invention, the extracts rich in large cell fragments according to the present invention, and the use of the microbial cell products according to the present invention.

[0022] Definition List

[0023] The following definitions are used in this specification and claims to define the subject matter stated. Other terms not referenced below have the meanings generally accepted in the art.

[0024] As used in this specification, "drying" refers to reducing the moisture content. The term drying includes partial drying, in which moisture can be retained in a reduced amount after drying, which can also be considered as concentration.

[0025] As used in this specification, “dry weight (DW)” and “cell dry weight” refer to weight measured in the absence of water. For example, a reference to microbial biomass containing a specific percentage of a particular component on a dry weight basis means that the percentage is calculated based on the weight of the biomass after substantially all water has been removed.

[0026] In the context of microbial cells, the term “disruption” as used in this specification is also referred to as “lysis” and means opening the cell to release cytoplasmic compounds (also referred to as “lysates”).

[0027] In the context of microbial cell decomposition, "decomposition" as used in this specification refers to cell fragmentation. This means that the average size of the resulting cell fragments must be smaller than the average cell size of the initial microbial cells. Decomposition can be viewed as a special type of disruption in which not only are cells opened up, but they are also broken down.

[0028] As used in this invention, "cytoplasmic material" or "cytoplasmic compound" refers to all substances normally contained within cells and surrounded by the cell membrane, except for the cell nucleus (if present). Cytoplasmic material is released from cells when they break down or are destroyed.

[0029] As used in this specification, "microbial cell" refers to a microorganism. This can be a single-celled organism, either eukaryotic or prokaryotic, and its community. Prokaryotes are cellular organisms lacking a nucleus surrounded by a membrane. In a three-domain system, based on molecular analysis, prokaryotes are divided into two domains: bacteria (formerly eubacteria) and archaea (formerly archaebacteria). Organisms with a nucleus reside in a third domain, namely eukaryotes. The microbial cells according to the invention also include algae and fungi such as yeast.

[0030] As used in this specification, "microorganism (microbe)" refers to any microscopic community or single-celled organism.

[0031] The term "microbial cell products" as used in this specification refers to products derived from microbial cells obtained by processing microbial cells in a certain manner.

[0032] As used in this invention, "extract rich in small cell fragments (EESF)" refers to a microbial cell product obtained by separating an aqueous suspension containing decomposed microbial cells. In this separation process, the extract is separated from the aqueous suspension containing decomposed microbial cells, leaving an aqueous suspension containing decomposed microbial cells that has partially depleted its small cell fragments. In other words, the separation process produces an extract rich in small cell fragments (as the main product) and an aqueous suspension depleted of small cell fragments, the latter of which can and will also be referred to as an extract rich in large cell fragments. As used in this specification, "small cell fragments" refers to cell fragments with a size equal to or less than d50 < 500 nanometers (nm) obtained from the decomposition of microbial cells.

[0033] As used in this invention, "extract rich in large cell debris (EELF)" or "aqueous suspension depleted of small cell debris" refers to a microbial cell product obtained by separating an aqueous suspension containing decomposed microbial cells. In this separation process, the extract is separated from the aqueous suspension containing decomposed microbial cells, leaving a partially depleted aqueous suspension containing decomposed microbial cells. In other words, the separation process produces an extract rich in small cell debris (as the main product) and an aqueous suspension depleted of small cell debris (as a byproduct), the latter of which can and will be referred to as an extract rich in large cell debris. As used in this specification, "large cell debris" refers to cell debris with a size greater than d50 > 500 nanometers (nm) obtained from the decomposition of microbial cells.

[0034] As used in this specification, "microbial biomass" and "biomass" refer to substances produced through the growth and / or reproduction of microbial cells, or substances produced as byproducts of fermentation processes. Biomass may comprise cells and / or intracellular contents as well as extracellular substances. Extracellular substances include, but are not limited to, compounds secreted by cells.

[0035] As used in this specification, "bead milling" refers to the agitation of microbial cells in a suspension using small abrasive particles (beads). Cells are broken down by shear forces, abrasion between beads, and collisions with each other. The shear forces generated by the beads damage the cells and cause decomposition, releasing cellular compounds in the process.

[0036] As used in this manual, "centrifugation" refers to the separation of particles from a solution using centrifugal force based on parameters such as particle size, shape, density, viscosity of the medium, and rotor speed. The centrifugation rate is typically specified as angular velocity, expressed as revolutions per minute (RPM), or acceleration, expressed as g. The conversion factor between RPM and g depends on the radius of the centrifuge rotor. The general formula for calculating the centrifuge's revolutions per minute (RPM) is:

[0037]

[0038] Where g represents the corresponding force of the centrifuge, and r represents the radius from the center of the rotor to a point in the sample. However, depending on the model of the centrifuge used, the corresponding angle and radius of the rotor may vary, so the formula will need to be modified. The most commonly used formula for calculating relative centrifugal force is:

[0039]

[0040] Where r is the radius in mm. Attached Figure Description

[0041] The invention is described below with reference to the accompanying drawings, in which embodiments of the invention are shown and similar reference numerals denote the same or similar elements.

[0042] Figure 1 An overview of the different (optional) steps of the method according to the invention and the obtained (intermediate) products is shown. This overview illustrates many different pathways for obtaining microbial cell products according to the invention.

[0043] Figure 2 shows an overview of native conformational proteins in extracts rich in small cell debris obtained at different pH values ​​(left), and the solubility of proteins in extracts rich in small cell debris at different pH values ​​(right).

[0044] Figure 3The dissolution of proteins and carbohydrates in an extract rich in small cell debris was shown during the bead milling test.

[0045] Figure 4 The particle size distribution of cell fractions after bead milling or after bead milling and centrifugation is shown.

[0046] Figure 5 The effect of centrifugal force on the maximum gel strength [Pa] after purification is shown.

[0047] Figure 6 The effect of membrane retention on the gelation behavior of purified extracts rich in small cell debris is shown, as measured by gel strength G'max [Pa].

[0048] Figure 7 The equilibrium surface tensions of the obtained byproducts (P, left panel) and the purified extract rich in small cell debris (R, right panel) at the air-water (left) and oil-water (right) interfaces are shown compared to the surface tensions of egg white protein isolates and whey protein isolates.

[0049] Figure 8 A simplified process flow diagram for preparing microbial cell products according to an embodiment of the present invention is shown.

[0050] Figure 9 The particle size distribution of yeast under different disruption conditions is shown.

[0051] Figure 10A and 10B The study shows the changes in specific surface area and particle size distribution of yeast over time during the disruption process.

[0052] Figure 11 The gel hardness of microbial cell products at low, medium, and high separation strengths was shown.

[0053] Figure 12 The particle size distribution of different fractions of yeast preparations under different separation intensities is shown.

[0054] Figure 13 The specific surface area and concentration of different fractions of yeast preparations under different separation intensities are shown.

[0055] Figure 14 The gel stiffness of EESF and EELF of yeast preparations isolated under different dilution conditions is shown.

[0056] Figure 15 The water-holding and oil-holding capacities of EELFs from isolated yeast preparations under different dilution conditions are shown.

[0057] Figure 16The particle size distribution of EESF in yeast preparations separated under different dilution conditions is shown.

[0058] Figure 17 The specific surface area and concentration of different fractions of the isolated yeast preparation under different dilution conditions are shown.

[0059] Figure 18 The particle size distribution of yeast preparations separated under different dilution conditions is shown.

[0060] Figure 19 The oil-holding capacity of microbial preparations derived from different microorganisms after separation under different dilution conditions was demonstrated.

[0061] Figure 20 The gel hardness, oil-holding capacity, and water-holding capacity of the yeast preparation were shown after disruption at different pH levels.

[0062] Figure 21A and 21B The particle size distribution of yeast that was destroyed over time at different pH levels is shown -d50 and D[3,2]. Detailed Implementation

[0063] As described above, the present invention relates in a first aspect to a method for preparing a microbial cell product, the method comprising: i) providing an aqueous suspension containing microbial cells; ii) subjecting the suspension to mechanical cytokinesis to obtain an aqueous suspension containing decomposed microbial cells; iii) separating the suspension to provide an extract rich in small cell fragments and an extract rich in large cell fragments; and v) combining at least a portion of each extract to provide a microbial cell product. The mechanical cytokinesis can be carried out at a temperature ranging from 1°C to 45°C (i.e., from 1°C to a maximum of and including 45°C), preferably from 15°C to 35°C, more preferably from about 25°C, to obtain the aqueous suspension containing decomposed microbial cells.

[0064] exist Figure 1 In the diagram, the step of providing the aqueous suspension (step i) is shown in gray on the left. Step ii) is shown in gray slightly to the left of center. After step i), the aqueous suspension can be directly subjected to step ii) or pretreated (step ia) first. For each of the different approaches (in Figure 1(Disclosed herein and as disclosed below), an optional pretreatment step may be present. The product from step ii) is an aqueous suspension containing decomposed microbial cells. The aqueous suspension is then separated (iii) to provide an extract rich in small fragments (EESF) and an extract rich in large cell fragments (EELF), as well as soluble compounds, each fraction suspended in the soluble compounds. A portion of each enriched extract is then combined (v) to provide the microbial cell product. This may be referred to herein in some cases as a mixed extract. Furthermore, Figure 8 The process is illustrated in the diagram.

[0065] The method may optionally include any one of steps ia), iv), purification, vi), drying, and vii). For example, the method may include steps i) and ii); or steps i), ia), and ii); or steps i), ia), ii), and iii); or steps i), ia), ii), iii), and iv); or steps i), ia), ii), iii), iv), and v); or steps i), ia), ii), iii), iv), v), and iv); or steps i), ia), ii), iii), iv), v), vi), and vii).

[0066] The microbial cell products that can be obtained by the method according to the present invention include: mixed extracts, mixed purified extracts, dried mixed extracts, dried mixed purified extracts, mixed dried extracts, and mixed dried purified extracts.

[0067] Microbial cell products refer to both purified and dried microbial cell products.

[0068] The purified microbial cell products that can be obtained by the method according to the present invention include mixed purified extracts, dried mixed purified extracts, and mixed dried purified extracts.

[0069] The dried microbial cell products that can be obtained by the method according to the present invention include dried mixed extracts, dried mixed purified extracts, mixed dried extracts, and mixed dried purified extracts.

[0070] Certain steps of the methods described herein can be implemented to obtain alternative products to the mixed extracts obtained from the methods of this invention. For example, refer to Figure 1 The steps in the above steps, after which a purified suspension of decomposed microbial cells is obtained, are performed in the following subsequent steps: steps i), ii), and iv); or steps i), ia), ii), and iv).

[0071] The dried, purified, and decomposed microbial cells are obtained after the following subsequent steps: steps i), ii), iv), and vi); or steps i), ia), ii), iv), and vi).

[0072] The dried, decomposed microbial cells are obtained after the following subsequent steps: steps i), ii), and vi), or steps i), ia), ii), and vi).

[0073] An extract rich in small cell fragments is obtained after performing the following subsequent steps: steps i), ii), and iii); or steps i), ia), ii), and iii).

[0074] An extract rich in large cell fragments is obtained after the following subsequent steps: steps i), ii), and iii); or steps i), ia), ii), and iii).

[0075] A dried extract rich in small cell debris is obtained after performing the following subsequent steps: steps i), ii), iii) and step vi) on the extract rich in small cell debris, or steps i), ia), ii), iii) and step vi) on the extract rich in small cell debris.

[0076] A dried extract rich in large cell debris is obtained after performing the following subsequent steps: steps i), ii), iii) and step vi) on the extract rich in large cell debris; or steps i), ia), ii), iii) and step vi) on the extract rich in large cell debris.

[0077] A purified extract rich in small cell debris is obtained after performing the following subsequent steps: steps i), ii), iii) and step iv) of the extract rich in small cell debris; or steps i), ia), ii), iii) and step iv) of the extract rich in small cell debris.

[0078] A purified extract rich in large cell debris is obtained after performing the following subsequent steps: steps i), ii), iii) and step iv) of the extract rich in large cell debris; or steps i), ia), ii), iii) and step iv) of the extract rich in large cell debris.

[0079] A dried, purified extract rich in small cell debris is obtained after performing the following subsequent steps: steps i), ii), iii), and step vi), and step vi); or steps i), ia), ii), iii), and step vi), and step vi).

[0080] A dried, purified extract rich in large cell debris is obtained after performing the following subsequent steps: steps i), ii), iii), and step vi), and step vi); or steps i), ia), ii), iii), and step vi), and step vi).

[0081] The mixed extract (microbial cell product according to the invention) is obtained after performing the following subsequent steps: steps i), ii), iii) and step v) of mixing a portion of the extract rich in small cell fragments and a portion of the extract rich in large cell fragments; or steps i), ia), ii), iii) and step v) of mixing a portion of the extract rich in small cell fragments and a portion of the extract rich in large cell fragments.

[0082] A dried mixed extract (microbial cell product according to the invention) is obtained after performing the following subsequent steps: steps i), ii), iii), step v), mixing a portion of the extract rich in small cell fragments and a portion of the extract rich in large cell fragments, and step vi); or steps i), ia), ii), iii), step v), mixing a portion of the extract rich in small cell fragments and a portion of the extract rich in large cell fragments, and step vi).

[0083] The mixed purified extract (microbial cell product according to the invention) is obtained after performing the following subsequent steps: steps i), ii), iii), step vi), and step v), mixing a portion of the purified extract rich in small cell fragments and a portion of the purified extract rich in large cell fragments; or steps i), ia), ii), iii), step vi), and step v), mixing a portion of the purified extract rich in small cell fragments and a portion of the purified extract rich in large cell fragments.

[0084] The dried mixed purified extract (microbial cell product according to the invention) is obtained after performing the following subsequent steps: steps i), ii), iii), step vi), mixing a portion of the purified extract rich in small cell fragments and a portion of the purified extract rich in large cell fragments, and step vi); or steps i), ia), ii), iii), step vi), mixing a portion of the purified extract rich in small cell fragments and a portion of the purified extract rich in large cell fragments, and step vi).

[0085] The mixed dried extract (microbial cell product according to the invention) is obtained after performing the following subsequent steps: steps i), ii), iii), step vi), and step v), mixing a portion of the dried extract rich in small cell fragments and a portion of the dried extract rich in large cell fragments; or steps i), ia), ii), iii), step vi), and step v), mixing a portion of the dried extract rich in small cell fragments and a portion of the dried extract rich in large cell fragments.

[0086] The following subsequent steps are performed to obtain a mixed dried purified extract (microbial cell product according to the invention): steps i), ii), iii), step vi), step vi), step v), and step v) of mixing a portion of the dried purified extract rich in small cell fragments and a portion of the dried purified extract rich in large cell fragments; or steps i), ia), ii), iii), step vi), step v) of mixing a portion of the dried purified extract rich in small cell fragments and a portion of the dried purified extract rich in large cell fragments; or steps i), ia), ii), iii), step vi), step vi), step v) of mixing a portion of the dried purified extract rich in small cell fragments and a portion of the dried purified extract rich in large cell fragments.

[0087] like Figure 1 As shown, different byproducts can be obtained. The first and / or second byproducts can be further processed, for example, by drying or concentration, to obtain dried or concentrated byproducts.

[0088] Microbial biomass containing microbial cells has traditionally been used to produce a wide range of industrial products or directly for various applications. Most industrial or commercial applications use a group of microbial biomass strains selected from the domains of bacteria, yeast, fungi, and algae. Generally, the products obtained from microbial biomass are either intracellular or extracellular. Extracellular products are excreted by the cells into a large volume of media, typically an aqueous phase. Conversely, intracellular products remain inside the cells. To obtain intracellular products, additional processing is required to release these products from the cells (by disrupting the cell membrane or cell wall) and to further separate the compounds of interest from the remaining biomass and other impurities.

[0089] In specific food applications, microbial biomass has been used as a source of protein (single-cell protein - SCP), a nutritional supplement, or for the production of various ingredients and additives.

[0090] Microbial biomass is typically used in the form of extracts, which requires the destruction / decomposition of the microbial cells that form the biomass. Extracts prepared from several different starting materials are known, such as fungal extracts, algal extracts, and yeast extracts. Of these, the most commonly used is the yeast-derived extract, also known as yeast extract (hereinafter referred to as "YE"). YE is (and can) used in a wide range of products, from growth media for laboratory cell culture to nutritional supplements and flavor enhancers for the food industry. The production process of YE is well known. Generally, yeast cells, primarily from the genus *Saccharomyces*, are destroyed (=decomposed) through heat-induced or chemically induced autolysis (or plasmolysis), followed by an incubation step at high temperatures (>50°C) to activate endogenous enzymes that break down (=digest) large intracellular products (such as proteins and nucleic acids) into their smaller components (such as peptides, amino acids, and nucleotides). The resulting digestive slurry is then further purified and replenished according to the final application to provide an extract that can be commercially available as YE.

[0091] Cell degradation methods can generally be categorized as either non-mechanical or mechanical. Non-mechanical degradation methods can be further subdivided into three categories: physical degradation (e.g., by decompression, osmotic shock, pyrolysis, ultrasound, or freeze-thaw), chemical degradation (e.g., by using solvents, detergents, dissociation agents, acids and bases, or chelates), and enzymatic degradation (e.g., by autolysis, phage lysis, or lyases). This invention relates only to mechanical degradation methods. An example of a mechanical degradation method is a ball mill, including a bead mill and a homogenizer.

[0092] The homogenizer operates under high pressure and is essentially a positive displacement pump, forcing the cell suspension through valves and then impacting the impingement rings at high speed. Typically, this process requires multiple passes under high pressure, which can lead to temperature rise and consequently, localized denaturation of unstable molecules.

[0093] Ball mills (including bead mills) can be vertical or horizontal and use abrasive media present in the grinding chamber. A motor drives a rotor to rotate the cell suspension at high speed. The cell suspension and the abrasive material (e.g., beads) generate shear forces that break down the cells. This results in the release of intracellular material into the aqueous suspension and also leads to cell fragmentation (i.e., decomposition). As the rotor speed increases, the shear force increases and cell fragmentation increases. Cell fragmentation generally increases as the size of the abrasive material decreases. Other parameters affect the performance of the decomposition process. Those skilled in the art can select the appropriate parameters and variables according to the present invention.

[0094] Examples of cell decomposition methods according to the prior art are as follows. US3888839A discloses a method for obtaining protein isolates from yeast cells, wherein the yeast cells are ruptured by high-pressure homogenization (mechanical decomposition) and subsequent incubation. EP1199353A1 discloses a method for producing yeast extract by treating a yeast suspension or yeast paste and separating insoluble components, wherein the yeast suspension or yeast paste is subjected to a high-voltage electrical pulse (physical decomposition). EP2774993A1A discloses the use of a cell wall degrading enzyme without protease (enzymatic decomposition), followed by heat treatment of the product at 70°C to 80°C for 10 to 20 minutes.

[0095] Microbial cells present in microbial biomass suspensions primarily contain proteins, carbohydrates, lipids, and minerals. Proteins and other unstable molecules undergo unfolding, denaturation, and degradation when exposed to high temperatures, prolonged incubation, extreme pH values, solvents, salts, and other irritating chemicals. When proteins and other functional molecules denature (loss of tertiary and quaternary structures), they lose (a portion) of their functional activity. After denaturation (unfolding), proteins lose their ability to interact with both hydrophilic and hydrophobic surfaces, and their ability to rearrange and form network structures after heat-cooling treatment is also affected. (Protein) functionality is crucial in many commercial applications, particularly for applications requiring gelling properties (similar to those of egg white). The inventors have observed that mechanical decomposition under the conditions described herein (e.g., within a specified temperature and pH range) is sufficiently mild to prevent the unfolding, denaturation, and / or degradation of proteins and other unstable molecules, and is therefore necessary to maintain functional properties, particularly gelling behavior.

[0096] In one embodiment, the mass concentration of microbial cells in the suspension in step i) ranges from 1% to 25%, preferably from 5% to 15%. The mass concentration is a percentage of dry weight, and 10% dry weight is equivalent to 100 g / L.

[0097] Aqueous suspensions containing microbial cells may also contain cytoplasmic material or other extracellular substances produced during reproduction or fermentation.

[0098] In one embodiment of the method according to the invention, the microbial cells are selected from single-celled or community-sized prokaryotic and eukaryotic cells, and one or more combinations thereof. In a preferred embodiment, the microbial cells are selected from the group consisting of yeast, algae, bacteria, fungi, and one or more combinations thereof. In a specific embodiment, the microbial cell is yeast.

[0099] In one embodiment, the method further includes: after step i) and before step ii), pretreating the aqueous suspension containing microbial cells in step ia) to obtain a pretreated aqueous suspension containing microbial cells and byproducts. In a specific embodiment, the pretreatment is selected from decantation, centrifugation, filtration (such as membrane filtration), and one or more combinations thereof. Prior to the pretreatment, the aqueous suspension containing microbial cells may be washed. The combination of washing and then pretreatment can be considered a dual pretreatment. Washing may be performed, for example, with water, acid, or alkali, or with a solvent (such as ethanol). In this embodiment, the steps are in the following sequence: step i), step ia), step ii), wherein the pretreated suspension obtained in step ia) is used in step ii) where the suspension is subjected to mechanical cellular degradation.

[0100] In one embodiment, the mechanical cell decomposition in step ii) is performed using a bead mill. In one embodiment, the bead milling is performed for a period ranging from 1 minute to 3 hours. The milling time can also be used to adjust the amount of cytoplasmic compounds and other cell-derived compounds (such as proteins and carbohydrates) released into the aqueous suspension. The cell decomposition process can be carried out at 15°C to 35°C (preferably about 25°C) to reduce metabolic activity, preserve protein function, and reduce cooling costs. The decomposition process is preferably carried out at pH 7 to pH 11, preferably around pH 9. Alternatively, cell decomposition can be carried out at T < 25°C for > 3 hours and at pH 11.

[0101] Preferred decomposition conditions are selected to provide the desired particle size distribution. As discussed further herein, altering the particle size distribution can allow for changes in the final properties of the blended product, such as to suit a specific intended use. The decomposition process is run in a manner that yields a bimodal particle size distribution (PSD), displaying peaks of intact cells and cell debris. In the case of yeast, a ~6 μm intact cell peak and a ~0.8 μm cell debris peak were obtained. See also Figure 4 and Figure 9 .

[0102] During cell degradation, PSD changes, showing a decrease in the intact cell peak followed by an increase in the cell debris peak. The degradation process is run until a specific PSD is obtained. In yeast, for example, specific surface area increases over time, while D[3,2], D[4,3], D10, D50, and D90 decrease over time—see [link to relevant documentation]. Figure 10A , 10B For yeast, the desired target PSDs are D10 < 0.5 μm, D50 < 4.5 μm, D90 < 7.5 μm, and D[3,2] < 2, D[4,3] < 4.5. Technicians can adjust the parameters of specific decomposition methods / equipment to achieve the desired PSDs. This can be done, for example, with bead mills, homogenizers, or other equipment and specific settings, as widely described in professional literature.

[0103] The bead size can be in the range of 0.1mm to 5mm, preferably in the range of 0.5mm to 1mm. Suitable bead materials include, but are not limited to, zirconium and glass.

[0104] Based on the total usable volume of the bead mill chamber, the appropriate bead filling percentage (the percentage of beads filling the bead mill chamber) can be considered to be in the range of 40% to 90%, preferably in the range of 65% to 80%.

[0105] A suitable rotational speed can be considered to be in the range of 1 m / s to 20 m / s. Depending on the configuration and geometry of each bead mill, a technician can estimate the corresponding rotor speed in rpm. A suitable rotational speed in rpm is, for example, 500 rpm to 5000 rpm, preferably 1000 rpm to 3000 rpm.

[0106] The appropriate microbial cell concentration can be considered to be in the range of 2% to 25% dry weight.

[0107] In one specific implementation, a Dyno-mill research laboratory (CB Mills) bead mill is used to break down microbial cells. Cells can also be broken down by shear forces, such as using blending (e.g., using a high-speed or Waring agitator), a French press, or even centrifugation where the cell walls are thin.

[0108] In one implementation, cell decomposition occurs without the addition of chemicals and / or solvents.

[0109] In one embodiment, the pH value during step ii) is in the range of 4 to 11. In a preferred embodiment, the pH value during step ii) is in the range of 7.5 to 9.

[0110] In one embodiment, the temperature during step ii) is below 45°C, preferably equal to or below 35°C, for example below 35°C or between 15°C and 25°C.

[0111] In one embodiment, the decomposition step is performed in such a manner that at least 10% of the particles have a particle size ≤0.5 μm and at least 50% of the obtained cell debris has a size ≤4.5 μm.

[0112] The method further includes step iii) following step ii), which involves subjecting the aqueous suspension containing decomposed microbial cells obtained in step ii) to a separation process to obtain an extract rich in small cell debris and an extract rich in large cell debris as microbial cell products, as well as a soluble compound suspended in the soluble compound. In other words, the aqueous suspension containing decomposed microbial cells is separated into two extracts. These may be referred to herein as EESF (extract rich in small fragments) and EELF (extract rich in large fragments). The separation process can be a solid-liquid separation. The separation process can be carried out using any suitable method known to those skilled in the art. In a preferred embodiment, the separation process is selected from the group consisting of decantation, centrifugation, filtration (such as membrane filtration), and one or more combinations thereof. Suitable separation methods also include sedimentation, deposition, flocculation / coagulation, precipitation, decantation, (hydraulic) cyclone separation, and (air) flotation. Specific separation conditions can also be fine-tuned to obtain enriched extracts with specific preferred properties, as explained herein.

[0113] Extracts rich in small cell debris may primarily contain soluble cytoplasmic material and small cell debris, while extracts rich in large cell debris may primarily contain large cell debris and some soluble cytoplasmic material.

[0114] EESF is preferably obtained through centrifugation at low and medium intensities. Low and medium intensities mean short time or low g-force or a combination thereof. An example of low intensities is centrifugation at <4000 rcf for <2 minutes in a benchtop centrifuge. An example of medium intensities is centrifugation at <4000 rcf for <15 minutes in a benchtop centrifuge. An example of high intensities is centrifugation at >20000 rcf for >20 minutes.

[0115] To increase EESF yield, low and medium separation strengths are preferred. Furthermore, low and medium separation strengths result in excellent functionality of EESF.

[0116] In one embodiment, the separation process in step iii) is centrifugation at a centrifugal force equal to or less than 4000 relative centrifugal force (rfc).

[0117] In one embodiment, the centrifugation occurs for a period of time equal to or less than 20 minutes. In another specific embodiment, the centrifugation occurs for a period of time equal to or less than 15 minutes.

[0118] In one embodiment, the extract rich in small cell debris comprises microbial cell debris and soluble cytoplasmic compounds, wherein at least d50 < 500 nm.

[0119] In one embodiment, compared with an aqueous suspension of small cell fragments obtained simultaneously (i.e., an extract rich in large cell fragments), the extract rich in small cell fragments contains at least 1%, preferably at least 10%, for example at least 20%, or even at least 30% or at least 50% more small cell fragments.

[0120] Separation parameters can be adjusted to achieve preferential recovery of particles with a preferred size distribution. For example, in the case of yeast, preferential recovery of particles in the 0.1 μm to 3 μm range (at least d50 < 500 nm) for EESF and preferential recovery of particles in the 3 μm to 10 μm range for EELF may be the goal.

[0121] The particle size distribution of EESF varies depending on the separation intensity in the centrifuge.

[0122] In some implementations, the water content of the decomposed cell preparation can be adjusted prior to the separation step. Changing the dilution level can modify the functional properties of EESF and EELF.

[0123] In one embodiment, the method further includes incubating the EESF prior to the mixing step v). It has been found that during cell degradation, the pH decreases (e.g., from about 9 to about 6.5). Under these conditions, a significant amount of CO2 can be released. This provides a leavening effect suitable for a variety of food applications. However, this may not always be desired, and therefore the EESF can be subjected to an incubation step to deplete excess CO2. For example, the EESF can be incubated at below 25°C, under mild or no-stirring and aerobic conditions for at least 60 minutes. In one embodiment, the EESF, as a liquid suspension containing >5% DW, is placed under stirring for >60 minutes at T < 25°C. During the incubation process, excess gas is released without affecting the functional properties of the EESF. After the incubation process, the leavening effect is significantly reduced. Furthermore, to the inventors' surprise, if the pH is slightly increased, for example from pH < 6.5 after degradation to pH > 6.9 (e.g., pH 6.9 or pH 7.0), the leavening effect can be significantly reduced.

[0124] In one embodiment, the method further includes iv) purifying at least one of the microbial products obtained in step ii) or iii) to obtain at least one purified microbial cell product. The purification treatment can be performed using any suitable method known to those skilled in the art. Examples of purification methods are adsorption, chromatography, filtration (e.g., percolation or membrane filtration), crystallization, flocculation / coagulation, precipitation, two-phase extraction, subcritical and supercritical extraction, and solvent extraction. In a preferred embodiment, the purification treatment is selected from the group consisting of washing, filtration (e.g., membrane filtration or percolation), adsorption, chromatography, crystallization, flocculation / coagulation, precipitation, two-phase extraction, subcritical and supercritical extraction, solvent extraction, and one or more combinations thereof. If the microbial products obtained in steps ii) and iii) are both purified, these purification treatments are selected independently.

[0125] In one embodiment, the purification process in step iv) is membrane filtration, wherein the membrane used has a rejection range of 1 kDa to 20000 kDa, preferably 10 kDa to 1000 kDa. In another embodiment, the membrane used has a rejection range of 0.1 μm to 2 μm.

[0126] The method includes v) mixing at least a portion of the extract rich in small cell debris obtained in step iii) and at least a portion of the extract rich in large cell debris obtained in step iii) to obtain a mixed extract as a microbial cell product, or mixing at least a portion of the purified extract rich in small cell debris obtained in step iv) and at least a portion of the purified extract rich in large cell debris obtained in step iv) to obtain a mixed purified extract as a microbial cell product. This mixing can be used to further refine the product. For example, mixing can be applied when the separation seems too “thorough” for the intended purpose, i.e., the enrichment of the extract is too high. For example, when the amount of small cell debris in the extract rich in small cell debris is higher than the optimal amount for a specific purpose, a portion of the extract rich in large cell debris can be mixed with the extract rich in small cell debris to obtain a mixed extract.

[0127] It is important to note that this provides a mixture with properties that differ from EESF or EELF, and also from cell-degrading preparations, because at least some soluble components are removed during the separation step. As will be apparent from the details provided herein, specific EESF and EELF extracts can be produced under slightly different conditions and in slightly different ways to obtain the desired properties, and the proportions to be combined can then be varied to obtain the desired properties in the mixture.

[0128] In one embodiment, the method further includes vi) subjecting at least one of the microbial products obtained in steps ii), iii), iv), or v) to drying to obtain dried microbial cell products. In a preferred embodiment, the drying is performed using a method selected from spray drying, freeze drying, and fluidized bed drying. Drying may also be concentration. Preferably, the water content of the dried microbial cell products is 10% by weight or less, or more preferably 5% by weight or less, based on the total weight of the dried microbial cell products. Spray drying and fluidized bed drying at temperatures above 150°C and below 200°C produce products with neutral color, odor, and taste. This is because volatile compounds are eliminated during the drying process. Freeze drying is a preferred technique for preserving odor, color, and taste because they are retained during sublimation.

[0129] To further preserve the functional properties of EESF and EELF, concentration is preferred. Concentration is preferably carried out using methods known in the art, wherein the product is not exposed to temperatures >50°C or <0°C. An example of such a method is membrane concentration. EESF and EELF are preferably concentrated to achieve a DW content >20%.

[0130] In one embodiment, the method further includes vii) mixing at least a portion of the dried extract rich in small cell fragments obtained in step vi) and at least a portion of the dried extract rich in large cell fragments obtained in step vi) to obtain a mixed dried extract as a microbial cell product, or mixing at least a portion of the dried purified extract rich in small cell fragments obtained in step vi) and at least a portion of the dried purified extract rich in large cell fragments obtained in step vi) to obtain a mixed dried purified extract as a microbial cell product. Alternatively, one of the dried extracts may be purified before mixing, without purifying the other. For simplicity, Figure 1 This option is not described in the document.

[0131] When concentrating rather than drying EESF and EELF Figure 1 All the mixing strategies described in [the document] also apply. For simplicity, Figure 1 This option is not described in the document.

[0132] In one aspect, the present invention relates to an extract rich in small cell debris that can be obtained by a method comprising at least steps i), ii), and iii), wherein the extract contains soluble proteins whose native conformations have a molecular weight greater than 10 kDa, preferably greater than 50 kDa, and more preferably greater than 60 kDa. In one embodiment, at least 95% of the native conformations of all soluble proteins in the extract have a molecular weight greater than 10 kDa, preferably greater than 50 kDa, and more preferably greater than 60 kDa.

[0133] In one embodiment of this aspect, the extract rich in small cell debris comprises microbial cell debris and soluble cytoplasmic compounds, wherein at least 80% of the cell debris has a d50 < 500 nm.

[0134] In one embodiment of this aspect, compared with an aqueous suspension of small cell fragments obtained simultaneously (i.e., an extract rich in large cell fragments), the extract rich in small cell fragments contains at least 1%, preferably at least 10%, for example at least 20%, or even at least 30% or at least 50% more small cell fragments.

[0135] In one embodiment, the microbial cell product provides egg-like adhesive, moisturizing, water- and oil-retaining, and / or emulsifying properties. In some cases, the microbial cell product is used as an adhesive. In some cases, the microbial cell product is used as a humectant. In some cases, the microbial cell product is used as an emulsifier. In some cases, the microbial cell product is used as a foaming agent. In some cases, the microbial cell product is used as a water- or oil-retaining agent. In some cases, the microbial cell product is used as a gelling agent.

[0136] In some embodiments, microbial cell products can be used as substitutes for egg yolks, egg whites, or whole eggs in the preparation of equal amounts of products using equal or unequal amounts of eggs. In a preferred embodiment, the microbial cell products are used as substitutes for egg whites.

[0137] In some respects, the present invention provides food products comprising a mixture of microbial cell products as described herein.

[0138] In one embodiment, the pH value during steps iii) and / or iv) is equivalent to the pH value at the end of step ii) of the method. In one embodiment, the temperature during steps iii) and / or iv) is the same as the temperature during step ii) of the method.

[0139] Although this invention is described primarily with reference to preparations obtained from yeast cells, it is not limited thereto. Various other microorganisms may be used. In embodiments, the microorganisms may be selected from fungi, including yeasts (preferably *Saccharomyces*, more preferably *Saccharomyces* var. *saccharomyces* or *Bakeryomyces* var. *saccharomyces*); plants, particularly microalgae (including *Tetraselmis* sp. or *Chlorella* sp., such as *C. vulgaris*); and cyanobacteria (including *Arthrospira* var. *platensis*). The microorganisms may also be selected from bacteria, such as lactic acid bacteria.

[0140] Those skilled in the art, when practicing the claimed invention, can understand and implement other variations of the disclosed embodiments by studying the drawings, disclosure, and appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used advantageously.

[0141] The scope of this invention is defined by the appended claims. One or more objects of this invention are achieved by the appended claims.

[0142] method

[0143] Provides an aqueous suspension containing microbial cells

[0144] In the example of providing an aqueous suspension, the aqueous suspension is prepared by adding baker's yeast (dry yeast or as a wet suspension) to a known volume of water under gentle stirring and constant temperature to achieve a concentration of 100 g / L (10% dry weight). Stirring is performed until a homogeneous suspension is obtained.

[0145] pH adjustment

[0146] pH adjustment can be achieved, for example, by slowly adding a 5M NaOH solution to an aqueous suspension containing microbial cells under constant temperature and stirring until the desired pH is reached. Other solutions of NaOH and other types of alkali (e.g., Ca(OH)2) can be used to adjust the pH of the microbial suspension. Similarly, suitable acidic solutions can be used to lower the pH of the microbial suspension.

[0147] Preprocessing

[0148] In one example, the pretreatment of microbial biomass can be carried out by washing, which includes four steps:

[0149] 1) Centrifuge the homogeneous yeast suspension (100g / L) at 3000rcf for 5 minutes at 15℃.

[0150] 2) Remove the obtained supernatant and replace it with an equal amount of water.

[0151] 3) Gently mix the obtained suspension until a uniform suspension is obtained.

[0152] 4) Repeat steps 1-3 2 to 3 times. The removed supernatant can be collected separately or mixed to obtain a byproduct (byproduct 1 in this invention).

[0153] 5) Steps 1-4 can be performed by a person skilled in the art using process configurations known in the art. An example of such a configuration is countercurrent washing or cocurrent washing.

[0154] Cellular decomposition

[0155] In the example of yeast cell decomposition, cell decomposition was performed using a bead mill. This bead milling was conducted by the Dyno Mill Research Laboratory (Willy A. Bachofen AG). A 200 ml solution (pH 8.9) containing 100 g / L (10% DW) of Saccharomyces cerevisiae was treated for 15 minutes in batch recirculation mode. A constant speed of 2039 rpm was used, and the temperature was controlled within the range of 24°C to 26°C. The mill contained 0.5 mm spherical zirconium beads with a 65% fill factor. This yielded an aqueous suspension containing decomposed microbial cells.

[0156] Separation Processing

[0157] In the separation example, separation was accomplished by centrifugation in an Allegra X-22R benchtop centrifuge equipped with a basket. The sample was centrifuged at 4000 rcf for 15 minutes at 15°C. This yielded an extract rich in small cell debris, as well as a residue, which was an aqueous suspension partially depleted of small cell debris, also known as an extract rich in large cell debris.

[0158] Purification

[0159] In the purification example, purification was accomplished by filtration using a laboratory-scale TTF-system (Millipore). Filtration was performed at a constant transmembrane pressure of <1 bar and in recirculation mode to achieve a concentration factor of 3 to 6 times. For each experiment, the mixture was diluted 1-fold with water before each filtration cycle. Therefore, purification was also performed as a percolation process.

[0160] Purification was accomplished by ultrafiltration or microfiltration, i.e., using an ultrafilter or microfilter in the methods described above. Ultrafiltration was performed using a hydrophilic membrane capsule (Biomax) in the range of 10 kDa to 1000 kDa. Microfiltration was performed using a 0.2 μm hollow fiber membrane unit (GE).

[0161] dry

[0162] In the drying example, drying was carried out using a spray dryer (Ollital Technology) at 180°C to achieve a final moisture content of <5% by weight. Alternatively, a freeze dryer (Zirbuss GmbH 2x3x3) can be used at -20°C and <1 mbar for 48 hours.

[0163] Example

[0164] The present invention is further illustrated by the following embodiments, which are merely illustrative and not intended to limit the invention.

[0165] Example 1

[0166] An aqueous suspension containing microbial cells from baker's yeast was prepared as described above. The pH of the aqueous suspension was adjusted to pH 7 before cell degradation.

[0167] Cell decomposition is performed as described above, and an aqueous suspension containing the decomposed microbial cells is obtained.

[0168] Example 2

[0169] An aqueous suspension containing microbial cells from baker's yeast was prepared as described above. The pH of the aqueous suspension was adjusted to pH 7 before cell degradation.

[0170] Cell decomposition and subsequent separation were performed as described above to obtain extracts rich in small cell fragments and extracts rich in large cell fragments.

[0171] Example 3

[0172] An aqueous suspension containing microbial cells from baker's yeast was prepared as described above. The pH of the aqueous suspension was adjusted to pH 9 before cell degradation.

[0173] As described above, cell decomposition, subsequent separation, and subsequent purification of the extract rich in small cell debris by ultrafiltration are performed to obtain a purified extract rich in small cell debris.

[0174] Example 4

[0175] An aqueous suspension containing microbial cells from baker's yeast was prepared as described above. The pH of the aqueous suspension was adjusted to pH 9 before cell degradation.

[0176] As described above, cell decomposition, subsequent separation, and subsequent purification of the extract rich in small cell debris by microfiltration were performed to obtain a purified extract rich in small cell debris.

[0177] Example 5

[0178] An aqueous suspension containing microbial cells from baker's yeast was prepared as described above. The pH of the aqueous suspension was adjusted to pH 9 before cell degradation.

[0179] As described above, the cell decomposition, subsequent separation, and subsequent purification of the extract rich in small cell debris by ultrafiltration, followed by drying, are performed to obtain a dried and purified extract rich in small cell debris.

[0180] Examples of temperature and pH changes in different steps

[0181] Table 1 shows examples of pH and temperature at different process steps according to this method.

[0182]

[0183]

[0184] Influence of processing temperature during bead milling

[0185] Temperature during bead milling affects the gelation behavior of the obtained microbial cell products.

[0186] This was investigated by repeating Example 1, except that the temperature varied from 20°C to 55°C. Another experiment (*) was performed, in which a 1-hour extended decomposition was conducted without temperature control. The highest temperature recorded was 60°C. The results are shown in Table 2.

[0187] Prior to bead milling, the temperature was adjusted to the desired value. The effect of temperature on cell degradation was estimated using a flow cytometer (BD Accuri C6). Before measurement, the sample was diluted 80-fold with water and analyzed using flow cytometry. Cell degradation was determined using forward scattering data. A biomass suspension at T = 20°C was used as a reference to estimate the fraction of cells destroyed.

[0188] For all samples, the resulting extracts, rich in small cell debris, were collected, dried, and analyzed to determine their gelling properties.

[0189] The gelation properties were determined as follows: An aqueous solution containing 200 g / L (20% DW) was prepared. Further dilution in water was performed to achieve 150 g / L (15% DW) and 100 g / L (10% DW). The suspension was heated at 95°C for 10 minutes using a block heater, and then cooled to ambient temperature (T = 25°C) in a water bath. The gelation properties of the resulting substance were analyzed according to the following ratings: 0 indicates no gelation observed, 1 indicates paste observed, 2 indicates droplet-like paste observed, 3 indicates very soft gel observed, 4 indicates soft gel observed, and 5 indicates hard gel observed.

[0190] Table 2: Gelation properties of extracts rich in small cell fragments produced at different temperatures during bead milling.

[0191]

[0192] The effect of pH on protein conformation and protein solubility at different pH values

[0193] Example 2 was repeated, but the pH of the suspension was adjusted to different pH values ​​before bead milling. Extracts rich in small cell debris were extracted and analyzed as follows:

[0194] Non-denaturing gel electrophoresis: Samples were added to pre-constructed gels with 4% to 12% crosslinking using 10×Tris-glycine buffer as the electrophoresis buffer. Electrophoresis was performed at 125V for 70 minutes and compared with native protein markers. Protein bands were stained with Coomassie blue.

[0195] Protein solubility: Protein concentration in extracts rich in small cell debris was measured at 280 nm using a spectrophotometer as an indicator of protein content. Data were normalized for ease of interpretation.

[0196] from Figure 2B It is clear that, at alkaline pH values, the proteins present in the extract rich in small cell debris have higher solubility. From Figure 2A It can be seen that at alkaline pH values, especially in the range of 4 to 11 (preferably 7 to 9), this increased solubility does not affect the native conformation of proteins and other molecular complexes. Figure 2A It is a non-denaturing gel electrophoresis method. If a protein exists in its native conformation, it will show bands. If the protein denatures, the corresponding bands will disappear completely or new bands of different molecular weights will be generated.

[0197] Effect of residence time in bead mill

[0198] Example 2 was repeated, and samples were taken at different time points during the bead milling process for further analysis. Extracts rich in small cell debris were collected for further analysis.

[0199] BSA was used as the standard protein, and proteins were determined according to Lowry's method (publications: Lowry, OH; Rosebrough, NJ; Farr, AL; Randall, RJ (1951). "Protein measurement with the Folin phenol reagent". Journal of Biological Chemistry. 193(1): 265-75). Glucose was used as the standard sugar, and carbohydrates were determined according to Dubois's method (publications: MICHEL DUBOIS, KAGILLES, JKHAMILTON, PAREBERS, and FRED SMITH. Colorimetric Method for Determination of Sugars and Related Substances. ANALYTICAL CHEMISTRY. Vol. 28, No. 3, March 1956).

[0200] Figure 3 The graph shows the amount of protein (left vertical axis) and carbohydrate (right vertical axis) dissolved as a function of residence time in the bead mill (horizontal axis). Figure 3 This demonstrates how residence time in a bead mill affects the dissolution of proteins and carbohydrates. Because individual molecules dissolve at different rates, the shear force and residence time in the bead mill can be adjusted to obtain extracts with different compositions, thereby achieving different functional activities.

[0201] This shows that the solubility levels of proteins and carbohydrates can be controlled by adjusting the residence time during the decomposition step. Figure 3 An example is provided illustrating how choosing two different residence times can result in two extracts having significantly different compositions. When the residence time was 5 minutes (see arrow at P1), the resulting extract was found to contain 30% less soluble carbohydrates compared to a residence time of 20 minutes (see arrow at P2).

[0202] Furthermore, the particle size distribution can be adjusted according to the residence time. This is particularly important in this invention because the functional properties of the final product depend on the particle size distribution.

[0203] Particle size distribution of cell fractions after bead milling

[0204] Example 2 was repeated, but with different grinding time values ​​(residence time in the bead mill). The resulting aqueous suspension (also known as slurry) containing decomposed microbial cells was collected for analysis. The extract rich in small cell fragments obtained after subsequent separation was also collected for further analysis.

[0205] Particle size distribution was measured using a Zeta-Sizer Ultra (Malvern Panalytical) with a disposable folded capillary. Samples were prepared in water to achieve a concentration of <10 g / L (1% DW). The sample was added to the measurement cell, ensuring no air bubbles formed. The sample was measured three times, with a 30-second waiting time, at a temperature of 25°C and a refractive index of 1.33.

[0206] The values ​​for D99, D70, and D50 are calculated based on particle size distribution data. D99 indicates that 99% of the particles are larger than this value. D70 indicates that 70% of the particles are larger than this value, and the same applies to D70 (70%) and D50 (50%). The percentage of particles in each size category is determined by the total number of particles, and the corresponding size for each interval is estimated. Table 3 shows the particle range after decomposition and separation treatment.

[0207] Table 3: D99, D70, and D50 values ​​of decomposed microbial biomass suspension (after decomposition) and extract rich in small particles (after separation).

[0208] 15 minutes after decomposition 15 minutes after separation 3 hours after decomposition 3 hours after separation D99(nm) 92.9 32.3 79.9 68.7 D70(nm) 488.7 169.9 197.6 361.3 D50(nm) 660.3 361.3 361.3 488.7

[0209] Figure 4 The particle size distribution of cell fragments in aqueous suspensions containing decomposed cells after bead milling for 15 minutes (“15-minute slurry” – black solid line) and 3 hours (“3-hour slurry” – gray solid line) is shown. The horizontal axis shows the diameter of cell debris in nanometers, and the vertical axis shows the signal intensity. These solid lines show peaks in the range of 200 nm to 400 nm (small cell debris), as well as peaks in the region between 500 nm and 1500 nm and near 5500 nm. Furthermore, the particle size distribution of the extract rich in small cell debris was determined after separation (centrifugation at 4000 rcf) (15-minute SN and 3-hour SN; where SN = supernatant). Figure 4 As shown, the result of the separation process is that centrifugation selectively maintains cell debris clusters with an average size in the range of 200 nm to 400 nm. In other words, these small cell debris enters the extract, while larger cell debris remains in the initially disrupted suspension (or an aqueous suspension depleted of small cell debris or an extract rich in large cell debris). This clearly demonstrates the effect of the separation step of the present invention. To the inventors' surprise, such small cell debris (and thus the extract rich in small cell debris) exhibits functional properties comparable to those of high-purity components (e.g., protein isolates) specified in the literature.

[0210] For extracts rich in small fragments, this invention typically refers to a particle size of 500 nm. For clarity and simplicity, this value refers to d50.

[0211] A key element of this invention is the fact that the extracts rich in small fragments and the extracts rich in large fragments exhibit a multimodal distribution. Therefore, the above extracts and products were further characterized using a Mastersizer 2000 (Malvem Panalytical), with a refractive index of 1.33 for the dispersant (water) and a particle refractive index of 1.34 for yeast. All analyses were performed using the Mie scattering model.

[0212] Influence of centrifugal force

[0213] Example 2 was repeated, but with different centrifugal force values ​​used during the separation step. The resulting extract, rich in small cell debris, was collected for further analysis.

[0214] The maximum gel strength (G′[Pa]) was measured using a rheometer (AnthonParr). The sample was subjected to a heat-cooling process (25°C, then 90°C, then 25°C). The G′max (also known as the storage modulus) value was obtained at the end of the cooling step (at a temperature of approximately 28°C).

[0215] Figure 5 Bar graphs of several centrifugal forces (RFC) and their effects on maximum gel strength are shown. Figure 5 As shown, the inventors have creatively discovered that adjusting the centrifugal force has a direct impact on the functional properties of the resulting extract after separation. Using a low centrifugal force allows the extract, rich in small cell debris (supernatant), to exhibit better gelling properties. Based on the invention disclosed herein, those skilled in the art can adjust the specific value of the centrifugal force and the time required to achieve the desired solid-liquid separation while maintaining the cell debris cluster within the range of 200 nm to 400 nm. Figure 5 In the given example, the preferred centrifugal force is <4000 rfc and the time is <15 minutes.

[0216] Effects of membrane retention on the gelation behavior of extracts rich in small cell debris

[0217] Repeat Example 3, but use a different membrane cutoff value for the filter used in the purification process.

[0218] The purified extract, rich in small fragments, was collected for further analysis.

[0219] The maximum gel strength (G′[Pa]) was measured using a rheometer (Anthon Parr). The sample was subjected to a heat-cooling process (25°C, then 90°C, then 25°C). The G′max[Pa] value was obtained at the end of the cooling step (at a temperature of approximately 28°C).

[0220] Figure 6The bar graph shows the purified extract rich in small cell fragments according to the invention compared with other protein-rich sources (such as egg white protein isolate, whey protein isolate, and pea protein isolate), as measured by gel strength. Figure 6 This demonstrates that membrane-based filtration can be used as a purification process to significantly enhance the gelling properties of extracts rich in small cell debris. The numbers 0.2, 100, and 10 represent membrane cutoff values, i.e., 2 μm, 100 kDa, and 10 kDa. Specifically, membrane-based filtration can be used to purify extracts rich in small cell debris into a retentative phase (purified extract rich in small cell debris) and an osmotic phase (byproducts). Figure 6 Comparative extracts rich in small cell debris were also shown, which were decomposed at pH 7 (enrichment fraction pH 7) or pH 9 (enrichment fraction pH 9). Figure 6 The purified extract (retention fraction) rich in small cell debris demonstrated good gelling properties. Furthermore, the extract rich in small cell debris also exhibited gelling properties, especially the extract at pH 9.

[0221] Surface tension

[0222] Example 3 was repeated, but with a different membrane cutoff value used for the filter during purification. The resulting byproducts and the purified extract rich in small fragments were collected for further analysis as follows.

[0223] Surface tension was measured using an automated droplet tensiometer (ADT, Teclis Tracker). Air-water and hexadecane-water (oil-water) samples were used as reference samples.

[0224] Figure 7 A bar graph showing the equilibrium surface tension of the byproduct (P) obtained at the air-water interface (left) and the purified extract (R) rich in small cell debris at the oil-water interface (right). Values ​​for egg white protein isolate and whey protein isolate are used for comparison. Equilibrium surface tension reflects the degree of surface stability; lower values ​​are obtained from samples with superior activity. The numbers 0.2, 1000, 100, and 10 represent membrane cutoff values, i.e., 2 μm, 1000 kDa, 100 kDa, and 10 kDa used for filtration in the purification step. The purification process according to some embodiments of the invention provides a method for selectively fractionating an extract rich in small cell debris into a purified extract and byproducts rich in small cell debris. Figure 7 As shown, in addition to gelling behavior, both extracts also exhibit excellent surface activity. The inventors have found that this surface activity is also reflected in the foaming and emulsifying activities of the extracts according to the invention, which are comparable to or superior to those of typical protein isolates.

[0225] Example composition of extract

[0226] Example 3 was performed, and the resulting extract was further analyzed.

[0227] The extracts obtained after separation and processing, rich in small cell debris and rich in large cell debris, can contain abundant proteins and carbohydrates. Table 4 shows example compositions of the extracts rich in small cell debris and the extracts rich in large cell debris. In the table, "ash" refers to any inorganic substances, such as minerals.

[0228] Table 4: Example Composition of Extracts (in DW%)

[0229] Extracts rich in small cell fragments Extracts rich in large cell fragments protein 57％ 43％ carbohydrate 22％ 37％ Ash 13％ 14％ lipids 8％ 6％

[0230] Further research

[0231] The effects of different processing and preparation methods on the properties of microbial preparations were further investigated. Figure 9 As can be seen, the particle size distribution changes with the duration of the decomposition process. In the case of yeast, a peak of ~6 μm intact cells and a peak of ~0.8 μm cell debris were obtained. With increasing decomposition time, the peak of intact cells decreased and the peak of cell debris increased. Therefore, the decomposition process can be run until the desired particle size distribution is obtained. For yeast, the required target PSDs are D10 < 0.5 μm, D50 < 4.5 μm, D90 < 7.5 μm, and D[3,2] < 2, D[4,3] < 4.5. Figure 10 shows the various measurements over time.

[0232] The effect of changing separation conditions, such as Figures 11 to 13 As shown. For these examples, the damaged yeast preparations are separated by centrifugation at low, medium, and high intensities. Low to medium intensities can increase the yield of EESF. For example, using low to medium centrifugation intensities compared to high centrifugation intensities can yield >10% (DW) of EESF.

[0233] Furthermore, the gel-forming properties of EESF decrease at high strength, while they improve at medium and low strength. Figure 11 Using high-intensity separation results in poor functionality of EESF. Medium separation intensity is preferred to ensure excellent functionality of EESF. In this context, "functionality" refers to gel hardness—for example, a gel hardness of 0.08 N to 0.1 N obtained under low-intensity separation, and a gel hardness of 0.05 N to 0.07 N obtained under high-intensity separation.

[0234] It is believed that different separation intensities will alter the particle content of EESF or EELF; see [link / reference]. Figure 12 and 13Increasing the separation intensity increases the specific surface area, but significantly reduces the number of particles. Low separation intensity is preferred for enriching EESFs with particles in the range of 0.1 μm to 0.3 μm. Under high-intensity separation conditions, the main soluble compounds are retained in the liquid phase with a small number of particles in the range of 0.1 μm to 0.3 μm. See also Figure 12 By increasing the separation intensity, the specific surface area increases, but the number of particles decreases significantly. See also... Figure 13 These particles are crucial for enhancing the overall yield and functional properties of EESF; therefore, a balance must be found between low and high separation strength.

[0235] EELF preferably contains fragments in the range of 2 μm to 10 μm. Cellular fragments in the range of 2 μm to 10 μm are preferred to enhance water-holding capacity without impairing oil-holding capacity. See the table below, which shows the water-holding and oil-holding capacities of EESF and EELF, as well as the preferred particle size distribution.

[0236] [g / g] WHC OHC EESF 0.5 1.5 EELF 2.5 1.6

[0237] Note that there is overlap in the psd values ​​of EESF and EELF, especially in the 1-4 μm range. This is more pronounced when using low-intensity separation (see...). Figure 12 Therefore, a medium-intensity separation is preferred.

[0238] Separation can also be performed at different dilutions to modify the functional properties of the fractions. See also Figures 14 to 18 For example, separation can be carried out at dry weight (DW) contents ranging from 20% to 2.5%. By reducing the DW content before separation (equivalent to increasing the degree of dilution), the gelling properties and water-holding capacity of the EELF are enhanced. Consequently, the functional properties in the EELF deteriorate. See also Figure 14 Conversely, when separation is carried out at low DW content, the water-holding and oil-holding capacities in the EELF are particularly improved. See also Figure 15 .

[0239] When different dilution levels were applied, the PSD ranges of EESF and EELF remained virtually unaffected. Only the relative contribution of the main peak in the multimodal distribution changed. The contribution of particles with peaks at approximately 0.2 μm compared to particles with broad peaks at approximately 1.5 μm to 2 μm increased with higher dilution levels. See also Figure 16 Similarly, higher dilution levels result in higher total specific surface area (SSA) (e.g. Figure 17 As shown), D[3,2] and D[4,3] decrease with increasing dilution level (e.g. Figure 18 (As shown).

[0240] Other microorganisms

[0241] Although the examples to date have all used yeast as the starting microorganism, the described process can also be applied to other microorganisms to produce functionally active cell fragments. The table below shows the stiffness of EESF gels obtained from two microalgal species and one cyanobacterial species under the isolation conditions according to the invention.

[0242]

[0243] *psd: Particle size distribution after decomposition

[0244] **High dilution:** The DW content before S / L separation is adjusted to 2.5%.

[0245] ***No gel: Although gelation was observed, the material could not be measured using texture profile analysis (TPA).

[0246] Furthermore, higher dilution prior to S / L separation is beneficial for enhancing the functional properties of EELF, such as gel hardness:

[0247] Gel hardness [N] of EELF

[0248]

[0249] *No gel: Although gelation was observed, the resulting material could not be measured using texture profile analysis (TPA).

[0250] Furthermore, adjusting the dilution level prior to the S / L separation step can be advantageously used to modulate other functional properties, such as oil holding capacity (OHC). See also Figure 19 .

[0251] Prolonged cell breakdown

[0252] The cell degradation step can be further extended to enhance the functional properties of EELF. For example, the PSD can be modified within a 240-minute timeframe to produce EELF with excellent gel stiffness, water-holding capacity (WHC), and oil-holding capacity (OHC).

[0253] EELF

[0254]

[0255] The functional properties of EELF were significantly enhanced by cell degradation under alkaline conditions, preferably at pH > 9, and S / L separation at low DW content (< 5%). See also Figure 20 However, at alkaline pH, the PSD during prolonged cell degradation processes exhibits a unique trend. Contrary to the expected stable reduction in particle size during micronization, particles tend to aggregate. This effect is particularly pronounced at pH 1 – see Figure 21.

[0256] Compared to conventional β-glucan extraction processes for yeast biomass, an extended cell degradation process at pH 11, followed by S / L at high dilution levels (DW < 5%), yields EELF with superior functionality. For reference, a process involving cell autolysis, cell homogenization, alkali extraction, acid extraction, and water extraction (Aut + Hom + Extr) is compared to the currently proposed method (extended degradation at pH 11 and high dilution).

[0257]

[0258] The reference process has been reported by: Saowanee Thammakiti, Manop Suphantharika, Thanapom Phaesuwan, Cornel Verduyn. Preparation of spent brewer's yeast β-glucans for potential applications in the food industry. Food Science and Technology. Vol. 39, No. 1, January 2004, pp. 21-29.

[0259] Concentration compared to drying

[0260] The functional properties of EESF and EELF deteriorate due to thermal processes (such as evaporation and drying) and can also freeze. Therefore, it is preferable to use both fractions (EESF and EELF) as wet components and avoid processes that may cause thermal degradation. Preferably, these two fractions should not undergo processes at temperatures above 45°C or below 0°C.

[0261] Component blending

[0262] Once prepared, EELF and EESF can be mixed to obtain the microbial cell preparation according to the invention. Combining / blending the dry or wet components (EESF and EELF) allows for customized functionality to provide texture, etc., in food applications. It is important to note that these ratios are not equal to the ratios in the disrupted slurry; in other words, the individual streams must be separated (using centrifugation as explained above) and blended back in specific ratios. Examples of this include:

[0263] Alternatives to the texture in burgers: Maintaining the same total concentration of ingredients, but altering the ratio between EESF and EELF results in changes in firmness, elasticity, and juiciness versus dryness. This provides food formulators with a range of options to achieve the texture they are looking for. For example, mixing them separately at ratios from 100:0 to 0:100 shows that a 70% EESF:30% EELF blend produces a firm, elastic, but not dry burger, while a 40% EESF:60% EELF blend produces a softer, juicier, and "greasy" burger. Therefore, the ratio can be optimized for specific markets.

[0264] Egg Substitutes in Bakeries: The intended use of the ingredient in bakery applications was to replace egg whites or whole eggs with EESF. This did indeed prove to work well, but surprisingly, a 50:50 mixture of EESF and EELF provided better texture and performance than whole eggs. This is counterintuitive, as the primary functionality required here is heat-setting gelation, and given EESF's superior gelling properties, the expected outcome was that EESF would perform better than the combination.

Claims

1. A method for preparing microbial cell products, the method comprising: i) Provide an aqueous suspension containing microbial cells; and ii) subject the suspension to mechanical cellular degradation in a temperature range of 15°C to 35°C and a pH range of 7 to 11 to obtain an aqueous suspension containing decomposed microbial cells; iii) The suspension is separated to provide an extract rich in small cell debris and an extract rich in large cell debris, wherein the extract rich in small cell debris has a particle size distribution of d50 equal to or less than 500 nm, wherein the size of the debris ranges from 0.1 to 3 μm; and the extract rich in large cell debris has a particle size distribution of d50 greater than 500 nm, wherein the size of the debris ranges from 3 to 10 μm.

2. The method according to claim 1, further comprising step v) after step iii): combining at least a portion of the extract rich in small cell fragments and at least a portion of the extract rich in large cell fragments.

3. The method according to claim 1, further comprising: After step i) and before step ii), step ia) pretreats the aqueous suspension containing microbial cells to obtain a pretreated aqueous suspension containing microbial cells and byproducts, wherein the pretreatment is preferably selected from decantation, centrifugation, filtration such as membrane filtration and one or more combinations thereof.

4. The method according to claim 1, wherein the separation in step iii) is selected from the group consisting of sedimentation, flocculation / coagulation, precipitation, decantation, (hydraulic) cyclone separation, (air) flotation, centrifugation, filtration, and one or more combinations thereof.

5. The method according to claim 1, further comprising step iv) after step ii) or step iii), wherein step iv) is to subject at least one of the microbial products obtained in step ii) or step iii) to purification treatment to obtain at least one purified microbial cell product, said purification treatment being independently selected from the group consisting of washing, filtration such as membrane filtration or percolation, adsorption, chromatography, crystallization, flocculation / coagulation, precipitation, two-phase extraction, subcritical extraction, supercritical extraction, solvent extraction distillation, and one or more combinations thereof.

6. The method according to claim 5 further includes a step vi) following step ii), iii), iv), or v), wherein step vi) involves subjecting at least one of the microbial products obtained in step ii), iii), iv), or v) to drying to obtain dried microbial cell products, wherein the drying is preferably performed using a method selected from spray drying, freeze drying, and fluidized bed drying, and preferably wherein, based on the total weight of the dried microbial cell products, the moisture content of the dried microbial cell products is 10% by weight or less, or more preferably 5% by weight or less.

7. The method according to claim 1, wherein the microbial cells are selected from single-celled or community-sized prokaryotic and eukaryotic cells and one or more combinations thereof, preferably the microbial cells are selected from the group consisting of yeast, algae, bacteria, fungi and one or more combinations thereof.

8. The method according to claim 1, wherein the mass concentration of microbial cells in the suspension in step i) is in the range of 1% to 25%, preferably 5% to 15%.

9. The method according to claim 1, wherein the mechanocellular decomposition in step ii) is performed using a bead mill, preferably, a) Performing the bead milling to obtain a predetermined particle size distribution of the suspension, wherein more preferably, the microbial cells are yeast, and the predetermined particle size distribution after step ii) is D10 < 0.5 μm, D50 < 4.5 μm, D90 < 7.5 μm and D[3,2] < 2, D[4,3] < 4.5, and / or b) The bead milling is carried out at pH > 9 for a period of time sufficient to cause particle aggregation, and the dry weight content prior to separation is at least 2.5%.

10. The method according to claim 1, wherein the separation process in step iii) is centrifugation at a centrifugal force equal to or less than 4000 relative centrifugal force (rfc); preferably, the centrifugation occurs for a period of time equal to or less than 20 minutes, more preferably less than 15 minutes; preferably, the water content of the decomposed cell suspension is adjusted before separation step iii).

11. The method of claim 2, further comprising incubating the extract rich in small cell debris at a temperature below 25°C for at least 60 minutes prior to mixing step v).

12. Microbial cell products that are directly obtained or obtainable by the method according to any one of the preceding claims.

13. A microbial cell product comprising an extract rich in small cell fragments, wherein the extract comprises small cell fragments with a particle size distribution of d50 equal to or less than 500 nm, and wherein the size of the small cell fragments ranges from 0.1 to 3 μm. Compared to unseparated, decomposed microbial cell biomass, the extract rich in small cell debris has a higher ratio of small cell debris to large cell debris.

14. A microbial cell product comprising an extract rich in large cell fragments, wherein the extract contains large cell fragments with a particle size distribution d50 greater than 500 nm, and wherein the size of the large cell fragments ranges from 3 to 10 μm. Compared to unseparated, decomposed microbial cell biomass, the extract rich in large cell debris has a higher ratio of large cell debris to small cell debris.

15. The microbial cell product according to claim 13 or 14, wherein the microbial cell product further comprises soluble cytoplasmic material.

16. The microbial cell product according to claim 13 or 14, wherein the extract is derived from yeast, preferably baker's yeast.

17. The microbial cell product according to claim 13 or 14, wherein the microbial cell product is dried.

18. The microbial cell product according to claim 13 or 14, wherein the dry weight content of the microbial cell product is at least 20%.

19. Food, animal food, or cosmetic, comprising the microbial cell products according to claim 12.

20. The use of the microbial cell product of claim 17 as an emulsifier, foaming agent, binder, leavening agent, thickener, humectant, adhesive, browning agent, clarifying agent, gelling agent, crystallization control agent, moisturizer, tenderizing agent, aeration agent, structure modifier, coagulant, coating agent, colorant, gloss agent, flavoring agent, freezing agent, heat preservation agent, texture modifier, pH buffer, shelf life extender, fat substitute, meat filler, preservative, antimicrobial agent, food spoilage inhibitor, malic lactic acid fermentation inhibitor, texture modifier, egg substitute, or any combination thereof; or Used in edible egg-free emulsions, egg analogues, egg-free scrambled eggs, egg-free pies, egg-free pound cakes, egg-free angel food cakes, egg-white meat substitutes, egg-free meat substitutes, egg-yolk cakes, egg-free and dairy-free cream cheese, egg-free pasta dough, egg-free custard, egg-free ice cream, or in dairy-free milk.

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