Method for producing single cell protein

Abstract

The current invention relates to a method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a). The current invention also relates to a fungal strain, to an SCP product comprising protein from biomass of such fungal strain, and to a food or feed product comprising such biomass or SCP product.

Description

[0001] METHOD FOR PRODUCING SINGLE CELL PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for producing single cell protein (SCP) using a fungal strain. The invention further also relates to such fungal strain, to an SCP product comprising protein from biomass of such fungal strain, and to a food or feed product comprising such biomass or SCP product.

[0004] BACKGROUND

[0005] For the production of single cell protein (SCP) with fungi, the choice of which strain to work with is crucial for safety reasons. As many Basidio- and Ascomycete strains produce a diverse array of metabolites, including (myco)toxins and antibiotics, the number of strains to choose from is limited. Mucorales strains, however, present an appealing alternative due to their typically non-toxin-producing nature, making them highly attractive for SCP production.

[0006] During submerged fermentation in bioreactors fungi are subjected to shear stress. Excessive shear stress can damage the mycelium and thereby inhibit their viability and productivity. In industrial stirred bioreactors, this sensitivity to shear stress imposes limitations to stirrer speed settings, thereby affecting oxygen transfer, and consequently overall reactor productivity.

[0007] While Basidio- and Ascomycete fungi possess segmented hyphae with septa, limiting the spread of damage from shear stress to individual cells, Mucorales strains lack such septa. Consequently, a single perforation in a Mucorales strain can lead to substantial loss of intracellular fluids, potentially resulting in lethal consequences for the fungus.

[0008] Recent discoveries have shed light on specialized wound healing proteins produced by at least two mucoromycete strains, which demonstrate remarkable efficacy in stopping the leakage of intracellular fluids within milliseconds during solid-state fermentation on agar plates. These proteins offer a promising solution for mitigating the effects of shear stress during submerged fermentation. Interestingly, genes encoding these wound healing proteins are found in nearly all Mucorales strains, including Rhizopus microsporus. However, the effectiveness of these proteins during submerged fermentation remains an area requiring further investigation. There remains a need in the art for an improved safe fungal strain, for example one belonging to the Mucoromycetes class, having a reduced susceptibility to shear stress, and a method for producing SCP using such fungal strain.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a method for producing single cell protein (SCP) according to claim 1.

[0011] In particular, said method comprises: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a). The fungal strain is a strain deposited as Deposit ID No CBS 152105, a mutant of said deposited strain, or a strain having at least 98 % genomic identity with said deposited strain.

[0012] Preferred embodiments of the method are shown in any of the claims 2 to 7.

[0013] In a second aspect, the present invention relates to a fungal strain according to claim 8 or 9. In another aspect, the present invention relates to the use of a fungal strain according to claim 10. In another aspect, the present invention relates to an SCP product according to claim 11. In another aspect, the present invention relates to a food or feed product according to claim 12.

[0014] In an aspect, the present invention also relates to a method for producing SCP comprising growing a fungal strain under conditions of high shear stress according to claim 13. Preferred embodiments of the method are shown in any on the claims 14 to 17.

[0015] DESCRIPTION OF FIGURES

[0016] Figure 1 shows the total biomass in gram dry matter of a Thermomucor indicae- seudaticae (deposited as Deposit ID No CBS 152105), a Rhizopus microsporus, and a Rhizomucor pusilus strain after 14-17.5 hours of batch fermentation in a bioreactor, as in Example 1. Black = unblended preculture, Gray = 5 seconds blended preculture.

[0017] Figure 2 shows the average growth rates in h^-l of a Thermomucor indicae- seudaticae (deposited as Deposit ID No CBS 152105), a Rhizopus microsporus, and a Rhizomucor pusilus strain during 14-17.5 hour batch fermentations in bioreactors, as in Example 1. Black = unblended preculture, Gray = 5 seconds blended preculture.

[0018] DEPOSIT INFORMATION

[0019] The fungal strain of the current invention was deposited under the terms of the Budapest Treaty on 12 July 2024 in the restricted CBS collection of Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT, the Netherlands, having received Deposit ID No. CBS 152105.

[0020] The biological material shall be made available as provided for under Rule 13bis.6 PCT and Rule 32(1) EPC only by the issuance of a sample to an Expert.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention concerns the production of single cell protein. In particular, the invention relates to a method for producing single cell protein wherein a biomass of a fungal strain is produced as single cell protein. The invention further concerns a fungal strain suitable for use in the production of SCP, preferably capable of growing in high shear stress conditions.

[0023] In an embodiment, the invention relates to a method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor, preferably in an industrial setting, in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a).

[0024] The fungal strain is preferably: a) a strain deposited as Deposit ID No CBS 152105, or b) a mutant of a strain deposited as Deposit ID No CBS 152105, wherein said mutant is still capable of growing under said conditions of high shear stress, or c) has at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, and is still capable of growing under said conditions of high shear stress.

[0025] Most preferably, the fungal strain is a strain deposited as Deposit ID No CBS 152105. Definitions

[0026] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0027] As used herein, the following terms have the following meanings:

[0028] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0029] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0030] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0031] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0032] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0033] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0034] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0035] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0036] The term "single cell protein" will be abbreviated "SCP" and is herein understood to refer to biomass consisting essentially of cells of microorganisms, which may include unicellular or multicellular forms of bacteria, yeasts, algae, and fungi including filamentous forms of fungi. SCP biomass, preferably in partly dewatered or dried form, is suitable as dietary source of protein or protein supplement in human food or animal feed. The expression "microorganism" as used herein refers to any organism that is microscopic in size and may include unicellular or multicellular entities. This encompasses bacteria, fungi, yeasts, algae, protozoa, and viruses. Microorganisms may exist as individual cells, colonies, or populations, and can include naturally occurring organisms, genetically modified organisms, or infectious agents such as viruses.

[0037] "Fungi" are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina. The term fungus includes both filamentous fungi and yeast.

[0038] The term "fungal strain" refers to a specific lineage or genetic variant of a fungal species, characterized by unique genetic and phenotypic traits. A fungal strain may be isolated from natural sources, evolved over time through natural selection and environmental adaptation, or generated through genetic modification techniques including mutagenesis or further techniques generally known in the art. It represents a distinct population of fungal cells that share common ancestry and exhibit similar physiological and biochemical properties.

[0039] In the present context, "shear stress" refers to the mechanical force exerted on microorganisms when they are subjected to fluid flow. In the context of growing microorganisms in a bioreactor, shear stress occurs primarily due to agitation, stirring, passing oxygen or other gasses through the medium, or blending of the culture medium in which the microorganisms grows, including many techniques used in bioprocess operations used to promote mass transfer of nutrients and oxygen to the microorganisms. When a culture of microorganisms is blended or stirred, it creates shear forces within the liquid medium. These forces can cause physical damage to the delicate structures of microorganisms, such as cell walls or membranes, leading to reduced cell viability and / or growth, altered metabolic activity, and / or reduced productivity of the bioprocess.

[0040] In addition, shear stress affects the mycelial macromorpholgy which in turn affects productivity of submerged fermentation processes. In the production of SCP, mycelium is ideally grown in a dispersed macromorphology, as in this form the mycelium grows rapidly and is less limited regarding nutrient transport. However, in this form they are more susceptible to shear stress. They furthermore provoke a higher medium viscosity, which in turn lowers the oxygen transfer rate. In a response to high shear stress, fungi may form a pelleted macromophology. The advantage of pellets is that they cause a low viscosity of the cultivation broth or medium and are more resistant to shear stress in comparison to the mycelium structure / disperse form. Pellets, however, may have dense and inactive cores due to poor oxygen diffusion, which may lead to cell lysis and a loss of the interior pellet structure. Overall, pelleting generally leads to reduced growth rates and thus reduced growth- associated product formation, such as SCP.

[0041] Having a strain that is less susceptible or has an improved resistance to shear stress can further be advantageous in certain bioprocessing applications. Strains that are more resistant to shear stress are less likely to suffer damage to their cellular structures during agitation, stirring, or passing oxygen or other gasses through the medium in bioreactors. Such agitation, stirring, or passing of gasses through the medium will promote single cell growth and prevent or at least reduce pellet formation. This can lead to more stable bioprocess conditions and a more predictable and consistent production of desired compounds or biomass. This can further lead to a better growth and thus productivity of the strains. Typically, bioprocesses using strains with such improved resistance to shear stress are easier to design and furthermore easier scalable, such as from laboratory scale to industrial scale, leading to smoother transitions between different production scales. Moreover, improved resistance to shear stress may reduce the need for costly measures to mitigate its effects, such as lowering agitation speeds or using specialized equipment. This can contribute to overall cost savings in bioprocess operations.

[0042] Shear stress can be measured directly or indirectly using various techniques, depending on the specific conditions and requirements of the bioprocess. Direct measurement can be performed using a rheometer or force sensor.

[0043] Rheometers are devices used to measure the flow properties of fluids. In bioprocessing, rheometers can be equipped with specialized probes to directly measure the shear stress exerted on the microbial culture as it flows past the probe, and measures generally in units of Pascal (Pa) or dynes per square centimeter (dyn / cm2).

[0044] Force sensors or load cells can be installed in bioreactor systems to directly measure the forces exerted by agitation or mixing on the culture medium. These sensors provide real-time data on shear stress levels during operation, and measure in units of Newton per square meter (N / m2) or Pa.

[0045] Indirect measurement can be measured using viscometry, particle image velocimetry (PIV) or computational fluid dynamics (CFD). Shear stress can be indirectly estimated by measuring the viscosity of the culture medium using viscometers. Changes in viscosity under different agitation speeds or shear rates can provide insights into the shear stress experienced by the microorganisms. Viscosity is typically measured in units of Pascal-second (Pa-s) or centipoise (cP). Shear stress can be calculated using the formula T = p * du / dy, where p is viscosity and du / dy is the velocity gradient.

[0046] PIV is an optical technique used to visualize fluid flow patterns within a bioreactor. By analyzing the velocity gradients in the fluid, shear stress levels can be inferred, and expressed in units of Pa or dyn / cm2.

[0047] CFD simulations can model fluid flow patterns and calculate shear stress distributions within a bioreactor based on its geometry, operating conditions, and fluid properties. Shear stress is calculated numerically and can be reported in units Pa or dyn / cm2.

[0048] The term "conditions of high shear stress" as used herein refers to shear stress as obtained by stirring the medium in the bioreactor with a tip speed of at least 1.5 m / s, preferably with an aeration rate of 0.75 volumes of air per volume of liquid per minute (VVM) or higher. The term may be defined by a tip speed of above 1.6 m / s, of above 1.7 m / s, of above 1.8 m / s, of above 1.9 m / s, of above 2.0 m / s, of above

[0049] 2.1 m / s, of above 2.2 m / s, of above 2.3 m / s, of above 2.4 m / s, of above 2.5 m / s, of above 2.6 m / s, of above 2.7 m / s, of above 2.8 m / s, of above 2.9 m / s, or of above 3.0 m / s, of above 3.1 m / s, of above 3.2 m / s, of above 3.3 m / s, of above 3.4 m / s, of above 3.5 m / s, of above 3.6 m / s, of above 3.7 m / s, of above 3.8 m / s, of above

[0050] 3.9 m / s, of above 4.0 m / s, of above 4.1 m / s, of above 4.2 m / s, of above 4.3 m / s, of above 4.4 m / s, of above 4.5 m / s, of above 4.6 m / s, of above 4.7 m / s, of above

[0051] 4.8 m / s, of above 4.9 m / s, or of above 5.0 m / s, of above 5.1 m / s, of above 5.2 m / s, of above 5.3 m / s, of above 5.4 m / s, of above 5.5 m / s, of above 5.6 m / s, of above 5.7 m / s, of above 5.8 m / s, of above 5.9 m / s, or even of above 6.0 m / s, of above

[0052] 6.1 m / s, of above 6.2 m / s, of above 6.3 m / s, of above 6.4 m / s, of above 6.5 m / s, of above 6.6 m / s, of above 6.7 m / s, of above 6.8 m / s, of above 6.9 m / s, or even of above 7.0 m / s, and / or optionally gradually increasing and / or decreasing over time. Preferably, the aeration rate may be a rate of 0.15 VVM or higher, 0.25 VVM or higher, 0.5 VVM or higher, 0.75 VVM or higher, 1.0 VVM or higher, 1.25 VVM or higher, 1.5 VVM or higher, 1.75 VVM or higher, 2.0 VVM or higher, 2.25 VVM or higher, 2.5 VVM or higher, 2.75 VVM or higher, or 3.0 VVM or higher.

[0053] Optionally, this condition of high shear stress can be maintained during the entire fermentation process. Optionally, this condition of high shear stress can be applied periodically, such as for a period of at least 2 seconds, at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 1 min, at least 2 min, at least 3 min, at least 4 min, at least 5 min, at least 7.5 min, at least 10 min, at least 15 min, at least 20 min, at least 30 min, at least 45 min, at least 1 hour, at least 2 hours, at least 3 hours, or at least 5 hours at a time. In a further embodiment, this period may be repeated one, twice, or multiple times during the growing step a).

[0054] Optionally, this condition of high shear stress can be implemented using primary and supplementary stirrers.

[0055] The term "capable of growing under conditions of high shear stress" in the present context can be defined as the ability of the fungal strain to exhibit viable growth, metabolic activity, and / or biomass accumulation when cultivated in a bioreactor within an industrial setting under the specified conditions of high shear stress. Specifically, the fungal strain demonstrates sustained proliferation and biomass production in the submerged culture medium containing a fermentable feedstock, despite the presence of elevated shear forces induced by stirring at a speed exceeding a tip speed of 1.5 m / s, or one of the tip speeds as defined above, and preferably an aeration rate 0.75 VVM or higher - or any combination of the speeds and / or aeration rates as defined above. Successful growth is evidenced by observable increases in biomass concentration, viable cell counts, O2 consumption, and / or other relevant growth parameters over the course of the fermentation process. Preferably, said increase of biomass concentration, O2 consumption, and / or of viable cell counts, relates to an increase of at least 5 %, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, or at least 150%. Said increase may also be a fold change increase of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 12.0, at least 14.0, at least 16.0, at least 18.0, at least 20.0, at least 22.0, at least 24.0, at least 26.0, at least 28.0, at least 30.0, at least 35.0, at least 40.0, at least 45.0, at least 50.0, at least 55.0, at least 60.0, at least 65.0, at least 70.0, at least 75.0, at least 80.0, at least 85.0, at least 90.0, at least 95.0, or at least 100.0.

[0056] Detailed description

[0057] In a first aspect, the invention provides a method for producing single cell protein (SCP). In particular, the method comprises the steps of: a) growing a fungal strain in a bioreactor, preferably in an industrial setting, in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a).

[0058] SCP, as used herein, refers to biomass consisting essentially of cells of microorganisms, which may include unicellular or multicellular forms of bacteria, yeasts, algae, and fungi including filamentous forms of fungi. SCP biomass, preferably in partly dewatered or dried form, is suitable as dietary source of protein or protein supplement in human food or animal feed. In the present context, this SCP is biomass obtained from the fungal strain used in the method.

[0059] In a preferred embodiment, the fungal strain is a thermophilic fungal strain.

[0060] In another or further embodiment, the fungal strain used in the method is a strain from which biomass can be obtained with a high protein content. Preferably the protein content of the biomass is at least 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65% (w / w) on dry matter basis. The high protein strains most likely have a lower content of carbon reserve and / or storage compounds, such as e.g. trehalose, glycogen and / or lipids.

[0061] In another or further embodiment, the fungal strain used in the method is a strain of which the proteins in the biomass contain one or more of the essential amino acids. Preferably the proteins are rich in such essential amino acids. Essential amino acids are herein understood to include at least one or more of lysine, phenylalanine, tyrosine, threonine, methionine, cysteine, valine, arginine, histidine, tryptophan, isoleucine and leucine, of which, lysine, threonine, methionine are most preferred.

[0062] As the SCP product is intended for use in food or feed for human or animal consumption, the production of mycotoxins, such as e.g. Ochratoxin A and Fumonisins, by the fungal strain to be applied is undesirable. Therefore a fungal strain for use in the invention preferably is selected that does not produce any mycotoxins. This screening can for instance be done by genetic means, by verifying e.g. with PCR or with whole genome sequencing, the absence of the presence of genes in mycotoxin pathways, and in the case such genes are present, by verifying that, under process conditions used, these genes are not expressed and / or these toxic compounds are not produced.

[0063] In a preferred embodiment, the fungal strain is of the order Mucorales, preferably of the genus Thermomucor. Preferably, the fungal strain is a Thermomucor indicae- seudaticae strain.

[0064] Fungal SCPs derived from Thermomucor indicae-seudatica may have a sum of total essential amino acids of 15% higher than that of soybean protein, where soybean protein comprises, according to literature, about the following composition : Lysine (6%), Methionine (1%), Cysteine (2%), Tryptophan (1%), Isoleucine (5%), Arginine (8%), Phenylalanine (5%), Histidine (3%), Leucine (8%), Tyrosine (4%), Valine (5%), Alanine (4%), Aspartic acid (12%), Glutamic acid (18%), Glycine (4%), Proline (5%), Serine (5%), Threonine (4%). Thus, the total amount of essential amino acids in soybean is 6% (Lysine) + 1% (Methionine) + 2% (Cysteine) + 1% (Tryptophan) + 5% (Isoleucine) + 5% (Phenylalanine) + 3% (Histidine) + 8% (Leucine) + 5% (Valine) + 4% (Threonine) = 40%. All values are approximate values.

[0065] In addition, the SCP from Thermomucor indicae-seudatica has a lysine content of more than 8%, even more than 8,5% of total amino acids, a leucine content of at least 8%, preferably at least 8.5%, and a phenylalanine + tyrosine content of at least 7%, preferably at least 8% of total amino acids. SCP from Thermomucor strains thus not only has a high protein content but also a high content of numerous essential amino acids. Thermomucor SCP thus has a surprisingly high nutritional value.

[0066] Mucorales strains are typically non-toxin-producing nature, making them highly attractive for use in the present method for SCP production.

[0067] Most preferably, in view of their high nutritional value and safe properties, the fungal strain is a Thermomucor indicae-seudaticae strain.

[0068] In an embodiment, the method comprises in step a) growing the fungal strain in the medium, under conditions of high shear stress. Such shear stress may for instance be generated by agitation, stirring, blending, passing one or more gasses through the medium, optionally with application of baffles, or pumping of the culture medium within the bioreactor.

[0069] In bioreactors and / or fermentation processes, shear stress occurs primarily due to agitation, stirring, passing oxygen or other gasses through the medium, or blending of the culture medium in which the microorganisms grows, including many techniques used in bioprocess operations used to promote mass transfer of nutrients and oxygen to the microorganisms. Such conditions could for instance be obtained when stirring the medium in a stirred tank reactor, either by primary or secondary stirrers, or when distributing (compressed) air or gas in a reactor.

[0070] The term "conditions of high shear stress" as used herein refers to shear stress as obtained by stirring the medium in the bioreactor with a tip speed of 1.5 m / s, preferably with an aeration rate of 0.75 VMM or higher; or as defined above.

[0071] The invention therefore also relates to a method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a), wherein the growing of the fungal strain comprises stirring the medium in the bioreactor at a with a tip speed of 1.5 m / s, preferably with an aeration rate of 0.75 VVM or higher; or as defined above.

[0072] In another or further embodiment, the fungal strain used in the method is capable of growing under conditions of high shear stress.

[0073] The term "capable of growing under conditions of high shear stress" in the present context is as defined above.

[0074] Shear stress can cause physical damage to the delicate structures of microorganisms, such as cell walls or membranes, leading to reduced cell viability and / or growth, altered metabolic activity, and / or reduced productivity of the bioprocess. Shear stress is of particular relevance to Mucorales strains, as such strains do not comprise segmented hyphae with septa, the latter which can limit the spread of damage from shear stress to individual cells in for instance Basidio- and Ascomycete fungi. Consequently, a single perforation in a Mucorales strain can lead to substantial loss of intracellular fluids, potentially resulting in lethal consequences for the fungus.

[0075] Shear stress is further of particular relevance when the strain is grown in a dispersed micromorphology, as is preferably the case for the present method.

[0076] Having a strain that is less susceptible or has an improved resistance to shear stress can be advantageous in bioprocessing applications. Strains that are more resistant to shear stress are less likely to suffer damage to their cellular structures during agitation, stirring, or passing oxygen or other gasses through the medium in bioreactors. This can lead to more stable bioprocess conditions and a more predictable and consistent production of desired compounds or biomass. This can further lead to a better growth and thus productivity of the strains. Typically, bioprocesses using strains with such improved resistance to shear stress are easier to design and furthermore easier scalable, such as from laboratory scale to industrial scale, leading to smoother transitions between different production scales. Moreover, improved resistance to shear stress may reduce the need for costly measures to mitigate its effects, such as lowering agitation speeds or using specialized equipment. This can contribute to overall cost savings in bioprocess operations.

[0077] In an embodiment, the fungal strain is grown under conditions of high shear stress as defined above in any one of the embodiments, thereby ensuring disperse mycelium morphology of the strain in contrast to pellet formation.

[0078] In an embodiment, and as surprisingly discovered by the inventors, the strain deposited under the regulations of the Budapest Treaty at the Westerdijk Fungal Biodiversity Institute Utrecht, The Netherlands (formerly referred to as Centraalbureau voor Schimmelcultures, CBS) on 12 July 2024 and assigned the accession number or Deposit ID No CBS 152105 is capable of growing under conditions of high shear stress.

[0079] Therefore, the strain used in either one of the described methods is preferably a strain : deposited as Deposit ID No CBS 152105: a mutant of a strain deposited as Deposit ID No CBS 152105, a strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105. In another or further embodiment, the mutant of the strain deposited as Deposit ID No CBS 152105 and / or the strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, are still capable of growing under said conditions of high shear stress, preferably in a manner similar to said strain deposited as Deposit ID No CBS 152105.

[0080] In a further embodiment, the fungal strain is a strain having at least 98 %, more preferably at least 98.1 %, more preferably at least 98.2 %, more preferably at least

[0081] 98.3 %, more preferably at least 98.4 %, more preferably at least 98.5 %, more preferably at least 98.6 %, more preferably at least 98.7 %, more preferably at least

[0082] 98.8 %, more preferably at least 98.9 %, more preferably at least 99 %, more preferably at least 99.1 %, more preferably at least 99.2 %, more preferably at least

[0083] 99.3 %, more preferably at least 99.4 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least

[0084] 99.8 %, more preferably at least 99.9 % genomic sequence identity with a with a strain deposited as Deposit ID No CBS 152105.

[0085] In an embodiment, the method further comprises a mixing step, performed using a mixing device, said mixing step being carried out either: during step a) while the fungal strain is growing in the bioreactor, or prior to step a) on a preculture of the fungal strain.

[0086] The invention thus also relates to a method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting, preferably under conditions of high shear stress, in a medium containing a fermentable feedstock, optionally a fermentable carbon- rich feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a), wherein the method further comprises a mixing step, performed using a mixing device, said mixing step being carried out either: during step a) while the fungal strain is growing in the bioreactor, or prior to step a) on a preculture of the fungal strain. As used herein, 'mixing' or 'blending' in the present context refers to the mechanical treatment of the fungal strain, either in the form of growing said strain in the bioreactor or a preculture thereof, wherein the fungal biomass is exposed to high- energy fluid shear using a device such as an immersion blender, rotor-stator homogenizer, or high-shear mixer.

[0087] This mixing or blending treatment typically involves a short-duration application, optionally repeated one or more times such as repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or even 50 times or more, generally ranging from 1 to 60 seconds, more preferably from 2 to 45 seconds, even more preferably from 3 to 30 seconds, most preferably from 3 to 10 seconds. In one embodiment, the mixing step is carried out for at least 1 second, preferably at least 2 seconds, preferably at least 3 seconds, more preferably at least 4 seconds, more preferably at least 5 seconds, more preferably at least 6 seconds, more preferably at least 7 seconds, more preferably at least 8 seconds, more preferably at least 9 seconds, more preferably at least 10 seconds, more preferably at least 15 seconds, more preferably at least 20 seconds, and most preferably at least 25 seconds. In an embodiment, the mixing step is carried out for no more than 60 seconds, preferably no more than 45 seconds, more preferably no more than 30 seconds, more preferably no more than 20 seconds, most preferably no more than 10 seconds.

[0088] Therefore, the mixing or blending treatment may comprise one or more consecutive round of mixing or blending, with settings (including duration, rpm, and or W) as defined above or below.

[0089] The mixing or blending treatment is performed at high rotational speed, typically involving one or more devices, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more devices, each operating at a speed in the range of 1,000 to 50,000 rpm, more preferably in the range of 2,000 to 35,000 rpm, more preferably in the range of 3,000 to 30,000 rpm, more preferably in the range of 4,000 to 25,000 rpm, more preferably in the range of 5,000 to 20,000 rpm, more preferably in the range of 5,000 to 15,000 rpm, more preferably in the range of 5,000 to 10,000 rpm, and most preferably in the range of 6,500 to 10,000 rpm. In one embodiment, the mixing speed is at least 1,000 rpm, preferably at least 5,000 rpm, more preferably at least 10,000 rpm, more preferably at least 15,000 rpm, more preferably at least 20,000 rpm, and most preferably at least 25,000 rpm. The rotational speed is preferably selected such that the shear conditions are sufficient to fragment the mycelial biomass into smaller fragments or dispersed hyphal elements, while preserving viability and avoiding complete cell lysis.

[0090] In an embodiment, the treatment is carried out at a power input of at least 100 W, preferably at least 150 W, more preferably at least 200 W, more preferably at least 250 W, more preferably at least 300 W, more preferably at least 350 W, more preferably at least 400 W, more preferably at least 450 W, more preferably at least 500 W, more preferably at least 550 W, more preferably at least 600 W, and most preferably at least 650 W.

[0091] The mixing or blending may be applied prior to inoculation of a (production-scale) bioreactor (i.e., on a preculture), or during cultivation or growing within the bioreactor itself.

[0092] The mixing or blending treatment as defined herein is distinct from conventional stirring or aeration commonly applied during fermentation. It constitutes an additional, high-energy mechanical stress step, intended to simulate extreme shear conditions either before or during fermentation.

[0093] While conventional stirring in bioreactors aims to provide gentle circulation and mass transfer throughout the vessel, blending involves concentrated and intense shear forces generated by sharp-edged blades operating at high rotational speeds and minimal wall clearance. Although both methods may operate at comparable tip speeds, blending produces significantly higher local shear stress due to its geometry, blade design, and energy density.

[0094] As such, not all stirring qualifies as blending: blending represents a specific subclass of high-shear interventions that cannot be equated with standard agitation. The differences in flow dynamics, turbulence scale, and energy dissipation profiles between these processes are substantial and translate into markedly different biological effects on fungi, particularly filamentous fungi, particularly in the context of mycelial morphology and viability.

[0095] Non-limiting examples of the differences between blending and conventional stirring are provided below for illustrative purposes only and are not intended to further restrict or define the terms "mixing," "blending," or "stirring" as used herein. Focus lays on shear stress, bulk flow pattern, and turbulence intensity as described below.

[0096] "Shear stress" depends not only on speed, but also on the abruptness of velocity changes near solid surfaces. A sharp blade rotating close to the vessel wall generates extreme velocity gradients; a blender is specifically designed to create such localized and intense shear zones.

[0097] The "bulk flow pattern" refers to the overall circulation and movement of fluid within a vessel or bioreactor, driven by mechanical agitation or mixing. The bulk flow pattern is fundamentally different depending on blade shape and orientation. Sharp, angled blades of a blender push fluid downward and outward, designed to create a high-speed vortex. Even at equal tip speeds, the blender generates a concentrated, high-energy flow core, whereas the bioreactor distributes energy more broadly, with baffles helping to suppress vortex formation and ensure overall circulation. "Turbulence intensity" refers to the magnitude of chaotic velocity fluctuations within a moving fluid, relative to its average flow speed. It is a key factor in determining how aggressively fluid forces act on suspended particles or cells. Although the Reynolds number is high in both blending and bioreactor stirring, the energy dissipation rate (e) and power density (W / m3) are far greater in a blender, which makes the turbulence more violent and significantly more damaging to delicate fungal structures.

[0098] By way of alternative or additional example, an not intended to limit the scope of the application, the blending treatment may be performed using an immersion blender with a blade tip diameter of 2-4 cm, operating at 650 Watt and / or a rotational speed of 6,500 to 10,000 rpm. The resulting shear conditions are preferably sufficient to fragment the mycelial biomass into smaller fragments or dispersed hyphal elements, without causing complete cell lysis or loss of viability of the specific strain or species blended.

[0099] In another or further embodiment, the fungal strain used in the method is capable of growing in the bioreactor after mixing in the bioreactor and / or after mixing of its preculture.

[0100] The term "capable of growing after mixing" or "capable of growing after blending", as used herein, refers to the ability of the fungal strain to maintain viability, metabolic activity and / or biomass accumulation when cultivated in a bioreactor after the fungal strain has been subjected to mechanical disruption by mixing or blending, either during growth in the bioreactor or to said growing step on a pre-culture of the strain, i.e. prior to inoculation of the strain in the bioreactor, preferably as defined above. In particular, the fungal strain(s) demonstrate(s) sustained proliferation and biomass production under submerged culture conditions, regardless of whether the preculture has been exposed to a blending treatment prior to inoculation or whether blending / mixing was applied during growth in the bioreactor, such as by high-energy mechanical disruption conditions including immersion blending at a power of at least 500 W for a period of at least 3 seconds, and / or at least 10.000 rpm for at least 3 seconds, or as defined above in any one of the embodiments. Successful growth is evidenced by observable increases in biomass concentration, viable cell counts, oxygen consumption and / or other relevant growth parameters during the fermentation process as known by the skilled person. Preferably, the fungal strain maintains at least 70%, more preferably at least 75%, 80%, 85%, 90%, or even 95% of the growth rate or final biomass yield as compared to growth obtained without such blending or mixing treatment, under otherwise identical growing and / or fermentation conditions.

[0101] Blending of precultures or the growing strain in the bioreactor can cause significant shear-induced damage to the hyphal structure of filamentous fungi, especially members of the Mucorales. Such damage can result in reduced viability, impaired germination, and limited growth capacity in the production bioreactor. This is of particular relevance to Mucorales strains, which lack septated hyphae and are therefore more vulnerable to uncontrolled loss of intracellular fluids upon membrane perforation. Consequently, a short blending step prior to fermentation, or a mixing step during fermentation, which may mimic extreme shear conditions, can critically impact the performance of most strains.

[0102] The ability of a fungal strain to withstand blending or mixing without compromising growth performance offers significant advantages in industrial processes. It simplifies upstream handling, allows for more homogeneous preculture preparation, and enables more robust and scalable seed train strategies. Furthermore, blendingresistant strains allow for reduced constraints on preculture transfer methods, contributing to improved reproducibility, fermentation control, and cost-efficiency in large-scale SCP production.

[0103] In an embodiment, the fungal strain is grown in a submerged culture that was inoculated with a blended preculture, as defined above in any one of the embodiments.

[0104] In another embodiment, the fungal strain is grown in a submerged culture that is subjected to a mixing step during the growth phase in the bioreactor. In either case, preferably, said fungal strain retains its ability to grow in a dispersed micromorphology following the blending or mixing step, as opposed to forming dense pellets, which further supports rapid growth and protein production.

[0105] In an embodiment, and as surprisingly discovered by the inventors, the strain deposited under the regulations of the Budapest Treaty at the Westerdijk Fungal Biodiversity Institute Utrecht, The Netherlands (formerly referred to as Centraalbureau voor Schimmelcultures, CBS) on 12 July 2024 and assigned the accession number or Deposit ID No CBS 152105 is capable of growing after mixing, preferably as defined above.

[0106] Therefore, the strain used in the present method comprising a mixing or blending step is preferably a strain: deposited as Deposit ID No CBS 152105: a mutant of a strain deposited as Deposit ID No CBS 152105, a strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105.

[0107] In another or further embodiment, the mutant of the strain deposited as Deposit ID No CBS 152105 and / or the strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, is still capable of growing after mixing or blending, preferably in a manner similar to said strain deposited as Deposit ID No CBS 152105.

[0108] In a further embodiment, the fungal strain is a strain having at least 98 %, more preferably at least 98.1 %, more preferably at least 98.2 %, more preferably at least

[0109] 98.3 %, more preferably at least 98.4 %, more preferably at least 98.5 %, more preferably at least 98.6 %, more preferably at least 98.7 %, more preferably at least

[0110] 98.8 %, more preferably at least 98.9 %, more preferably at least 99 %, more preferably at least 99.1 %, more preferably at least 99.2 %, more preferably at least

[0111] 99.3 %, more preferably at least 99.4 %, more preferably at least 99.5 %, more preferably at least 99.6 %, more preferably at least 99.7 %, more preferably at least

[0112] 99.8 %, more preferably at least 99.9 % genomic sequence identity with a with a strain deposited as Deposit ID No CBS 152105.

[0113] In an embodiment, in step a) the fungal strain is a thermophilic strain and / or is grown at a temperature of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 °C or more. In an embodiment, in step a) the fungal strain can grow in low pH condition, such as a pH of 3.8, 3.75, 3.74, 3.73, 3.72, 3.71, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, or 2.0, or less. Preferably, in step a) the fungal strain is grown at a pH of 3.8, 3.75, 3.74, 3.73, 3.72, 3.71, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, or 2.0, or less.

[0114] In an embodiment, in step a) the fungal strain is grown under non-sterile conditions.

[0115] In an embodiment, the fermentable feedstock may comprise a large variety of substances that can be used as a carbon source. These include sugars (e.g., glucose, galactose, mannose, trehalose, sucrose, arabinose, mannose, xylose, fructose, etc.), glycerol, starch, carbohydrates, lignocellulosic feedstock, waste streams, and combinations thereof.

[0116] In an embodiment, the fungal strain used in the method can grow on carbon-rich, i.e. energy-dominated, feedstocks. These include both simple sugars such as sucrose and glucose, fructose, as well as polymeric sugars such as starch, inulin, cellulose, hemicellulose, chitin, and pectin. Additionally, organic acids such as lactic acid, acetic acid, formic acid, and alcohols like ethanol and methanol (often formed in silage processes or derived from pectin and hemicellulose) can be utilized. Lipids present in the form of triglycerides or phospholipids are also suitable. Other sugars present in hemicellulose, such as rhamnose, fucose, galactose, xylose, arabinose, mannose, galacturonic acid, glucuronic acid, raffinose, melibiose, stachyose, etc., can enhance the growth or protein formation of the fungus and fermentation by the fungus can minimize carbon burden from a filtrate, which has to go to waste water treatment or biogas installation. Additionally and preferably, the conversion of betaine, ferulic acid, and coumaric acid by the fungus maximizes yield.

[0117] Examples of such feedstocks include but are not limited to co-products from the dairy, bioethanol, starch, biofuel, potato, brewing, distilling, fruit, vegetable, juice processing, alcohol, ethanol, sugar, paper, pulp, vegetable processing, canning, wet milling, fish processing, and manure industries.

[0118] In an embodiment, the concentration of the feedstock is maintained below a level at which toxic compounds in the feedstock reduce the growth rate of the fungal strain. The feedstock can be fed to the medium at a rate that keeps the concentration below this toxic threshold, defined as the highest concentration that does not cause a reduction in at least one of the rate of CO2 production and the rate of O2 consumption by the fungal strain. Preferably, the feedstock concentration and / or feedstock organic matter in the medium is less than 5, 4, 3, or 2% (w / v) dry matter.

[0119] In an embodiment, the fungal strain used in the method can grow on carbon-limited, i.e. energy-deficient, feedstocks. These include sources with very low concentrations of readily available carbon, such as certain types of wastewater, industrial effluents with minimal organic content, and nutrient-depleted soils. Specific examples include effluents from the dairy or bioethanol industries post-fermentation, secondary- treated municipal wastewater, and agricultural runoff with minimal organic matter. Other potential carbon-limited substrates include minimal media formulations with trace amounts of simple sugars or organic acids, and lignocellulosic residues pretreated to remove most soluble carbohydrates.

[0120] In an embodiment, the medium comprises essential macronutrients: nitrogen (N), phosphorus (P), calcium (Ca), magnesium (Mg), carbon (C), potassium (K), and sulfur (S). Trace nutrients such as iron (Fe), boron (B), chromium (Cr), copper (Cu), selenium (Se), manganese (Mn), molybdenum (Mo), vanadium (V), and zinc (Zn) can also be added to the medium.

[0121] Additional nutrient additions can be incorporated into the media. Examples include carbohydrates (e.g., monosaccharides, polysaccharides), amino acid donors (e.g., amino acids, polypeptides), and combinations thereof. Compounds facilitating the pretreatment of carbon sources, such as acidification materials, manganese donors, nutrients, and pH buffering materials, can also be added to the media.

[0122] In a preferred embodiment, the medium comprises or is fed a nitrogen source. Suitable nitrogen sources include one or more of ammonia, urea, ammonium nitrate (H4NO3), ammonium sulfate (H4SO4), nitrate salts (e.g., KNO3), ammonia salts (e.g., H4SO4), organic nitrogen sources (e.g., proteins, peptides, amino acids), industrial waste streams high in nitrogen, and combinations thereof. Ammonia or H2NO3 can be used to control pH in the fermenter, while urea serves as a pH-independent nitrogen source. Nitrogen sources from waste streams are also preferred, including amines from burden condensates of molasses, sugar beet or cane vinasses, vinasses from the wine industry, grape residues, potato protein liquor (PPL), corn steep liquor (CSL), ammonia from animal farm exhaust gas cleaning scrubbers, and the thin fraction of manure processing.

[0123] In an embodiment, the fungal strain used in the method may be a cellulolytic and / or hemi-cellulolytic fungal strain. In an embodiment, for example when the SCP is to be applied in animal feed, the fermentable feedstock encompasses a diverse range of sources, including byproducts or waste from agriculture or food production, silage, and the organic fraction of municipal solid waste (MSW). Preferably, the fermentable feedstock comprises by-products from the biofuel industry, such as residues from biodiesel and bioethanol production (e.g., glycerin, vinasse), by-products from the beverage industry (e.g., spent grains from breweries), sugar beet pulp, liquid C-starch from grain processing, vegetable waste from the production of peeled, cut vegetables or rejected vegetables, palm mill residues (e.g., palm oil mill effluent (POME), empty fruit bunches (EFB), palm fronds), dedicated crops grown for biofuel or bioethanol production (e.g., switchgrass, miscanthus, willow, soybeans, rapeseed, sunflower, barley, oats, sugarcane, cassava), and vegetable products suitable for food application (e.g., corn, potato, wheat, rice, cassava, sugarcane or sugarcane juice, sugar beet or sugar beet juice or thick juice, glucose syrups).

[0124] In an embodiment, nitrogen limitation is avoided, and the fungal strain is preferably grown under carbon limitation. This maximizes the protein content of the biomass produced and avoids the accumulation of carbon reserve and / or storage compounds, such as trehalose, glycogen, and / or lipids, which result from carbon excess.

[0125] In a preferred embodiment, the method is a fed-batch process, a repeated fed-batch process (where a part of the fermentation broth is harvested repeatedly), or a continuous process.

[0126] In an embodiment, the bioreactor is used at a large volume, such as in 50,000 - 200,000 L working volume bioreactors. When preparing material at such volumes the culture must pass through a successive series of larger bioreactors, any bioreactor being inoculated at 0.5 - 15% of the working volume according to the parameters of the seed train. A typical process would pass a culture from master culture, optionally to (Petri) plates, to flasks, to seed bioreactors to the final main bioreactor when scaling the method of the present invention. To reach large volumes, multiple seeds may be used. The media of the seed can be the same or different as the media in the main. In one embodiment, foaming is minimized by use of antifoam, such as on the order of 0.01 to 2.5 g / L of media, such as for instance 0.05, or 0.5, or 1, or 1.5, or 2.0 g / L (organic or inorganic antifoam may be used), depending on the need for such antifoam product.

[0127] Subsequently, according to step b) of the method, SCP is recovered from the medium in the form of biomass of said fungal strain grown in step a). In an embodiment, the biomass is preferably recovered from the medium by at least one of sieving, filtration, pressing, and decantation. More preferably, the biomass is recovered from the medium by at least one of rotating drum filtration, a filter press, a belt filter, a decanter centrifuge, a screw press, and sieving. Preferably biomass is recovered using a press such as a filter press or a screw press. Preferably, biomass is recovered using a press followed by sieving on a sieve or a screen, preferably with 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0,25, 0.3, 0.4, 0.5, 0.7, 1 or 2 mm diameter of pores. More preferably, the biomass is recovered by at least two, three or four consecutive rounds of sieving on a sieve or screen whereby a smaller diameter of pores is applied in each subsequent round of sieving. E.g. a first round of sieving using 2 mm pore diameter, followed by subsequent rounds of 1, 0.5 and / or 0.1 mm.

[0128] In an embodiment, the dry matter concentration of the sieved, filtered, pressed, or decanted biomass (cake) is at least 12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 52%, 53%, 54%, or 55% (w / v). Optionally, dry matter concentration of the sieved, filtered or decantated biomass (cake) is further increased by further removal of water, i.e. drying.

[0129] In an embodiment, the biomass recovered from the medium can be processed according to a variety of methods. In one embodiment, the biomass may be pasteurized or sterilized before further drying, and may be powdered subsequent to the drying step.

[0130] In an embodiment, the sieved, filtered, pressed, or decanted biomass (cake) can e.g. be further dried by pressing residual water out, for example by pressing (more of) the residual water out using e.g. compressed air using a pneumapress, using a hydraulic press and / or mechanical pressing, using e.g. a belt press or a screw press. In an embodiment, such pressing can be used as an initial recovering step as well, as alternative to or as additional step to sieving, filtration and decantation described above, and which may be performed before or after said sieving, filtration, or decantation.

[0131] In warmer climates the biomass (cake) can simply dried to the air (in the sun). After pressing the biomass to a cake, optionally the cake can be milled or extruded e.g. to enable drying, preferably air drying. Preferably, the particle size of the pressed mycelial biomass cake is reduced by physical means to enable (more efficient) drying of the pressed cake. This can optionally done by extrusion of the mycelial cake through holes with a diameter of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mm, using extruders that are known in the art per se. If however the dry matter concentration of the pressed cake after pressing is so high, that extrusion of the pressed cake is no longer possible (e.g. when the cake is too firm to allow for extrusion), the particle size of the cake can be reduced by a combination of milling and sieving. As a milling step any type of mill known in the art per se can be used, such as e.g. a knife mill or a hammer mill, etc. To obtain homogeneous particle size of the milled pressed cake, the larger particles still present after milling can be removed before drying by sieving with a pore diameter size in the sieve of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mm. The resulting milled cake would have preferably a particle size between 1 - 3 mm before drying. By reducing the particle size, evaporation of water from the pressed cake is more efficient and faster. Alternatively, vacuum drying or dewatering methods, such as using a vacuum belt press, can also be employed to further enhance the drying process.

[0132] In an embodiment, drying of the cake is done by using waste heat, e.g. from a plant where hot water is obtained after condensation of gas (e.g. ethanol distillation, potato cooking, steam-pealing of potatoes, etc.). The air can be heated using a heat exchanger to heat up dry air with hot water from the heat source.

[0133] In an embodiment, drying of the extruded or milled cake is preferably done at temperatures of 30 - 70 °C. The hot air can then dry the cake in a gentle and cost effective way in a belt dryer or fluid bed dryer. However, higher temperatures may also be used if required, though caution is advised. Steam drying at high temperatures (e.g. > 80 °C) is preferably avoided as it can negatively influence digestibility of the proteins by denaturing and baking, and even chemical decomposition of the amino acids by Maillard reactions.

[0134] In another or further embodiment, drying can be done in a desiccator, vacuum dryer, conical dryer, spray dryer, fluid bed or any method known in the art.

[0135] In another or further embodiment, heat produced during the growing, and thus fermentation, of the fungal strain, which preferably is a thermophilic strain, is reused for the drying of the biomass recovered from the medium. Preferably, a heat exchanger or heat pump is used to capture the heat from a gas flow discharged during the growing and preferably aerobic fermentation of the strain, and heating an air flow subsequently used for the drying process. This gas flow is preferably condensed before used in the drying process.

[0136] Preferably, drying methods are chosen that yield a dried SCP product (e.g., a powder) with the greatest digestibility and bioavailability. The dried product can be optionally blended, pestled, milled or pulverized, or other methods as known in the art.

[0137] In a preferred embodiment, the water fraction that is obtained after sieving, filtering, decanting and / or further pressing the biomass (cake) is partly recycled and / or reused. For example, the water fraction may be recycled back to the fermentation and / or used for further fermentation batches. In a further preferred method of the invention the fermenter is operated without any cooling device that requires input energy.

[0138] In a second aspect, the invention relates to a fungal strain.

[0139] A person of ordinary skill in the art will appreciate that elements of the aspect of the method as described above return in the aspect of the fungal strain, the use of the fungal strain, the SCP product, and the food or feed product of the invention. Consequently, all aspects of the present invention are related. All features as described in one of the aspects, as described above as well as below, can relate to any of these aspects, even if they are described in conjunction with a specific aspect.

[0140] In an embodiment, the fungal strain is a thermophilic strain. Preferably, the fungal strain is of the genus Thermomucor. Most preferably, in view of their high nutritional value and safe properties, the fungal strain is a Thermomucor indicae-seudaticae strain. Most preferably, the fungal strain is a Thermomucor indicae-seudaticae strain deposited as Deposit ID No CBS 152105. Advantages of such strains are as described above.

[0141] In another or further embodiment, the fungal strain is capable of growing under conditions of high shear stress and / or after mixing, as defined above.

[0142] In an embodiment, and as surprisingly discovered by the inventors, the strain deposited as Deposit ID No CBS 152105 is particularly capable of growing under conditions of high shear stress and / or after mixing.

[0143] Moreover, the present invention also relates to a mutant of a strain deposited as Deposit ID No CBS 152105, and to a strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, as described above in relation to the method.

[0144] In another or further embodiment, the mutant of the strain deposited as Deposit ID No CBS 152105and / or the strain having at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, are still capable of growing under said conditions of high shear stress and / or after mixing, preferably similar as and / or even better than the strain deposited as Deposit ID No CBS 152105.

[0145] The fungal strain may be a fungal strain as described above in any one of the embodiments.

[0146] In a third aspect, the invention relates to a use of the fungal strain as described in any one of the embodiments, for the production of SCP.

[0147] In an embodiment, the fungal strain is grown in a medium containing a fermentable feedstock, preferably grown in a submerged culture, and preferably under condition of high shear stress and / or after mixing. Subsequently, SCP is recovered from the medium in the form of biomass of said fungal strain, as described above in any one of the embodiments.

[0148] In another aspect, the invention relates to an SCP product. The SCP product preferably comprises protein from biomass of at least one fungal strain as described above in any one of the embodiments. Preferably, the strain is a strain as deposited as Deposit ID No CBS 152105, a mutant thereof, or a strain having at least 98 % genomic sequence identity with said strain, and preferably, said strain is capable of growing under conditions of high shear stress and / or after mixing.

[0149] In an embodiment, the SCP product is edible. In another or further embodiment, the SCP product is free from mycotoxins. Preferably, the fungal biomass, or at least the resulting SCP product, is safe for animals and / or humans to eat.

[0150] In another or further embodiment, the SCP product comprises essential amino acids. Essential amino acids are herein understood to include at least one or more of lysine, phenylalanine, tyrosine, threonine, cysteine, methionine, valine, arginine, histidine, tryptophan, isoleucine and leucine, of which, lysine, threonine, methionine are most preferred.

[0151] In another or further embodiment, the SCP product has a lysine content of more than 8,5% of total amino acids, a leucine content of at least 8.5%, and a phenylalanine + tyrosine content of at least 8% of total amino acids. SCP from Thermomucor strains thus not only has a high protein content but also a high content of numerous essential amino acids. Thermomucor SCP thus has a surprisingly high nutritional value.

[0152] In an embodiment, the SCP product has a sum of total essential amino acids of 15% higher than that of soybean protein, and as described above.

[0153] In an embodiment, the fungal biomass or the resulting SCP product may be used as or in foodstuff such as, but not limited to, food for consumption of humans, animal feed, fodder, compound feed, petfood, fish feed and / or aquafeed. Alternatively, or additionally, the fungal biomass or SCP product is foodstuff or is a protein source for use in a foodstuff. Alternatively, the fungal biomass or SCP product is animal feed or is protein source for use in an animal feed.

[0154] In another aspect the invention relates to a food or feed product comprising protein from biomass of a fungal strain deposited as Deposit ID No CBS 152105, and / or the SCP product as described above in any one of the embodiments.

[0155] The SCP obtained in a method according to the invention can e.g. be used to supplement feed or as an ingredient in feed for a variety of different livestock animal types, including pigs, poultry, ruminant livestock as well as aquatic fish and crustacean species. For the application of the SCP as fish feed, preferably the feed is enriched with a source of omega- fatty acids such as fish oil, or a lipid rich algae, such as Cryptocodinium cohnii, or Traustochytrium aureum. An additional advantage of the SCP obtained in a method according to the invention is that the acidic pH at which the SCP is produced will prevent contamination of the SCP by problematic bacteria such as E.coli, Salmonella, Bacillus cereus, Enterobacteriaceae, Listeria etc., which may be present in SCP produced in other methods.

[0156] Alternatively or in addition to the above, the SCP obtained in a method according to the invention can be used as pet food or pet food ingredient. Preferably, said SCP can be used as pet food or pet food ingredient for animals, such as but not limited to dogs, cats, rabbits, guinea pigs, hamsters, ferrets, birds (such as parakeets, cockatiels), reptiles (such as turtles, lizards, snakes), fish (such as goldfish, bettas), amphibians (such as frogs, newts, salamanders), or combinations thereof.

[0157] Alternatively or in addition to the above, the SCP can be utilized as a high-quality protein ingredient in the livestock industry, particularly for animals with a vulnerable gastrointestinal system. This includes young animals, whose gastrointestinal systems may be still under development, such as piglets, chicks, calves, lambs, poults, ducklings, and young goats, as well as fish like salmon. SCP is also particularly beneficial for animals sensitive to typical plant-related allergens or anti- nutritional factors.

[0158] Alternatively, the SCP obtained in a method according to the invention can be used as a food or food ingredient. As mentioned, fungal SCPs derived from Thermomucor indicae-seudatica has a surprisingly high nutritional value, and is a very useful as food or food additive.

[0159] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0160] EXAMPLES AND / OR DESCRIPTION OF FIGURES

[0161] Example 1: The effect of blending precultures of Mucorales fungi on their successive performance during batch fermentations in bioreactors

[0162] Summary

[0163] The experiment focused on the resilience of three Mucorales strains to shear stress. Firstly, 1-day-old precultures of Thermomucor indicae-seudaticae, Rhizopus microsporus, and Rhizomucor pusillus were prepared in shake flasks from spore solutions. Subsequently, these precultures were either 1) directly introduced into a 3.5L bioreactor for a 14-hour batch fermentation, or 2) subjected to a 5-second blending with an immersion blender before inoculation into the bioreactor setup. The bioreactors were then harvested, and the total biomass production was quantified. All three strains exhibited robust growth following standard inoculation. Intriguingly, T. indicae-seudaticae demonstrated comparable growth performance during batch fermentation even after blending its preculture. Conversely, the performance of R. microsporus and R. pusillus was significantly hampered post-blending. These findings suggest that T. indicae-seudaticae possesses superior resistance to shear stress compared to the other Mucorales strains.

[0164] Introduction

[0165] For the production of single cell protein (SCP) with fungi, the choice of which strain to work with is crucial for safety reasons. As many Basidio- and Ascomycete strains produce a variety of (myco)toxins and antibiotics, the number of strains to choose from is partly limited. Mucorales strains, however, present an appealing alternative due to their typically non-toxin-producing nature, making them highly attractive for SCP production.

[0166] During submerged fermentation in bioreactors, fungi are susceptible to shear stress, which can significantly impact their viability and productivity. In industrial stirred bioreactors, this sensitivity to shear stress imposes limitations to stirrer speed settings, also affecting oxygen transfer, and, consequently overall reactor productivity.

[0167] While Basidio- and Ascomycete fungi possess segmented hyphae with septa, limiting the spread of damage from shear stress to individual cells, Mucorales strains lack such septa. Consequently, a single perforation in a Mucorales strain can lead to substantial loss of intracellular fluids, potentially resulting in lethal consequences for the fungus.

[0168] Recent discoveries have shed light on specialized wound healing proteins produced by at least two mucoromycete strains, which demonstrate remarkable efficacy in stopping the leakage of intracellular fluids within milliseconds during solid-state fermentation on agar plates. These proteins offer a promising solution for mitigating the effects of shear stress during submerged fermentation. Interestingly, genes encoding these wound healing proteins are found in nearly all Mucorales strains, including Rhizopus microsporus. However, the effectiveness of these proteins during submerged fermentation remains an area requiring further investigation.

[0169] In this context, our first experiment focuses on assessing the suitability of three distinct Mucorales strains - Thermomucor indicae-seudaticae deposited as Deposit ID No CBS 152105, Rhizopus microsporus, and Rhizomucor pusillus - for efficient SCP production. We propose a comprehensive evaluation methodology, including batch fermentation trials following a high shear stress pretreatment, to determine their performance and viability as SCP production strains.

[0170] Materials and methods

[0171] Preculture 150 mL precultures of a Thermomucor indicae-seudaticae deposited as Deposit ID No CBS 152105, a Rhizopus microsporus and a Rhizomucor pusillus strain were prepared in shake flasks. Briefly, a medium at pH 3.5, composed of 20 g / L yeast extract, 100 g / L glucose. laq, 13.6 g / L KH2PO4, 1 g / L Tween-80 and 10 g / L of a mineral mixture was transferred to shake flasks and inoculated with spore solutions of the respective strains. Subsequently, they were incubated for 24 hours at 49.5°C and shaken orbitally at 180 RPM.

[0172] Batch fermentation

[0173] 100 mL of the precultures was used as inoculant for batch fermentations in a 3.5L bioreactor. In treatment 1, pre-cultures were used directly as inoculant; in treatment 2, pre-cultures were blended with an immersion blender for five seconds and then immediately used in order to avoid recovery of the strain from the blending step. The remaining 50 mL of the precultures was used to determine the total biomass at time 0 (tO). All batch fermentations were performed on a minimal medium (comprising 44 g / L glucose. laq, 8.8 g / L ammonium sulphate, 10 g / L mineral solution, 1 g / L chloramphenicol (20 g / L) solution), at 49.5°C. The pH was kept at 3.5 using sulphuric acid and sodium hydroxide as titrants. After 14-17.5 hours (timeend or t end) the fermentations were terminated and the biomasses were harvested by pouring the suspensions over a cheese cloth. Subsequently, the biomasses were washed in the cheesecloth using tab water and pressed manually. Hereafter, the total biomasses were weighed and their masses corrected for their respective dry matter contents. Average growth rates were calculated with pavg= LN( X t end / X t0) / total fermentation time.

[0174] Results

[0175] As can be observed in figure 1, all three strains were able to grow in treatment 1, i.e. during the batch fermentations with the unblended precultures. For treatment 2, i.e. the blended precultures, however, reduced growth was observed by the R. microsporus and the R. pusillus, resulting in a factor 5 to 9 lower biomass. The performance of the T. indicae-seudaticae however, remained unaffected by blending.

[0176] From Figure 1, one could suspect that the growth rate of the T. indicae-seudaticae is lower compared to the other two strains. However, as the biomass concentration of the T. indicae-seudaticae was relatively low at tO, the actual average growth rate of the T. indicae-seudaticae was similar to that of the R. microsporus (figure 2). Interestingly, Figure 2 shows that the growth rates of both the blended Rhizopus and Rhizomucor were heavily affected by blending, while this was not the case for the Thermomucor strain. For the Rhizopus, the growth rate after blending was close to zero (i.e. the biomass at the end of the fermentation was the same as that of the inoculum) while that of the Rhizomucor was halved. Poor growth was reflected in inclined Cb / off gas measurements as well.

[0177] Discussion and conclusion

[0178] These results clearly indicate that the performance of the T. indicae-seudaticae is unaffected or at least much less affected by high shear stress caused by blending, whereas the performance of the R. microsporus and that of the R. pusillus is strongly inhibited by this shear stress. It is expected based on literature that all three Mucorales strains, including T. indicae-seudaticae , have the wound healing proteins encoded in their DNA, indicating that further research should be performed to understand the reason for the T. indicae-seudaticae to outperform the other two strains.

[0179] It is expected that such T. indicae-seudaticae strain with reduced susceptibility to shear stress could outperform other strains in industrial stirred tank bioreactors, where shear stress is induced by stirring, which would result in an important economical advantage. In addition, the use of such strain can also lead to more stable bioprocess conditions and a more predictable and consistent production of desired compounds or biomass.

Claims

33CLAIMS1. A method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a), wherein the fungal strain: a) is a strain deposited as Deposit ID No CBS 152105, or b) is a mutant of a strain deposited as Deposit ID No CBS 152105, wherein said mutant is capable of growing under conditions of high shear stress, or c) has at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, and is capable of growing under conditions of high shear stress.

2. The method according to claim 1, wherein said strain is a strain deposited as Deposit ID No CBS 152105.

3. The method according to claim 1 or 2, wherein the fungal strain is grown in the bioreactor under conditions of high shear stress.

4. The method according to any one of the previous claims, wherein the fungal strain is grown at a temperature higher than 45 °C and / or a pH of less than 3.8.

5. The method according to any one of the previous claims, wherein the fungal strain is grown under non-sterile conditions.

6. The method according to any one of the previous claims, wherein the fermentable feedstock is selected from co-products from one or more of the dairy, bioethanol, starch, biofuel, potato, brewing, distilling, fruit, vegetable, juice processing, alcohol, ethanol, sugar, paper, pulp, canning, wet milling, fish processing, and manure industries.

7. The method according to any one of the previous claims, wherein the SCP is subjected to downstream processing steps such as dewatering, drying, milling, or extraction to obtain a desired form or concentration.

8. A fungal strain capable of growing under conditions of high shear stress wherein the fungal strain : a) is a strain deposited as Deposit ID No CBS 152105, or34 b) is a mutant of a strain deposited as Deposit ID No CBS 152105, wherein said mutant is still capable of growing under said conditions of high shear stress, or c) has at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, and is still capable of growing under said conditions of high shear stress.

9. The fungal strain of claim 8, wherein said strain is deposited as Deposit ID No CBS 152105.

10. Use of a fungal strain according to claim 8 or 9, in the production of SCP, wherein preferably the fungal strain is grown in a medium containing a fermentable feedstock and grown in a submerged culture, preferably under condition of high shear stress, and SCP is recovered from the medium in the form of biomass of said fungal strain.

11. An SCP product comprising protein from biomass of a fungal strain according to claim 8 or 9.

12. A food or feed product comprising protein from biomass of a fungal strain according to claim 8 or 9, and / or the SCP product according to claim 11.

13. A method for producing single cell protein (SCP), the method comprising: a) growing a fungal strain of the genus Thermomucor in a bioreactor in an industrial setting under conditions of high shear stress in a medium containing a fermentable feedstock, wherein the fungal strain is grown in a submerged culture, and b) recovering SCP from the medium in the form of biomass of said fungal strain grown in step a).

14. The method of claim 13, wherein the conditions of high shear stress refer to shear stress as obtained by stirring the medium in the bioreactor at a tip speed of above 1.5 m / s, preferably with an aeration rate of 0.75 volumes of air per volume of liquid per minute (VVM) or higher.

15. The method of claim 13 or 14, wherein the fungal strain: a) is a strain deposited as Deposit ID No CBS 152105, or b) is a mutant of a strain deposited as Deposit ID No CBS 152105, wherein said mutant is still capable of growing under said conditions of high shear stress, or c) has at least 98 % genomic sequence identity with a strain deposited as Deposit ID No CBS 152105, and is still capable of growing under said conditions of high shear stress.

16. The method according to any one of claims 13-15, wherein said strain is a strain deposited as Deposit ID No CBS 152105.

17. The method according to any one of claims 13-16, wherein the shear stress is generated by agitation, stirring, blending, passing one or more gasses through the medium, or pumping of the culture medium within the bioreactor.

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