Microbial extracts, uses and applications

Abstract

The present invention relates to a method for producing an extract of microbial cells with improved functional properties. The invention further relates to an extract of microbial cells obtained or obtainable by said method. The invention further relates to the use of said microbial cell extract with improved functionality, which can be applied to gelling agents, thickeners, blowing agents, emulsifiers, texturizing agents and other suitable applications.

Description

Technical Field

[0001] The present invention relates to a method for improving the functionality of a microbial cell extract. The present invention further relates to a microbial cell extract with improved functionality obtained or obtainable by the method. The present invention further relates to the use of the microbial cell extract with improved functionality, which can be applied to gelling agents, thickeners, foaming agents, emulsifiers, texturizing agents and other suitable applications. Background Art

[0002] Microorganisms play an essential role in the food industry, not only as biotransformers in traditional foods and beverages such as cheese, beer, wine, tofu and tempeh, but also as sources of biomass, nutrients and functional ingredients. Recently, technological developments have driven the advancement of cultured meat products, in which animal cells are cultured and grown in bioreactors to simulate animal muscle tissue.

[0003] The most common example of the direct use of microorganisms in food in the form of microbial biomass is the so-called single-cell protein (SCP), especially in the 1940s, when the production of bacterial, fungal and yeast biomass accelerated in order to fill the protein gap and supply sufficient food worldwide. In addition to SCP, microorganisms can also be used for the targeted production of functional proteins with high market value. Examples of this are the expression of milk and egg proteins in host organisms for the large-scale production of dairy products and egg-containing ingredients without animal origin.

[0004] In addition, microorganisms can be processed to purify certain components of interest or to extract target compounds present inside the cell, in the cell wall or membrane, or in specialized organelles. Examples of such compounds are lipids and pigments from algae (Silva et al., 2020), polysaccharides from macroalgae, or proteins from yeast (Kinsella and Shetty, 1978).

[0005] In the specific case of yeast and yeast-derived proteins, there are a large number of products developed on a commercial scale, the most notable of which are yeast extracts and yeast proteins. The production of yeast extracts and yeast proteins is traditionally accomplished through a series of processes involving cell lysis, separation and protein purification (US3888839A and EP3670646A1). The most commonly used cell lysis methods are autolysis and plasmolysis, in which intrinsic biochemical reactions are responsible for the breakdown of the cell wall. After this, a separation step is performed to produce a soluble fraction and an insoluble fraction. The methods frequently used for this step are centrifugation and filtration. In addition, protein purification is also carried out using methods such as isoelectric precipitation (Kinsella and Shetty, 1978). Traditional yeast processing leads to protein denaturation and hydrolysis due to high temperature and long-term exposure, extreme changes in pH, and the use of salts or solvents. Even if the final protein fraction is of high purity, its functional properties are limited (Kinsella and Shetty, 1978). In many food applications including baked goods, confectionery, simulated meat and simulated dairy products, functional properties such as foaming, emulsification and gelling are essential for technological applications.

[0006] Vananuvat and Kinsella (1975) and Kinsella and Shetty (1978) provided a comprehensive overview of the nutritional and functional properties of proteins from microbial biomass, especially from yeast. The authors clearly pointed out that the functional properties of yeast proteins are still limited under traditional extraction methods. In addition, the authors proposed methods to improve their functionality, mainly by functionalizing proteins through chemical reactions.

[0007] US3887431 proposes a method for producing a soluble protein isolate from yeast that exhibits technical functionality, including gelling. Similarly, US3888839 describes a method for producing yeast protein that can be used as a meat extender. GB1578235A also claims a method for preparing yeast protein with functionality similar to egg protein, but does not disclose further information about the functional properties of these proteins or methods for improving functional properties. US3867554 and US 5756135 describe methods for preparing yeast polysaccharides / solid extracts that can be used as fat substitutes. US10407600B2 and US2603630A propose methods for preparing yeast protein extracts with adhesive properties. WO2006067145A1 and WO2018002505A1 claim methods for producing yeast protein extracts and their use in stabilizing wine and controlling beer turbidity. EP 3670646 A1 describes an ultrafiltration method for purifying soluble yeast proteins which exhibit thermogelling properties.

[0008] Recently, a new method was reported in which soluble yeast proteins were purified using ultrafiltration technology (WO2020127951A2) to obtain protein concentrates showing gel-like properties. Patent application PCT / EP2021 / 075137 proposes a method for producing aqueous microbial extracts enriched in large and small fragments that show unique functional properties.

[0009] Despite numerous scientific reports and inventions related to microbial proteins (especially yeast proteins) and many claims about their functional properties and wide applications, the performance of microbial proteins as foaming agents, emulsifiers and gelling agents has not been explicitly explored, and no new methods have been proposed to improve their functionality, except for chemical functionalization strategies that are well known in the art.

[0010] Therefore, there is a need to develop methods to improve the functional properties of microbial cell extracts, particularly those related to foaming, emulsification and gelling. The present invention addresses this need. Summary of the invention

[0011] The present invention describes a method for preparing a microbial cell extract, the method comprising: providing a microbial biomass; decomposing the microbial biomass; subjecting the resulting broken biomass to solid-liquid separation to obtain a light fraction and a heavy fraction. The method may then optionally further comprise subjecting the heavy fraction and / or the light fraction and / or the combination of the light fraction and the heavy fraction to a purification step, and / or a concentration step, and / or preparing an alkaline suspension, and / or further solid-liquid separation, and / or emulsification, and / or dilution and / or pH adjustment, and / or stirring, and / or drying and / or heat treatment.

[0012] In one aspect of the present invention, there is provided a method for preparing a microbial cell extract, the method comprising:

[0013] a) providing a microbial biomass in an alkaline aqueous suspension at a pH of 7-11;

[0014] b) mechanically decomposing the microbial biomass at a temperature below 40°C using a non-denaturing process such that the decomposed biomass consists of a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a particle size distribution (psd) with an average of about D50 < 4.5 μm;

[0015] c) subjecting the decomposed biomass to a solid-liquid separation process to separate the decomposed biomass into a light fraction (also referred to herein as an extract enriched in small fragments) and a heavy fraction (also referred to herein as an extract enriched in large fragments), wherein the light fraction consists of a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a psd with a D50 of about 0.5 um or less; and the heavy fraction consists of a population of soluble compounds and suspended fragments having a psd with a D50>0.5 um; and

[0016] d) further processing one or both of the light fraction and the heavy fraction to optimize one or more functional properties of the fractions.

[0017] In some embodiments, the heavy fraction consists of a population of soluble compounds and suspended debris with a psd of D50>4 um.

[0018] In one embodiment, any combination of the fractions obtained as described above may be combined in any ratio depending on the specific application of the microbial cell extract.

[0019] As will be described herein, the one or more functional properties may be selected from gelling properties, foaming properties, emulsifying properties, textural properties, glossing properties and / or browning properties. The further processing of step d) is a non-chemical deep processing step; and preferably, the chemical structure of the components of the fraction is not changed. Shetty and Kinsella (1978) and Vananuvat and Kinsella (1975) describe in detail the functional properties of proteins from yeast. These authors emphasize the limited functionality of yeast proteins, particularly foaming, and indicate that functional properties can be improved by protein functionalization, such as succinylation and phosphorylation. One of the key aspects of the present invention is that a method is provided to enhance the functionality of light fractions and / or heavy fractions without the need for biochemical reactions or complex purification methods. In general, unless otherwise indicated, the different processing steps described herein can be combined when processing a given fraction.

[0020] The functional properties described herein are primarily those desired for use in food manufacturing (including animal feed), but it will be appreciated that such functional properties, including gelling, emulsifying and / or foaming may also be desired in other applications such as cosmetic production.

[0021] Microbial biomass contains microbial cells, which are traditionally used to produce a variety of products with industrial value, or directly used in a variety of applications. Most industrial or commercial applications utilize a group of microbial biomass strains selected from the fields of bacteria, yeast, fungi and algae. In general, the products obtained from microbial biomass are either intracellular or extracellular. Extracellular products are secreted by cells into the main culture medium, which is usually an aqueous phase. In contrast, intracellular products remain inside the cells. In order to obtain intracellular products, additional processing is required to release these products from the cells (by destroying the cell membrane or cell wall), and further separate the compounds of interest from the remaining biomass and other impurities.

[0022] In the specific field of 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.

[0023] Microbial biomass is often used in the form of extracts, for which it is necessary to destroy / decompose the microbial cells that form the biomass. Extracts prepared from several different starting materials are known, such as fungal extracts, algae 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 has been (and can be) widely used in various products, from growth media for laboratory cultured cells to nutritional supplements and flavor enhancers for the food industry. The production process of YE is well known. In general, the destruction (= decomposition) of yeasts mainly from the genus Saccharomyces ( Saccharomyces ) of yeast cells, followed by an incubation step at high temperature (>50°C) to activate endogenous enzymes that break down (= digest) large intracellular products such as proteins and nucleic acids into their smaller components, such as peptides, amino acids and nucleotides. The digested slurry obtained is then further purified and supplemented, depending on the final application, to provide an extract that can be commercialized as YE.

[0024] The microbial biomass used in the present invention can be obtained from several types of microorganisms, including microalgae, yeast, bacteria and fungi. An example of a genus from which the microbial biomass that can be used to produce a microbial extract can be derived is Saccharomyces ( Saccharomyces ) and Pichia ( Pichia ) (yeast), Tetraselmis ( Tetraselmis ), Chlorella ( Chlorella ), Arthrospira ( Arthrospira ) (algae), Fusarium spp. Fusarium ) (fungi), Methylobacterium ( Methylobacterium ) (bacteria) and Lactobacillus ( Lactobacillus ) (bacteria). Preferably, the microbial biomass is derived from yeast, more preferably from the genus Saccharomyces and / or Pichia. Yeasts that can be used in the present invention include Saccharomyces, such as Saccharomyces cerevisiae ( S. cerevisiae )、Saccharomyces cerevisiae ( S. chevalieri )、Saccharomyces boulardii( S. boulardii )、Bayan yeast ( S. bayanus )、Italian yeast( S. italicus )、Saccharomyces cerevisiae ( S. delbrueckii )、Rossaccharomyces cerevisiae( S. rosei )、Saccharomyces cerevisiae( S. micro-ellipsodes ), Carlsberg yeast ( S. carlsbergensis )、Bisporus bisporus( S. bisporus ), fermentative yeast ( S. fermentati )、Pasteurella S. pastorianis ), Saccharomyces rouxii ( S. rouxii ) or Saccharomyces cerevisiae ( S. uvarum ); belongs to the genus Schizosaccharomyces ( Schizo-saccharomyces ) yeast, such as Schizosaccharomyces japonicus ( S. japonicus ), Cambodian fission yeast ( S. kambucha ), Schizosaccharomyces cerevisiae ( S. octo-sporus ) or Schizosaccharomyces pombe ( S. pombe ) ; belongs to the genus Hansenula ( Hansenula ) yeast, such as Hansenella wencheirosporum ( H. wingei ), arni Hansen yeast ( H. arni ), Hansenula henselae ( H. henricii ), Hansenula americana ( H. americana )、Canada Hansen yeast( H. canadiensis ), Hansenula mycelium H. capsulata ) or Hansenula polymorpha ( H. polymorpha ); belongs to the genus Candida ( Candida ) yeasts, such as Candida albicans ( C. albicans ), Candida utilis ( C. utilis ), Candida boidinii ( C. boidinii ), Candida asteroidea ( C. stellatoidea )、Candida anonymida ( C. famata ), Candida tropicalis ( C. tropicalis ), Candida glabrata ( C. glabrata ) or Candida parapsilosis ( C. parapsilosis );Belongs to the genus Pichia ( Pichia ) yeast, such as Pichia pastoris ( P. pastoris )、Pichia kluyveri( P. kluyveri )、Pichia polymorpha( P. polymorpha)、Pichia pastoris( P. barkeri )、Pichia pastoris P. cactophila )、Pichia rhodendron P. rhodanensis ), Pichia cissibiricum ( P. cecembensis ), Pichia cephalosporin ( P. cephalocereana )、Pichia thermophila( P. eremophilia ), fermentation of Pichia pastoris ( P. fermentans ) or Pichia kudriavzevii ( P. kudriavzevii ); belongs to the genus Kluyveromyces ( Kluyveromyces ) yeast, such as Kluyveromyces marxianus ( K. marxianus ); and Torulopsis ( Torulopsis ) yeast, such as Torulopsis bovis ( T. bovina ) or Torulopsis glabrata ( T. glabrata ).

[0025] In a preferred embodiment, the microbial biomass is free of contaminants - for example, the biomass can be purified by centrifugation followed by washing and resuspension; multiple rounds of washing and resuspension can be used. In another preferred embodiment, the microbial biomass is prepared in an alkaline aqueous suspension having a pH range of 7-11, more preferably a pH of about 9, and optionally the concentration of the microbial biomass in the aqueous suspension is about 50-150 g / L, preferably about 100 g / L.

[0026] In general, cell disintegration methods can be divided into non-mechanical methods and mechanical methods. Non-mechanical disintegration methods can be further subdivided into three categories: physical disintegration (e.g., by decompression, osmotic shock, pyrolysis, ultrasound or freeze-thaw), chemical disintegration (e.g., by using solvents, detergents, chaotropes, acids and bases or chelating agents) and enzymatic disintegration (e.g., by autolysis, phage lysis or lytic enzymes). The present invention preferably relates to mechanical disintegration methods. Examples of mechanical disintegration methods include ball mills (including bead mills) and homogenizers.

[0027] Ball mills (including bead mills) can be vertical and horizontal and use grinding media present in the 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 causes the intracellular material to be released into the aqueous suspension and will also cause cell disruption (i.e., decomposition). As the rotor speed increases, the shear force increases therewith, and cell disruption also increases. As the size of the grinding material decreases, cell disruption will usually increase therewith. Other parameters also affect the performance of the decomposition process. Those skilled in the art can select the correct parameters and variables according to the present invention.

[0028] 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 stream at high speed onto an impaction ring. Typically, multiple passes are required at high pressure, which can result in elevated temperatures that can cause local denaturation of unstable molecules.

[0029] Preferably, the mechanical disintegration step is performed by bead milling or high pressure homogenization. Most preferably, the mechanical disintegration step is performed by bead milling. Preferably, the disintegration step is performed under the following conditions, namely, the pH range is 7-11 (optionally 8-10 or 8.5-9.5, or most preferably 9), and the temperature range is about 10-30°C, more preferably about 15-25°C, even more preferably 20-25°C, and most preferably about 25°C. The technical effect of performing the disintegration step within the pH and temperature range is to prevent the denaturation of proteins and other unstable molecules (that is, this is a non-denaturing process), thereby preventing the activation of lytic enzymes, proteases or other hydrolases present in the microbial biomass. The skilled person is able to adjust the process parameters of the disintegration method (speed, flow rate, filling rate, bead size, pressure, etc.) to preferably maintain the temperature <25°C and achieve the desired PSD target.

[0030] The disintegration step produces a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a particle size distribution (psd) averaging about D50 < 4.5 μm. It will be appreciated that one skilled in the art will be able to adjust the parameters used in the disintegration step accordingly to achieve the desired goals for their application. During the cell disintegration process, the psd changes, showing a decrease in the peak for intact cells and an increase in the peak for cell fragments. The disintegration process is run until a specific psd is achieved. Using yeast as an example, the specific surface area increases over time, while D[3,2], D[4,3], D10, D50, and D90 decrease over time. Other psd parameters may also be considered; for example, for yeast, a desired target psd may be D10 < 0.5 um, D50 < 4.5 um, D90 < 7.5 um and D[3,2] < 2 um, D[4,3] < 4.5 um.

[0031] In the art, the particle size distribution is reported as a volume distribution. Take D50 as an example, which is also known in the art as Dv50; the terms D50 and Dv50 are used interchangeably herein. D50 or Dv50 is defined in the art as the maximum particle size measured by diameter, below which 50% of the sample volume is below, also known as the median particle size (diameter) calculated by volume. Fig.15 This concept is illustrated in Figure 1. There are many analytical techniques and methods for particle size analysis.

[0032] A particle size analyzer is an analytical instrument used to measure, display and report the size distribution of a given population of particles or droplets. Laser diffraction particle size analyzers calculate particle size based on the angles of light scattered by a stream of particles passing through a laser beam. This technique allows for continuous measurement of bulk materials over a wide range of sizes. The size limit and sensitivity of a laser diffraction particle analyzer depends on the number and location of detectors in the instrument. Dynamic light scattering particle analyzers are primarily used to analyze particles in solution. Dynamic light scattering determines size based on the fluctuations in scattered laser intensity resulting from the Brownian motion of the particles. Inductive grating particle size analyzers determine the size of small particles in solution by electrically arranging the particles and then measuring their diffusion.

[0033] In the context of the present invention, D50 is determined using a laser diffraction particle size analyzer Malvern Mastersizer 2000, wherein the dispersant RI is 1.33 and the particle / material RI is 1.34, using the general analysis model MS2000 and the Mie scattering model. As described above, a decomposition method is used to obtain a bimodal distribution and a particle size distribution (psd) with an average of about D50<4.5 μm. Examples of cell decomposition methods according to the prior art are as follows. US3888839A discloses a method for obtaining a protein isolate from yeast cells, wherein the yeast cells are broken by high-pressure homogenization (mechanical decomposition) and subsequent incubation. EP1199353A1 discloses a method for producing a yeast extract by treating a yeast suspension or yeast paste and separating out insoluble components, wherein high-voltage electric pulses are applied to the yeast suspension or yeast paste (physical decomposition). EP2774993A1A discloses the use of a cell wall degrading enzyme (enzymatic decomposition) without a protease, followed by a heat treatment of the product at 70-80°C for 10 to 20 minutes.

[0034] The microbial cells present in the microbial biomass suspension mainly contain proteins, carbohydrates, lipids and minerals. Proteins and other unstable molecules will unfold, denature and degrade when exposed to high temperatures, long incubation times, extreme pH values, solvents, salts and other harsh chemicals. When proteins and other functional molecules denature (lose tertiary and quaternary structures), their (partial) functional activity is lost. When denatured (unfolded), proteins lose the ability to interact with hydrophilic and hydrophobic surfaces, and their ability to rearrange and form a network structure after heat-cooling treatment is also affected. The inventors have observed that under the conditions described herein (e.g., in a specified pH range), the use of mechanical decomposition is mild enough to prevent the unfolding, denaturation and / or degradation of proteins and other unstable molecules, and therefore, this is necessary to maintain functional properties, in particular gelling behavior, water holding capacity and oil holding capacity.

[0035] The aqueous suspension comprising microbial biomass may further comprise cytoplasmic material or other extracellular material produced during proliferation or fermentation.

[0036] In one embodiment of the method according to the invention, the microbial biomass comprises microbial cells selected from unicellular or colony prokaryotes and eukaryotes and one or more combinations thereof. In a preferred embodiment, the microbial cells are selected from the group consisting of yeast, algae, bacteria, fungi and one or more combinations thereof. In a specific embodiment, the microbial cells are yeast.

[0037] The bead size range that can be considered is 0.1-5 mm, with a preferred range of 0.5-1 mm. Suitable bead materials include, but are not limited to, zirconium and glass. Depending on the total available volume of the bead mill chamber, a suitable bead filling (percentage of the bead mill chamber filled with beads) can be considered to be in the range of 40-90%, with a preferred range of 65-80%, and more preferably 75%.

[0038] It can be considered that the appropriate rotation speed range is 1-20 m / s. According to the configuration and geometry of each bead mill, a person skilled in the art can estimate the corresponding rotor speed in rpm. A suitable rotation speed in rpm is, for example, 500-5000 rpm, preferably 1000-3000 rpm.

[0039] It is believed that a suitable microbial cell concentration range is 2-25% dry weight.

[0040] In one embodiment, a Dyno-mill research laboratory (WAB) bead mill is used to break up microbial cells. Cells can also be broken up by shear forces, such as by stirring (e.g., with a high speed or Waring stirrer as an example), French press, or even by centrifugation in the case of fragile cell walls, to break up cells.

[0041] In one embodiment, cell disruption is performed without the addition of chemicals and / or solvents.

[0042] The decomposition step preferably produces a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a particle size distribution (psd) averaging about D50 < 4.5 μm. It should be appreciated that those skilled in the art will be able to adjust the parameters used in the decomposition step accordingly to achieve the desired objectives of their application.

[0043] In a preferred embodiment, the solid-liquid separation step is selected from methods known in the art, including but not limited to centrifugation, decantation and filtration. More preferably, separation is carried out by centrifugation, and in a most preferred embodiment, separation is carried out using centrifugation with a gentle centrifugal field. An example of a gentle centrifugal field is separation at a speed of <4000 rcf in a desktop centrifuge <15 minutes. In one embodiment, the separation step is centrifuged under a centrifugal force equal to or less than 4000 relative centrifugal forces (RFC). In one embodiment, the centrifugation lasts for a period of time equal to or less than 20 minutes. In a specific embodiment, the centrifugation lasts for a period of time equal to or less than 15 minutes. In another specific embodiment, centrifugation is carried out at about 15 ℃. It should be understood that those skilled in the art can adjust the centrifugation parameters according to the type of equipment used, to achieve target properties (e.g., psd) in two extracts.

[0044] The solid-liquid separation step produces a light fraction (an extract enriched in small fragments) and a heavy fraction (an extract enriched in large fragments), as described in patent application PCT / EP2021 / 075137.

[0045] In one embodiment, the separation step of the method produces a light fraction comprising small cell fragments ranging from about 0.1-3 μm and a heavy fraction comprising large cell fragments >1 μm in size.

[0046] Preferably, separation produces such a volume ratio, wherein the maximum volume ratio of the light phase to the total starting fraction is 0.1, more preferably <0.05. Preferably, the volume ratio of the heavy fraction of the heavy phase to the total starting fraction is at least>0.65, more preferably>0.9. An estimated value of the volume ratio obtained from the separation is listed in Example 1. Those skilled in the art will be able to adjust separation parameters, such as centrifugal parameters, such as but not limited to time, g force and / or sigma factor of any value. Those skilled in the art will recognize that the adjustment mode of the parameters will vary according to the centrifugal unit adopted to achieve the desired classification target.

[0047] In one embodiment of the invention, the light and / or heavy fractions are further processed by purification and / or concentration and / or pH adjustment and / or by an emulsification process. Examples of concentration methods include, but are not limited to, filtration, evaporation, freeze concentration, and pervaporation. Examples of purification methods include, but are not limited to, isoelectric precipitation, coagulation, adsorption, and filtration. It is known in the art that filtration methods such as reverse osmosis (RO), nanofiltration (NF), ultrafiltration (UF), and microfiltration (MF) have been used to concentrate and purify proteins from yeast. Examples of such advances are given by Levesley et al. (2000), Shetty and Kinsella (1978), and Kollar et al. (1992).

[0048] In a preferred embodiment, the light fraction is purified and / or concentrated such that the processed light fraction has a dry weight (DW) content of at least 10%, preferably at least 20%, even more preferably at least 30%. Preferably, the processed light fraction has a dry weight (DW) content of 10-30%, 20-30% or about 30%.

[0049] In a preferred embodiment, filtration is used to concentrate the light fraction. More preferably, diafiltration is used to concentrate the light fraction. Diafiltration is a process for separating and purifying a target product from a main solution containing other small molecular weight (MW) substances, such as salts, sugars and amino acids. The term "diafiltration" refers to a combination of "dilution" and "filtration". A buffer solution or demineralized water is added to the concentrate or retentate to make up for the permeate water lost during the filtration process, in this way keeping the concentration of the excluded compound (i.e., the target product) constant while diluting the unwanted small MW compounds so that they are gradually "washed out" through the filtration cycle. Preferably, diafiltration is used to concentrate the light fraction, wherein the ratio of water to light fraction is about 1:1, and wherein preferably, the membrane used for diafiltration ranges from 10 kDa–0.2 μm, more preferably 100 kDa–1000 kDa.

[0050] In one embodiment, the light fraction as described above is purified and / or concentrated to retain fragments <0.3 μm and large molecular weight compounds >100 kDa.

[0051] The light fraction purified and / or concentrated and / or pH adjusted and / or emulsified as described above is hereinafter referred to as processed light fraction.

[0052] The inventors have determined that the processed light fraction has improved foaming and / or emulsifying and / or heat setting gelling properties compared to the unprocessed light fraction. Preferably, the improved heat setting gelling properties of the processed light fraction are stable for at least 5 days without adding stabilizers and / or other preservatives when the processed light fraction is preferably stored at a temperature of about 4°C and preferably protected from light. When applied as a wet ingredient, the processed light fraction exhibits its strongest heat setting gelling ability; when the processed light fraction is used as a wet ingredient, the processed light fraction has a DW content>5%, preferably DW reaches about 10%, 15%, 20%, 25%, 30%, 35%, 40% to be considered "wet".

[0053] The method may further include a step of drying the processed light fraction or heavy fraction to remove moisture; preferably, the drying step is performed to achieve a moisture content of <15%, <10% or preferably <5%. In one embodiment, drying the light fraction from a moisture content of at least 85% to a lower moisture content of <15% enhances the functionality of the processed light fraction.

[0054] The method may further comprise the step of adjusting the pH of the processed light fraction to a pH range of 5 to 8, more preferably a pH range of 6 to 7. These pH ranges provide excellent heat set gelling properties.

[0055] The processing step may include preparing an emulsion of the fraction; this may be an emulsion of the heavy fraction, the light fraction, or (preferably) a processed light fraction that has previously been concentrated and / or purified as described herein. Alternatively, the emulsion is a combination of the light fraction and / or the heavy fraction and / or the processed light fraction. Preferably, the emulsion comprises a suspension of the fraction in an oil:water mixture having a mass ratio of at least 0.25 and less than 2.5. More preferably, the ratio of oil to water is about 1. In a preferred embodiment, the fraction is first dispersed in the oil phase, then the water phase is added, and sufficient shear is applied to ensure uniform dispersion. The emulsion system prepared according to this method can produce excellent gel hardness, whether as a single ingredient or as a functional ingredient added to foods such as simulated meat and cheese analogs. What is "sufficient shear" may depend on the type of equipment used to prepare the emulsion, but can be easily determined by a person skilled in the art. For example, an emulsion can be prepared at a laboratory scale using a rotor-stator homogenizer, the mixing intensity is adjusted, and the corresponding droplet size of the oil phase dispersed in water is measured. For example, if the average droplet size of the oil droplets in the emulsion does not change significantly between 40 and 65 rpm, then the shear generated by the mixer at 40 rpm can be considered sufficient to prepare the emulsion.

[0056] In one embodiment, the emulsion has an oil:water ratio of at least 0.25, preferably about 1, further preferably less than 2, and even less than 1.5. An oil:water ratio of at least 0.25 provides better heat setting gelling properties, including at least one of increased gel hardness, excellent gel texture, and improved juiciness. In addition, an emulsion having an oil:water ratio of at least 0.25 reduces the sensory intensity of the concentrated light fraction.

[0057] In one embodiment, the emulsion has an oil:water ratio > 2.5, preferably less than 5, less than 4, less than 3. This provides a more porous and / or firmer texture to the resulting heat-set gel compared to lower ratios.

[0058] The processing step may further comprise adjusting the pH of the fraction to an alkaline pH; this may be the pH of the heavy fraction, the light fraction or (preferably) the processed light fraction which has previously been concentrated and / or purified as described herein. Alternatively, this may be a combination of the heavy fraction and / or the light fraction and / or the processed light fraction. In one embodiment, adjusting the pH of at least one fraction or a combination of fractions to an alkaline pH enhances the thickening activity of the fraction. In one embodiment, when the pH is adjusted to alkaline, the emulsifying properties of the processed light fraction and / or heavy fraction are considered to be improved compared to the fraction without pH adjustment. In a preferred embodiment, the pH is adjusted to a range of 7–11, more preferably a range of 7.5-8.5. In particular, the processed light fraction and heavy fraction exhibit unique viscosity behavior as a function of temperature and concentration. This property can be used to prepare emulsions and food systems. In particular, since the heavy fraction has significantly greater viscosity than the processed light fraction, relatively low concentrations can be used to simulate foods with high fat content. Mixtures of these fractions may be prepared for specific food compositions and textures (eg, yogurt, mayonnaise, and tofu).Thus, the method may further comprise combining a portion of the heavy fraction with a portion of the light fraction or a processed light fraction.

[0059] In further embodiments, the light fraction, which may have been concentrated and / or purified as described herein, may be further processed by the following steps:

[0060] Adjust the DW content of the processed light fraction to 5%–15%, preferably 10%-15%;

[0061] subjecting the product of step i) to solid-liquid separation to obtain a substantially hydrophobic phase and a substantially hydrophilic phase;

[0062] The hydrophilic phase obtained from step ii) is collected (optionally the hydrophilic phase is lightly dried, and / or the resulting powder is optionally resuspended in water to a concentration of 0.1-200 g / L), and the pH value of the collected hydrophilic phase is adjusted to a range of 2-5 or a range of 3.5-4.5, or preferably a range of 4-5.

[0063] It has been observed that this additional processing improves the foaming properties of the (processed) light fraction, as the resulting product, in addition to an improved heat-set gelling power, has significant foaming and foam stability, making it suitable for food products that would not be achieved without the present method. Examples of such food products include cakes and confectionery products, where both foaming and gelling power are required.

[0064] The solid-liquid separation in step ii) produces two phases; the top phase contains mainly hydrophobic compounds, while the bottom phase contains mainly hydrophilic compounds. The top (hydrophobic) phase may be white, and / or may have a creamy texture to the touch.

[0065] In a preferred embodiment, the solid-liquid separation in step ii) is performed using a centrifugal separator. Examples of centrifugal separators include, but are not limited to, cream separators, centrifugal sterilizers, and tabletop centrifuges. A person skilled in the art will be able to select suitable equipment and process conditions to obtain two phases, wherein the top phase can be skimmed off and separated from the bottom phase.

[0066] In another embodiment, processing step d) comprises preparing an aqueous suspension comprising a heavy fraction at 1-15% DW, preferably 5-15%, more preferably 10-15% DW, and the pH value of the heavy fraction aqueous suspension is adjusted to pH 2-5, more preferably 4-5. It has been observed that this improves foaming properties, including foaming ability and / or foam stability. This result is particularly unexpected, because those skilled in the art would expect that the foaming properties of the heavy fraction would be poor, since the heavy fraction is mainly composed of cell fragments of size>1 μm, and D50>0.5 (or in some embodiments,>4.0). The heavy fraction of this processing can produce stable foam, and is particularly suitable as a partial substitute or ingredient in food products, such as, but not limited to, xanthan gum and / or carrageenan. Alternatively, the heavy fraction of processing can be used as a texturizing agent in food products, and / or as a fat substitute, for example, in imitation meat.

[0067] In yet another embodiment of the invention, the processing step may comprise heat treating a fraction; this may be the heavy fraction, the light fraction or (preferably) a processed light fraction that has previously been concentrated and / or purified as described herein; alternatively, this may be a combination of the light fraction, the heavy fraction and / or a processed light fraction. Heat treating may comprise drying; and / or heating. Drying may include, but is not limited to, freeze drying and spray drying. Drying may be performed to obtain a powder, preferably with a moisture content of <10%. Preferably, the dried powder is then exposed to a heat source under controlled conditions. Preferably, the heat source has a temperature in the range of 40-80°C, more preferably in the range of 70-80°C. In a preferred embodiment, the dried powder is exposed to the heat source for a period of more than 5 days, more preferably >10 days, even more preferably >15 days. The resulting heat treated product has unique functional properties; for example, a higher water holding capacity and / or a higher heat setting gelling capacity compared to the untreated product. Preferably, the water holding capacity of the product is increased by at least 5 times and / or the heat setting gelling capacity of the product is increased by at least 2 times. Furthermore, the gel obtained using the heat-treated product was improved compared to the gel obtained using the untreated product; the improved gel was structurally more porous, drier, calmer and harder than the gel obtained using the untreated product, whereas the gel obtained using the untreated product was smoother, wetter and more fragile.

[0068] In another embodiment of the present invention, a gel-like structure can be prepared from the processed light fraction by any of the following steps:

[0069] Stirring the processed light fraction in an aqueous suspension having a DW content of >5% at an acidic pH, preferably in the pH range of 3-6, more preferably in the range of 3.5-4.5, until a first gel-like structure is formed; or

[0070] The processed light fraction is stirred in an aqueous suspension having a DW content >5% at an alkaline pH, preferably in the pH range of 8-11, more preferably in the range of 9-11, thereby producing a firm gel-like structure.

[0071] The aqueous suspension comprises a processed light fraction at >5% DW, more preferably >10% DW, most preferably >15%, and less than 25%, even less than 20%.

[0072] Preferably, the pH of the aqueous suspension in step i) is adjusted to pH < 5, more preferably < 4. Preferably, the pH in step ii) is adjusted to pH > 9, more preferably > 11. The hard gel-like structure thus produced has an appearance similar to gelatin.

[0073] In another embodiment of the present invention, the processed light fraction can be used as a glazing agent and / or film former and / or browning agent by subjecting the processed light fraction to the following steps:

[0074] preparing an aqueous suspension of a processed light fraction, wherein the processed light fraction is at >1% DW;

[0075] adding said aqueous suspension to a food product, externally and / or within said food product;

[0076] The food is subjected to heat treatment.

[0077] In another embodiment, the heavy fraction and / or the light fraction can be subjected to extended cell decomposition under alkaline conditions as described in PCT / EP2021 / 075137 (WO2022 / 058287) and PCT / EP2023 / 056907. The surprising properties of the resulting heavy phase are reported in PCT / EP2021 / 075137 (WO2022 / 058287) and PCT / EP2023 / 056907. However, when subjected to extended decomposition, the light phase exhibits unexpected functional properties, including higher water holding capacity, gel hardness, foaming stability, and emulsion stability. This higher functionality is described in Example 2.

[0078] Examples of suitable food products include, but are not limited to, baked goods, such as bread and imitation meat. Heat treatments include, but are not limited to, baking and / or frying. The resulting food product may have a coating having a waxy texture and / or appearance on the surface of the food product, exhibiting a glazing effect. Alternatively or additionally, the food product may have a browning effect, and may also increase crispness. Glazing and browning are both highly desirable properties required of food products, and are traditionally obtained from proteins of plant and animal origin.

[0079] The present invention describes a method of producing a microbial cell extract and further processing steps to enhance the functional properties of the microbial cell extract. The method described in the present invention results from unexpected findings that are particularly unique because no biochemical functionalization is required as is commonly reported in the scientific literature.

[0080] One aspect of the present invention also provides an oil:water emulsion comprising a fraction obtained by a method as described herein. The present invention also provides a food ingredient for providing a desired functional property to a food selected from gelling, foaming, glazing, browning, texture, emulsification; the food ingredient comprises a fraction obtained by a method as described herein. There is further provided a food comprising such a food ingredient; and / or a food comprising such a fraction. The food may be selected from imitation meat; imitation dairy products, including cheese, yogurt, butter or cream analogs; tofu analogs; tempeh analogs; mayonnaise analogs.

[0081] Although the present invention is mainly described with respect to products obtained from yeast cells, the present invention is not limited thereto. Various other microorganisms may also be used. In embodiments, the microorganism may be selected from fungi, including yeast (preferably Saccharomyces species, more preferably brewer's yeast or baker's yeast, or Pichia species); plants, in particular microalgae (including Tetraselmis species or Chlorella species, such as Chlorella vulgaris ( C. vulgaris )); and cyanobacteria (including Arthrospira species, preferably Arthrospira platensis ( A. platensis )). The microorganisms may also be selected from bacteria, such as Methylobacterium species or lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 -Particle size distribution and particle size after cell dissociation by bead beating.

[0083] Figure 2 - Particle size distribution and particle size after solid-liquid separation (classification) of disrupted microbial biomass. The red line represents the light phase, while the green line represents the heavy phase.

[0084] Figure 3 - Gel hardness of gels prepared from a light phase concentrated at several diafiltration ratios using membranes with different cut-off values.

[0085] Figure 4 - Gelling properties of concentrated light fraction as wet and dry component at different concentrations.

[0086] Figure 5 - Stability of concentrated light fraction (wet, 17% DW) from 0 to 15 days.

[0087] Figure 6 -Gel hardness of concentrated light fraction over a range of pH values.

[0088] Figure 7 - Emulsion system that enhances the gelling properties of concentrated light fractions.

[0089] Figure 8 -Viscosity of concentrated light phase in emulsion system at different shear rates.

[0090] Fig. 9 - Emulsion stability and emulsion texture achieved by compositions comprising a concentrated light phase at different pH values.

[0091] Fig.10 - Emulsion systems obtained with a composition comprising concentrated light and heavy phases at low oil content and without heating.

[0092] Fig.11- Emulsion systems obtained with compositions comprising concentrated light and heavy phases at high oil content and with heating.

[0093] Fig.12 - Emulsion systems obtained with compositions comprising concentrated light and heavy phases at high oil content and without heating.

[0094] Fig.13 - Gel-like structure obtained from concentrated light phase at alkaline pH (middle), acidic pH (right) and original suspension (left). The conditions for gel preparation were <4.5 in the acidic range and >10 for the alkaline range.

[0095] Fig.14 - Glazing and browning of products coated with different concentrations (5, 10, 20% DW) of concentrated light and heavy phases and subjected to 2 different heating regimes (25 and 35 minutes at 200°C). Egg white and uncoated products were used as controls.

[0096] Fig.15 - Graphical representation of D10, D50, D90 of a volume based bimodal distribution.

[0097] Fig.16 - Enrichment of particles from a fragmented suspension with a population of fragments ( Fig.16 a), and at high centrifugal force ( Fig.16 b) Medium centrifugal force ( Fig.16 c) and low centrifugal force ( Fig.16 d) into a light phase and a heavy phase. The phase separation is indicated by a horizontal line.

[0098] Fig.17 - psd of yeast biomass (circles), disrupted biomass (squares), fraction enriched in small fragments (triangles) and fraction enriched in large fragments (inverted triangles) according to the invention.

[0099] Fig.18 - PSD of the light phase after cell dissociation and centrifugation at several intensities (g-force): high intensity 20,000 x g for 15 min (triangles), medium intensity 4,000 x g for 15 min (squares), and low intensity 1,000 x g for 5 min (circles). The dashed box shows the expected range of particle enrichment.

[0100] Fig.19 - Gel hardness at different reconstitution ratios of light phase and heavy phase according to the invention.

[0101] Fig. 20 - According to the invention, psd derived from decomposed biomass, light phase and heavy phase of Methylobacterium sp.

[0102] Fig.21 -Viscosity of emulsions prepared with several ratios of light and heavy phases.

[0103] Fig. 22 - Gel hardness of gels prepared in emulsions and aqueous suspensions containing different weight ratios of light phase (ME1) and heavy phase (ME2) (inclusion level was 10-12 wt % for all samples). DETAILED DESCRIPTION OF THE INVENTION

[0105] The following embodiments apply to all aspects of the invention.

[0106] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Each aspect so defined can be combined with any other one or more aspects or one or more embodiments, unless the opposite situation is clearly pointed out. Specifically, any feature that is indicated as preferred or advantageous can be combined with any other one or more features that are indicated as preferred or advantageous.

[0107] The following definitions are used in this specification and claims to define the subject matter. Other terms not mentioned below are meant to have art-recognized meanings.

[0108] As used in this specification, "drying" means reducing the moisture content. The term drying includes partial drying, in which a small amount of moisture may remain after drying, which can also be regarded as concentration.

[0109] 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 specified percentage of a particular component by dry weight refers to that percentage calculated based on the weight of the biomass after substantially removing all water.

[0110] "Disruption / rupture" as used in the present specification in the context of microbial cells is also referred to as "lysis" and means opening the cells to release cytoplasmic compounds (also referred to as "lysate").

[0111] As used in this specification, "disintegration" in the context of microbial cell disintegration refers to the breaking of the cells. 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 specific type of disruption in which the cells are not only opened but also broken.

[0112] As used herein, "cytoplasmic material" or "cytoplasmic compound" refers to all materials 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 is broken down or destroyed.

[0113] "Microbial cell" as used in this specification refers to: microorganisms. This can be eukaryotic and prokaryotic single-cell organisms and colonies thereof. A prokaryote is a cellular organism that lacks a nucleus surrounded by an envelope. In the three-domain system, prokaryotes are divided into two domains based on molecular analysis: bacteria (formerly known as eubacteria) and archaea (formerly known as archaea). Organisms with a nucleus are placed in the third domain, the eukaryotic domain. Microbial cells according to the present invention also include algae and fungi, such as yeast.

[0114] As used herein, "microorganism" and "microorganism" refer to any microscopic colony or single-cell organism.

[0115] The "microbial cell product" used in this specification refers to a product derived from microbial cells obtained by processing microbial cells in some manner.

[0116] "Light fraction" and "light phase" as used in this specification refer to a phase of a microbial cell extract (EESF) rich in small cell fragments ranging from about 0.1-3 μm. "Small cell fragments" as used in this specification refer to cell fragments with a size equal to or less than d50 ≤ 500 nanometers (nm) obtained from decomposing microbial cells. Light fraction and small fraction can be used interchangeably herein.

[0117] "Heavy fraction" and "heavy phase" as used in this specification refer to the phase of the microbial cell extract (EELF) enriched in large cell fragments of size>1 μm. "Large cell fragments" as used in this specification refer to cell fragments with a size greater than d50 ≥ 500 nanometers (nm) obtained from decomposing microbial cells. Heavy fraction and large fraction can be used interchangeably herein.

[0118] As used in this specification, "enriched" or "enriched / enriched" means that the particles selectively move to one of the two separated phases, i.e., the light phase or the heavy phase; Fig.16 illustrates this concept. When high centrifugal forces are used, all particles / insoluble matter are transferred to the heavy phase ( Fig.16 b); If the centrifugal force used is too low, the separation between the light and heavy phases is poor and the fragments are not clearly separated ( Fig.16 d). However, the use of mild or moderate centrifugal forces results in small fragments being preferentially concentrated in the light phase (the extract enriched in small fragments), while large fragments are preferentially concentrated in the heavy phase (the extract enriched in large fragments), e.g. Fig.16 c. The concept of enrichment is further described in Example 14. Note that this is different from simply separating the soluble and insoluble fractions, as the insoluble material remains in both the light and heavy phases.

[0119] "Microbial biomass" and "biomass" as used in this specification refer to substances produced by the growth and / or proliferation of microbial cells, or substances produced as by-products of fermentation processes. Biomass can contain cells and / or intracellular contents as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by cells.

[0120] As used herein, "bead milling" refers to the agitation of suspended microbial cells with small grinding particles (beads). The cells are broken by shear forces, grinding between beads, and collisions between / with beads. The shear forces generated by the beads disrupt the cells and cause cell breakdown, while releasing cellular compounds.

[0121] As used in this specification, "centrifugation" means the separation of particles from a solution by applying a centrifugal force based on parameters such as particle size, shape, density, medium viscosity, and rotor speed. Centrifugal rate is expressed in terms of angular velocity, usually expressed in revolutions per minute (RPM), or in terms of 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

[0122]

[0123] in g represents the corresponding force of the centrifuge, and r represents the radius from the center of the rotor to a point in the sample. However, depending on the centrifuge model used, the corresponding rotor angle and radius may be different, so the formula also needs to be modified. The most commonly used formula for calculating relative centrifugal force is:

[0124]

[0125] where r is the radius in mm.

[0126] As used herein, "water holding capacity (WHC)" refers to the amount of water that a sample can hold per unit weight.

[0127] The "oil holding capacity (OHC)" used in this specification refers to the amount of oil that a sample can hold per unit weight.

[0128] The present invention relates to methods for producing microbial cell extracts, methods for improving the functional properties of said microbial cell extracts and the use of said microbial cell extracts in technical applications. In one embodiment, the present invention relates to the use of microbial cell extracts in foaming agents, emulsifiers, thickeners, texturizing agents, gelling agents or any other suitable applications. Example

[0129] The invention is now described by way of the following non-limiting examples.

[0130] Example 1 - Preparation of microbial extracts

[0131] Microbial cell extracts were prepared according to the process described in PCT / EP2021 / 075137. In summary, a yeast suspension of the genus Saccharomyces free of foreign contaminants was bead milled under the following conditions:

[0132] : The biomass suspension concentration was about 100 g / L, and the pH was adjusted from about 4.5-5.5 to about 9 using NaOH.

[0133] The suspension was bead milled in batch recirculation mode at approximately 20 °C, a tip speed of 14 m / s, a bead filling factor of 75%, and 0.5-1 mm zirconium beads.

[0134] The temperature of the final disrupted suspension was about 23°C and the final pH was 5-6.5.

[0135] The particle size distribution (PSD) and particle size after bead milling are shown in Table 1. Figure 1 shown.

[0136] After decomposition, the disrupted microbial biomass is subjected to solid-liquid separation, also known as classification step, to separate the decomposed biomass into a light phase (also known as small fragments-rich extract) and a heavy phase (also known as large fragments-rich extract), wherein the small fragments-rich extract (light phase) consists of a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a psd of about 0.5 μm or less; and the large fragments-rich extract (heavy phase) consists of a population of soluble compounds and suspended fragments with a psd of D50>4 um. This separation is performed by centrifugation using a batch centrifuge, with a volume of 1 L at 4000xg for 15 minutes at 15°C. Particle size distribution (psd) and particle size of the light phase (red) and heavy phase (green) as shown in Figure 2. Figure 2 As shown in Table 1 below:

[0137] Table 1: PSD of light and heavy phases

[0138] The light phase or light fraction obtained after centrifugal classification is also called a microbial cell extract rich in small cell debris. The heavy phase or heavy fraction is also called a microbial cell extract rich in large cell debris.

[0139] Both the light and heavy phases were then centrifuged for 10 minutes using a benchtop centrifuge, with a 10 ml volume at 4000 x g for 15 minutes at approximately 15°C. After this additional centrifugation step, new light and heavy phases were formed, and the volume ratios were calculated as follows:

[0140] Light

[0141] Feed volume (Vf) – 10 ml

[0142] Heavy phase volume (Vh) – 0.5 ml

[0143] Light phase volume (Vl) – 9.5 ml

[0144] Volume ratio =

[0145] Reappearance

[0146] Feed volume (Vf) – 10 ml

[0147] Heavy phase volume (Vh) – 9.0 ml

[0148] Light phase volume (Vl) – 1.0 ml

[0149] Volume ratio =

[0150] The resulting microbial cell extracts, i.e., the light phase and the heavy phase, were then further characterized. The compositions of the light phase and the heavy phase are shown in Table 2 below. The composition of each extract was analyzed five times and the standard deviation was calculated.

[0151] Table 2: Average composition (%DW) of microbial extracts, i.e., light and heavy phases (n=5, and SD=standard deviation)

[0152] Example 2 - Functionality of the light phase after extended bead milling

[0153] The present embodiment describes the functional properties of the light phase of the "extended decomposition" process reported in PCT / EP2021 / 075137 (WO2022 / 058287) and PCT / EP2023 / 056907. Surprisingly, compared with the functional properties described by the reference process (reference, pH 9) of the light phase, the light phase obtained by the extended decomposition process also shows improved functional properties (Table 3). As a reference, the process involving cell autolysis, cell homogenization, alkali extraction, acid extraction and aqueous extraction is compared with the currently proposed method. The following literature reports the reference process, i.e. Saowanee Thammakiti, Manop Suphantharika, Thanaporn Phaesuwan, Cornel Verduyn. Preparation of spent brewer's yeast β-glucans for potential applications in the foodindustry. Food Science and Technology. Vol. 39, No. 1, January 2004, pp. 21-29.

[0154] Table 3: Functional properties of the light phase before further processing. Example 3 - Concentration of the light phase

[0155] The light phase is mixed with distilled water in different proportions, and the resulting suspension is filtered through a hydrophilic membrane PES with a cutoff of 10 kDa and 100 kDa, respectively, maintaining a transmembrane pressure of <1 bar and a temperature of <25°C. The filtration process is carried out until a 5-fold concentration factor is obtained. For example, starting from 100 ml of feed, filtration is carried out until about 20 ml of retentate and about 80 ml of permeate are obtained. The filtration is carried out without pH adjustment. After filtration, the retentate fraction is collected for analysis of heat-set gelling properties. This retentate is referred to herein as "concentrated light fraction" or light phase, or "processed light fraction" or light fraction; and may also be referred to as "ME1s" in the examples and drawings.

[0156] The heat-set gelling properties were measured by a double compression test on samples at a constant DW content using a texture analyzer after heating at 90 °C for 30 min followed by cooling to room temperature for 20 min.

[0157] like Figure 3As shown, the experimental data indicate that gel hardness (one of the most important parameters determining the performance of heat-set gelling) is at its highest level at a percolation ratio of approximately 1:1. It is also evident that the 100 kDa membrane is optimal compared to the 10 kDa membrane.

[0158] The gelling properties (measured as gel hardness) of the light fraction obtained after concentrating the light fraction as a wet fraction are higher compared to the dry and resuspended fractions of the same DW content. Figure 4 As shown, the gel hardness at different concentrations does not follow a linear trend, but decays exponentially.

[0159] like Figure 5 As shown, the stability of the gelling properties of the concentrated light fraction (wet 17% DW) was monitored for 15 days when stored at about 4°C protected from light, as measured by gel hardness, to assess whether the heat-set gelling properties of the light phase could be maintained over extended periods of time without the addition of stabilizers or preservatives. As can be seen here, the overall stability was high, with gel hardness up to 5.5 days very similar to that observed prior to storage. During the 15 days of storage, the gel hardness did not drop below 60% of that achieved prior to storage, although the gel hardness did begin to steadily decrease after 5.5 days.

[0160] like Figure 6 As shown, the effect of pH on the gelling properties (measured as gel hardness) of the concentrated light fraction was evaluated over a wide pH range. It can be seen that the gelling properties are optimal at pH 5.3-7.

[0161] Example 4 - Emulsion system for enhancing the gelling properties of concentrated light phase

[0162] Prepare concentrated light phase in different suspensions, to evaluate its gelling properties.For each suspension tested, the concentrated light phase is dried to form a powder, then dispersed in the relevant medium using a medium speed shearing agitator, so that in each suspension, the DW content that the light phase has is 20%.Then each suspension is carried out to standard thermal gelling test, and the gelling hardness of the obtained gel is measured using a texture analyzer. The obtained results are shown in Table 4 below.

[0163] Table 4: Gels obtained from suspensions and emulsions containing a light phase at 20% DW

[0164] The results in Table 4 show that the gel hardness of the concentrated light phase in the emulsion system is significantly higher than when it is in aqueous suspension alone or in oil suspension alone. In addition, there is a clear advantage in preparing the emulsion by adding the concentrated light phase to the oil before adding water, as this can achieve better gel hardness.

[0165] The gelling properties of emulsions containing concentrated light phase are also affected by the oil:water ratio in the emulsion. The concentrated light phase was added at 20% DW to emulsions with an oil:water ratio ranging from 0 to 2.25 and the gelling properties were measured by gel hardness using a texture analyzer. Figure 7 As shown in Figure 2, gel hardness increases almost four-fold as the oil:water ratio increases from 0 to about 1. Gel hardness increases even further when the oil:water ratio exceeds 1, but the resulting gels at these higher ratios are darker, more porous, more brittle, and have an uneven texture. In contrast, gels produced at an oil:water ratio of about 1 are relatively elastic, smooth, springy, and light beige in color.

[0166] Example 5 - Unique properties of a mixture of light fraction (ME1) and heavy fraction (ME2)

[0167] This example demonstrates the unique and unexpected properties of the microbial fractions obtained as described in the present invention. The powders obtained after drying the light and heavy fractions as described in Example 1 were added to an emulsion using a high-speed shear homogenizer (UltraTurrax, t50, IKA) with an oil:water ratio of 1:1 and a dry weight content of 5-7% ( Fig.21 The obtained emulsions were stable (no phase separation was observed) for more than 12 hours. Fig.21 As shown in Figure 2, the viscosity of the resulting emulsion depends strongly on the ratio of the light fraction to the heavy fraction, even if the total dry weight content varies only in the range of 5–7%. -1 The viscosity is measured at a shear rate of 1.

[0168] Furthermore, the emulsion was heated in a water bath at 90°C for 30 minutes and then allowed to cool to room temperature. The resulting gel was then analyzed using a texture analyzer (TA plus, Lloyd). Fig. 22 The results in show that in the case of emulsions, the gelling properties of the microbial fraction are significantly enhanced and that there is an optimal ratio of light to heavy fraction that optimizes gel hardness. Note that the optimal ratio for gelling is not the same as the ratio for maximum viscosity of the emulsion. This example clearly shows that the ratio of light to heavy phase can be used to achieve different textural properties.

[0169] Example 6 - Emulsion properties of concentrated light and heavy phases

[0170] The emulsification behavior of the concentrated light and heavy phases was studied under several different conditions.

[0171] Effect of shear on viscosity

[0172] The samples containing concentrated light phase were prepared in an emulsion system containing 1:5.6:20 concentrated light phase:water:oil. The viscosity of the emulsion was then measured under various shear treatments. Overall, a strong correlation was observed between shear and viscosity (e.g. Figure 8 ), which indicates that the concentrated light extract behaves as a shear thickening material.

[0173] Effect of pH value on emulsion

[0174] The emulsifying power and emulsion stability of emulsions prepared using microbial extracts (concentrated light phase and / or heavy phase) can be improved by adjusting the pH of the suspension they contain. The concentrated light phase was used at 10% DW and the experiments were conducted at pH values ​​ranging from 3.5 to 10. The resulting emulsions at pH 3.5 were soft and creamy, while those at pH 5.3, 7 and 10 were thick and creamy and could even be sliced. A thicker emulsion was produced at pH 10. In addition, the emulsion stability was evaluated at different pH levels. The stability was measured in terms of phase separation (the time before the emulsion phases start to separate). Emulsion stability and emulsion texture are shown in Table 1. Fig. 9 shown.

[0175] Effect of processing conditions on the texture of emulsions containing concentrated light and / or heavy phases

[0176] Several prototypes were prepared containing concentrated light and heavy phases alone and in combination, with different oil contents (high oil content = 52% and low oil content = 13%), the content of microbial extracts was tested at 4% and 8%, and the effect of heat treatment during emulsification was also evaluated. (Note that "ME1s" refers to concentrated light phases, while "ME2" refers to heavy phases).

[0177] Under the conditions of low oil content (13%) and no heating during emulsification, different textures ranging from cream to skim milk and butter were obtained, such as Fig.10 shown.

[0178] At high oil content (52%) and with heat during emulsification, the resulting texture was more viscous and thicker. Textures comparable to yogurt, mayonnaise and tofu were obtained. It is also evident that the contribution of the heavy phase (“ME2”) to the overall texture provides a more pronounced thickening behavior than the concentrated light phase (“ME1s”), as shown in Figure 2. Fig.11 shown.

[0179] At high oil content (52%) and without heating during emulsification, several textures were obtained, such as stirred yogurt and thick mayonnaise. Again, the thickening properties of the heavy phase were shown to play a key role in achieving the desired thick mayonnaise texture ( Fig.12). From these results it is clear that the heavy phase can be used as a fat replacer in the production of food products such as mayonnaise which usually require an oil content of at least 80%. Example 7 - Foaming stability

[0180] When subjected to strong mechanical shear and / or bubbling, aqueous suspensions containing concentrated light phases and / or heavy phases can form foams. However, the foam stability is generally poor. It was found that by adjusting the pH value of the suspension containing concentrated light phases and / or heavy phases, the foam stability of the concentrated light phase and heavy phase can be significantly prolonged, as shown in Table 5 below:

[0181] Table 5: Half time of foams generated at different pH values ​​comprising concentrated light phase ("ME1s") and heavy phase ("ME2") compared to an egg white control.

[0182] In order to further improve the foam properties of the concentrated light phase, an aqueous suspension of the concentrated light phase containing about 15% DW was centrifuged at 3000xg at 15 ° C for 50 minutes in a table centrifuge. After centrifugation, a two-phase system is formed, wherein the top phase is about 5% v / v, and the bottom phase is about 95% v / v. The top layer can be easily skimmed off using a mechanical element. The bottom phase is collected and the pH value is adjusted to about 4.1, and the resulting suspension is then used to prepare meringue using a standard recipe. As a control sample, a concentrated light phase with the same DW content is used, but without pH adjustment or centrifugation steps. During the whipping step, the adjusted concentrated light phase shows a volume increase of at least 500%, and remains stable. In contrast, the concentrated light phase without pH adjustment and centrifugation only shows a volume increase of about 200%, and is unstable. During the baking step, the meringues made with the conditioned concentrated light phase had a volume and structure comparable to those made with egg whites, whereas the meringues made with the unconditioned concentrated light phase (no pH adjustment and no centrifugation) were flat and soft.

[0183] Example 8 - Microbial Cell Extracts as Foam Stabilizers

[0184] Since the heavy phase has the characteristics of a thickener, it can be used as a foam stabilizer. Experiments were conducted using a standard kitchen milk foam machine. An aqueous suspension containing a heavy phase was prepared in a concentration range of 1-10% DW and 3 foaming cycles were performed. The resulting foam was transferred to a graduated cylinder where the stability of the foam over time was measured, measured as a discharge. Xanthan gum (XG) with 2% DW was used as a control. The foaming power was determined indirectly by measuring the weight of 100 ml of foam produced by the foaming machine. The foaming power and discharge (foam stability) results are shown in Table 6 below.

[0185] Table 6: Foaming capacity (weight) and foam stability (weight) of aqueous suspensions containing 1-10% DW heavy phase and control xanthan gum (XG)

[0186] Example 9 - Foaming properties of the heavy fraction (ME2)

[0187] A heavy fraction (ME2) was produced according to Example 1 and lightly dried before being used in a model formulation to produce a meringue-like product. ME2 was dispersed in water and the pH was adjusted to about 4, followed by stirring and slow addition of sugar until a glossy foam-like texture was obtained. A control experiment was also performed using egg white as a positive control. As shown in Table 7, even at lower inclusion levels, the unique ratio of sugar to ME2 resulted in the formation of a foam-like structure with the desired properties of a positive control (c+).

[0188] Table 7: Foaming test of ME2

[0189] Example 10 - Heat Treatment of Concentrated Light Phase

[0190] The powder of concentrated light phase containing about 4.1% water was placed in an aluminum tray and exposed to a heat source at several temperatures. The treatment was carried out for 0 days (untreated), 10 days and 20 days. At the end of the treatment, the powder was resuspended in water at 20% DW and measured using a standard gel test in a texture analyzer to determine water holding capacity (WHC) and gelling properties. The results in Table 8 show that both gel hardness and water holding capacity increased significantly at high temperatures and exposure time. After 20 days of treatment at 80°C, the gel structure was more porous, more brittle and harder.

[0191] Table 8: Effect of heat treatment on gel hardness and water holding capacity (WHC) of powders containing concentrated light phase

[0192] Example 11 - Gel-like structure comprising a concentrated light phase

[0193] An aqueous suspension containing about 15% DW of the concentrated light phase was prepared and the pH of the suspension was adjusted using NaOH or HCl under gentle mechanical stirring. After the gel-like texture was formed, stirring was stopped ( Fig.13 ); The figure shows the gel-like structures obtained under alkaline pH (middle), acidic pH (right) and original suspension (left). The conditions for gel preparation were <4.5 in the acidic range and >10 for the alkaline range.

[0194] Example 12 - Use of microbial cell extracts as glazing agents, browning agents and coating agents

[0195] The aqueous suspension comprising concentrated light phase and / or heavy phase can be used as glazing agent, browning agent and / or coating agent in various food products. The preparation comprises concentrated light phase and / or heavy phase aqueous suspension in the range of 5-20% DW, and it is spread / coated on the surface of standard dough, and baked in an oven at 200 ℃ for 25 minutes and 35 minutes subsequently. Egg white coating and no coating at all are used as control samples. After baking, it is observed that the browning of the product comprising microbial extract is related to the content of concentrated light phase and heavy phase (the higher the concentration, the darker the color produced). The color intensity is comparable to the browning effect obtained from egg white, and the longer the baking time, the darker the color produced ( Fig.14 In another example, an aqueous suspension comprising a concentrated light phase is coated on the surface of simulated meat and used as a binder for breading for preparing chicken analogs.

[0196] Example 13 - Formulations and Foods

[0197] This example shows several food products in which the microbial cell extracts and fractions described herein provide unique and surprising functionality.

[0198] Imitation meat

[0199] The light and heavy phases obtained according to the present invention can be used to replace binders such as hydrocolloids (e.g., methylcellulose) and proteins (e.g., egg white and potato protein) in burgers (veggie burgers, raw burgers, rare burgers) and other simulated meats (e.g., chicken and meatballs). After drying, burgers prepared using the light and heavy phases (ME1 and ME2) were rated as having textural properties comparable to reference burgers containing methylcellulose (Table 9). Moreover, when the light and heavy phases were added in the form of an emulsion, the resulting burgers were juicier, firmer, and presented better flavor characteristics.

[0200] Table 9: Burger recipe containing the light fraction (ME1) and the heavy fraction (ME2) obtained according to the invention and methylcellulose (MC).

[0201] sausage

[0202] The light fraction (ME1) and the heavy fraction (ME2) of the microbial cell extract can be added to the mixture to make vegetarian sausages. Table 10 shows the qualitative evaluation of sausages prepared using the light phase and the heavy phase compared to egg white as a reference binder. This example shows that a mixture of dry light and heavy phases added in the form of an emulsion can be used to replace egg white to produce vegetarian sausages.

[0203] Table 10: Qualitative evaluation of sausages prepared using light phase (ME1) and heavy phase (ME2) compared to using egg white.

[0204] pasta

[0205] Microbial cell extract light fraction (ME1) and heavy fraction (ME2) have been used to replace egg white in pasta.

[0206] Cheese analogs

[0207] Microbial cell extract light fraction (ME1) and heavy fraction (ME2) have been used to prepare vegetarian cheese analogs. As shown in Table 11, cheese analogs containing dried light fraction (ME1) and heavy fraction (ME2) were prepared and compared with cheese analogs containing potato protein. When the microbial cell extract was added in the form of an emulsion, the resulting analog showed a hardness comparable to the reference. In addition, the inclusion level and preparation method of the light phase (ME1) and the heavy phase (ME2) can be adjusted to prepare other types of cheese analogs, such as semi-hard cheese, cream or cheese fillings.

[0208] Table 11: Recipes for preparing cheese analogs containing a light phase (ME1) and a heavy phase (ME2) and the corresponding hardness.

[0209] Methylcellulose Alternatives

[0210] The microbial cell extracts of the present invention can be used to replace methylcellulose and similar hydrocolloids (including HPMC / CMC) in potato products to maintain their shape during frying. Addition of dried light phase (ME1) and / or heavy phase (ME2) (0.1–4% wt) to hot mashed potatoes yields a stable product with improved browning and crispness compared to reference samples containing 0.2-1% methylcellulose.

[0211] Other Products

[0212] Microbial cell extract light and heavy fractions can be used to replace binders and emulsifiers in a wide variety of formulations, food matrices and food products. Example 14 - Enrichment

[0213] In one embodiment showing enrichment, microbial biomass having a D50 of about 7.49 μm (composed of Fig.17 The circles in the figure decompose into a suspension with a bimodal distribution, which consists of Fig.17The decomposed biomass is then separated into an extract rich in small fragments, represented by triangles, with a D50 of about 0.35 μm, and a fraction rich in large fragments, represented by inverted triangles ( Fig.17 ), D50 is about 5.41 μm. Therefore, the light phase will be rich in small fragments with a size range of 0.1-3 μm (D50<0.5 μm). Correspondingly, the heavy phase will be rich in large fragments with a size of >0.3 μm (D50>0.5 μm).

[0214] We have demonstrated that there is a range of centrifugal forces that results in optimal functionality of the fraction enriched in small fragments. Fig.18 It is shown that moderate centrifugal forces lead to better enrichment of fragments in the range of 0.1–3 μm, represented by squares. Strong centrifugal forces (triangles) produce PSDs in the range of 0.1–0.4 μm, while low centrifugal forces produce PSDs with fragments spanning up to 10 μm (circles). The desired enrichment of small fragments is represented by the dashed squares. If too high or too low centrifugal forces are used, this can lead to different PSDs and unexpectedly poor functional performance, highlighting that obtaining a PSD as defined in the present invention is critical.

[0215] Using different centrifugal forces affects the dry matter content of the light phase and the heavy phase. As described above, samples were prepared according to the method of the present invention, but mild and high centrifugal forces were used for separation. After separation, the samples were subjected to an oven method known in the art, in which the samples were kept in an oven at 100°C until they reached constant weight. The results are shown in Table 12 below. Significant differences in dry matter content were observed when different centrifugal forces were used. When a mild centrifugal force of 4000 xg was used for 15 minutes, additional dry matter appeared in the light phase due to more small particles retained in the suspension. This is the advantage of using mild centrifugal forces to achieve separation.

[0216] Table 12: Dry matter content of light and heavy phases after centrifugation of disrupted biomass using high and light centrifugal forces.

[0217] In previous publications, it was reported that the production of microbial protein concentrates and protein isolates from soluble fractions after cell lysis and centrifugation required a centrifugal force in the range of 10000-30000 x g to produce such soluble fractions and remove all insoluble compounds and particles. Traditionally, soluble fractions with higher purity are associated with high functionality and excellent performance. However, unexpectedly, the inventors found that selective enrichment of fractions with small fragments produced excellent functionality as described above.

[0218] Example 15 - Recombination of light and heavy phase functionality

[0219] Fig.19 Several reconstitution ratios of the fraction enriched in small fragments and the fraction enriched in large fragments are shown. The fractions reconstituted in various ratios by weight were dried using spray drying and evaluated based on gel hardness.

[0220] The gel hardness was measured after heat setting gel in a water bath (15% DW suspension, heated at 90°C for 30 minutes, then cooled at room temperature for 20 minutes and the hardness was measured using a texture analyzer Lloyd TA-Plus).

[0221] Fig.19 It was shown that the optimal gel hardness was achieved when the ratio of EESF to EELF was 15:85. For reference, in this example, an approximate ratio of the two fractions before reconstitution of 40:60 produced a gel hardness of about 8 N.

[0222] Surprisingly, there is a ratio where there is a synergy between the two fractions resulting in excellent functionality, in this example gel hardness.

[0223] Example 16 - Functionality of microbial cell extracts derived from Methylobacterium species

[0224] Microbial cell extracts are prepared according to the method described in PCT / EP2021 / 075137 (WO2022 / 058287). In summary, the microbial cell extract is produced by the following steps: i) providing an aqueous suspension containing microbial cells; ii) subjecting the suspension to mechanical cell decomposition to obtain an aqueous suspension containing decomposed microbial cells; and iii) separating the suspension to provide an extract rich in small cell fragments ("light phase") and an extract rich in large cell fragments ("heavy phase"). It is noteworthy that, optionally, at least a portion of each extract can be recombined to provide a recombinant microbial cell product.

[0225] In this example, an aqueous suspension containing microbial cells contained about 100 g / L of biomass of the genus Methylobacterium, the pH was adjusted to about 9 with NaOH, and the cells were broken down by bead milling using 0.3 mm zirconium beads, with a filling rate of 65%, a stirring speed of 2039 rpm, and a temperature of about 20°C.

[0226] The particle size distribution of the decomposed biomass is as follows: Fig.17 As shown and represented by the triangles, it can be seen here that a D50 of about 1.03 μm is achieved.

[0227] After the disintegration step, the resulting microbial suspension was centrifuged using a batch centrifuge at 4000 xg for 15 minutes at 15° C. This resulted in the formation of a light phase and a heavy phase of the microbial suspension (also referred to as an extract enriched in small cell debris and an extract enriched in large cell debris, respectively).

[0228] The particle size distribution and particle size of the light fraction and heavy fraction obtained by centrifugal separation are as follows: Fig. 20 As shown, the squares represent the heavy fraction and the circles represent the light fraction. The D50 of the light fraction is about 0.57 μm. Meanwhile, the D50 of the heavy fraction is about 0.9 μm.

[0229] The resulting fractions were then analyzed based on their functional properties as shown below in Table 13. The results here show that both the oil holding capacity and gelling properties were significantly improved in both EESF and EELF compared to the decomposed biomass.

[0230] Table 13: Functional properties of decomposed Methylobacterium species and the resulting EESF and EELF

[0231] Although the present invention is mainly described with respect to products obtained from yeast cells, the present invention is not limited thereto. Various other microorganisms may also be used. In embodiments, the microorganism may be selected from fungi, including yeast (preferably Saccharomyces species, more preferably brewer's yeast or baker's yeast, or Pichia species); plants, in particular microalgae (including Tetraselmis species or Chlorella species, such as Chlorella vulgaris ( C. vulgaris )); and cyanobacteria (including Arthrospira species, preferably Arthrospira platensis ( A. platensis )). The microorganisms may also be selected from bacteria, such as Methylobacterium species or lactic acid bacteria.

[0232] Other variations of the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention by studying the drawings, the present 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 a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0233] Although the foregoing disclosure provides a general description of the subject matter included within the scope of the present invention, including methods of making and using the present invention and the best mode thereof, the following examples are provided in order to further enable those skilled in the art to practice the present invention and provide a complete written description thereof. However, those skilled in the art will recognize that the specific contents of these examples should not be interpreted as limitations on the present invention, and the scope of the present invention should be understood from the claims attached to this disclosure and their equivalents. In view of this disclosure, various other aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0234] The scope of the present invention is defined by the appended claims. One or more objects of the invention are achieved by the appended claims.

[0235] References

[0236] JE Kinsella, KJ Shetty. Yeast Proteins: Recovery, Nutritional and Functional Properties. Nutritional Improvement of Food and Feed Proteins pp. 797-825.

[0237] WO2020127951A2.FUNCTIONAL YEAST PROTEIN CONCENTRATE.

[0238] Vananuvat and Kinsella. Some Functional Properties of Protein Isolates from Yeast, Saccharomyces fragilis. J. Agric. Food Chem., Vol. 23, No. 4, 1975.

[0239] US 3888839. Isolated yeast protein product with intact RNA and aprocess for making the same.

[0240] US 3887431 Yeast protein isolate with reduced nucleic acid content and process of making same.

[0241] GB1578235A. Process for preparing functional yeast proteins using alkaline conditions

[0242] US 5756135. Water insoluble yeast solids product and process of making same

[0243] US10407600B2. Yeast-based adhesive extract, composition and method

[0244] US2603630A. Method for extracting proteins from yeast

[0245] WO2006067145A1. New mannoprotein with full solubility in wine and its application in the stabilization of wine

[0246] WO2018002505A1 Use of a yeast protein extract to stabilize beer haze

[0247] Samara C. Silva, Isabel C. F. R. Ferreira, Madalena M. Dias and M. Filomena Barreiro, Cristina González-Fernandez, Academic Editor. Microalgae-Derived Pigments: A 10-Year Bibliometric Review and Industry and Market Trend Analysis. Molecules. August 2020; 25(15): 3406.

[0248] Gabriela Vollet Marson, Débora Tamires Vitor Pereira, Mariana Teixeira da Costa Machado, M. Di Luccio, Julian Martínez, M. Belleville, M. Hubinger. Ultrafiltration performance of spent brewer's yeast protein hydrolysate: Impact of pH and membrane material on fouling. 2021. Journal of Food Engineering 302(4):110569.

[0249] K J Shetty, J E Kinsella. Effect of thiol reagents on extractability of protein from yeast. Biotechnol Bioeng, May 1978;20(5):755 - 66.

[0250] R. Kollar, E. Sturdik, J. Sajbidor. Complete fractionation of saccharomyces cerevisiae biomass. Food Biotechnology. Volume 6, 1992 - Issue 3.

[0251] J. A. Levesley, M. SeggiannI.M. Hoare. Microfiltration of Protein Precipitate from Clarified Yeast Cell Homogenate for the Recovery of a Soluble Product. Separation Science and Technology. Volume 35, 2000 - Issue 5.

Claims

1. A method for preparing a microbial cell extract, the method comprising: include: a) providing microbial biomass in an alkaline aqueous suspension at a pH of 7-11; b) mechanically decomposing the microbial biomass at a temperature below 40° C. using a non-denaturing process such that the decomposed biomass consists of a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a particle size distribution (psd) with an average of about D50 < 4.5 μm; c) subjecting the decomposed biomass to a solid-liquid separation process to separate the decomposed biomass into a light fraction (also referred to herein as an extract enriched in small fragments) and a heavy fraction (also referred to herein as an extract enriched in large fragments), wherein the light fraction consists of a population of soluble compounds and suspended fragments characterized by a bimodal distribution and a psd with a D50 of about 0.5 μm or less; and the heavy fraction consists of a population of soluble compounds and suspended fragments with a psd with a D50>0.5 μm; and d) further processing one or both of the light fraction and the heavy fraction to optimize one or more functional properties of the fractions, preferably for use in food manufacturing or cosmetic manufacturing.

2. The method of claim 1, wherein the microbial biomass is derived from yeast, more preferably from the genus Saccharomyces ( Saccharomyces ) and / or Pichia ( Pichia ).

3. The process according to any one of the preceding claims, wherein step d) comprises further processing of the light fraction by purification and / or concentration.

4. The method of claim 3, wherein the light fraction is purified and / or concentrated such that the processed light fraction has a dry weight (DW) content of at least 20%.

5. The method of claim 3 or 4, wherein diafiltration is used to concentrate the light fraction, the ratio of water to the light fraction being about 1:1 during diafiltration, and wherein the membrane used for diafiltration is in the range of 10 kDa - 1000 kDa.

6. The method of any one of claims 3-5, further comprising the step of drying the processed light fraction to remove moisture.

7. The method of any one of claims 3-6, further comprising adjusting the pH of the processed light fraction to a range of 5-8.

8. The method of any of the preceding claims, further comprising combining two or more fractions selected from the light fraction, the heavy fraction and the processed heavy fraction.

9. A method as claimed in any preceding claim, wherein the processing step comprises preparing an emulsion of the fraction.

10. The method of claim 9, wherein the emulsion comprises a suspension of the fraction in an oil:water mixture having a mass ratio of 0.25 to 2.

5.

11. The method of claim 9 or 10, wherein the emulsion has an oil:water ratio of 0.25 to 1.

5.

12. The method of any one of the preceding claims, wherein the processing step comprises adjusting the pH of the fraction to an alkaline pH.

13. The method of any one of claims 3 to 7, comprising further processing the processed light fraction by: adjusting the DW content of the processed light fraction to 5%-15%, preferably 10%-15%; The product of step i) is subjected to solid-liquid separation to obtain a mainly hydrophobic phase and a mainly hydrophilic phase; The hydrophilic phase obtained from step ii) is collected, and the pH value of the collected hydrophilic phase is adjusted to a range of 2-5, preferably a range of 4-5.

14. The method according to any one of the preceding claims, wherein the processing step d) comprises preparing an aqueous suspension comprising 1-15% DW of the heavy fraction and adjusting the pH of the heavy fraction aqueous suspension to pH 2-5.

15. A method as claimed in any preceding claim, wherein the processing step d) comprises heat treating the fraction.

16. The method of claim 15, wherein the heat treatment comprises drying to obtain a powder, preferably with a moisture content < 10%, and exposing the dried powder to heating in the range of 40-80°C for at least 1 day.

17. The method of claim 15 or 16, further comprising combining two or more fractions before heat treating the combination.

18. The method of any one of claims 3 to 7, comprising further processing the processed light fraction by any of the following steps: stirring the processed light fraction in an aqueous suspension having a DW content of >5% at an acidic pH until a first gel-like structure is formed; or The processed light fraction in aqueous suspension with DW content >5% was stirred at alkaline pH to produce a firm gel-like structure.

19. The method of any one of claims 3-7, comprising further processing the processed light fraction by: preparing an aqueous suspension of the processed light fraction, wherein the processed light fraction is at >1% DW; adding said aqueous suspension to a food product, either externally and / or within said food product; The food is subjected to heat treatment.

20. The method of any preceding claim, further comprising adding the product of step d) or any subsequent step to a food ingredient or food product.

21. An oil:water emulsion comprising a fraction obtained by the process as claimed in any one of the preceding claims.

22. A food ingredient for providing a food with a desired functional property selected from gelling, foaming, glazing, browning, texture, emulsifier; the food ingredient comprising a fraction obtained by the method as claimed in any one of claims 1 to 19.

23. A food comprising the food ingredient as claimed in claim 22; and / or a fraction obtained by the method as claimed in any one of claims 1 to 19.

24. The food product of claim 23 selected from the group consisting of simulated meat; simulated dairy products including cheese, yogurt, butter or cream analogs; tofu analogs; tempeh analogs; and mayonnaise analogs.

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