Microbial cell products, methods for obtaining microbial cell products, and uses of microbial cell products

Abstract

The present invention relates to a method for preparing a microbial cell product, comprising: providing an aqueous suspension comprising microbial cells; subjecting the suspension to mechanical cell disruption at a temperature ranging from 15°C to 35°C, preferably at a pH ranging from 9 to 11, to obtain an aqueous suspension comprising disrupted microbial cells; separating the suspension to provide an extract enriched in small cell fragments and an extract enriched in large cell fragments; and combining at least a portion of each extract to provide the microbial cell product. The present invention further relates to a microbial cell product obtained or obtainable by the method. The present invention further relates to the use of the product in food, for example as an egg white substitute; in animal food; and / or in cosmetic formulations.

Description

Technical Field

[0001] The present invention relates to a method for obtaining a microbial cell product. The present invention further relates to a microbial cell product obtained or obtainable by the method. The present invention further relates to the use of the microbial cell product. Background Art

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

[0003] Egg white makes up about two-thirds of an egg's weight. It's primarily composed of about 90% water, with about 10% protein (including albumin, mucin, and globulin) dissolved in this 90%. Unlike the yolk, which is rich in lipids (fat), egg white contains virtually no fat and less than 1% carbohydrates. Egg white contains about 56% of the protein in an egg. Egg white has many uses in foods (e.g., meringues, mousses) and many others (e.g., in vaccines, such as the flu shot).

[0004] There are many disadvantages to using eggs. For example, eggs contain high levels of cholesterol and saturated fat, which can increase the risk of cardiovascular disease and obesity. Consumers who otherwise benefit from the high nutritional content and enjoy egg-containing products may be prevented from doing so due to food allergies or other dietary restrictions. For example, an estimated 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 housing egg-laying birds. Furthermore, industrial chicken farming has negative environmental impacts and raises several important humanitarian concerns.

[0005] Additionally, eggs have a limited shelf life and pose a risk of carrying infectious pathogens such as Salmonella, E. coli, and other pathogens that could pose a threat to public health.

[0006] Many attempts have been made to create egg substitutes (whole egg substitutes or egg white substitutes) that recapitulate the desirable characteristics of natural eggs while minimizing the undesirable characteristics of eggs. Egg substitutes aim to achieve binding, moisturizing, emulsifying and / or leavening properties similar to those of eggs or egg whites.

[0007] There are many substitutes based on home cooking, such as mashed banana, applesauce, aquafaba (chickpea cooking liquid), 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] In addition, industrially produced plant-based egg substitutes are known from, for example, WO2013067453A1, which discloses beans or peas as starting materials. WO2017102535A1 discloses a method for obtaining a gluten-free natural rapeseed protein isolate comprising <10 ppm of gliadin, which can be used for any human nutritional food application, including as a foaming agent to replace egg white. WO2016077457A1 discloses an alternative egg-free egg white protein production method 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, whole microalgal cells and / or microalgal oil in combination with one or more other edible ingredients.

[0009] The above-mentioned whole eggs and egg white substitutes only partially possess the desired combination of egg white properties which allow a wide range of uses of the compositions 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] It is an object of the present invention to provide egg-free compositions that can be used as binders, gelling agents, thickeners, foaming agents, humectants, emulsifiers, and / or oil / water binders. It is a further object of embodiments of the present invention to provide improved compositions for use in food products, such as as egg white substitutes; in animal food products, such as granules, dry / powdered, semi-moist, or wet feed formulations; and / or in cosmetic formulations, such as solutions, creams, lotions, suspensions, ointments / pastes, powders, and gels.

[0012] In a first aspect, the present invention relates to a method for preparing a microbial cell product. The method of the invention comprises: i) providing an aqueous suspension comprising microbial cells; ii) subjecting the suspension to mechanical cell disruption to obtain an aqueous suspension comprising disrupted microbial cells; iii) separating the suspension to provide an extract enriched in small cell fragments and an extract enriched in large cell fragments; and v) combining at least a portion of each extract to provide a microbial cell product.

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

[0014] Further details of this fine-tuning and other steps of the method are described in more detail herein.

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

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

[0017] In another aspect, the present invention relates to a microbial cell product directly obtained or obtainable by a method according to the invention.

[0018] In another aspect, the present invention relates to an extract enriched in small cell fragments and an extract enriched in large cell fragments obtainable by the method according to the invention.

[0019] In another aspect, the present invention relates to the use of a microbial cell product according to the present invention as an emulsifier, foaming agent, binder, leavening agent, thickener, humectant, adhesive, browning agent, clarifier, gelling agent, crystallization controller, wetting agent, tenderizer, aerating agent, texturizing agent, coagulant, coating agent, colorant, glossing agent, flavoring agent, freezing agent, warming agent, mouthfeel improver, pH buffer, shelf life extender, fat replacer, meat filler, preservative, antimicrobial agent, food spoilage inhibitor, malolactic fermentation inhibitor, texture improver, egg replacer, or any combination thereof.

[0020] In a further aspect, the present invention relates to the use of a microbial cell product according to the present invention in an edible egg-free emulsion, an egg analog, an egg-free scrambled eggs, an egg-free pie, an egg-free pound cake, an egg-free angel food cake, an egg-free meat substitute, an egg-free meat substitute, an egg-free yolk pie, an egg-free and dairy-free cream cheese, an egg-free pasta dough, an egg-free custard, an egg-free ice cream, or in a dairy-free milk.

[0021] The corresponding embodiments disclosed for the method also apply to the microbial cell product according to the invention, the extract enriched in small cell fragments according to the invention, the extract enriched in large cell fragments according to the invention, and the use of the microbial cell product according to the invention.

[0022] Definition List

[0023] The following definitions are used in this specification and claims to define the recited subject matter.Other terms not cited below are referred to as having meanings generally accepted in the art.

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

[0025] As used herein, "dry weight (DW)" and "dry cell weight" refer to weight measured in the relative absence of water. For example, reference to a microbial biomass comprising a particular component in a particular percentage by dry weight 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, "disruption" as used herein is also referred to as "lysis" and refers to the opening of cells to release cytoplasmic compounds (also referred to as "lysate").

[0027] In the context of microbial cell disruption, "disintegration," as used herein, refers to cell disruption. This means that the average size of the resulting cell fragments must be smaller than the average cell size of the initial microbial cells. Disintegration can be considered a special type of disruption in which cells are not only opened but also disrupted.

[0028] As used herein, "cytoplasmic material" or "cytoplasmic compounds" refers to all substances normally contained within a cell and surrounded by a cell membrane, excluding the cell nucleus (if present). Cytoplasmic material is released from the cell when the cell breaks down or is destroyed.

[0029] As used herein, the term "microbial cell" refers to a microorganism. This can be eukaryotic and prokaryotic single-cell organisms and their communities. Prokaryotes are cellular organisms that lack a nucleus surrounded by a membrane. In the three-domain system, prokaryotes are divided into two domains based on molecular analysis: bacteria (formerly eubacteria) and archaea (formerly archaebacteria). Organisms with a nucleus are located in the third domain, the eukaryotes. Microbial cells according to the present invention also include algae and fungi such as yeast.

[0030] As used herein, "microorganism" or "microbe" refers to any microscopic colony or single-cell organism.

[0031] The "microbial cell product" used in this specification refers to a product derived from microbial cells obtained by treating the microbial cells in a certain manner.

[0032] As used herein, "extract enriched in small cell fragments (EESF)" refers to a microbial cell product obtained by separating an aqueous suspension containing decomposed microbial cells. During this separation process, an extract is separated from the aqueous suspension containing decomposed microbial cells, leaving an aqueous suspension containing decomposed microbial cells partially depleted of small cell fragments. In other words, the separation process produces an extract enriched in small cell fragments (as the primary product) and an aqueous suspension depleted of small cell fragments, the latter of which can and will be referred to as an extract enriched in large cell fragments. As used herein, "small cell fragments" refers to cell fragments obtained by decomposing microbial cells that have a size equal to or less than d50 ≤ 500 nanometers (nm).

[0033] As used herein, an "extract enriched in large cell fragments (EELF)" or "aqueous suspension depleted of small cell fragments" refers to a microbial cell product obtained by separating an aqueous suspension containing decomposed microbial cells. In this separation process, an extract is separated from the aqueous suspension containing decomposed microbial cells, leaving an aqueous suspension containing decomposed microbial cells partially depleted of small cell fragments. In other words, the separation process produces an extract enriched in small cell fragments (as a primary product) and an aqueous suspension depleted of small cell fragments (as a by-product), the latter of which can and will be referred to as an extract enriched in large cell fragments. As used herein, "large cell fragments" refers to cell fragments obtained by decomposing microbial cells that have a size greater than d50 ≥ 500 nanometers (nm).

[0034] As used herein, "microbial biomass" and "biomass" refer to materials produced by the growth and / or reproduction of microbial cells, or as a byproduct of a fermentation process. Biomass may include cells and / or intracellular contents as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by cells.

[0035] As used herein, "bead milling" refers to the agitation of microbial cells in suspension using small abrasive particles (beads). Cells are disrupted by shear forces, abrasion between beads, and collisions with beads. The shear forces generated by the beads disrupt the cells and cause their disintegration, releasing cellular compounds.

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

[0037]

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

[0039]

[0040] where r is the radius in mm. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0042] Figure 1 An overview of the different (optional) steps of the method according to the invention and the (intermediate) products obtained is shown. The overview shows many different ways to obtain microbial cell products according to the invention.

[0043] Figure 2 shows the profiles of native conformation proteins of extracts enriched in small cell debris obtained at different pH values ​​(left) and the solubility of proteins in extracts enriched in small cell debris at different pH values ​​(right).

[0044] Figure 3Shown is the solubilization of proteins and carbohydrates in extracts enriched in small cell debris during the bead beating experiment.

[0045] Figure 4 Shown are the particle size distributions of the cell fractions after bead beating or after bead beating and centrifugation.

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

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

[0048] Figure 7 Shown are the equilibrium surface tensions of the obtained by-product (P, left) and the purified extract enriched in small cell fragments (R, right) at the air-water (left) and oil-water (right) interfaces compared to the surface tensions of egg white protein isolate and whey protein isolate.

[0049] Figure 8 A simplified process flow diagram for preparing a microbial cell product 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 specific surface area and particle size distribution of yeast during disruption are shown as a function of time.

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

[0053] Figure 12 Shown are the particle size distributions of different fractions of a yeast preparation at different separation intensities.

[0054] Figure 13 The specific surface area and concentration of different fractions of the yeast preparation at different separation strengths are shown.

[0055] Figure 14 Shown are the gel firmness of EESF and EELF of isolated yeast preparations at different dilution conditions.

[0056] Figure 15 Shown are the water- and oil-holding capacities of the EELF of isolated yeast preparations at different dilution conditions.

[0057] Figure 16Shown are the particle size distributions of EESF of isolated yeast preparations at different dilution conditions.

[0058] Figure 17 Shown are the specific surface areas and concentrations of different fractions of the isolated yeast preparation at different dilution conditions.

[0059] Figure 18 Shown are the particle size distributions of isolated yeast preparations at different dilution conditions.

[0060] Figure 19 Shown are the oil holding capacities of microbial preparations from different microorganisms after separation under different dilution conditions.

[0061] Figure 20 Shown are the gel hardness and the oil and water holding capacities of yeast preparations after disruption at different pH levels.

[0062] Figure 21A and 21B Shown are the particle size distributions - d50 and D[3,2] - of yeast disrupted at different pH levels over time. DETAILED DESCRIPTION

[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 comprising microbial cells; ii) subjecting the suspension to mechanical cell disruption to obtain an aqueous suspension comprising disrupted microbial cells; iii) separating the suspension to provide an extract enriched in small cell fragments and an extract enriched in large cell fragments; and v) combining at least a portion of each extract to provide a microbial cell product. Mechanical cell disruption can be carried out at a temperature in the range of 1°C to 45°C (i.e., in the range of 1°C to up to and including 45°C), preferably 15°C to 35°C, more preferably about 25°C, to obtain an aqueous suspension comprising disrupted microbial cells.

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

[0065] The method may optionally include any one of steps ia) pretreatment, iv) purification treatment, vi) drying, and vii) mixing. 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 obtainable by the method of 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 also refer to purified microbial cell products and dried microbial cell products.

[0068] The purified microbial cell products obtainable by the method of the present invention include mixed purified extracts, dried mixed purified extracts, and mixed dried purified extracts.

[0069] The dried microbial cell products obtainable by the method of 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 may be implemented to obtain alternative products to the mixed extracts obtained from the methods of the present invention. Figure 1 The steps of the present invention are performed to obtain a purified suspension of decomposed microbial cells after performing the following subsequent steps: step i), step ii) and step iv); or step i), step ia), step ii) and step iv).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0086] The mixed dried purified extract (microbial cell product according to the present invention) is obtained after performing the following subsequent steps: step i), step ii), step iii), performing step vi on both the extract enriched in small cell fragments and the extract enriched in large cell fragments), performing step vi) on both the purified extract enriched in small cell fragments and the extract enriched in large cell fragments, and step v) of mixing (a portion of) the dried purified extract enriched in small cell fragments and (a portion of) the dried purified extract enriched in large cell fragments; or step i), step ia), step ii), step iii), performing step vi on both the extract enriched in small cell fragments and the extract enriched in large cell fragments), performing step vi) on both the purified extract enriched in small cell fragments and the purified extract enriched in large cell fragments, and step v) of mixing (a portion of) the dried purified extract enriched in small cell fragments and (a portion of) the dried purified extract enriched in large cell fragments.

[0087] like Figure 1 As shown, different by-products can be obtained. The first and / or second by-product can be further processed by, for example, drying or concentrating to obtain a dry or concentrated by-product.

[0088] The microbial biomass that comprises microbial cells has been used to produce a wide range of industrial products traditionally, or has been directly used in multiple applications.Most industrial or commercial applications use a group of microbial biomass strains selected from the domain of bacteria, yeast, fungi and algae.In general, the product obtained from the microbial biomass is intracellular or extracellular.Extracellular products are excreted in a large amount of media by cells, typically aqueous phase.On the contrary, intracellular products are retained in the cell interior.In order to obtain intracellular products, it is necessary to carry out additional processing to discharge these products (by making cell membrane or cell wall damage) from the cell and further separate the compound of interest from remaining biomass and other impurities.

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

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

[0091] Generally, cell disintegration methods can be divided into non-mechanical or mechanical. Non-mechanical disintegration methods can be further divided into three categories: physical disintegration (e.g., by decompression, osmotic shock, pyrolysis, ultrasound or freeze-thaw), chemical disintegration (e.g., by using solvents, detergents, chaotropic agents, acids and bases, or chelates) and enzymatic disintegration (e.g., by autolysis, phage lysis or lytic enzymes). The present invention relates only to mechanical disintegration methods. Examples of mechanical disintegration methods are ball mills, including bead mills and homogenizers.

[0092] The homogenizer operates at high pressure and is essentially a positive displacement pump that forces the cell suspension through a valve and then impacts the flow at high speed onto an impact ring. Typically, multiple passes are required at high pressure, which can result in elevated temperatures that can cause local denaturation of unstable molecules.

[0093] Ball mills (including bead mills) can be vertical or horizontal and use grinding media present in a grinding chamber. The motor drives the rotor to rotate the cell suspension at high speed. The cell suspension and the grinding material (e.g., beads) generate shear forces to break the cells. This results in the release of intracellular material into the aqueous suspension, which will also cause cell fragmentation (i.e., decomposition). As the rotor speed increases, the shear force increases and cell breakage increases. As the size of the grinding material decreases, cell breakage generally increases. Other parameters affect the performance of the decomposition process. Technicians can select the correct parameters and variables based on the present invention.

[0094] Examples of prior art cell disintegration methods are as follows. US Pat. No. 3,888,839 A discloses a method for obtaining protein isolates from yeast cells, wherein the yeast cells are disrupted by high-pressure homogenization (mechanical disintegration) and subsequent incubation. EP 1,199,353 A1 discloses a method for producing yeast extracts by treating a yeast suspension or yeast paste and separating insoluble components, wherein the yeast suspension or yeast paste is subjected to high-voltage electric pulses (physical disintegration). EP 2,774,993 A1A discloses the use of protease-free cell wall-degrading enzymes (enzymatic disintegration) followed by heat treatment of the product at 70°C to 80°C for 10 to 20 minutes.

[0095] Microbial cells present in a suspension of microbial biomass primarily contain proteins, carbohydrates, lipids, and minerals. When exposed to high temperatures, prolonged incubation, extreme pH values, solvents, salts, and other harsh chemicals, proteins and other unstable molecules can unfold, denature, and degrade. When proteins and other functional molecules denature (loss of tertiary and quaternary structure), (a portion of) their functional activity is lost. After denaturation (unfolding), proteins lose their ability to interact with hydrophilic and hydrophobic surfaces, and their ability to rearrange and form network-like structures after heat-cooling treatment is also affected. (Protein) functionality is very important in many commercial applications, particularly for applications requiring gelling properties similar to those of egg white. The inventors have observed that mechanical disintegration under the conditions described herein (e.g., within the specified temperature and pH ranges) is sufficiently gentle to prevent the unfolding, denaturation, and / or degradation of proteins and other unstable molecules, which is 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 material produced during the growth or fermentation process.

[0098] In one embodiment of the method according to the present invention, the microbial cell is selected from the group consisting of unicellular or colony prokaryotic cells and eukaryotic cells and one or more combinations thereof. In a preferred embodiment, the microbial cell is 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 comprises: after step i) and before step ii), ia) pretreating the aqueous suspension comprising microbial cells to obtain a pretreated aqueous suspension comprising microbial cells and by-products. In a specific embodiment, the pretreatment is selected from decantation, centrifugation, filtration (such as membrane filtration) and one or more combinations thereof. Before the pretreatment, the aqueous suspension comprising microbial cells can be washed. The combination of washing and then pretreatment can be regarded as a double pretreatment. Washing can, for example, be carried out with water, acid or alkali, or with a solvent (such as ethanol). In the present embodiment, the order of steps is as follows: step i), step ia), step ii), wherein in the step ii) where the suspension is subjected to mechanical cell decomposition, the pretreated suspension obtained in step ia) is used.

[0100] In one embodiment, the mechanical cell decomposition of step ii) is carried out using a bead mill. In one embodiment, a bead mill is used for a time period in the range of 1 minute to 3 hours. The grinding time can also be used to regulate 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, retain protein function and reduce cooling costs. The decomposition process is preferably carried out at pH 7 to pH 11, preferably at about pH 9. Alternatively, the cell decomposition is carried out at T < 25°C for a time of > 3 hours and is carried out at pH 11.

[0101] The disintegration conditions are preferably selected to provide the desired particle size distribution. As discussed further herein, varying the particle size distribution can allow the final properties of the mixed product to be varied, for example to suit a particular intended use. The disintegration process is run in such a way that a bimodal particle size distribution (PSD) is obtained, showing an intact cell peak and a cell debris peak. In the case of yeast, an intact cell peak of ∼6 μm and a cell debris peak of ∼0.8 μm are obtained. See Figure 4 and Figure 9 .

[0102] During the cell disintegration process, the psd changes, showing a decrease in the intact cell peak followed by an increase in the cell debris peak. Run the disintegration process until a specific psd is obtained. For example, in yeast, the specific surface area increases over time, while D[3,2], D[4,3], D10, D50, and D90 decrease over time - see 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. A skilled artisan is able to adjust the parameters of a specific disintegration method / device to achieve the desired PSD. This can be performed, for example, using a bead mill, homogenizer, or other equipment and specific settings, as widely described in the specialized literature.

[0103] Bead sizes in the range of 0.1 mm to 5 mm are contemplated, preferably in the range of 0.5 mm to 1 mm. Suitable bead materials include, but are not limited to, zirconium and glass.

[0104] Based on the total available volume of the bead mill chamber, a suitable bead filling degree (percentage of the bead mill chamber filled with beads) can be considered to be in the range of 40% to 90%, preferably in the range of 65% to 80%.

[0105] Suitable rotational speeds can be considered to be in the range of 1 m / s to 20 m / s. A skilled person can estimate the corresponding rotor speed in rpm, depending on the configuration and geometry of the respective bead mill. Suitable rotational speeds in rpm are, for example, 500 rpm to 5000 rpm, preferably 1000 rpm to 3000 rpm.

[0106] Suitable microbial cell concentrations are believed to be in the range of 2% to 25% dry weight.

[0107] In one embodiment, a Dyno-mill research laboratory (CB Mills) bead mill is used to disintegrate microbial cells. Cells can also be disrupted by shear forces to disintegrate cells, such as using blending (e.g., using a high speed or Waring blender), a French press, or even centrifugation in the case of weak cell walls.

[0108] In one embodiment, cell disruption 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, such as below 35°C or between 15°C and 25°C.

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

[0112] The method also includes a step iii) after step ii), wherein step iii) is to subject the aqueous suspension of the decomposed microbial cells obtained in step ii) to a separation process to obtain an extract rich in small cell fragments and an extract rich in large cell fragments as a microbial cell product, as well as soluble compounds, the extract being suspended in the soluble compounds. In other words, the aqueous suspension of the decomposed microbial cells is separated into two extracts. These can be referred to as EESF (extract rich in small fragments) and EELF (extract rich in large fragments) in this article. The separation process can be a solid-liquid separation. The separation process can be carried out using any suitable method known to the skilled person. 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, settling, flocculation / coagulation, precipitation, decantation, (hydraulic) cyclone separation, and (air) flotation. Specific separation conditions can also be fine-tuned to obtain an enriched extract with specific preferred properties, as explained herein.

[0113] An extract enriched in small cell debris may contain primarily soluble cytoplasmic material as well as small cell debris, whereas an extract enriched in large cell debris may contain primarily large cell debris as well as some soluble cytoplasmic material.

[0114] EESF is preferably obtained by centrifugation at low or medium intensity. Low or medium intensity means short time or low g-force or a combination. An example of low intensity is separation at <4000 rcf in a benchtop centrifuge for <2 minutes. An example of medium intensity is separation at <4000 rcf in a benchtop centrifuge for <15 minutes. An example of high centrifugation is separation at >20,000 rcf for >20 minutes.

[0115] To increase the yield of EESF, low and medium separation strengths are preferred. In addition, low and medium separation strengths lead to EESF with excellent functionality.

[0116] In one embodiment, the separation treatment of 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 shorter than 20 minutes. In a specific embodiment, the centrifugation occurs for a period of time equal to or shorter than 15 minutes.

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

[0119] In one embodiment, the extract enriched in small cell fragments comprises at least 1%, preferably at least 10%, such as at least 20% or even at least 30% or at least 50% more small cell fragments than the simultaneously obtained aqueous suspension depleted of small cell fragments (i.e., an extract enriched in large cell fragments).

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

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

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

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

[0124] In one embodiment, the method further comprises iv) subjecting at least one of the microbial products obtained in step ii) or step iii) to a purification process to obtain at least one purified microbial cell product. The purification process can be carried out using any suitable method known to the skilled person. Examples of purification processes are adsorption, chromatography, filtration (e.g., diafiltration or membrane filtration), crystallization, flocculation / coagulation, precipitation, two-phase extraction, subcritical and supercritical extraction, and solvent extraction. In a preferred embodiment, the purification process is selected from the group consisting of washing, filtration (e.g., membrane filtration or diafiltration), 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 step ii) and step iii) are both subjected to purification processes, these purification processes are selected independently.

[0125] In one embodiment, the purification treatment in step iv) is membrane filtration, wherein the cut-off value of the membrane used is in the range of 1 kDa to 20,000 kDa, preferably in the range of 10 kDa to 1,000 kDa. In one embodiment, the cut-off value of the membrane used is in the range of 0.1 μm to 2 μm.

[0126] The method includes v) mixing at least a portion of the extract enriched in small cell fragments obtained in step iii) with at least a portion of the extract enriched in large cell fragments obtained in step iii) to obtain a mixed extract as the microbial cell product, or mixing at least a portion of the purified extract enriched in small cell fragments obtained in step iv) with at least a portion of the purified extract enriched in large cell fragments obtained in step iv) to obtain a mixed purified extract as the microbial cell product. This mixing can be used to further adjust the product. For example, mixing can be applied when the separation appears too "thorough" for the intended purpose, that is, when the enrichment of the extract is too high. For example, when the amount of small cell fragments in the extract enriched in small cell fragments is higher than the optimal amount for a specific purpose, a portion of the extract enriched in large cell fragments can be mixed with the extract enriched in small cell fragments to obtain a mixed extract.

[0127] It is important to note that this provides a mixed product that has properties that are different from those of EESF or EELF, and additionally different from those of the disaggregated cell preparation, since 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 in slightly different ways under slightly different conditions to achieve desired properties, and the proportions to be combined can then be varied to achieve the desired properties in the mixed product.

[0128] In one embodiment, the method further comprises vi) subjecting at least one of the microbial products obtained in step ii), step iii), step iv) or step v) to drying to obtain a dried microbial cell product. In a preferred embodiment, drying is performed by 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 product is 10% by weight or less, or more preferably 5% by weight or less, based on the total weight of the dried microbial cell product. 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 due to the elimination of volatile compounds during the drying process. Freeze drying is a preferred technique for preserving odor, color and taste because they are retained during the sublimation process.

[0129] To further preserve the functional properties of the EESF and EELF, concentration is preferred. Concentration is preferably performed using methods known in the art, without exposing the product to temperatures greater than 50°C or less than 0°C. An example of such a method is membrane concentration. The EESF and EELF are preferably concentrated to a DW content greater than 20%.

[0130] In one embodiment, the method further comprises vii) mixing at least a portion of the dry extract enriched in small cell fragments obtained in step vi) with at least a portion of the dry extract enriched in large cell fragments obtained in step vi) to obtain a mixed dry extract as the microbial cell product, or mixing at least a portion of the dry purified extract enriched in small cell fragments obtained in step vi) with at least a portion of the dry purified extract enriched in large cell fragments obtained in step vi) to obtain a mixed dry purified extract as the microbial cell product. Alternatively, one of the dry extracts may be purified before mixing, while the other dry extract may not be purified. For simplicity, Figure 1 This option is not described in .

[0131] When EESF and EELF are concentrated rather than dried, Figure 1 All the mixed strategies described in also apply. For simplicity, Figure 1 This option is not described in .

[0132] In one aspect, the present invention relates to an extract enriched in small cell debris obtainable by a method comprising at least steps i), ii), and iii) above, wherein the extract comprises soluble proteins having a molecular weight in their native form greater than 10 kDa, preferably greater than 50 kDa, more preferably greater than 60 kD. In one embodiment, at least 95% of all soluble proteins in the extract have a molecular weight in their native form greater than 10 kDa, preferably greater than 50 kDa, more preferably greater than 60 kD.

[0133] In one embodiment of this aspect, the small cell fragment-enriched extract comprises microbial cell fragments and soluble cytoplasmic compounds, wherein at least 80% of the cell fragments have a d50 ≤ 500 nm.

[0134] In one embodiment of this aspect, the extract enriched in small cell fragments comprises at least 1%, preferably at least 10%, such as at least 20% or even at least 30% or at least 50% more small cell fragments than the simultaneously obtained aqueous suspension depleted of small cell fragments (i.e., an extract enriched in large cell fragments).

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

[0136] In some embodiments, the microbial cell product can be used as a substitute for egg yolk, egg white, or whole egg in the preparation of an equivalent product prepared using an equal or unequal amount of egg. In a preferred embodiment, the microbial cell product is used as a substitute for egg white.

[0137] In some aspects, the present invention provides a food product comprising a mixed microbial cell product as described herein.

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

[0139] Although the present invention is primarily described with reference to preparations obtained from yeast cells, the invention is not limited thereto. Various other microorganisms may be used. In an embodiment, the microorganism may be selected from fungi, including yeast (preferably Saccharomyces spp., more preferably brewer's yeast or baker's yeast); plants, in particular microalgae (including Tetraselmis sp. or Chlorella sp., such as C. vulgaris); and cyanobacteria (including Arthrospirasp sp., preferably A. platensis). The microorganism may also be selected from bacteria, such as lactic acid bacteria.

[0140] Other variations to the disclosed embodiments will be apparent to those skilled in the art in practicing the claimed invention by studying the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude 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 to advantage.

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

[0142] method

[0143] Providing 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 at 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] Adjustment of pH is accomplished, for example, by slowly adding a 5 M NaOH solution to an aqueous suspension containing microbial cells under constant gentle stirring and at a constant temperature until the desired pH is reached. Other solutions of NaOH and other types of bases (e.g., CaOH ) can be used to adjust the pH of a microbial suspension. Similarly, suitable acid solutions can be used to lower the pH of a microbial suspension.

[0147] Preprocessing

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

[0149] 1) A homogeneous yeast suspension (100 g / L) was subjected to centrifugation at 3000 rcf for 5 minutes at a temperature of 15°C.

[0150] 2) The resulting supernatant was removed and replaced with an equal amount of water.

[0151] 3) The obtained suspension was gently mixed until a homogeneous suspension was obtained.

[0152] 4) Steps 1 to 3 are repeated 2 to 3 times. The removed supernatant can be collected separately or blended to obtain a by-product (by-product 1 in the present invention).

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

[0154] Cell breakdown

[0155] In the example of yeast cell disintegration, the cell disintegration was carried out using a bead mill. This bead mill was carried out by the Dyno Mill research laboratory (Willy A. Bachofen AG). 200 ml of a solution (pH 8.9) containing 100 g / L (10% DW) of Saccharomyces cerevisiae was treated in batch recirculation mode for 15 minutes. A constant speed of 2039 rpm was used, and the temperature was controlled in the range of 24°C to 26°C. The mill contained 0.5 mm spherical zirconium beads with a filling rate of 65%. This resulted in an aqueous suspension containing the disintegrated microbial cells.

[0156] Separation processing

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

[0158] Purification treatment

[0159] In the example of purification, 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. For each experiment, dilution was performed 1-fold with water before each filtration cycle. Thus, purification was also performed as a diafiltration process.

[0160] Purification was accomplished by ultrafiltration or microfiltration, i.e., using an ultrafilter or microfilter in the above-described method. Ultrafiltration was performed using a hydrophilic membrane cassette in the range of 10 kDa to 1000 kDa (Biomax). 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 a final moisture content of <5% by weight. Drying was also carried out using a freeze dryer (Zirbuss GmbH 2x3x3) at −20° C. and <1 mbar for 48 hours.

[0163] Example

[0164] The present invention is further illustrated based on the following examples, which are merely illustrative and are not to be construed as limiting the present 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 dissociation.

[0167] Cell disintegration is performed as described above, and an aqueous suspension containing disintegrated 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 dissociation.

[0170] Cell disaggregation and subsequent separation were performed as described above, and an extract enriched in small cell debris and an extract enriched in large cell debris were obtained.

[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 dissociation.

[0173] Cell dissociation, subsequent separation and subsequent purification of the extract enriched in small cell fragments by ultrafiltration are performed as described above, and a purified extract enriched in small cell fragments is obtained.

[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 dissociation.

[0176] Cell disruption, subsequent separation and subsequent purification of the extract enriched in small cell debris by microfiltration are performed as described above, and a purified extract enriched in small cell debris is obtained.

[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 dissociation.

[0179] Cell dissociation, subsequent separation and subsequent purification of the extract enriched in small cell fragments by ultrafiltration, and subsequent drying are performed as described above, and a dried purified extract enriched in small cell fragments is obtained.

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

[0181] Table 1 shows examples of pH and temperature throughout the different process steps according to the present method.

[0182]

[0183]

[0184] Effect of processing temperature during bead milling

[0185] The 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 was varied within the range of 20°C to 55°C. Another experiment (*) was conducted in which the decomposition was carried out for an extended period of 1 hour without temperature control. The maximum temperature recorded was 60°C. The results are shown in Table 2.

[0187] Before bead milling, the temperature was adjusted to the desired value. A flow cytometer (BD Accuri C6) was used to estimate the effect of temperature on cell lysis. Before measurement, the samples were diluted 80 times with water and analyzed using a flow cytometer. Cell lysis was determined by forward scatter data. A biomass suspension at T = 20°C was used as a reference to estimate the fraction of damaged cells.

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

[0189] The gelation was determined as follows. An aqueous solution containing 200 g / L (20% DW) was prepared. Further dilutions were made in water to reach 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 material were analyzed according to the following scale (score): 0 means no gelation was observed, 1 means a paste was observed, 2 means a dripping paste was observed, 3 means a very soft gel was observed, 4 means a soft gel was observed, and 5 means a hard gel was observed.

[0190] Table 2: Gelation properties of extracts enriched in small cell debris generated at different temperatures during bead beating.

[0191]

[0192] 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 ​​of the suspension before bead milling. An extract enriched in small cell debris was obtained and analyzed as follows:

[0194] Native gel electrophoresis: Samples were loaded onto precast gels with 4% to 12% cross-linking using 10× Tris-glycine buffer. Electrophoresis was performed at 125 V for 70 minutes and compared with native protein markers. Protein bands were stained with Coomassie blue.

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

[0196] from Figure 2B It can be clearly seen that for alkaline pH values, the solubility of proteins present in the extract enriched in small cell debris is higher. Figure 2A It can be seen that at alkaline pH values, particularly 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, if the protein exists in its native conformation, then the band will be displayed. If the protein is denatured, then the corresponding band will disappear completely or a new band of a different molecular weight will appear.

[0197] Influence of residence time in a bead mill

[0198] Example 2 was repeated and samples were taken at different time points during the bead beating process for further analysis. The extracts enriched in small cell debris were collected for further analysis.

[0199] Protein was determined using BSA as a standard protein according to the method of Lowry (Lowry, OH; Rosebrough, NJ; Farr, AL; Randall, RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry. 193(1): 265–75). Carbohydrates were determined using glucose as a standard sugar according to the method of Dubois (Michael Dubois, Kagilles, J. K. Hamilton, Parebers, and Fred Smith. Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry. Vol. 28, No. 3, March 1956).

[0200] Figure 3 Graph showing the solubilization amount of protein (left vertical axis) and carbohydrate (right vertical axis) as a function of residence time in the bead mill (horizontal axis). Figure 3 The results show how the residence time in the bead mill affects the solubilization of proteins and carbohydrates. Since 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 and thus different functional activities.

[0201] This shows that by adjusting the residence time during the digestion step, the solubilization levels of proteins and carbohydrates can be controlled. Figure 3 An example is provided of how choosing two different residence times can result in two extracts with significantly different compositions. When the residence time was 5 minutes (see arrow at P1), the extract obtained was found to contain 30% less soluble carbohydrates than when the residence time was 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 the present invention because the functional properties of the final product depend on the particle size distribution.

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

[0204] Example 2 was repeated, but using different values ​​for the grinding time (residence time in the bead mill). The aqueous suspension obtained, containing the decomposed microbial cells (also referred to as slurry), was collected for analysis. The extract enriched 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. The sample was prepared in water to a concentration of <10 g / L (1% DW). The sample was added to the measuring cell, ensuring that no bubbles formed. The sample was measured three times with a waiting time of 30 seconds, a temperature of 25°C, and a refractive index of 1.33.

[0206] D99, D70, and D50 values ​​are calculated from particle size distribution data. The D99 particle size indicates that 99% of the particles are larger than this value. The D70 particle size indicates that 70% of the particles are larger than this value, and similarly for 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 of each interval is estimated. Table 3 shows the particle ranges after decomposition and separation processing.

[0207] Table 3: D99, D70 and D50 values ​​of decomposed microbial biomass suspension (after decomposition) and extract enriched 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 the cell fraction in an aqueous suspension containing disintegrated cells after 15 minutes ("15 min slurry" - black solid line) and 3 hours ("3 h slurry" - grey solid line) of bead beating is shown. The horizontal axis shows the diameter of the cell debris in nanometers and the vertical axis shows the intensity of the signal. The 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 around 5500 nm. In addition, the particle size distribution of the extract enriched in small cell debris was determined after separation (centrifugation at 4000 rcf) (15 min SN and 3 h SN; where SN = supernatant). As Figure 4 As shown, the result of the separation process is that the centrifugation method selectively maintains a population of cell fragments with an average size in the range of 200 nm to 400 nm. In other words, these small cell fragments enter the extract, while larger cell fragments remain in the original disrupted suspension (either an aqueous suspension depleted of small cell fragments or an extract enriched in large cell fragments). This clearly demonstrates the effectiveness of the separation step of the present invention. To the inventors' surprise, such small cell fragments (and the extracts enriched in small cell fragments therefrom) exhibit functional properties comparable to those of highly purified components (e.g., protein isolates) specified in the literature.

[0210] For extracts rich in small fragments, the present invention generally refers to a particle size of 500 nm. For the sake of clarity and simplicity, this value is referred to as d50.

[0211] An important element of the present invention is the fact that the extracts enriched in small fragments and the extracts enriched in large fragments exhibit multimodal distributions. Therefore, the extracts and products described above were further characterized using a Mastersizer 2000 (Malvern Panalytical), using 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] The influence of centrifugal force

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

[0214] The maximum gel strength (G' [Pa]) was measured using a rheometer (Anthon Parr). The sample was subjected to a heating-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 about 28°C).

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

[0216] Effect of membrane retention on the gelation behavior of extracts enriched in small cell fragments

[0217] Example 3 was repeated, but using different membrane cut-off values ​​for the filters used in the purification process.

[0218] The resulting purified extract enriched 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 heating-cooling treatment (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 about 28°C).

[0220] Figure 6Bar graph showing a purified extract enriched in small cell fragments according to the present invention compared to other protein enriched sources such as egg white protein isolate, whey protein isolate and pea protein isolate as measured by gel strength. Figure 6 It is shown that membrane-based filtration can be used as a purification process to significantly enhance the gelling properties of an extract enriched in small cell debris. The numbers 0.2, 100, and 10 represent the cutoff values ​​of the membrane, i.e., 2 μm, 100 kDa, and 10 kDa. In particular, membrane-based filtration can be used to purify an extract enriched in small cell debris into a retentate phase (purified extract enriched in small cell debris) and a permeate phase (byproduct). Figure 6 Also shown are comparative extracts enriched for small cell debris, which were resolved at pH 7 (enriched fraction pH 7) or pH 9 (enriched fraction pH 9). Figure 6 As demonstrated, the purified extract enriched in small cell debris (retentate fraction) exhibited good gelling properties. In addition, the extract enriched in small cell debris also exhibited gelling properties, especially the extract at pH 9.

[0221] surface tension

[0222] Example 3 was repeated, but using different membrane cut-off values ​​for the filters used in the purification process. The resulting byproducts and the purified extract enriched in small fragments were collected for further analysis as follows.

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

[0224] Figure 7 Bar graphs showing the equilibrium surface tension of the by-product (P) obtained at the air-water interface (left) and the purified extract enriched in small cell fragments (R) at the oil-water interface (right). The values ​​for egg white protein isolate and whey protein isolate are used for comparison. The equilibrium surface tension reflects the degree of surface stabilization; lower values ​​are obtained from samples with superior activity. The numbers 0.2, 1000, 100 and 10 represent the cut-off values ​​of the membranes, 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 present invention provides a way to selectively fractionate an extract enriched in small cell fragments into a purified extract enriched in small cell fragments and by-products. As Figure 7 As shown, in addition to the 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 better than those of typical protein isolates.

[0225] Example composition of extracts

[0226] Example 3 was performed and the resulting extracts were further analyzed.

[0227] The small cell fragment-enriched extract and the large cell fragment-enriched extract obtained after the separation process can be rich in protein and carbohydrates. Table 4 shows an example composition of the small cell fragment-enriched extract and the large cell fragment-enriched extract. In the table, "ash" refers to any inorganic matter, such as minerals.

[0228] Table 4: Example composition of extracts (in % DW)

[0229] Extracts enriched in small cell fragments Large cell fragment-enriched extract protein 57％ 43％ carbohydrate 22％ 37％ ash content 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 studied. Figure 9 As can be seen in Figure 10, the particle size distribution changes as the disruption process is run for longer periods of time. In the case of yeast, a peak of intact cells of ~6 μm and a peak of cell debris of ~0.8 μm are obtained. As disruption time increases, the intact cell peak decreases and the cell debris peak increases. Therefore, the disruption process can be run until the desired particle size distribution is achieved. For yeast, the desired target particle size distributions (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 effects of varying separation conditions such as Figures 11 to 13 For these examples, disrupted yeast preparations were separated by centrifugation at low, medium, and high intensities. Low and medium intensities can increase EESF yield. For example, using low and medium centrifugation intensities can achieve >10% EESF yield (DW) compared to high centrifugation intensities.

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

[0234] It is believed that different separation strengths will change the particle content of the EESF or EELF; see Figure 12 and 13By increasing the separation intensity, the specific surface area increases, but the number of particles decreases significantly. It is preferred to use a low separation intensity in order to enrich the EESF with particles in the range of 0.1 μm to 0.3 μm. Under high-intensity separation conditions, the main soluble compounds remain in the liquid phase with a small number of particles in the range of 0.1 μm to 0.3 μm. See Figure 12 By increasing the separation strength, the specific surface area increases, but the number of particles decreases significantly. Figure 13 These particles are crucial to enhancing the overall yield and functional properties of EESF; therefore, a balance between low and high separation strength must be found.

[0235] EELF preferably contains fragments in the range of 2 μm to 10 μm. Cell fragments in the range of 2 μm to 10 μm are preferred to enhance water holding capacity without compromising oil holding capacity. See the table below, which shows the water holding capacity and oil holding capacity 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 PSDs of the EESF and EELF, especially in the 1–4 μm range. This is more apparent when using low-intensity separation (see Figure 12 ). Therefore, it is preferred to use a medium intensity separation.

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

[0239] The psd range of the EESF and EELF remains virtually unaffected when different dilution levels are applied. Only the relative contribution of the main peaks in the multimodal distribution changes. The contribution of particles with a peak at approximately 0.2 μm compared to the broad peak at approximately 1.5 μm to 2 μm increases with higher dilution levels. Figure 16 Likewise, higher dilution levels lead to higher total specific surface area (SSA) (e.g. Figure 17 As shown in Figure 2 ), D[3,2] and D[4,3] decrease with increasing dilution levels (as shown in Figure 2 ). Figure 18 shown).

[0240] Other microorganisms

[0241] Although the examples so far have 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 hardness of gels obtained from EESF of two microalgae species and one cyanobacterium species under the separation conditions according to the present invention.

[0242]

[0243] *psd: particle size distribution after decomposition

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

[0245] ***No gel: Although gelation was observed, the resulting material could not be measured using Texture Profile Analysis (TPA).

[0246] Furthermore, higher dilution before S / L separation was beneficial for enhancing the functional properties of EELF, such as gel firmness:

[0247] EELF gel hardness [N]

[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 adjust other functional properties, such as oil holding capacity (OHC). Figure 19 .

[0251] Prolonged cell breakdown

[0252] The cell dissociation step can be further extended to enhance the functional properties of EELF. For example, the PSD time period can be modified to 240 min to produce EELF with excellent gel hardness, water holding capacity (WHC), and oil holding capacity (OHC).

[0253] EELF

[0254]

[0255] By performing cell dissociation under alkaline conditions, preferably at pH > 9, and S / L separation at low DW content (< 5%), the functional properties of EELF are significantly enhanced. Figure 20 However, at alkaline pH, the PSD during the extended cell disintegration process showed a unique trend. Instead of the steady decrease in particle size expected during micronization, the particles tended to aggregate. This effect was particularly pronounced at pH 11 – see Figure 21.

[0256] Compared to the traditional beta-glucan extraction process for yeast biomass, an extended cell lysis process at pH 11 followed by S / L at high dilution levels (DW < 5%) produced EELF with superior functionality. As a reference, a process involving cell autolysis, cell homogenization, alkaline extraction, acid extraction, and water extraction (Aut+Hom+Extr) was compared with the currently proposed method (extended lysis at pH 11 and high dilution).

[0257]

[0258] The reference process has been reported by Saowanee Thammakiti, Manop Suphantharika, Thanaporn 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] Concentrated vs. Dry

[0260] The functional properties of EESF and EELF deteriorate due to thermal processes such as evaporation and drying, but they can also freeze. Therefore, it is preferable to use both fractions (EESF and EELF) as wet ingredients and avoid processes that could cause thermal denaturation. Preferably, these two fractions should not be subjected to processes above 45°C or below 0°C.

[0261] Ingredient blending

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

[0263] Texturizers in alternative meat burgers: Keeping the total ingredient concentration the same but varying the ratio between EESF and EELF resulted in variations in firmness, springiness, and juiciness versus dryness. This provides food formulators with a range of options to achieve the texture they're looking for. For example, mixing at ratios ranging from 100:0 to 0:100 showed that a blend of 70 EESF:30 EELF produced a firm, springy, but not dry burger, while a blend of 40 EESF:60 EELF resulted in a softer, juicier, and "fattier" burger. Thus, the ratio can be optimized for specific markets.

[0264] Egg Replacement in Bakery: The intended use of the ingredient in bakery was to use EESF in place of egg whites or whole eggs in bakery applications. This proved successful, but surprisingly, a 50:50 blend of EESF and EELF provided better texture and performance as a whole egg replacement. This was counterintuitive, as the primary functionality required here was heat-set gelation, and since EESF has superior gelling properties, the expected result would be that EESF would perform better than the combination.

Claims

1. A method for preparing a microbial cell product, the method comprising: i) providing an aqueous suspension comprising microbial cells; and ii) subjecting the suspension to mechanical cell disintegration at a temperature in the range of 15° C. to 35° C. and at a pH in the range of 7 to 11 to obtain an aqueous suspension comprising disintegrated microbial cells; iii) separating the suspension to provide an extract enriched in small cell fragments and an extract enriched in large cell fragments, wherein the extract enriched in small cell fragments has a particle size distribution of d50 equal to or less than 500 nm, wherein the small cell fragments have a size range of 0.1 to 3 μm; and the extract enriched in large cell fragments has a particle size distribution of d50 greater than 500 nm, wherein the large cell fragments have a size range of 3 to 10 μm; and v) combining a portion of each extract to provide the microbial cell product.

2. 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 a by-product, wherein the pretreatment is selected from decantation, centrifugation, filtration, and one or more combinations thereof.

3. The method according to claim 1, wherein the separation of step iii) is selected from the group consisting of flocculation, sedimentation, decantation, air flotation, centrifugation, filtration, and one or more combinations thereof.

4. The method according to claim 1, wherein the separation in step iii) is hydrocyclone separation.

5. The method according to claim 1 , further comprising a step iv) after step ii) or after step iii), wherein step iv) is subjecting at least one of the microbial products obtained in step ii) or step iii) to a purification treatment to obtain at least one purified microbial cell product, wherein the purification treatment is independently selected from the group consisting of washing, filtration, adsorption, chromatography, crystallization, flocculation, 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 comprising a step vi) after step ii), step iii), step iv) or step v), wherein step vi) is to subject at least one of the microbial products obtained in step ii), step iii), step iv) or step v) to drying to obtain a dried microbial cell product, wherein the drying is performed by a method selected from the group consisting of spray drying, freeze drying and fluidized bed drying, wherein The dried microbial cell product has a water content of 10 wt% or less based on the total weight of the dried microbial cell product. 7 . The method according to claim 6 , wherein the dried microbial cell product has a moisture content of 5 wt % or less based on the total weight of the dried microbial cell product.

8. The method of claim 1, wherein the microbial cells are selected from the group consisting of algae, bacteria, fungi, and one or more combinations thereof.

9. The method of claim 8, wherein the microbial cell is yeast.

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

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

12. The method according to claim 1, wherein the mechanical cell disruption of step ii) is performed using bead beating.

13. The method according to claim 12, wherein bead milling is performed to obtain a predetermined particle size distribution of the suspension.

14. The method according to claim 13, wherein the microbial cell is 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.

15. The method of claim 12, wherein bead milling is performed at a pH > 9 for a period of time sufficient to cause agglomeration of the particles and wherein the dry weight content prior to separation is at least 2.5%.

16. The method according to claim 1, wherein the separation treatment in step iii) is centrifugation at a centrifugal force equal to or less than 4000 relative centrifugal force (rcf).

17. The method of claim 16, wherein the centrifugation occurs for a period of time equal to or shorter than 20 minutes.

18. The method of claim 16, wherein the centrifugation occurs for a period of time less than 15 minutes.

19. The method of claim 1, further comprising adjusting the water content of the disintegrated cell suspension prior to the separation step iii).

20. The method of claim 1, further comprising incubating the small cell debris enriched extract at less than 25°C for at least 60 minutes prior to the mixing step v).

21. A microbial cell product obtained by a method according to any one of the preceding claims.

22. Food, animal feed or cosmetic comprising the microbial cell product according to claim 21.

23. Use of the microbial cell product of claim 21 as a leavening agent, a gelling agent, a texture improver, an egg replacer, or any combination thereof.

24. Use of the microbial cell product of claim 21 in an edible egg-free emulsion, egg analog, egg-free scrambled eggs, egg-free pie, egg-free pound cake, egg-free angel food cake, egg-free meat substitute, egg-free meat substitute, egg-free yolk pie, egg-free and dairy-free cream cheese, egg-free pasta dough, egg-free custard, or egg-free ice cream, or in dairy-free milk.

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